Camp. &o&em. Pfiysiol. Vol. WC, No. 2, pp. 233-239, 1987 0306-4492/87 $3.00 + 0.00 Printed in Great Britain Q 1987 Pergamon Journals Ltd

RECEPTOR TURNOVER AND THE ACTION OF 5HYDROXYTRYPTAMINE ON THE SALIVARY

GLANDS OF THE BLOWFLY CALLJPHORA ~RY~~RUC~~~A~A, THE HOUSEFLY MUS‘CA DOMEXICA AND FROG SKIN EPITHELIUM

TERENCE DALTON

School of Biobgieal Sciences, Queen Mary College, Mile End Road, London Et 4NS, UK (Telephone: 01-980 4811)

(Received I1 December 1986)

Abatrati-t. Continual stimulation of frog skin epitbelium and the salivary glands of the insects ~u~~~p~~~~ and Mrpsca with S-hydroxytryptami~e (5-HT) leads to desensitisation, i.e. the tissue fails to respond to the application of further 5-HT.

2. Incubation of desensitised frog skin and Musca salivary glands with either N-acetyl neuraminic acid or inositol partially restored the 5-HT responses whilst incubation with a combination of N-acetyl neuraminic acid and inositol gave additive effects on the recovery of the 5-HT responses.

3. Incubation of desensitised salivary glands of Ca~/~~i~ru with inositol totally restored the T-NT response whilst incubation with N-acetyl neuraminic acid had no effect.

4. It is concluded that desensitisation involves depletion of secondary messenger from the tissues coupled with receptor degradation and that considerable differences exist in the turnover of 5-HT receptors, the receptors in Musea sahvary glands being highly labile, those in CuNiphora salivary glands highly stable and those of frog skin epithelium being intermediate In their stability.

INTRODUCI’ION

Whilst there is an enormous literature cm the bio- chemistry and pharmacology of 5-HT (Essman, 1978) and its physiolo~~a1 effects (Fuller, 1980) knowledge of the biochemistry of 5-HT receptors and the cellular basis of S-HT action remains relatively vague. A deeper understanding of MIT receptor function has, however, been achieved by the study of model systems-pa~i&ularly smooth muscle preparations (Horn, 197.5), the salivary glands of the blowfly Culliphora and the frog skin epithelium.

5-HT has 2 effects on ion transport across the frog skin epithelium: it stimulates active sodium transport and decreases the passive chloride permeability (Dal- ton, 1976a). Both of these responses appear to be mediated via a single MIT specific receptor type (Dalton, 1979a) which appears to be intimately asso- ciated with membrane gangliosides (glyco-sphingo lipids). The selective removal of N-acetyl neuraminic acid specifically abolishes the 5-HT response (Dalton, 1979b).

5-HT stimulates fluid secretion by the salivary glands of the blowfly Cal&how erythrcxvphalu and the S-HT receptors of this tissue show very similar pha~acola~ical characteristics to frog skin receptors (Berridge, 1972; Dalton, 1977, 1979a) although 2 distinct receptor types do appear to exist: one linked to adenylate cyclase and the other linked to some form of calcium ionophore (Berridge and Heslop, 1981). Activation of this latter receptor type leads to an increased hydrolysis of membrane phospho- inositides to give 2 productsdiacyl glycerol and inositol tri-phosphate-both of which act as second- ary messengers in the mediation of the physiological

response. Inositol tri-phosphate appears to mobilise calcium ions whilst diacyl glycerol stimulates protein phospho~l~tion (Berridge, 1984).

Continual stimulation of ~u~ii~~~r~ salivary glands with MIT leads to desensitisation, i.e. the tissue either fails to respond, or responds with a much reduced response, to the application of further 5-HT (Fain and Berridge, 1979). Such a reduction in re- sponse could be due to receptor breakdown, de- pletion of secondary for subsequent) cellular messen- ger molecules or a combination of these.

In this study the relative importance of both these processes in regulating the 5-HT response in 3 tissues-the salivary glands of the insects C~~~~~h~r~ ~blow~y~ and M.~sx fhouseffy) and the frog skin epithelium-has been investigated.

MATERfAiS AND METHODS

Frogs, Rana temporaria, were rapidly pithed, the ventral skin removed, stretched across a double Using-type cham- ber and incubated in Ringer solution (composition in mM): Na, If3.5, K, 3.5, CI, 116.5, HCO,, 2.4, Ca, 1.9 (pH7.8), aerated with compressed air. The tissue was incubated for 45 min, the skin washed with fresh Ringer and incubated for a further 15 min prior to experimentation.

E#ect of calcium ions on 5-HT responses

In the experiments to show the dependence of the 5-HT response on the presence of calcium ions the tissue was incubated as above in Ca2+-free Ringer solution containing 1 mM EDTA for 45 min followed by a 15 min incubation in Ca2+-free Ringer solution. S-HT (IO-‘M) was then added to

233

234 TERENCE DALTON

the serosal medium and after 15 min 1.9 mM calcium chlor- ide (final concentration) added to either the mucosal or serosal solution.

Effect of multiple applications qf S-HT

After 60 min pre-incubation, 5-HT ( 10m5 M), dissolved in Ringer solution, was added to the serosal medium of one half chamber, the other half acting as control. The changes in membrane potential and short-circuit current (see) were recorded manually using conventional techniques (Ussing and Zerahn, 195 I) for 60 min. The Ringer solutions in all 4 half chambers were then replaced and a further dose of 5-HT (10e5 M) added to the serosal chamber of the experi- mental tissue. This procedure was repeated 4 times. On the final addition of 5-HT (IO-’ M) it was added to the serosal medium of both the experimental and control tissues.

Effecr of N-acetyl neuraminic acid and inositol

After pre-incubation, 5-HT (10m5 M) was added to the serosal medium of both half chambers. The Ringer was replaced after 60 min with fresh Ringer and 5-HT (IO-’ M) added to the serosal medium of both half chambers. This procedure was repeated until the skin in both half chambers had received a total of 4 applications of 5-HT. The Ringer was renewed again and the tissue in one half chamber incubated for 2 hr with either N-acetyl neuraminic acid (10m4 M), inositol (10m4 M) or a combination of these in the mucosal and serosal media. 5-HT (10e5 M) was then added to the serosal medium of both half chambers.

Calliphora and Musca salivary glands

A breeding stock of Musca domesrica, obtained from the World Health Organisation, Geneva, and Calliphora ery- throcephala were maintained at a temperature of 26 & 1°C.

The salivary glands of 3-day-old CaNiphora erythro- cephala or Musca domestica were set up for the measure- ment of fluid secretion using the method of Berridge and Pate1 (1968). Each gland was isolated in a 10 ~1 droplet of Ringer solution under dense liquid paraffin. The cephalic end of the gland was drawn out of the droplet and attached, under the liquid paraffin, to a small glass rod. The rate of fluid secretion was measured by removing the secreted droplet of fluid from the cephalic end at frequent intervals, measuring the droplet diameter, and obtaining the volume by calculation. All glands were incubated in normal Ringer solution for 30min prior to experimental treatment.

The Ringer solution used for Calliphora salivary glands had the composition (mM): NaCI, 120, KCl, 20, NaH,PO,, 8, CaCI,, 2, MgCl,, 2, trehalose, 5, glucose, 5, glutamine, 2, sodium glutamate, 2, proline, 2, alanine, 2, glycine, 2, malic acid, 2, citric acid, 2, fumaric acid, 2 and pH adjusted to 7.2 using KOH.

The Ringer solution used for Musca salivary glands had the composition (mM): NaCI, 94, KCI, 20, NaH,PO,, 2.2, Na,HPO,, 3.8, NaOH, 2.5, MgCI,, 10, CaCI,. 3, trehalose, 4, glucose, 4, sodium pyruvate, 4.5, sodium malate, 3, sodium-a-ketoglutarate, 1.5, disodium succinate, 0.2 and sodium fumarate, 0.2 (Dalton and Windmill, 1980, 1981).

Effect of multiple applications of 5-HT

In one series of experiments the tissue was incubated for 2 hr in the presence of 5-HT (10-s M), washed with Ringer solution and re-challenged with 5-HT. In a second series of experiments, glands were alternately incubated with 5-HT (1Om8 M) or Ringer solution for lo-min periods.

Effect of N-acetyl neuraminic acid and inosilol

Calliphora glands were incubated for 2 hr in S-HT (IO-‘M), washed in Ringer solution and incubated for 2 hr in either N-acetyl neuraminic acid (10m4 M), inositol (10m4 M) or a combination of these. The response to 5-HT (IO-* M) was then determined.

Musca salivary glands were isolated in either normal Ringer solution or Ringer solution containing N-acetyl neuraminic acid (1O-4 M), inositol (10m4 M) or a combina- tion of these and incubated for 2 hr. The response to the addition of S-HT (lo-* M) was then determined.

All chemicals used were obtained from Sigma Chemical Company, 5-HT creatinine-sulphate complex was used throughout this study and all solutions were freshly pre- pared prior to use, especially the 5-HT, as it has been shown to rapidly lose its activity in solution (Dalton, 1976b).

RESULTS

Short-circuit current (see) and transepithelial re-

sistance across frog skin epithelium increase on the

application of 5-HT to the serosal medium (Fig. 1). It has been shown (Dalton, 1976a) that the increase in see is equal to an increase in active sodium transport across the skin and the total increase in transepithelial resistance is equal to a decrease in mucosal-serosal chloride permeability. The effect of 5-HT on chloride permeability (resistance) is crit- ically dependent upon the presence of calcium ions in

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Fig. 1. The response of frog skin to the serosal addition of S-hydroxytryptamine (5-HT) (lOmSM). (a) The change in membrane potential and see in control tissue (membrane potential (-_O-), see (-¤-)) and in response to the addition, at time zero, of 5-HT (membrane potential (- @--), see (-_O-)). (b) The response to 5-HT plotted as the % change in membrane potential (-a-) and see (-0---). (c) The change in transepithelial resistance+ calculated from the membrane potential and see by Ohm’s Law, in control tissue (-a---) and in response to 5-HT (-_O-). (d) The resistance response to the addition of 5-HT calculated as the % change. Each point represents the mean of 6 experiments.

SHT-R turnover in insects and frog 235

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Fig. 2. The effect of adding Ca*+ ions to the mucosal medium on membrane potential (-•-_), see (-_O-) and transepithelial resistance (lower graph) (-_O-) measured across frog skin treated with 5-HT (lo-’ M) in the absence of Ca2+ ions. 5-HT was added at time zero and Cat& (1.9mM) added at maximum 5-HT response. Each point

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the mucosal medium (Fig. 2). Removal of calcium ions from the incubation medium has no effect on the XC response of frog skin to S-HT but has a marked effect on the transepithelial-resistance response. In the absence of calcium ions there is little change in skin resistance. Replacement of calcium ions to the mucosal medium at peak response leads to an in- crease in resistance but no change in see. Addition of calcium to the serosal medium has no significant effect.

Repeated applications of maximal stimulatory con- centrations of 5-HT stimulate progressively smaller changes in both see and transepithelial resistance (Fig. 3). Such desensitised skin does partially recover if incubated in Ringer solution for 2 hr (Fig. 4) however, incubation with N-acetyl neuraminic acid enhances the recovery of both the see and transep- ithelial responses of the skin to 5-HT. Incubation with inositol also partially restores both responses but has a larger effect on the recovery of the chloride permeability response whilst incubation with a com- bination of N-acetyl neuraminic acid and inositol gives additive effects and totally restores the chloride permeability response (Fig. 4).

Cdiphora salivary glands respond maximally to several repeated applications of 5-HT (Fig. 5A), although the response does finally decline. Constant incubation with S-HT for 2 hr results in a decline of the stimulated secretion rate to basal levels with 5-HT having no eflect on such glands (Fig. 5B). “Desensi- tised” glands do not show any recovery when incu- bated in either Ringer solution or N-acetyl neura- minic acid whilst incubation with inositol totally restores the response (Fig. 6A).

The salivary glands of Musca, unlike Cufliphora exhibited a highly variable secretion rate when iso- lated in Ringer solution (Fig. 7). In addition, the

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Fig. 3. The response of frog skin to the repeated application of 5-HT (10m5 M) to the serosal medium. The upper graph shows the change in see control tissue (-_O-) and in tissue treated with 5-HT (-_O-) and the lower graph shows the change in transepithelial resistance in control (-¤-) and experimental tissue (-a-). At time zero 5-HT (lo-’ M) was added to the serosal medium of the experimental tissue and at the times indicated by the arrows both experimental and control tissues were washed with Ringer solution. 5-HT (10m5 M) was added to the serosal medium of the experimental tissue and carrier to the serosal medium of the control tissue. On the final application 5-HT was added to the serosal medium of

both the experimental and control tissues. Each point represents the mean of 3 experiments.

236 TERENCE DALTON

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Fig. 4. The recovery of the 5-HT responses in desensitised frog skin. Tissue was desensitised by 4 applications of 5-HT (IOm5 M), at 60min intervals, in the serosal medium. The see response (a) and transepithelial resistance response (b) to the serosal addition of 5-HT (10m5 M) was measured either immediately, i.e. no recovery time (histograms a) or foliowing 2 hr incubation in either Ringer solution (histograms b), N-acetyl neuraminic acid (IO-” M) (histograms c), inositol (IO-‘M) (histograms d) or a combination of N-acetyl neuraminic acid and inositol (histograms e). The responsescan be compared with the normal responses induced by 5-HT in fresh frog skin (histograms f). Each histogram shows the mean

response k SEM N = 8.

glands were highly variable in their response to S-HT secretion. Stimulated rates of secretion were never as although, as shown in Fig. 7, glands with a low initial high as those achieved by stimulated Calliphoru sali- rate of fluid secretion did exhibit larger responses to vary glands although the dose-response character- applied S-HT than glands with initial high rates of istics of the 2 tissues are the same (Dalton, un-

published data). Subsequent washing and addition of a further dose

of 5-HT to h4usca salivary glands sometimes induced a much reduced response or, more usually, failed to stimulate any further response. Glands isolated in Ca*+-free Ringer showed a similar wide range of initial secretion rates and all failed to respond to the addition of 5-HT (Fig. 7). Glands isolated and incu- bated in N-acetyl neuraminic acid showed stable low secretion rates at the end of the incubation and these glands responded to the application of S-HT. Glands incubated in inositol, exhibited an increased secretion rate upon the application of 5-HT although this

2 30 increase is significantly less than glands incubated

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z ; 3 10 DISCUSSION

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cl 0 frog skin appears to be mediated by an influx of CL 0 20 40 60 so loo I20 140 calcium ions into the tissue across the mucosal bor-

Time (mins) der. In many tissues where calcium acts as messenger

Fig. 5. The effect of 5-HT on fluid secretion by isolated in mediating the physiological response interaction of

salivary glands of CaNiphora eryrhrocephala. The upper a primary messenger with its receptor stimulates the

graph shows the effect of adding S-HT, ( lOA M), at time activity of the enzyme phospholipase C which acts on

zero, on secretion rate. After 120 min the tissue was washed membrane phospho-inositides to produce diglyceride

with Ringer solution and re-challenged with S-HT (IO-* M). and water-soluble inositol phosphates (Nishizuka,

The lower graph shows the effect of alternately adding 5-HT 1984; Troyer et al., 1986). Berridge (1984) proposed (IO-&M) (open arrows) and washing with Ringer solution that, in many tissues, inositol tri-phosphate acts as (closed arrows) on secretion rate. Each point represents the secondary messenger to increase cytosolic calcium

mean of 3 experiments. levels.

SHT-R turnover in insects and frog 237

a ) Calliphara

( b) Musca

Fig. 6. The recovery of S-HT responses in desensitised Calliphora salivary glands (a) and in Musca salivary glands (b). Calliphora salivary glands were desensitised by incubating in 5-HT (lo-* M) for 2 hr. The rate of fluid secretion was then measured in the presence of S-HT (IO-* M) either immediately (histogram a) or following 2 hr incubation in either Ringer solution (histogram b), N-acetyl neuraminic acid (lo-” M) (histogram c) or inositol (10e4M) (histogram d). These responses can be compared with the normal 5-HT-induced response in freshly isolated salivary glands (histogram e). Musca salivary glands were isolated and the rate of fluid secretion in the presence of S-HT (10m8M) determined following 2 hr incubation in Ringer solution (histogram a), N-acetyl neuraminic acid (10m4 M) (histogram b), inositol

(10e4 M) (histogram c) or a combination of N-acetyl neuraminic acid and inositol (histogram d).

Dalton (1979b) showed that treatment of frog skin with the enzyme N-acetyl neuraminidase, which re- moves N-acetyl neuraminic acid residues from glyco- lipids and glyco-proteins, specifically diminishes the responses of frog skin to 5-HT thus showing that S-HT receptor degradation involves the removal of N-acetyl neuraminic acid. Desensitisation of frog skin to the application of 5-HT could involve the degradation of the S-HT receptor by the removal of N-acetyl neuraminic acid, the depletion of the tissue of secondary messenger (in the case of the chloride

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Fig. 7. The effect of 5-HT on fluid secretion by isolated salivary glands of Musca domestica. Salivary glands were isolated in either Ringer solution (-a-) or Ca2+-free Ringer solution (-_O-) and the initial rate of fluid secre- tion measured over a 10min period. S-HT (lo-‘M) was then added to the incubation medium and the rate of

secretion monitored for a further 10 min.

permeability response of inositol phosphates), or a combination of both effects.

Incubation of desensitised frog skin for 2 hr in Ringer solution does lead to partial recovery of both the sodium transport and chloride permeability re- sponses, however, incubation with N-acetyl neura- minic acid enhances the recovery of both responses thus indicating that some receptor degradation does occur following 5-HT binding. The (reduced) recov- ery that occurs when the tissue is incubated in Ringer solution only would indicate that some receptor re-synthesis does take place under these conditions probably by utilising the N-acetyl neuraminic acid released in receptor breakdown since it has been shown that if the removed N-acetyl neuraminic acid is degraded using the enzyme N-acetyl neuraminic acid aldolase then receptor re-synthesis is inhibited (Dalton, 1979b). Addition of inositol to desensitised frog skin leads to a partial recovery of both responses but has a larger effect on the recovery of the chloride permeability response. These responses thus appear to be dependent upon inositol metabolism and the subsequent mobilisation of calcium ions and desensi- tisation is associated with the depletion of inositol derivatives as has been shown for Cdiphora salivary glands (Fain and Berridge, 1979). The larger effect of inositol incubation on the chloride permeability re- sponse relative to the sodium transport response would indicate that the chloride permeability re- sponse is more dependent upon inositol (and hence calcium ions) than the sodium transport response. This latter response is mediated (principally) by cyclic adenosine monophosphate (Dalton, unpublished data) as a secondary messenger, however, the data presented in Fig. 4 would indicate that the stimulated pathway leading to increased sodium transport is dependent at some point on inositol/calcium ions. Incubation in a combination of N-acetyl neuraminic acid and inositol totally restores the chloride perme-

ability response but only partially restores the sodium involves both receptor degradation (removal of N- transport response. Such an observation would be acetyl neuraminic acid) and the tissue depletion of expected if desensitisation of the tissue to 5-HT secondary messenger (inositol derivatives) whilst in involved both receptor degradation and depletion of the salivary glands of Culliphoru erythrocephalu de- secondary messenger (inositol derivatives) and if the sodium transport response (linked primarily to

sensitisation is associated with tissue depletion of secondary messenger only.

CAMP production) was critically dependent upon the number of functional receptors in the tissue and less

In Muscu salivary glands the receptors appear to be

dependent upon inositol and calcium whilst the chlor- highly labile with receptor degradation occurring rapidly following 5-HT stimulation with re-synthesis

ide permeability response, although dependent upon occurring at very low rates so that receptor numbers the number of functional receptors in the tissue, was more dependent upon the levels of available inositol

determine the magnitude of response to a greater

derivatives/calcium ions, i.e. secondary messenger. degree than cellular levels of secondary messenger.

The results obtained with Culliphora salivary 5-HT receptors in Culliphoru may either be very

stable with little degradation following 5-HT stimu- glands substantiate the observations of Fain and Berridge (1979) that the response of desensitised

lation or if receptor degradation does take place it is restricted to spare (excess non-physiological) recep-

glands can be totally restored with inositol incu- tors with tissue levels of secondary messenger deter- bation. Incubation in Ringer solution or N-acetyl neuraminic acid does not induce any recovery. Thus,

mining the magnitude of response.

assuming that the 5-HT receptors in CaNiphora con-

The 5-HT receptors in frog skin epithelium appear

tain N-acetyl neuraminic acid, little, if any, degra- to be intermediate in stability. Receptor degradation

dation of physiologically active 5-HT receptors oc- does follow 5-HT stimulation but receptor re- synthesis occurs at measurable rates. The number of

curs in the salivary glands following 5-HT stimu- lation and the observed reduction in response is due

active receptors appears to be more important in determining the 5-HT response on active sodium

to secondary messenger depletion. The lack of re- transport across this tissue whereas tissue levels of sponse from incubation with N-acetyl neuraminic acid does not preclude receptor degradation however,

inositol derivatives (as secondary messenger) and

since both a receptor threshold and reserve exist for their depletion appears to be more important in determining the magnitude of the reduced chloride

5-HT in the salivary glands (Dalton, 1977). Degra- permeability response. dation of receptors may be occurring but this may be associated only with the reserve-thus the population of physiologically active receptors would not be REFERENCES affected.

5-HT receptors in the salivary glands of Muscu are Berridge M. J. (1972) The mode of action of 5-hydroxy-

somewhat different. Salivary secretion on isolation tryptamine. J. cq. Viol. 56, 3 I I-323.

and the response to 5-HT are variable. It seems likely Berridge M. J. (1984) Inositol triphosphate and diacyl-

that the removal of the glands from the animal glycerol as secondary messengers. Biochem. J. 220, 345-360.

stimulates them to secrete at variable rates and that Berridge M. J. and Heslop J. P. (1981) Separate

this stimulation leads to variable degrees of desensi- 5-hydroxytryptamine receptors on the salivary gland of

tisation. Incubation of these glands in Ringer solu- the blowfly are linked to the generation of either cyclic

tion does not restore any response whilst incubation adenosine 3’-5’.monophosphate or calcium signals, Br. J.

in N-acetyl neuraminic acid does lead to a restoration Pharmuc. 73, 729 738.

of response. This suggests that the 5-HT receptors do Berridge M. J. and Pate1 N. G. (1968) Insect salivary glands:

contain N-acetyl neuraminic acid and that it is stimulation of fluid secretion by 5hydroxytryptamine and

removed from the receptor following 5-HT stimu- adenosine 3’-5’-monophosphate. Science, (Wash.) 162, 462463.

lation leading to suppression of subsequent re- sponses. Thus under normal conditions receptor re-

Dalton T. (1976a) The effect of 5-hydroxytryptamine creati-

synthesis appears to be very slow since incubation in nine sulphate on sodium transport across isolated frog skin. Camp. Biochem. Physiol. 56C, 4141.

Ringer solution does not restore any response; in frog Dalton T. (1976b) The loss of biological activity of

skin a similar incubation does lead to a small recov- 5-hydroxytryptamine creatinine sulphate. Experientia 32,

ery indicating a higher rate of receptor re-synthesis in 1421-1422.

this tissue. The 5-HT response of Musca salivary Dalton T. (1977) Threshold and receptor reserve in the

glands is dependent upon the presence of calcium action of 5-hydroxytryptamine on the salivary glands of

ions in the incubation medium, as is the response in Calliphoru er~throcephulu. J. Insect Physiol. 23, 62543 1.

CuIliphoru glands (Prince and Berridge, 1973) and Dalton T. (1979a) Pharmacology of 5-hydroxytryptamine

incubation of glands with inositol leads to a partial receptors in frog skin epithelium. Br. J. Pharmac. 65, 579-585.

restoration of 5-HT response although the restored Dalton T. (1979b) Selective destruction of 5_hydroxytrypt-

response is less than that obtained by incubation in amine receptors by neuraminidase. Biochem. biophys.

N-acetyl neuraminic acid-thus desensitisation ap- Acta 555, 362-365.

pears to be due more to receptor degradation than to Dalton T. and Windmill D. M. (1980) Fluid secretion by

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Dalton T. and Windmill D. M. (1981) The permeability

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Desensitisation of frog skin epithelium and salivary housefly Musca domestica Comp. Biochem. Physiol. 69A, 21 l-217.

238 TERENCE DALTON

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SHT-R turnover in insects and frog 239

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Fain J. N. and Berridge M. J. (1979) Relationship between phosphatidylinositol synthesis and recovery of 5- hydroxytryptamine responsive calcium flux in blowfly salivary glands. Biochem. J. 180, 655-661.

Horn A. S. (1975) SAR for neurotransmitter receptor agonists and antagonists. In Handbook of Psycho- pharmacology (Edited by Iversen L. L., Iversen S. D. and Snyder S. H.), Vol. 2, pp. 179-243. Plenum Press, New York.

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in the action of 5-hydroxytryptamine and cyclic AMP on salivary glands. J. exp. Biol. 58, 367-385.

Nishizuka Y. (1984) The role of protein kinase C in cell surface signal transduction and tumour production. Nature 308, 693498.

Troyer D. A., Schwertz D. W., Kreisberg J. I. and Ven- katchalam M. A. (1986) Inositol phospholipid metabo- lism in the kidney. Ann. Rev. Physiol. 48, 51-71.

Ussing H. H. and Zerahn K. (1951) Active transport of sodium as the source of electric current in the short- circuited isolated frog skin. Acfa physiol. Stand. 23, 1 l&127.