Proc. Nat!. Acad. Sci. USAVol. 85, pp. 3155-3159, May 1988Medical Sciences

Paradoxical relationship between atriopeptin plasma levels anddiuresis-natriuresis induced by acute volume expansion

(atrial natriuretic factor/vasopressin)

MORIYUKI SAKATA, JAMES E. GREENWALD, AND PHILIP NEEDLEMAN*Department of Pharmacology, Washington University School of Medicine, Saint Louis, MO 63110

Contributed by Philip Needleman, December 10, 1987

ABSTRACT Surgical removal of one or both atrial ap-pendages was employed in rats to reduce the intrinsic stores ofatriopeptin (AP). In conscious rats (with intact baroreceptorreflexes), bilateral or unilateral atrial appendectomy sup-pressed the diuresis and natriuresis produced by acute volumeexpansion. Surprisingly, volume expansion (with 4% bovineserum albumin in saline at 1.5 ml/kg per min for 15 min) didnot result in an increase in plasma AP immunoreactivity(APir) in control or atrial-appendectomized conscious rats.Previous studies demonstrated that acute volume expansion inanesthetized animals caused increased plasma APir. Indeed,we found that volume expansion causes comparable diuresis-natriuresis in conscious and chloral hydrate-anesthetized rats,but only the latter group exhibits an increase in plasma APir.Brattleboro rats, which are deficient in vasopressin, exhibitthe same response as Long-Evans controls in that acutevolume expansion in conscious animals produces a pronounceddiuresis and natriuresis but no APir release, but when thesesame animals are anesthetized, there is a simultaneous induc-tion of diuresis-natriuresis and APir release by volume expan-sion. Plasma AP does not increase in conscious rats despite alarge volume load, 3040% of the total blood volume given in15 min, and the natriuresis-diuresis appears to also be inde-pendent of vasopressin. On the other hand, the diuresisinduced by acute volume expansion in anesthetized rats seemsdependent on the elevated APir, since rats made autoimmuneto AP (which are nonresponsive to exogenous AP infusions)exhibit a diuresis in conscious but not anesthetized rats. Wetherefore conclude that the participation of AP in volumehomeostasis is more likely in pathophysiological states and thatanother mechanism or possibly another atrial factor mediatesthe diuresis-natriuresis induced by volume expansion in con-scious rats.

Atrial distension produced by elevation of atrial pressure orhead-out water immersion (1), volume expansion (2-5), andadministration of pressor agents (1, 6) lead to release ofatriopeptin (AP) from the atria. Many investigators havereported that AP immunoreactivity (APir) release appears tobe stimulated by atrial distension through acute volumeexpansion both in animals (2, 3, 7, 8) and in humans (4-9).Removal of right atrial appendages in anesthetized ratsattenuated the urinary volume (UV) and urinary sodiumexcretion (UNaV) in response to volume expansion (10).Similarly, conscious rats 2 weeks after bilateral atrial appen-dectomy exhibited reduced UV and UNaV during volumeexpansion (11). Villarreal et al. (12) measured plasma APir,UV, and UNaV during volume expansion in bilateral atrialappendectomized rats and demonstrated that removal ofboth atrial appendages in anesthetized rats attenuated the

release of plasma APir and the increase in UV and UNaVafter volume expansion.The current literature is replete with studies implicating a

critical role ofAP in volume homeostasis. There are numer-ous pharmacological confirmations of the ability of exoge-nous administration ofAP to induce diuresis and natriuresis.There are also a number of reports demonstrating simulta-neous alterations in diuresis and plasma APir concentrationsduring various manipulations (e.g., volume expansion, saltloading, water immersion, vasoconstrictors) but few demon-strations of the actual physiological role of AP in volumehomeostasis.

In the present study we examined the changes in plasmaAPir in atrial appendectomized rats. We decided to performthese experiments in conscious precannulated animals be-cause we had observed that basal plasma APir levels wereconsiderably higher in chloral hydrate-anesthetized animalscompared to unanesthetized controls. The choice of theseconditions led us to an unanticipated finding: the diuresiscaused by volume expansion of conscious rats appears to beindependent of AP.

METHODSAtrial Appendectomy. Male Sprague-Dawley rats (Chap-

pell Breeders, Saint Louis) weighing between 300 and 450 gwere anesthetized with chloral hydrate (350 mg/kg, i.p.) andfour different groups of animals were prepared: sham-operated and bilateral, right, and left atrial appendecto-mized. Animals were mechanically ventilated and an inci-sion was made between the second and third ribs. The rightand left atrial appendages were exposed and gently extendedto their maximal length. They were then ligated around theirbase with 3-0 silk ligatures, and the appendage was excised.Sham operations were performed by exposing the atria,placing ligatures around the base, and then removing theligatures without excising the atrial appendages. A drainagetube (PE-360, Clay Adams) was placed in the thoraciccavity, and the chest was closed while applying negativepressure to the drainage tube until the rats were consciousand breathing spontaneously. They were then maintained onnormal diet and water ad lib. Three to 4 weeks after atrialappendectomy, animals were anesthetized with diethylether, and an indwelling polyethylene cannula (PE-50, ClayAdams) was placed in the femoral vein for subsequentadministration of fluids or drugs. A similar indwelling can-nula was placed in the femoral artery for blood sampling andmeasurement of mean blood pressure and in the urinarybladder for the collection of urine. The rats were allowed torecover completely from the ether for 2 hr. Urine volumewas determined gravimetrically, and urine sodium concen-

Abbreviations: AP, atriopeptin; APir, AP immunoreactivity; AP24and AP28, 24- and 28-amino acid residue forms of AP; UV, urinaryvolume; UNaV, urinary sodium excretion.*To whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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trations were measured on a flame photometer (IL 943automatic flame photometer; Instrumentation Laboratory,Lexington, MA). After two 15-min control collection peri-ods, blood volume was expanded with 4% bovine serumalbumin in normal saline infused intravenously at a rate of1.5 ml/kg per min for 15 min. Arterial blood samples werecollected into V1o vol of 50 mM EDTA 15, 30, 45, and 60 minafter the beginning of the volume expansion. Plasma sampleswere immediately frozen and stored at - 70'C until assayedby enzyme immunoassay (13). The blood taken was replacedwith an equivalent volume of physiological saline. Atrialextracts were prepared and analyzed as previously described(14).

Effect of Anesthesia on Response to Volume Expansion.Normal animals were instrumented as described above.Blood volume was expanded with 4% bovine serum albuminin saline infused intravenously at a rate of 1.5 ml/kg per minfor 15 min. Arterial blood samples were collected 5, 10, 15,30, 45, and 60 min after the beginning of the volumeexpansion. Forty-five minutes after the end of the volumeexpansion period, the same rat was anesthetized with chloralhydrate (350 mg/kg, i.p.). Fifteen minutes later, volumeexpansion was repeated at the same rate and for the samelength of time.

Vasopressin-Deficient Animals. Brattleboro rats were pur-chased from Blue Spruce Farm (Altamont, NY) and com-pared to control vasopressin-replete Long-Evans rats fromthe same source. These animals were used to study theeffects of acute volume expansion in conscious and anesthe-tized animals as described above.AP Immunization Protocol and Titer Determination. Fe-

male and male Sprague-Dawley rats weighing 200-250 gwere initially inoculated subcutaneously with 200 ,ug of theconjugated rat AP28 in an emulsion of Freund's completeadjuvant (Calbiochem). To make AP immunogenic, AP28,the 28 amino acid residue form of AP, was covalently boundto bovine thyroglobulin (Sigma) by the carbodiimide method(15). Animals were boosted monthly with 100 ,ug of the ratAP28-thyroglobulin conjugate in an emulsion of Freund'sincomplete adjuvant until antibodies were produced. Anti-body titers were defined as the dilution of rat serum thatbound 50% of iodinated rat AP24 (10,000 cpm; AP24 is the24-residue form). Serum was incubated with iodinated pep-tide for 24 hr at 4°C, then free peptide was precipitated with2.5 mg of activated charcoal (Sigma) and radioactivity wasmeasured with an Apex y counter [Micromedic Systems,(Horsham, PA)].AP24 Infusions for Validation of the Autoimmunity. Six

rats that attained titers of 1:500 to 1:1000 and six nonimmunerats were selected for infusions of rat AP24. Arterial, ve-nous, and bladder access was achieved by surgical place-ment of PE-50 tubing into the femoral artery, femoral vein,and urinary bladder after rats were anesthetized with chloralhydrate (350 mg/kg, i.p.). Urine volume was determinedgravimetrically by collection into preweighed tubes. Fluidsand AP24 were infused into the femoral vein. The blood

volumes of all animals were expanded for 1 hr with 5%dextrose/0.25% sodium chloride (0.15 ml/min per kg) priorto basal urine collections. Initially, to validate that ourimmunized rats were indeed devoid of the effects of circu-lating AP, we measured the amount of urine produced inresponse to the exogenous administration of rat AP24.Infusion (15 min) of AP24 (300 ng/kg per min) into nonim-munized anesthetized rats produced a 600% increase in urineflow rate. Immunized rats produced no significant increasein urine output in response to 300 ng ofAP per kg per min (adose that effectively raises plasma AP to 1500 pg/ml, deter-mined by enzyme immunoassay), thus demonstrating thatthe circulating antibodies generated against AP in these ratsdo indeed physiologically inactivate AP.

RESULTS

Appendectomy Validation. Both atrial-appendectomizedand sham-operated animals gained weight normally andmaintained blood pressure during the 3- to 4-week postop-erative period (Table 1). In the bilateral atrial appendectomygroup, the APir content of each atrium was decreased by50-75% compared to the sham-operated group. In right andleft atrial appendectomy groups, the ipsilateral APir contentwas reduced by 60-70%, while the APir content of thecontralateral atrium was not affected (Table 1).We tested the competency of the cardiac baroreceptor

reflexes to establish that our surgical procedures did notinterfere with neuronal connections that might be essentialfor the stretch-induced AP release. Phenylephrine (30,g/kg) was administered intravenously into the conscioussham-operated control and the atrial-appendectomized ani-mals (n = 5 per group). No differences between the groupswere detected in either the phenylephrine-induced vasocon-striction (39 ± 4% increase in blood pressure) or the reflexbradycardia (31 ± 4% decrease in heart rate). Similarly,nitroprusside produced a comparable vasodilation (36 5%fall in blood pressure) and reflex tachycardia (9 + 3%increase in heart rate) in the sham-operated and atrial-appendectomized rats.

Effect of Atrial Appendectomy on Diuresis-Natriuresis andAPir in Conscious Rats. Diuresis (Fig. LA) and natriuresis(Fig. 1B) after volume expansion were attenuated by atrialappendectomy. Thirty minutes after volume expansion, theurine volumes of bilateral, right, and left atrial appendec-tomy groups were significantly less than the volume of thesham-operated group (Fig. LA). Inhibition of diuresis wascomparable in the right and bilateral atrial-appendectomizedrats at 30 min, suggesting that the right atrium is thedominant chamber in eliciting the subsequent renal responsein the rat. In bilateral atrial-appendectomized rats, in whichthe most pronounced suppressive effect on diuresis waselicited, UV was still significantly less than in the sham-operated control group at 45 and 60 min. Surprisingly,volume expansion did not increase plasma APir in thesham-operated or appendectomized groups (Fig. 1C).

Table 1. Characterization of the atrial-appendectomized rats

Body weight, g

3-4 wk MBP,Treatment Pre-op post-op mm Hg APir, ,ug/RA APir, ,ug/LA

Sham-operated (n = 8) 280 ± 11 369 ± 13 135 ± 4 (3) 6.8 ± 0.49 (4) 3.6 ± 0.6 (4)Right atrial-x (n = 9) 311 ± 10 381 ± 14 123 ± 4 (4) 2.8 ± 0.7** (4) 4.9 ± 0.5 (4)Left atrial-x (n = 10) 311 ± 9 367 ± 15 127 ± 8 (3) 6.7 ± 0.57 (4) 1.1 ± 0.19* (4)Bilateral atrial-x (n = 11) 292 ± 13 342 ± 12 123 ± 4 (4) 1.7 ± 0.34*** (5) 1.8 ± 0.5* (5)The values presented are the mean ± SEM; results of M-test analysis are indicated with asterisks (*, P < 0.05; **, P <

0.01; ***, P < 0.001; each vs. sham-operated). The number of animals in each group is indicated in parentheses. RA, rightatrium; LA, left atrium; atrial-x, atrial appendectomy; MBP, mean blood pressure.

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FIG. 1. Effects of volume expansion on UV (A), UNaV (B), and plasma AP release (C) in conscious rats. The stippled areas indicate the15-min periods of acute volume expansion. There were no significant differences in basal values of APir among the four groups [in pg/ml:sham-operated (o), 60 ± 10; bilateral atrial appendectomy (e), 105 ± 21; right atrial appendectomy (A), 64 ± 8; left atrial appendectomy (U),99 ± 14]. Each value represents the mean ± SEM for eight animals in each group. By Student's t test: *, P < 0.05; **, P < 0.01; ***, P <0.001; each vs. sham-operated group at the same time.

Effect of Anesthesia on APir Release. Since the literature isreplete with studies showing AP release when the bloodvolume of anesthetized animals is expanded (2, 12), wereevaluated our experiments to understand the discrepancy.Our protocols were designed to use each set of animals as itsown control and to compare conscious and anesthetizedanimals. After the infusion of 4% bovine serum albumin innormal saline, UV and UNaV were increased in consciousrats, but APir was not altered (Fig. 2). Volume expansionwas repeated after the administration of chloral hydrate in

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SEM for four rats. Basal values for the animals with expanded bloodvolume were as follows: UV, 0.07 ± 0.01 ml/15 min; UNaV, 5.7 ±

2 ,tEq/15 min; and plasma APir, 176 ± 77 pg/ml.

the same animals. Under anesthesia, infusion of 4% bovineserum albumin in saline produced a prolonged increase inboth UV and UNaV. Furthermore, in anesthetized animals,volume expansion increased plasma APir by 7-fold at 15 minafter volume expansion (153 ± 41 pg/ml to 1071 ± 159pg/ml) (Fig. 2).To rule out the possibility that hemodynamic changes or

an altered volume stasis of the experimental animals mightinappropriately influence the results, a number of controlexperiments were performed. As indicated above, the ani-mals were used as their own controls and therefore theirblood volumes were expanded twice (once when the animalswere conscious and once when they were anesthetized). Weestablished the following controls: (i) two consecutive acutevolume expansions in conscious rats did not increase plasmaAPir; (ii) volume expansion in anesthetized rats that werenot previously treated as conscious animals still elicited theprofound acute increase in plasma APir; and (iii) no markedchanges in UV, UNaV, or plasma APir were noted inconscious and subsequently anesthetized rats whose bloodvolume was not acutely expanded (Fig. 2). The mean arterialblood pressure in the conscious rats, 116 ± 4 mm Hg (n =

6), was lowered by the chloral hydrate anesthesia (i.v.) to 80± 4 mm Hg, and acute volume expansion did not signifi-cantly increase the blood pressure in either the conscious orthe anesthetized rats. On the other hand, the acute volumeexpansion comparably increased the right atrial pressure by2.2 ± 0.5 mm Hg in the conscious rats and 3.1 ± 0.6 mm Hgin the anesthetized rats (n = 6).

Influence of Endogenous Vasopressin on the Diuresis Pro-duced by Volume Expansion. Volume expansion is known tosuppress vasopressin release after atrial stretch by activationof a vagal afferent reflex. The above data in the consciousanimal suggest that the AP-independent diuresis produced inthe conscious rat could reflect vasopressin modulation. Wetested this hypothesis by repeating the above protocols inBrattleboro rats. To validate the vasopressin deficiency inthese purchased rats, we placed the animals in metaboliccages and measured UV and osmolarity after several days ofequilibration with the animals having free access to food andwater. The UV in control (Long-Evans) rats was 2.5 ± 0.2ml/kg per hr, and the urine osmolarity was 2000 ± 80milliosmoles/kg of H20; in the Brattleboro rats, urinaryexcretion was 10-fold higher, 23.2 ± 1.8 ml/kg per hr, andthe osmolarity was comparably suppressed, to 220 ± 100milliosmoles/kg of H20 (n = 6). Acute volume expansion

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resulted in the same levels of diuresis-natriuresis in theconscious Brattleboro and Long-Evans rats, and the levelswere not dependent on changes in plasma APir (Fig. 3).Furthermore, both groups exhibited simultaneous peptiderelease and increase in UV during anesthesia.AP-Dependent Diuresis in Anesthetized Rats. While the

above data indicate that plasma AP rises during volumeexpansion in chloral hydrate-anesthetized rats, they do notindicate that the diuresis-natriuresis and the increasedplasma APir are directly related phenomena. Unfortunately,there are no currently available AP antagonists to test thismechanism directly. We have developed animals that areautoimmune to AP by immunization with AP24 conjugatedto thyroglobulin. Animals that developed an adequate titer(1:1000 to 1:5000) were utilized. To validate the efficacy toimmunization, the animals were challenged by an infusion ofexogenous AP24 (Fig. 4). The immunized animals did notexhibit a diuresis (UV = 0.23 ml/15 min) when infused with300 ng/kg per min, while control rats responded with adiuresis of 0.83 ml/15 min.Acute volume expansion produced a diuresis that was

indistinguishable in the autoimmune and nonimmune con-scious rats (Fig. 4). However, after anesthesia the diuresis inresponse to either acute volume expansion or infusion ofexogenous AP was totally suppressed in the autoimmuneanimals.

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4) or -replete (Long-Evans; *; n = 3) rats to volume expansion.

FIG. 4. Response of rats made autoimmune to AP to acutevolume expansion. o, Normal rats (n = 6); e, autoimmune rats (n =8).

DISCUSSIONIn the present experiments we used a surgical method toremove 50-75% of the cardiac atria (Table 1). This atrialappendectomy blunted the diuresis and natriuresis producedby volume expansion. Surprisingly, in conscious rats, vol-ume expansion did not cause APir release into the circula-tion (Fig. 1). Removal of the atrial appendages blunted therenal response to acute volume expansion in conscious rats(11). Nemeh and Gilmore (16) have questioned the impor-tance of atrial stretch as a stimulus for AP release in primatesbecause they found no change in plasma APir in consciousmonkeys in spite of increases in left atrial pressure. Inconscious humans, APir levels were practically unalteredwhen saline was infused i.v. at the rate of about 1.0 ml/kgper min for 30 min (17). Goetz et al. (18) also suggested thatthe substantial increase in plasma APir induced by left atrialstretch is not responsible for the diuretic and natriureticeffects, since left atrial distension in cardiac denervatedconscious dogs still causes an increase in plasma APirwithout causing a natriuresis-diuresis. Therefore, the diu-resis that occurred in sham-operated control dogs during leftatrial distension was elicited by neural or humoral mecha-nisms that acted independently of AP. A comparable mech-anism may have been acting in our rats during acute volumeexpansion.Our data and that of others (12) indicated that acute

volume expansion in anesthetized rats caused a pronouncedrelease of APir in the plasma. Because we failed to observeAPir release after volume expansion in conscious rats, weexamined the effect of anesthesia on APir release. In thechloral hydrate-anesthetized rats, volume expansion didindeed simultaneously produce diuresis and an increase inAPir (Fig. 2). From the autoimmune rat experiments (Fig. 4),we conclude that the diuresis induced in chloral hydrate-treated animals seems dependent solely on AP release andresponse.Volume expansion did not stimulate plasma APir release

in conscious rats, but atrial appendectomy attenuated the

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diuresis. Thus, the diuresis induced by volume expansion inconscious rats appeared to be independent of AP regulationand may result from reduced sympathetic tone, suppressionof plasma renin activity, or inhibition of vasopressin. Wefurther found that the diuresis in conscious Brattleboro ratswas independent of vasopressin or AP changes (Fig. 3).Chemical sympathectomy with 6-hydroxydopamine did notmodify the diuresis or pattern of APir release induced byacute volume expansion in conscious or anesthetized rats(data not shown). The mechanism of the diuresis induced byacute volume expansion in the conscious rat is unknown, butit does appear dependent on an intact right atrium (Fig. 1).The paradoxical response unmasked by volume expansion inthe conscious rat may merely reflect a physiological alter-ation in hemodynamics or renal function that is masked byanesthesia. Another possibility is that the suppressed diure-sis-natriuresis induced by volume expansion in the con-scious atrial-appendectomized rats might reflect the loss ofafferent neuronal pathways involved in the subsequent pe-ripheral release of a putative noncardiac diuretic substance.Alternatively, an intriguing possibility that derives from ourexperiments is that another factor is present in the atria thatresults in the diuresis-natriuresis induced by volume expan-sion in the conscious rat. If such a substance existed in theatrium, it would not have been readily detected because ofthe presence of AP either in anesthetized rats whose bloodvolume had been expanded or in tests of atrial extracts invivo. At best, there is only circumstantial evidence for sucha possibility. Direct demonstration of such a putative mate-rial would put this hypothesis on a firmer foundation.

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