The Na+-K+-ATPase, or sodium pump, is the membrane-bound enzyme that maintains the Na+ and K+ gradients across the plasma membrane of animal cells. Because of its importance in many basic and specialized cellular functions, this enzyme must be able to adapt to changing cellular and physiological stimuli. This review presents an overview of the many mechanisms in place to regulate sodium pump activity in a tissue-specific manner. These mechanisms include regulation by substrates, membrane-associated components such as cytoskeletal elements and the γ-subunit, and circulating endogenous inhibitors as well as a variety of hormones, including corticosteroids, peptide hormones, and catecholamines. In addition, the review considers the effects of a range of specific intracellular signaling pathways involved in the regulation of pump activity and subcellular distribution, with particular consideration given to the effects of protein kinases and phosphatases.

in 1997, the Nobel Prize in Chemistry was shared by Danish researcher Jens C. Skou for his discovery of the Na+-K+-ATPase. Although the existence of an active “sodium pump” had been previously hypothesized, Skou was the first to suggest, in 1957, a link between transport of Na+ and K+ across the plasma membrane and a Na+- and K+-activated ATPase activity (307). The significance of this discovery is underscored by the subsequent publication, each year, of scores of reports relevant to various aspects of Na+-K+-ATPase structure and function. Although much information about the enzyme has become available in the years since its discovery, one area of pump research that is not completely understood, despite recent advances, is that of pump regulation.

The basic function of the Na+-K+-ATPase, or sodium pump, is to maintain the high Na+ and K+gradients across the plasma membrane of animal cells. In particular, the sodium pump is the major determinant of cytoplasmic Na+. As such, it has an important role in regulating cell volume, cytoplasmic pH and Ca2+ levels through the Na+/H+ and Na+/Ca2+exchangers, respectively, and in driving a variety of secondary transport processes such as Na+-dependent glucose and amino acid transport. The sodium pump, in turn, is the target of multiple regulatory mechanisms activated in response to changing cellular requirements. The requirement for modulators of the Na+-K+-ATPase is likely to be greatest in tissues in which perturbations of the intracellular alkali cation content underlie their specialized functions, in addition to those processes mentioned above (see below for specific references). Prime examples are the changes in sodium pump activity that occur in response to physiological stimuli such as nerve impulse propagation, exercise, and changes in diet. Expression of various isoforms of the sodium pump may fulfill some of the requirements for altered pump behavior (for recent discussion, see Ref. 42). However, direct tissue-specific modulation of the enzyme also underlies mechanisms of pump regulation.

One of the primary needs for sodium pump adaptation comes from changes in dietary Na+ and K+. The mediators of natriuresis and diuresis, namely, hormones that control the volume and ionic composition of blood and urine, often act directly on the sodium pump of the kidney and intestine. The function of the pump in absorption or reabsorption of Na+ and K+ and, secondarily, other solutes, requires tight regulation of the enzyme to maintain normal levels of Na+ and K+ during altered salt intake (for reviews, see Refs. 101, 160). In addition, because water and Na+ transport across epithelia are invariably linked, the work of the sodium pump is also critical to water absorption in the intestine and reabsorption in the kidney. Illustrating this are reports

that impairment of the sodium pump in kidney and small intestine can be associated with the pathophysiology of hypertension (168) and chronic diarrhea (123), respectively.

In excitable tissues such as neurons (141), skeletal muscle cells (82), and pacemaker fibers of the heart (320), the sodium pump must reestablish the electrical potential across the plasma membrane following excitation-induced depolarization. Although part of this function is undoubtedly fulfilled by the presence and distinct kinetics of the α3-isoform in neurons, regulatory events are also likely to be involved as evidenced by the multiple effects of various hormones on Na+-K+-ATPase activity in these tissues. In skeletal muscle, regulation of sodium pump activity has widespread physiological implications. Continuous stimulation of muscle fibers during exercise leads to dissipation of the cation gradient necessary for muscle contraction. To offset excessive release of K+from the muscle cells, rapid activation of Na+-K+-ATPase activity under these conditions is an essential means of delaying the onset of muscular fatigue and reducing potentially toxic levels of plasma K+.

Na+-K+-ATPase regulation in cardiac muscle is particularly critical to the myocardium, where the enzyme acts as an indirect regulator of contraction (45). Thus the sodium pump controls the steady-state cytoplasmic Na+concentration, which then determines Ca2+ concentration via the Na+/Ca2+ exchanger. Ca2+, in turn, is pumped into the sarcoplasmic reticulum (SR) by the sarco(endo)plasmic reticulum calcium (SERCA) pumps. Regulation of the sodium pump in these tissues is therefore paramount for determining the “set point” for cardiac muscle contraction and the steady-state contraction of vascular smooth muscle. Physiological regulators that act in a manner analogous to that ascribed to cardiac glycoside inhibitors of the Na+-K+-ATPase are likely to be critical for normal heart contraction. The aforementioned mechanism of increasing the force of contraction via increasing cell Na+ is considered to be the basis of digitalis therapy for cardiac insufficiency (329).

This monograph focuses on mechanisms of tissue-specific regulation of the sodium pump, with emphasis on two areas. One deals with mechanisms involving signaling pathways that result in modulations in pump activity, and the other deals with the regulation resulting from the interaction of the pump complex with other membrane components, which, in turn, may or may not be subject to modulation via other signaling cascades.

SUBSTRATE CONCENTRATIONS AS DETERMINANTS OF PUMP ACTIVITY

The simplest and most straightforward determinants of pump activity are the concentrations of substrates. The sodium pump is activated by Na+ and ATP at cytoplasmic sites and by K+ at extracellular sites. The most dramatic effects involve variations in cytoplasmic Na+ concentration. Half-maximal activation of the enzyme by intracellular Na+occurs at concentrations of ∼10–40 mM, which, depending on the tissue, are often at or above the steady-state Na+concentration (for example, see Ref. 309). Accordingly, small changes in the cytoplasmic Na+ concentration secondary to activation of either various Na+-dependent transporters or Na+ channels can have dramatic effects on sodium pump activity. As described below, some hormones

appear to alter sodium pump activity by changing its apparent affinity for Na+ (K Na ′). Aside from its direct effects on the Na+-K+-ATPase, Na+ has been shown to induce other mechanisms of upregulation of the sodium pump. For example, Na+ influx is thought to be the first signal leading to an increase in surface sodium pumps in one kind of aldosterone-mediated short-term regulation (302).

Whereas the high affinity of the enzyme for K+ at activating sites generally precludes an effect of variations in extracellular K+ concentrations on sodium pump activity except, perhaps, in some neuronal tissues (318), K+ has been shown to act as a competitive inhibitor of Na+ binding at cytoplasmic sites (134). Therefore, variation in cytoplasmic K+ concentration, or, more likely, in the affinity of the enzyme for K+ as an antagonist at cytoplasmic Na+-activating binding sites, is a plausible mechanism for determining the set point for the physiological concentration at half-maximal activation (K 0.5) for cytoplasmic Na+activation (327).

Because the K 0.5 of the Na+-K+-ATPase for ATP is between 300 and 800 mM (310), the ATP concentration in most cells is saturating for the enzyme. However, in some tissues and under certain conditions, ATP levels may fall to subsaturating levels. For example, cells of the kidney medulla are known to function under near anoxic conditions (56), and such conditions can lead to dramatic drops in ATP levels (310). Thus variations in ATP concentration or in the affinity of the sodium pump for ATP may be physiologically relevant mechanisms of pump regulation in this tissue.

MEMBRANE-ASSOCIATED COMPONENTS

Because the Na+-K+-ATPase is a membrane-embedded protein, the nature of constituents comprising the membrane components should be an important determinant of enzyme function. Unfortunately, this is an unclear aspect of pump research due mainly to the difficulty in separating such components from the enzyme complex. As a first step toward gaining some insight into the question of whether and to what extent tissue- rather than isoform-specific differences in kinetic pump behavior reflect pump modulation by components of the membrane, Munzer and co-workers (240,241) examined the kinetic behavior of kidney pumps delivered by polyethylene glycol-mediated fusion into another (red blood cell) environment. In the case of pumps incorporated into genetically low-K+ (LK) red blood cells, they obtained unequivocal evidence of kinetic changes effected by the Lpantigen of these red cells (see below; Ref. 353). Using the same membrane fusion system, Therien and Blostein (324) recently showed that the membrane environment has highly specific effects on the interaction of kidney pumps with Na+ and K+ on the cytoplasmic side; specifically, fusion of kidney pumps into dog red blood cells abrogates, at least partly, the relatively high susceptibility of kidney α1 pumps to K+/Na+ antagonism at cytoplasmic cation activation sites.

In general, there is little information on the nature and mechanistic basis of sodium pump modulation by specific membrane components. Many reports have focused on the role of membrane lipids. The main effects of lipids on the sodium pump are related to membrane fluidity and thickness. In general, lipids that promote bilayer formation of physiological thickness and increased fluidity tend to promote optimal Na+-K+-ATPase activity (172,186, 221), as do negatively charged lipids such as phosphatidylserine and phosphatidylglycerol (187). The effects of cholesterol on

enzyme activity are often also related to membrane fluidity (140), although specific effects of cholesterol on the sodium pump have been reported (356). Free fatty acids present in the membrane or as the products of phospholipase A2 (PLA2)-dependent regulatory pathway tend to inhibit the Na+-K+-ATPase (254).

The Lp Blood Group Antigen

A striking and mechanistically well-characterized tissue-specific modulator of the Na+-K+-ATPase is the Lp antigen of LK ruminant red cells, in particular those of sheep. The Lp antigen is so called because of its association with the L blood group antigens and its highly specific effects on the sodium pump (reviewed in Ref. 103). Evidence for the existence of this inhibitor was derived from studies on the effects of an antiserum specific for the Lp antigen; treatment with anti-Lp stimulates Na+-K+-ATPase of LK, but not of high-K+ (HK), erythrocytes (104). In addition, trypsinization of intact cells reverses the effects of anti-Lp (199), providing evidence that the inhibitor is a peptide distinct from the sodium pump itself and that the anti-Lp epitope is removed upon trypsin treatment. Experiments using anti-Lp and trypsin have led to a model of Lp-mediated inhibition of Na+-K+-ATPase whereby the antigen inhibits sodium pump activity in two distinct ways. One is secondary to an Lp antigen-induced increase in the susceptibility of pumps to noncompetitive inhibition by K+ (102) and the other to an increase in pump protein turnover during red cell maturation (352). In the pump/red cell fusion experiments mentioned above, it was observed that rat kidney pumps fused into LK red blood cells were stimulated by anti-Lp, providing unequivocal proof that the Lp antigen is a molecular entity distinct from the sodium pump. However, the exact molecular nature of the protein remains unknown.

Components of the Cytoskeleton

Interactions of the Na+-K+-ATPase with components of the cytoskeleton of cells are well documented. Specific cytoskeletal proteins thought to interact with the sodium pump, either directly or indirectly, include spectrin (182), actin (190), adducin (330), pasin (193), and ankyrin (245). Generally, ankyrin appears to mediate associations between the sodium pump and other cytoskeletal proteins, although direct associations of the enzyme with pasin and actin have also been observed. The two specific domains of the sodium pump that interact with ankyrin have been recently identified (96, 361). Of these, residues in the first cytoplasmic domain (142–166 of the rat α1-isoform) are especially intriguing because this region is highly conserved in all sodium pump isoforms and in H,K- and Ca2+-ATPases, suggesting interactions of these P-type ion pumps with ankyrin. Ankyrin binding to the second cytoplasmic loop is likely mediated by a four-residue motif (ALLK) that has homology to a sequence of the anion exchanger, another ankyrin-binding transporter (174).

The main consequence of interactions between the Na+-K+-ATPase and the cytoskeleton is believed to be the correct processing and targeting of sodium pumps to the appropriate membrane compartment. For example, disruptions in the cellular distribution of Na+-K+-ATPase, induced either by ATP depletion or hypoxia, are linked to alterations in cytoskeletal proteins (233, 262), and a spectrin-ankyrin complex is required for transport of pumps from the endoplasmic reticulum to the Golgi apparatus (97). Recently, a role for cytoskeletal proteins in regulating sodium pump activity has been suggested. For example, monomeric, but not polymerized,

actin has been shown to activate the sodium pump by a mechanism mediated by cAMP-dependent protein kinase (PKA) (60, 61). In addition, mutant forms of adducin have been shown to stimulate Na+-K+-ATPase activity in transfected NRK-52E cells (330).

The identification of genetic polymorphisms in adducin in Milan hypertensive strain rats and in humans has led Manunta et al. (219) to suggest that adducin variants may affect kidney function by modulating the overall cation transport in renal epithelia, both by affecting assembly of the cytoskeleton and by modulating sodium pump activity. In a recent report, they showed that both rat and human adducins stimulate Na+-K+-ATPase activity by increasing the apparent affinity for ATP (114). Interestingly, the mechanism appears to involve acceleration of the rate of the conformational change E2(K) → E1(Na) or E2(K).ATP → E1Na.ATP. Stimulation is specific in that a stimulatory effect noted also with ankyrin, which also binds Na+-K+-ATPase, is not additive. In general, these findings suggest a specific interaction between adducin and the Na+-K+-ATPase of the kidney. It is intriguing that the effect is similar to that effected by the γ-subunit of the pump (see below). Whether interaction of adducin with the pump involves the γ-subunit is relevant to the modulatory effect of adducin remains to be determined.

The γ-Subunit

The γ-subunit is a small transmembrane protein that specifically associates with the Na+-K+-ATPase in a tissue-specific manner. Though its existence had been previously suggested (282), it was Forbush and co-workers (124) who first demonstrated that this small hydrophobic peptide is specific to the sodium pump by showing that it is specifically labeled, along with the α-subunit, by a photoactive derivative of ouabain. Although the peptide was at first thought to represent a third component of the Na+-K+-ATPase, recent evidence suggests that it is not an integral part of the enzyme complex.

Following the report of Forbush et al. (124), who studied the pig enzyme, experiments using various ouabain derivatives resulted in the identification of a small sodium pump-associated proteolipid in various tissues (151, 214, 284,286). This peptide, initially referred to as “γ component” or “γ-subunit” (281), appeared to be present in approximately equimolar amounts compared with the α- and β-subunits (85, 155). The initial molecular cloning experiments indicated that the γ-subunit consisted of 58 amino acids and had a molecular mass of ∼6,500 Da (228). Since then, cDNAs for the human (185) and Xenopus laevis (25) γ-subunits have also been cloned and sequenced. Sequence comparisons show strong homology (75%) among different species, which is further increased to 93% when only mammalian sequences are compared. Structural analysis has revealed that the γ-subunit contains a single transmembrane domain, with an NH2 terminus-out, COOH terminus-in topology (25, 325). The NH2 terminus, at least that of the rat sequence, has since been shown to be somewhat longer and different than originally reported. (For details, see Ref. 326 and GenBank accession no.AF129400.1).

An intriguing feature of the γ-subunit structure is that it is detected as two species with similar amino acid composition irrespective of the protein separation methods used (for examples, see Refs. 85, 228, 304). Early evidence suggested that the two

bands detected on Western blots, henceforth referred to as γaand γb, are the products of a single mRNA species (228). Béguin and co-workers (25) later showed that in Xenopus, the two bands of γ are due to alternate usage of two distinct start codons in the γ-subunit message; only one appears relevant in vivo in this species. However, recent mass spectrometry analysis of the rat protein indicated that γa and γb are variants, most likely splice variants (194). γa has a mass of 7,184 Da, whereas the faster migrating γb species has a mass of 7,354 Da and contains only a different NH2 terminus (6- replacing 7-residues). In fact, their amino acid sequences indicate that they correspond exactly to two splice variants contained in the expressed sequence tag database as noted by Sweadner et al. (315). Recent expression studies show clearly that the γa and γb protein products of transcription/translation have the same mobilities as the upper and lower bands, respectively, of the kidney medulla (195). Depending on the cell line used, additional bands, presumably the products of posttranslational modification, are seen, namely, γ′a with higher apparent mobility than γa in HEK and γ′b with lower mobility than γb in HeLa, whereas only γaand γb are detected in HeLa and HEK, respectively.

The expression of γ-subunit mRNA has been investigated by Northern blot analysis in the rat, human, and X. laevis, and it was shown that the peptide is expressed in a tissue-specific manner in these species. Thus, in humans, γ-subunit mRNA was detected in kidney, pancreas, and fetal liver (185), and inXenopus, it was detected mainly in kidney and stomach, with trace amounts in heart, skin, and oocytes (25). In rats, the situation is more complex, because two distinct mRNA species were detected by using the rat γ-subunit cDNA as a probe (228). The larger of the two, at 1.5 kb, corresponds in size to the Xenopus mRNA and was detected mainly in kidney and spleen, with lower amounts in lung, heart, and brain. The smaller transcript migrated at 0.8 kb, a size similar to that of human γ-subunit message, and was detected at high levels in the kidney and at very low levels in the spleen, lung, and heart. Also in the rat, Therien and co-workers (325, 326) have recently shown that at the protein level, the γ-subunit is expressed only in kidney tubules, with very low levels found in the spleen.

Most available data indicate that the γ-subunit is not expressed at the plasma membrane without the Na+-K+-ATPase, except perhaps in very early development, as described below. For example, γ-subunit is expressed at the surface of Xenopusoocytes only on coinjection of cRNA for the α- and β-subunits (25); immunocytochemical analysis has shown that the expression patterns of α- and γ-subunits are identical in renal proximal tubules and collecting ducts (228), although γ-subunit appears to be absent in other parts of the kidney (13, 325). In addition, coimmunoprecipitation of the γ-subunit with both the α- and β-subunits has been demonstrated (228). On the other hand, in their study on the role of the γ-subunit in mouse blastocyst development, Jones and co-workers (173) have shown that the γ-subunit is expressed at high levels at the apical membrane, whereas the α- and β-subunits are present only at the basolateral membrane.

The first attempts at defining a functional role for the γ-subunit indicated that this peptide is not essential for normal enzyme function. For example, Hardwicke and Freytag (155) were able to show that separation of the γ-peptide from the αβ complex by nonionic detergent solubilization of shark rectal gland and avian salt gland membranes had no effect on Na+-K+-ATPase activity. More recently, it has been

shown that the presence of the γ-subunit is not necessary for functional expression of the sodium pump in insect cells (95), Xenopus oocytes (25), and yeast (296). In the latter system, the γ-subunit was shown to have no effect on either ouabain-sensitive Na+-K+-ATPase activity or86Rb+ influx. The failure to detect γ-subunit mRNA (25, 185, 228) or protein (325) in many tissues also supports the notion that the γ-subunit is not an essential component of the Na+-K+-ATPase.

Recent experiments have shown that the γ-subunit has a potentially important functional role in some systems. Treatment of mouse blastocysts with γ-subunit antisense oligodeoxynucleotide reduced the amount of expressed γ-subunit and caused a reduction in ouabain-sensitive 86Rb+ transport as well as delayed blastocoele formation (173). In experiments on cRNA-injected Xenopus oocytes, the γ-subunit has been shown to influence the apparent affinity of the Na+-K+-ATPase for K+ in a complex Na+- and voltage-dependent fashion (25), although the interpretation of these results remains unclear. A role of the γ-subunit in interactions of the Na+-K+-ATPase with K+ had previously been suggested by Or et al. (259), who showed that the γ-subunit is a component of the protein complex found in so-called “19-kDa membranes.” Such membranes are formed by tryptic digestion following occlusion of K+ (or Rb+) by the enzyme to form E2(K) (181). More recently, Arystarkhova et al. (13) reported that the γ-subunit decreases both Na+ and K+ affinities of the sodium pump when transfected into NRK-52E cells transfected with γa cDNA (13). However, the decrease in Na+ affinity is difficult to reconcile with the following:1) the increase in K′Na(∼10-fold) is much larger than that (2-fold) observed for kidney compared with γ-subunit-free tissues (324,327) if one takes into account the level of expression, and 2) a change in K′Na could only be detected with cells expressing both γ′aand γa, and not γa alone, despite the finding (195) that γ′a appears to be a cell-specific modification of γa. In another recent report, the human γ-subunit has been shown to induce ouabain-independent ion currents in injected Xenopus oocytes and 86Rb+ and 22Na+influx in baculovirus-infected Sf-9 cells (231). As described below, it is unclear whether this channel-like function is physiologically relevant, an artifact of high-level expression, or peculiar to human γ-subunit, for which the primary sequence at the extracellular amino terminus is notably different from that for several other species (231).

In addition to the aforementioned studies on baculovirus-infected Sf-9 cells, cRNA-injected Xenopus oocytes, and transfected NRK-52E cells, the possible functional role of the γ-subunit has recently been investigated in human HeLa and HEK cells. The initial approach was to test what effects, if any, an anti-γ antiserum had on the function of the sodium pump of rat kidney. A specific effect was evidenced in the finding that anti-γ inhibits Na+-K+-ATPase turnover in kidney, but not in tissues that do not express γ-subunit (325), and that a peptide corresponding to the epitope of the antiserum can abrogate the effect (326). Further analysis of the functional effects of anti-γ showed that it stabilizes the E2 form(s) of the enzyme. Thus the pH-dependence of the anti-γ-mediated inhibition of activity, together with the observation that Rb+ protects against tryptic digestion of the γ-peptide (325), are consistent with a role of anti-γ in shifting the equilibrium of the K+-deocclusion reaction [E2(K) ↔ E1] toward E2(K). On the basis of the well-documented effects of anti-Lp antigen on the kinetics of the LK sheep red blood cell Na+-K+-ATPase (see above), it was hypothesized that anti-γ mediates its effects by disrupting interactions between the Na+-K+-ATPase complex and the γ-subunit such that the role of the γ-subunit is to shift the aforementioned equilibrium toward E1. By transfecting the γ-subunit into HEK cells,

it was recently shown that this is indeed the case (326). These experiments with transfected cells showed that the γ-subunit stabilizes the E1 conformation of the Na+-K+-ATPase by increasing the affinity of the enzyme for ATP at its low-affinity site and that anti-γ reverses this increase in affinity in transfected cells (326). These findings, taken together with the observation that inhibition of Na+-K+-ATPase activity by anti-γ in the renal enzyme was increased at subsaturating concentrations of ATP, provide strong support for the conclusion that anti-γ reverses γ-subunit-mediated effects. It should be noted that a γ-subunit-mediated increase in the affinity of the enzyme for ATP may lead to a secondary decrease in its apparent affinity for K+ (328), which would agree with the results of Arystarkhova et al. (13) regarding γ-subunit-mediated decrease in K+ affinity. However, it is likely to be the change in ATP affinity that is physiologically important, as described below.

What is the physiological importance of a regulator of the affinity of the sodium pump for ATP? In most cells, ATP levels are sufficient to saturate the Na+-K+-ATPase, and therefore a modest shift in ATP affinity should not have dramatic effects. However, there are cases where ATP levels in intact cells are dramatically lowered, such as during anoxic shock. The relationship between anoxia, or hypoxia, and cellular ATP concentration has been studied in many tissues (15, 171, 191,210, 230, 260,310). As might be expected, dramatic decreases in ATP levels (30–90%) have been reported following brief periods of oxygen and/or glucose deprivation. In many cases, ATP concentration under anoxic conditions falls to a value that will affect Na+-K+-ATPase activity, assuming aK′ATP of 400–800 mM (310). For example, Koop and Cobbold (191) estimated that chemical hypoxia lowers the concentration of ATP in intact hepatocytes to 50–100 μM. In addition, Milusheva et al. (230) reported that incubation of rat striatal brain slices under glucose-free, hypoxic conditions for a relatively short period of time (30 min) can decrease cytoplasmic ATP levels to 10% of control, which, even assuming a relatively high starting concentration of 5 mM, translates to <500 μM. Finally, a direct correlation between hypoxia and sodium pump activity was provided by Aw and Jones (15), who observed a near total inhibition of sodium pump-mediated Rb+ uptake in hepatocytes under conditions where ATP levels dropped a mere 40%. It might be argued that in the aforementioned studies, anoxia was induced artificially, and that such conditions may not be relevant to situations in vivo. However, recent studies have shown that even in normal, disease-free organisms, at least one tissue, the kidney medulla, must function under near-anoxic conditions (reviewed in Refs. 56, 81). As is the case in most segments of the nephron, water and solute reabsorption and secretion in the medulla are under the control of the sodium pump. As such, continued pumping is crucial for proper kidney function. Therefore, the existence of a reversible regulator of Na+-K+-ATPase ATP affinity would allow for fine tuning of sodium pump activity under ATP-depleted conditions. This regulator should alter the affinity of the pump for the nucleotide only moderately, because an excessive increase would effect even greater decreases in ATP concentration (310), leading to compromised cell viability.

The γ-Subunit as a Member of a Family of Proteins

In recent years, several small single-transmembrane-domain peptides with high sequence homology to the γ-subunit have been identified. As such, studies on these peptides may reveal interesting information on the structure and function of the γ-subunit. To date, in addition to the γ-subunit itself, three members of this family have

been cloned: phospholemman (PLM) (263), channel-inducing factor (CHIF) (14), and mammary tumor-associated 8-kDa protein (Mat-8) (237). Cloned sequences of these peptides include PLM of the mouse (48), dog (263), rat, and human (72), CHIF of the rat (14), and Mat-8 of the human (238) and mouse (237). Two additional sequences with homology to the γ-subunit family of proteins are known, namely, a “phospholemman-like protein” in humans (HPLP; Ref. 17), and a “regulated ion channel homologue” (RIC; Ref. 128) in the mouse. The amino acid sequences of the rat γ-subunit, CHIF, PLM, and mouse Mat-8 are compared in Fig. 1. For the rat γ-subunit, the revised sequence of γa is shown (231, 326), whereas for PLM, CHIF, and Mat-8, the sequences for the mature proteins, after cleavage of their putative signal peptide (see below), are shown. As illustrated in Fig.1, the latter three proteins have 38–43% homology with the γ-subunit, and this value increases to 74–80% in the transmembrane domain and immediate flanking sequences (P18to C52 of the rat γ-subunit). There are several highly conserved motifs present in most of the known sequences of this family of proteins. With the use of numbering for the rat γ-subunit, these motifs include 1) P18FXYD in the extracellular domain, 2) G29G in the transmembrane domain, and3) S47X(R/K)C(R/K)C flanking the transmembrane domain on the cytoplasmic side. It should be noted, however, that in the γ-subunit, the third motif described above contains a Phe residue instead of the first Cys. Interestingly, Gly-30, Gly-41, and Ser-47 are 100% conserved among all known sequences. Of these, Ser-47 is especially intriguing because the nearby presence of positive charges (either K or R) make it a possible target for phosphorylation by protein kinase C (PKC).

Researchers have established the structure of a crucial enzyme -- the so-called sodium-potassium pump -- which forms part of every cell in the human body. The result may pave the way for a better understanding of neurological diseases.

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The figure shows the tunnel-like entry point to the binding sites of the sodium-potassium pump in the sodium-bound state. The three small sodium ions are bound inside the pump (violet spheres to the left), whereas there is not sufficient room for the larger potassium ions (green spheres to the right). The blue web shows the inner surface of the protein blocking the potassium ions. The letter code indicates the amino acids shown in the pump, which are essential to the binding process.

Credit: Bente Vilsen and Flemming Cornelius

[Click to enlarge image]

It's not visible to the naked eye and you can't feel it, but up to 40 per cent of your body's energy goes into supplying the microscopic sodium-potassium pump with the energy it needs. The pump is constantly doing its job in every cell of all animals and humans. It works much like a small battery which, among other things, maintains the sodium balance which is crucial to keep muscles and nerves working.

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The sodium-potassium pump transports sodium out and potassium into the cell in a fixed cycle. During this process the structure of the pump changes. It is well-established that the pump has a sodium and a potassium form. But the structural differences between the two forms have remained a mystery, and researchers have been unable to explain how the pump distinguishes sodium from potassium.

Structure solves the mystery

Thanks to the international collaboration between Professor Chikashi Toyoshima's group at the University of Tokyo and researchers from Aarhus University, the structure of the sodium-bound form of the protein has now been described. For the first time ever, the sodium ions can be studied at a resolution so high - 0.28 nanometres - that researchers can actually see the sodium ions and observe where they bind in the structure of the pump. In 2000, Professor Chikashi Toyoshima's group described the structure of a calcium-pump for the first time, and in 2007 and 2009 research groups from Aarhus University and Toyoshima's group described the potassium-bound form of the sodium-potassium pump.

"The new protein structure shows how the smaller sodium ions are bound and subsequently transported out of the cell, whereas the access of the slightly larger potassium ions is blocked. We now understand

how the pump distinguishes between sodium and potassium at the molecular level. This is a great leap forward for research into ion pumps and may help us understand and treat serious neurological conditions associated with mutations of the sodium-potassium pump, including a form of Parkinsonism and alternating hemiplegia of childhood in which sodium binding is defective," explains Bente Vilsen, a professor at Aarhus University who spearheaded the project's activities in Aarhus with Associate Professor Flemming Cornelius.

Impressed Nobel Prize winner

The vital pump was discovered in 1957 by Professor Jens Christian Skou of Aarhus University, who received the Nobel Prize for his discovery in 1997. The new result is the culmination of five or six decades of research aimed at the mechanism behind this vital motor of the cells.

"Years ago, when the first electron microscopic images were taken in which the enzyme was but a millimetre-sized dot at 250,000 magnifications, I thought, how on earth will we ever be able to establish the structure of the enzyme. The pump transports potassium into and sodium out of the cells, so it must be capable of distinguishing between the two ions. But until now, it has been a mystery how this was possible," says retired Professor Jens Christian Skou, who - even at 94 years of age - keeps up to date with new developments in the field of research which he initiated more than 50 years ago.

"Now, the researchers have described the structure that allows the enzyme to identify sodium and this may pave the way for a more detailed understanding of how the pump works. It is an impressive achievement and something I haven't even dared dream of," concludes Jens Christian Skou.

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The above story is based on materials provided by Aarhus University. Note: Materials may be edited for content and length.

From Wikipedia, the free encyclopedia

Flow of ions.

Alpha and beta units.

Sodium-potassium pump, E2-Pi state. Calculated hydrocarbon boundaries of the lipid bilayer are shown as blue (intracellular) and red (extracellular) planes

Na+

/K+

-ATPase (Sodium-potassium adenosine triphosphatase, also known as Na+

/K+

pump, sodium-potassium pump, or sodium pump) is an antiporter enzyme (EC 3.6.3.9) (an electrogenic transmembrane ATPase) located in the plasma membrane of all animal cells. The Na+

/K+

-ATPase enzyme pumps sodium out of cells, while pumping potassium into cells.

Contents

1 Sodium-potassium pumps 2 Function

o 2.1 Resting potential o 2.2 Transport o 2.3 Controlling cell volume o 2.4 Functioning as signal transducer o 2.5 Controlling neuron activity states

3 Mechanism 4 Regulation

o 4.1 Endogenous o 4.2 Exogenous

5 Discovery 6 Genes 7 See also 8 References 9 Additional images 10 External links

Sodium-potassium pumps

Active transport is responsible for cells' containing relatively high concentrations of potassium ions but low concentrations of sodium ions. The mechanism responsible for this is the sodium-potassium pump, which moves these two ions in opposite directions across the plasma membrane. This was investigated by following the passage of radioactively labeled ions across the plasma membrane of certain cells. It was found that the concentrations of sodium and potassium ions on the two sides of the membrane are interdependent, suggesting that the same carrier transports both ions. It is now known that the carrier is an ATP-ase and that it pumps three sodium ions out of the cell for every two potassium ions pumped in.

The sodium-potassium pump was discovered in the 1950s by a Danish scientist, Jens Christian Skou, who was awarded a Nobel Prize in 1997. It marked an important step forward in our understanding of how ions get into and out of cells, and it has a

particular significance for excitable cells such as nervous cells, which depend on this pump for responding to stimuli and transmitting impulses.

Function

The Na+

/K+

-ATPase helps maintain resting potential, avail transport, and regulate cellular volume.[1] It also functions as signal transducer/integrator to regulate MAPK pathway, ROS, as well as intracellular calcium. In most animal cells, the Na+

/K+

-ATPase is responsible for about 1/5 of the cell's energy expenditure.[citation needed] For neurons, the Na+

/K+

-ATPase can be responsible for up to 2/3 of the cell's energy expenditure.[2]

Resting potential

See also: Resting potential

In order to maintain the cell membrane potential, cells keep a low concentration of sodium ions and high levels of potassium ions within the cell (intracellular). The sodium-potassium pump moves 3 sodium ions out and moves 2 potassium ions in, thus in total removing one positive charge carrier from the intracellular space. Please see Mechanism for details.

The action of the sodium-potassium pump is not the only mechanism responsible for the generation of the resting membrane potential. Also, the selective permeability of the cell's plasma membrane for the different ions plays an important role. All mechanisms involved are explained in the main article on generation of the resting membrane potential.

Transport

Export of sodium from the cell provides the driving force for several secondary active transporters membrane transport proteins, which import glucose, amino acids, and other nutrients into the cell by use of the sodium gradient.

Another important task of the Na+

-K+

pump is to provide a Na + gradient that is used by certain carrier processes. In the gut, for example, sodium is transported out of the reabsorbing cell on the blood (interstitial fluid) side via the Na+

-K+

pump, whereas, on the reabsorbing (luminal) side, the Na+

-Glucose symporter uses the created Na+

gradient as a source of energy to import both Na+

and glucose, which is far more efficient than simple diffusion. Similar processes are located in the renal tubular system.

Controlling cell volume

Failure of the Na+

-K+

pumps can result in swelling of the cell. A cell's osmolarity is the sum of the concentrations of the various ion species and many proteins and other organic compounds inside the cell. When this is higher than the osmolarity outside of the cell, water flows into the cell through osmosis. This can cause the cell to swell up and lyse. The Na+

-K+

pump helps to maintain the right concentrations of ions. Furthermore, when the cell begins to swell, this automatically activates the Na+

-K+

pump.[citation needed]

Functioning as signal transducer

Within the last decade [when?], many independent labs have demonstrated that, in addition to the classical ion transporting, this membrane protein can also relay extracellular ouabain-binding signalling into the cell through regulation of protein tyrosine phosphorylation. The downstream signals through ouabain-triggered protein phosphorylation events include activation of the mitogen-activated protein kinase (MAPK) signal cascades, mitochondrial reactive oxygen species (ROS) production, as well as activation of phospholipase C (PLC) and inositol triphosphate (IP3) receptor (IP3R) in different intracellular compartments.[3]

Protein-protein interactions play a very important role in Na+

-K+

pump-mediated signal transduction. For example, Na+

-K+

pump interacts directly with Src, a non-receptor tyrosine kinase, to form a signaling receptor complex.[4] Src kinase is inhibited by Na+

-K+

pump, while, upon ouabain binding, the Src kinase domain will be released and then activated. Based on this scenario, NaKtide, a peptide Src inhibitor derived from Na+

-K+

pump, was developed as a functional ouabain-Na+

-K+

pump-mediated signal transduction.[5] Na+

-K+

pump also interacts with ankyrin, IP3R, PI3K, PLC-gamma and cofilin.[6]

Controlling neuron activity states

The Na+

-K+

pump has been shown to control and set the intrinsic activity mode of cerebellar Purkinje neurons.[7] This suggests that the pump might not simply be a homeostatic, "housekeeping" molecule for ionic gradients; but could be a computation element in the cerebellum and the brain. Indeed, a mutation in the Na+

-K+

pump causes rapid onset dystonia parkinsonism, which has symptoms to indicate that it is a pathology of cerebellar computation.[8] Furthermore, an ouabain block of Na+

-K+

pumps in the cerebellum of a live mouse results in it displaying ataxia and dystonia.[9] The distribution of the Na+

-K+

pump on myelinated axons, in human brain, was demonstrated to be along the internodal axolemma, and not within the nodal axolemma as previously thought.[10]

Mechanism

The pump, while binding ATP, binds 3 intracellular Na+

ions.[1]

ATP is hydrolyzed, leading to phosphorylation of the pump at a highly conserved aspartate residue and subsequent release of ADP.

A conformational change in the pump exposes the Na+

ions to the outside. The phosphorylated form of the pump has a low affinity for Na+

ions, so they are released. The pump binds 2 extracellular K +

ions. This causes the dephosphorylation of the pump, reverting it to its previous conformational state, transporting the K+

ions into the cell. The unphosphorylated form of the pump has a higher affinity for Na+

ions than K+

ions, so the two bound K+

ions are released. ATP binds, and the process starts again.

Regulation

Endogenous

The Na+

/K+

-ATPase is upregulated by cAMP.[11] Thus, substances causing an increase in cAMP upregulate the Na+

/K+

-ATPase. These include the ligands of the Gs-coupled GPCRs. In contrast, substances causing a decrease in cAMP downregulate the Na+

/K+

-ATPase. These include the ligands of the Gi-coupled GPCRs.

Note: Early studies indicated the opposite effect, but these were later found to be inaccurate due to additional complicating factors.[citation needed]

Exogenous

The Na+

-K+

-ATPase can be pharmacologically modified by administrating drugs exogenously.

For instance, Na+

-K+

-ATPase found in the membrane of heart cells is an important target of cardiac glycosides (for example digoxin and ouabain), inotropic drugs used to improve heart performance by increasing its force of contraction.

Contraction of any muscle is dependent on a 100- to 10,000-times-higher-than-resting intracellular Ca 2+ concentration, which, as soon as it is put back again on its normal level by a carrier enzyme in the plasma membrane, and a calcium pump in sarcoplasmic reticulum, muscle relaxes.

Since this carrier enzyme (Na+

-Ca2+

translocator) uses the Na gradient generated by the Na+

-K+

pump to remove Ca2+

from the intracellular space, slowing down the Na+

-K+

pump results in a permanently elevated Ca2+

level in the muscle, which may be the mechanism of the long-term inotropic effect of cardiac glycosides such as digoxin.

Discovery

Na+

/K+

-ATPase was discovered by Jens Christian Skou in 1957 while working as assistant professor at the Department of Physiology, University of Aarhus, Denmark. He published his work that year.[12]

In 1997, he received one-half of the Nobel Prize in Chemistry "for the first discovery of an ion-transporting enzyme, Na+

, K+

-ATPase."[13]

Vasopressin or anti diuretic hormone (ADH) is released from the posterior pituitary gland in response to low blood volume. ADH synthesis occurs at the supraoptic nucleus of the hypothalamos and is storage at the posterior pituitary. This hormone has three main functions: increase water reabsorption, vasoconstriction, and promotes release of coagulation factor VIII. The first function is achieved via activation of the V2 receptors at the level of the renal tubules. Via a G-protein couple pathway ADH increases the expression and function of the aquaporins, special proteins at the luminal side of the renal tubules that work in water reabsorption. The second function of ADH is achieved by activation of V1 receptors in the blood vessels. The activation of these receptors cause an increase in the intracellular calcium levels and as a result constriction of the smooth muscle cells of the blood vessels, causing vasoconstriction or increase in the peripheral vascular resistance.  Through these mechanisms ADH helps in the maintenance of a ‘normal’ blood volume and osmolarity and in the regulation of blood pressure. Diabetes insipidus (DI) is a pathological condition where there is diminished response to ADH. It can be secondary to a decreased release of the hormone (central or neurogenic DI) or to an inadequate response at the kidneys (nephrogenic DI). Patients with DI will have an increased urinary volume, and the urine will be hypotonic in comparison with the serum osmolarity. 

 

ADH is synthetised in the hypothalamus & is transported to the posterior pituitary.

ADH is a nonapeptide produced in the supraoptic and paraventricular nuclei and other areas of the hypothalamus. Its major role is in the regulation of water balance by its effect on the kidneys. ADH is also known as vasopressin because of the vasopressor response to pharmacological doses. Humans and most animals have arginine-vasopressin but pigs have the arginine replaced by a lysine.

ADH is produced from a much larger precursor protein (prepropressophysin). The gene for this precursor is located on human chromosome 20 and is very closely related to the oxytocin gene. These genes probably arose from an ancestral gene as a result of gene duplication about 350 million years ago. The ADH precursor protein contains sequences for three separate peptides into which it is split during transport down the nerve axon to the posterior pituitary. These are ADH, neurophysin & a glycopeptide. The physiological role of these later two peptides is unclear but neurophysin may have a role as a carrier or binding protein within the granules.

The secretory granules containing the ADH and neurophysin move down the axons (axonal transport) to the nerve terminals in the posterior pituitary from where they are secreted into the systemic circulation by a process of exocytosis (involving calcium).

Intravascular ADH has a half-life of only about 15 minutes being rapidly metabolised in the liver and kidney to inactive products.

5.6.2 Renal Actions of ADH

ADH acts on receptors in the basolateral membrane of cells in the cortical and medullary collecting tubules and not on the apical (or luminal) membrane. These membranes have different properties. The apical membrane of these cells is impermeable to water in the absence of ADH but the basolateral membrane is always permeable to water.

ADH initiates its physiological actions by combining with a specific receptor. These are two major types of vasopressin receptors: V1 & V2. The V1 receptors are located on blood vessels and are responsible for the vasopressor action.

The V2 receptors are in the basolateral membrane of the collecting tubule cells in the kidney. Various agonists and antagonists at these receptors have been developed. Desamino-d-arginine vasopressin (dDAVP) is a synthetic V2-agonist which is used clinically in treatment of diabetes insipidus.

The action at the V2 receptor activates adenyl cyclase and cyclic AMP (second messenger) is formed. This initiates a series of events which causes specific vesicles in the cytoplasm to move to and fuse with the apical membrane. The vesicles contain the water channels (aquaporin 2) which are now inserted in the apical (ie luminal) membrane rendering it permeable to water. Water moves into the cell through these channels in response to the osmotic gradient. It passes into the circulation across the basolateral membrane. The basolateral membrane is always freely permeable to water but the apical membrane is permeable only when the water channels are inserted. When intracellular cyclic AMP levels fall, the water channels are removed from the membrane and reform as vesicles.

The cycle of insertion of water channels into then removal from the luminal membrane is referred to as vesicular trafficking and is the final mediator of the ADH-dependent water permeability of the collecting duct cells.

The water channels are membrane proteins called aquaporins. Aquaporin-2 is the protein which is the vasopressin responsive water channel in the collecting duct. It is inserted into the apical membrane in reponse to cyclic AMP. The protein forms a tetrameric complex that spans the membrane and forms a channel which allows rapid water movement in response to an osmolar gradient.

Aquaporins 3 & 4 are the water channels located in the basolateral membrane. Their water permeability is not altered by ADH action and their presence means the basolateral membrane has a continuous water permeability.

Other interesting recent findings in this area are:

Mercurial diuretics bind to a specific site on aquaporin-2 and block water reabsorption. This is the mechanism of their diuretic action

The autosomal dominant form of nephrogenic diabetes insipidus is due to mutations in the aquaporin-2 gene

The X-linked form of nephrogenic diabetes is due to mutations in the gene for the V2 vasopressin receptor. (This receptor gene is on the X chromosome)

Lithium causes marked down-regulation of aquaporin-2 expression and causes a form of acquired nephrogenic diabetes insipidus

Overall Effects in the Kidney

In the absence of ADH, the apical membranes of the cells in the cortical and medullary collecting tubules have very low water permeability. Large volumes of hypotonic urine are produced. Up to 12% of the filtered load of 180l/day is excreted (urine volume up to 23 liters/day!)

In the presence of ADH, the cells are much more permeable to water. At maximal ADH levels, less then 1% of the filtered water is excreted (urine volume 500mls/day)

Feedback loop: Reabsorption of water reduces plasma [Na+] and this is detected by the osmoreceptors in the hypothalamus. This allows sensitive feedback control of ADH secretion. (Aquaporin 4 is found in the cells of the thirst centre in the hypothalamus and is probably involved in the mechanism which monitors plasma tonicity)

Maintaining Water Volume

Your kidneys have the ability to conserve or waste water. For example, if you drink a large glass of water, you'll find that you will have the urge to urinate within an hour or so. In

contrast, if you don't drink for a while, such as overnight, you will not produce much urine and it will usually be very concentrated (i.e. darker). How does your kidney know the

difference? The answer to this question involves two mechanisms:

The structure and transport properties of the loop of Henle in the nephron.

The anti-diuretic hormone (ADH), also called vasopressin, secreted by the pituitary gland.

The loop of Henle has a descending limb and an ascending limb. As filtrate moves down the loop of Henle, water is reabsorbed, but ions (Na,Cl) aren't. The removal of water serves to

concentrate the Na and Cl in the lumen. Now, as the filtrate moves up the other side (ascending limb), Na and Cl are reabsorbed, but water isn't. What these two transport

properties do is set up a concentration difference in NaCl along the length of the loop, with the highest concentration at the bottom and lowest concentration at the top. The loop of

Henle can then concentrate NaCl in the medulla. The longer the loop, the bigger the concentration gradient. This also means that the medulla tissue tends to be saltier than the

cortex tissue.

Now, as the filtrate flows through the collecting ducts, which go back down through the medulla, water can be reabsorbed from the filtrate by osmosis. Water moves from an area

of low Na concentration (high water concentration) in the collecting ducts to an area of high Na concentration (low water concentration) in the medullary tissue. If you remove water

from the filtrate at this final stage, you can concentrate the urine.

ADH, which is secreted by the pituitary gland, controls the ability of water to pass through the cells in the walls of the collecting ducts. If no ADH is present, then no water can pass through the walls of the ducts. The more ADH present, the more water can pass through.

Specialized nerve cells, called osmoreceptors, in the hypothalamus of the brain sense the Na concentration of the blood. The nerve endings of these osmoreceptors are located in the

posterior pituitary gland and secrete ADH. If the Na concentration of the blood is high, the osmoreceptors secrete ADH. If the Na concentration of the blood is low, they don't secrete

ADH. In reality, there is always some very low level of ADH secreted from the osmoreceptors.

Now let's look at how your kidneys maintain water volume.

Vasopressin, also known as arginine vasopressin (AVP), antidiuretic hormone (ADH), or argipressin, is a neurohypophysial hormone found in most mammals. Its two primary functions are to retain water in the body and to constrict blood vessels. Vasopressin regulates the body's retention of water by acting to increase water absorption in the collecting ducts of the kidney nephron.[1][2] Vasopressin is a peptide hormone that increases water permeability of the kidney's collecting duct and distal convoluted tubule by inducing translocation of aquaporin-CD water channels in the kidney nephron collecting duct plasma membrane.[3] It also increases peripheral vascular resistance, which in turn increases arterial blood pressure. It plays a key role in homeostasis, by the regulation of water, glucose, and salts in the blood. It is derived from a preprohormone precursor that is synthesized in the hypothalamus and stored in vesicles at the posterior pituitary. Most of it is stored in the posterior pituitary to be released into the bloodstream. However, some AVP may also be released directly into the brain, and accumulating evidence suggests it plays an important role in social behavior, sexual motivation and bonding, and maternal responses to stress. It has a very short half-life between 16-24 minutes.[2]

Contents

1 Physiology o 1.1 Function

1.1.1 Kidney 1.1.2 Cardiovascular system 1.1.3 Central nervous system

o 1.2 Controlo 1.3 Secretiono 1.4 Receptors

2 Structure and relation to oxytocin

3 Role in disease o 3.1 Lack of AVPo 3.2 Excess AVP

4 Pharmacology o 4.1 Vasopressin analogueso 4.2 The role of vasopressin analogues in cardiac arrest

4.2.1 Vasopressin vs. epinephrine 4.2.2 Vasopressin and epinephrine vs. epinephrine alone 4.2.3 2010 American Heart Association Guidelines

o 4.3 Vasopressin receptor inhibition 5 See also 6 References 7 Further reading 8 External links

Physiology

Function

One of the most important roles of AVP is to regulate the body's retention of water; it is released when the body is dehydrated and causes the kidneys to conserve water, thus concentrating the urine and reducing urine volume. At high concentrations, it also raises blood pressure by inducing moderate vasoconstriction. In addition, it has a variety of neurological effects on the brain, having been found, for example, to influence pair-bonding in voles. The high-density distributions of vasopressin receptor AVPr1a in prairie vole ventral forebrain regions have been shown to facilitate and coordinate reward circuits during partner preference formation, critical for pair bond formation.[4]

A very similar substance, lysine vasopressin (LVP) or lypressin, has the same function in pigs and is often used in human therapy.

Kidney

Vasopressin has two main effects by which it contributes to increased urine osmolarity (increased concentration) and decreased water excretion:

1. Increasing the water permeability of distal tubule and collecting duct cells in the kidney, thus allowing water reabsorption and excretion of more concentrated urine, i.e., antidiuresis. This occurs through insertion of water channels (Aquaporin-2) into the apical membrane of distal tubule and collecting duct epithelial cells. Aquaporins allow water to move down their osmotic gradient and out of the nephron, increasing the amount of water re-absorbed from the filtrate (forming urine) back into the bloodstream.V2 receptors, which are G protein-coupled receptors on the basolateral plasma membrane of the epithelial cells, couple to the heterotrimeric G-protein Gs, which activates adenylyl cyclases III and VI to convert ATP into cAMP, plus 2 inorganic phosphates. The rise in cAMP then triggers the insertion of aquaporin-2 water channels by exocytosis of intracellular vesicles, recycling

endosomes. Vasopressin also increases the concentration of calcium in the collecting duct cells, by episodic release from intracellular stores. Vasopressin, acting through cAMP, also increases transcription of the aquaporin-2 gene, thus increasing the total number of aquaporin-2 molecules in collecting duct cells.Cyclic-AMP activates protein kinase A (PKA) by binding to its regulatory subunits and allowing them to detach from the catalytic subunits. Detachment exposes the catalytic site in the enzyme, allowing it to add phosphate groups to proteins (including the aquaporin-2 protein), which alters their functions.

2. Increasing permeability of the inner medullary portion of the collecting duct to urea by regulating the cell surface expression of urea transporters,[5] which facilitates its reabsorption into the medullary interstitium as it travels down the concentration gradient created by removing water from the connecting tubule, cortical collecting duct, and outer medullary collecting duct.

Cardiovascular system

Vasopressin increases peripheral vascular resistance (vasoconstriction) and thus increases arterial blood pressure. This effect appears small in healthy individuals; however it becomes an important compensatory mechanism for restoring blood pressure in hypovolemic shock such as that which occurs during hemorrhage.

Central nervous system

Avp is expressed in the periventricular region of the hypothalamus in the adult mouse.[6] Allen Brain Atlases

Vasopressin released within the brain has many actions:

It has been implicated in memory formation, including delayed reflexes, image, short- and long-term memory, though the mechanism remains unknown; these findings are controversial. However, the synthetic vasopressin analogue desmopressin has come to interest as a likely nootropic.

Vasopressin is released into the brain in a circadian rhythm by neurons of the supraoptic nucleus.

Vasopressin released from centrally projecting hypothalamic neurons is involved in aggression, blood pressure regulation, and temperature regulation.

It is likely that vasopressin acts in conjunction with corticotropin-releasing hormone to modulate the release of corticosteroids from the adrenal gland in

response to stress, particularly during pregnancy and lactation in mammals.[7][8]

[9]

Selective AVPr1a blockade in the ventral pallidum has been shown to prevent partner preference in prairie voles, suggesting that these receptors in this ventral forebrain region are crucial for pair bonding.[4]

Recent evidence suggests that vasopressin may have analgesic effects. The analgesia effects of vasopressin were found to be dependent on both stress and gender.[10]

Evidence for this comes from experimental studies in several species, which indicate that the precise distribution of vasopressin and vasopressin receptors in the brain is associated with species-typical patterns of social behavior. In particular, there are consistent differences between monogamous species and promiscuous species in the distribution of AVP receptors, and sometimes in the distribution of vasopressin-containing axons, even when closely related species are compared.[11] Moreover, studies involving either injecting AVP agonists into the brain or blocking the actions of AVP support the hypothesis that vasopressin is involved in aggression toward other males. There is also evidence that differences in the AVP receptor gene between individual members of a species might be predictive of differences in social behavior. One study has suggested that genetic variation in male humans affects pair-bonding behavior. The brain of males uses vasopressin as a reward for forming lasting bonds with a mate, and men with one or two of the genetic alleles are more likely to experience marital discord. The partners of the men with two of the alleles affecting vasopressin reception state disappointing levels of satisfaction, affection, and cohesion.[12] Vasopressin receptors distributed along the reward circuit pathway, to be specific in the ventral pallidum, are activated when AVP is released during social interactions such as mating, in monogamous prairie voles. The activation of the reward circuitry reinforces this behavior, leading to conditioned partner preference, and thereby initiates the formation of a pair bond.[13]

Control

Vasopressin is secreted from the posterior pituitary gland in response to reductions in plasma volume, in response to increases in the plasma osmolality, and in response to cholecystokinin (CCK) secreted by the small intestine:

Secretion in response to reduced plasma volume is activated by pressure receptors in the veins, atria, and carotids.

Secretion in response to increases in plasma osmotic pressure is mediated by osmoreceptors in the hypothalamus.

Secretion in response to increases in plasma CCK is mediated by an unknown pathway.

The neurons that make AVP, in the hypothalamic supraoptic nuclei (SON) and paraventricular nuclei (PVN), are themselves osmoreceptors, but they also receive synaptic input from other osmoreceptors located in regions adjacent to the anterior wall of the third ventricle. These regions include the organum vasculosum of the lamina terminalis and the subfornical organ.

Many factors influence the secretion of vasopressin:

Ethanol (alcohol) reduces the calcium-dependent secretion of AVP by blocking voltage-gated calcium channels in neurohypophyseal nerve terminals.[14]

Angiotensin II stimulates AVP secretion, in keeping with its general pressor and pro-volumic effects on the body.[15]

Atrial natriuretic peptide inhibits AVP secretion, in part by inhibiting Angiotensin II-induced stimulation of AVP secretion.[15]

Secretion

The main stimulus for secretion of vasopressin is increased osmolality of plasma. Reduced volume of extracellular fluid also has this effect, but is a less sensitive mechanism.

The AVP that is measured in peripheral blood is almost all derived from secretion from the posterior pituitary gland (except in cases of AVP-secreting tumours). Vasopressin is produced by magnocellular neurosecretory neurons in the Paraventricular nucleus of hypothalamus (PVN) and Supraoptic nucleus (SON). It then travels down the axon through the infundibulum within neurosecretory granules that are found within Herring bodies, localized swellings of the axons and nerve terminals. These carry the peptide directly to the posterior pituitary gland, where it is stored until released into the blood. However there are two other sources of AVP with important local effects:

AVP is also synthesized by parvocellular neurosecretory neurons at the PVN, transported and released at the median eminence, which then travels through the hypophyseal portal system to the anterior pituitary where it stimulates corticotropic cells synergistically with CRH to produce ACTH (by itself it is a weak secretagogue).[16]

Vasopressin is also released into the brain by several different populations of smaller neurons.

Receptors

Below is a table summarizing some of the actions of AVP at its four receptors, differently expressed in different tissues and exerting different actions:

TypeSecond messenger systemLocationsActions

AVPR1APhosphatidylinositol/calcium

Liver, kidney, peripheral vasculature, brain

Vasoconstriction, gluconeogenesis, platelet aggregation, and release of factor VIII and von Willebrand factor; social recognition,[17] circadian tau[18]

AVPR1B or

Phosphatidylinositol/calciumPituitary gland, brain

Adrenocorticotropic hormone secretion in

AVPR3response to stress;[19] social interpretation of olfactory cues[20]

AVPR2Adenylate cyclase/cAMP

Basolateral membrane of the cells lining the collecting ducts of the kidneys (especially the cortical and outer medullary collecting ducts)

Insertion of aquaporin-2 (AQP2) channels (water channels). This allows water to be reabsorbed down an osmotic gradient, and so the urine is more concentrated. Release of von Willebrand factor and surface expression of P-selectin through exocytosis of Weibel-Palade bodies from endothelial cells [21] [22]

VACM-1Phosphatidylinositol/calcium

Vascular endothelium and renal collecting tubules

Increases cytosolic calcium and acts as an inverse agonist of cAMP accumulation[23]

Structure and relation to oxytocin

Chemical structure of the argipressin (indicating that this compound is of the vasopressin family with an arginine at the 8th amino acid position.

Chemical structure of oxytocin

The vasopressins are peptides consisting of nine amino acids (nonapeptides). (NB: the value in the table above of 164 amino acids is that obtained before the hormone is activated by cleavage). The amino acid sequence of arginine vasopressin is Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly, with the cysteine residues forming a disulfide bond. Lysine vasopressin has a lysine in place of the arginine.

The structure of oxytocin is very similar to that of the vasopressins: It is also a nonapeptide with a disulfide bridge and its amino acid sequence differs at only two positions (see table below). The two genes are located on the same chromosome separated by a relatively small distance of less than 15,000 bases in most species. The magnocellular neurons that make vasopressin are adjacent to magnocellular neurons that make oxytocin, and are similar in many respects. The similarity of the two peptides can cause some cross-reactions: oxytocin has a slight antidiuretic function, and high levels of AVP can cause uterine contractions.[24][25]

Below is a table showing the superfamily of vasopressin and oxytocin neuropeptides:

Vertebrate Vasopressin FamilyCys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2

Argipressin (AVP, ADH)

Most mammals

Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys-Gly-NH2

Lypressin (LVP)

Pigs, hippos, warthogs, some marsupials

Cys-Phe-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2

PhenypressinSome marsupials

Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2

Vasotocin†Non-mammals

Vertebrate Oxytocin Family

Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2

Oxytocin (OXT)

Most mammals, ratfish

Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Pro-Gly-NH2

Prol-Oxytocin

Some New World monkeys, northern tree shrews

Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Ile-Gly-NH2

Mesotocin

Most marsupials, all birds, reptiles, amphibians, lungfishes, coelacanths

Cys-Tyr-Ile-Gln-Ser-Cys-Pro-Ile-Gly-NH2

SeritocinFrogs

Cys-Tyr-Ile-Ser-Asn-Cys-Pro-Ile-Gly-NH2

IsotocinBony fishes

Cys-Tyr-Ile-Ser-Asn-Cys-Pro-Gln-Gly-NH2

GlumitocinSkates

Cys-Tyr-Ile-Asn/Gln-Asn-Cys-Pro-Leu/Val-Gly-NH2

Various tocins

Sharks

Invertebrate VP/OT Superfamily

Cys-Leu-Ile-Thr-Asn-Cys-Pro-Arg-Gly-NH2

Diuretic Hormone

Locust

Cys-Phe-Val-Arg-Asn-Cys-Pro-Thr-Gly-NH2

AnnetocinEarthworm

Cys-Phe-Ile-Arg-Asn-Cys-Pro-Lys-Gly-NH2

Lys-Connopressin

Geography & imperial cone snail, pond snail, sea hare, leech

Cys-Ile-Ile-Arg-Asn-Cys-Pro-Arg-Gly-NH2

Arg-Connopressin

Striped cone snail

Cys-Tyr-Phe-Arg-Asn-Cys-Pro-Ile-Gly-NH2

CephalotocinOctopus

Cys-Phe-Trp-Thr-Ser-Cys-Pro-Ile-Gly-NH2

OctopressinOctopus

†Vasotocin is the evolutionary progenitor of all the vertebrate neurohypophysial hormones.[26]

Role in disease

Lack of AVP

Decreased AVP release or decreased renal sensitivity to AVP leads to diabetes insipidus, a condition featuring hypernatremia (increased blood sodium concentration), polyuria (excess urine production), and polydipsia (thirst).

Excess AVP

High levels of AVP secretion may lead to hyponatremia. In many cases, the AVP secretion is appropriate (due to severe hypovolemia), and the state is labelled "hypovolemic hyponatremia". In certain disease states (heart failure, nephrotic syndrome) the body fluid volume is increased but AVP production is not suppressed for various reasons; this state is labelled "hypervolemic hyponatremia". A proportion of cases of hyponatremia feature neither hyper- nor hypovolemia. In this group (labelled "euvolemic hyponatremia"), AVP secretion is either driven by a lack of cortisol or thyroxine (hypoadrenalism and hypothyroidism, respectively) or a very low level of urinary solute excretion (potomania, low-protein diet), or it is entirely inappropriate. This last category is classified as the syndrome of inappropriate antidiuretic hormone (SIADH).[27]

SIADH in turn can be caused by a number of problems. Some forms of cancer can cause SIADH, particularly small cell lung carcinoma but also a number of other tumors. A variety of diseases affecting the brain or the lung (infections, bleeding) can be the driver behind SIADH. A number of drugs has been associated with SIADH, such as certain antidepressants (serotonin reuptake inhibitors and tricyclic antidepressants), the anticonvulsant carbamazepine, oxytocin (used to induce and stimulate labor), and the chemotherapy drug vincristine. It has also been associated

with fluoroquinolones (including ciprofloxacin and moxifloxacin).[2] Finally, it can occur without a clear explanation.[27]

Hyponatremia can be treated pharmaceutically through the use of vasopressin receptor antagonists.[27]

Pharmacology

Vasopressin analogues

Vasopressin agonists are used therapeutically in various conditions, and its long-acting synthetic analogue desmopressin is used in conditions featuring low vasopressin secretion, as well as for control of bleeding (in some forms of von Willebrand disease and in mild haemophilia A) and in extreme cases of bedwetting by children. Terlipressin and related analogues are used as vasoconstrictors in certain conditions. Use of vasopressin analogues for esophageal varices commenced in 1970.[28]

Vasopressin infusion has also been used as a second line of management in septic shock patients not responding to high dose of inotropes (e.g., dopamine or norepinephrine).

The role of vasopressin analogues in cardiac arrest

Injection of vasopressors for the treatment of cardiac arrest was first suggested in the literature in 1896 when Austrian scientist Dr. R. Gottlieb described the vasopressor epinephrine as an "infusion of a solution of suprarenal extract [that] would restore circulation when the blood pressure had been lowered to unrecordable levels by chloral hydrate."[29] Modern interest in vasopressors as a treatment for cardiac arrest stem mostly from canine studies performed in the 1960s by anesthesiologists Dr. John W. Pearson and Dr. Joseph Stafford Redding in which they demonstrated improved outcomes with the use of adjunct intracardiac epinephrine injection during resuscitation attempts after induced cardiac arrest.[29] Also contributing to the idea that vasopressors may be useful treatments in cardiac arrest are studies performed in the early to mid 1990's that found significantly higher levels of endogenous serum vasopressin in adults after successful resuscitation from out-of-hospital cardiac arrest compared to those who did not live.[30][31] Results of animal models have supported the use of either vasopressin or epinephrine in cardiac arrest resuscitation attempts, showing improved coronary perfusion pressure[32] and overall improvement in short-term survival as well as neurological outcomes.[33]

Vasopressin vs. epinephrine

Table 1. Meta-analysis of outcomes for patients treated with vasopressin versus epinephrine[32]

RR (95% CI)Failure of ROSC0.81 (0.58-1.12)Death before hospital admission0.72 (0.38-1.39)Death within 24 hours0.74 (0.38-1.43)

Death before hospital discharge0.96 (0.87-1.05)Number of deaths and neurologically impaired survivors1.00 (0.94-1.07)

Although both vasopressors, vasopressin and epinephrine differ in that vasopressin does not have direct effects on cardiac contractility as epinephrine does.[33] Thus, vasopressin is theorized to be of increased benefit over epinephrine in cardiac arrest due to its properties of not increasing myocardial and cerebral oxygen demands.[33] This idea has led to the advent of several studies searching for the presence of a clinical difference in benefit of these two treatment choices. Initial small studies demonstrated improved outcomes with vasopressin in comparison to epinephrine.[34] However, subsequent studies have not all been in agreement. Several randomized controlled trials have been unable to reproduce positive results with vasopressin treatment in both return of spontaneous circulation (ROSC) and survival to hospital discharge,[34][35][36][37] including a systematic review and meta-analysis completed in 2005 that found no evidence of a significant difference with vasopressin in five studied outcomes (see Table 1).[32]

Vasopressin and epinephrine vs. epinephrine alone

Table 2. Significant outcomes for combined vasopressin and epinephrine treatmentRR (95% CI)p value

ROSC[37]1.42 (1.14-1.77)

Survival to hospital admission[38]1.42 (1.02-2.04)

0.05

In subgroup: PEA [38] 1.30 (0.90-2.06)

0.02

In subgroup: Collapse to ED arrival time of 15–30 minutes[38]

1.22 (1.01-1.49)

0.05

In subgroup: Collapse to ED arrival time of 30–45 minutes[38]

1.11 (1.00-1.24)

0.05

Survival to hospital discharge[37]3.69 (1.52-8.95)

There is no current evidence of significant survival benefit with improved neurological outcomes in patients given combinations of both epinephrine and vasopressin during cardiac arrest.[32][35][39][40] A systematic review from 2008 did, however, find one study that showed a statistically significant improvement in ROSC and survival to hospital discharge with this combination treatment; unfortunately, those patients that survived to hospital discharge had overall poor outcomes and many suffered permanent, severe neurological damage.[37][40] A more recently published clinical trial out of Singapore has shown similar results, finding combination treatment to only improve the rate of survival to hospital admission, especially in the subgroup analysis of patients with longer "collapse to emergency department" arrival times of 15 to 45 minutes.[38] Table 2 lists all statistically significant findings of a correlation between combined treatment and positive outcomes found in these two studies.

2010 American Heart Association Guidelines

The 2010 American Heart Association Guidelines for Cardiopulmonary Resuscitation and Emergency Cardiovascular Care recommend the consideration of vasopressor treatment in the form of epinephrine in adults with cardiac arrest (Class IIb, LOE A recommendation).[41] Due to the absence of evidence that vasopressin administered instead of or in addition to epinephrine has significant positive outcomes, the guidelines do not currently contain vasopressin as a part of the cardiac arrest algorithms.[41] It does, however, allow for one dose of vasopressin to replace either the first or second dose of epinephrine in the treatment of cardiac arrest (Class IIb, LOE A recommendation).[41]

Vasopressin receptor inhibition

Main article: vasopressin receptor antagonist

A vasopressin receptor antagonist is an agent that interferes with action at the vasopressin receptors. They can be used in the treatment of hyponatremia.[42]

See also

Oxytocin Sexual motivation and hormones Vasopressin receptor Vasopressin receptor antagonists Copeptin