THE ROLE OF CALCIUM IN THE ACTION OF 5-HYDROXYTRYPTAMINE AND CYCLIC AMP ON SALIVARY GLANDS

Transcription

1 J. Exp. Biol. (1973). 58, With 14 text-figures Printed in Great Britain THE ROLE OF CALCIUM IN THE ACTION OF 5-HYDROXYTRYPTAMINE AND CYCLIC AMP ON SALIVARY GLANDS BY WILLIAM T. PRINCE AND MICHAEL J. BERRIDGE A.R.C. Unit of Invertebrate Chemistry and Physiology, Department of Zoology, Downing Street, Cambridge {Received 26 July 1972) INTRODUCTION The purpose of this paper is to examine the role of calcium in the control of fluid secretion by insect salivary glands. Intracellular calcium homeostasis is important in the control of many cellular processes. Calcium fluxes have been associated with action potentials in giant axons and with the action of many hormones (Baker, Hodgkin & Ridgway, 1971; Rasmussen, 1970, 1971). Changes in intracellular calcium concentration are known to effect changes in membrane permeability (Meech, 1972; Romero & Whittam, 1971). Also, calcium mediates excitation-contraction coupling in different kinds of muscle (Triggle, 1971). Because of these facts we have looked at the role of calcium in the functioning of the salivary gland and especially at the possibility that, like cyclic 3',5'-adenosine monophosphate (cyclic AMP), calcium might serve as an intracellular mediator of the action of 5-hydroxytryptamine (5-HT). 5-HT and cyclic AMP both stimulate fluid secretion in the isolated salivary glands of the adult blowfly (Calliphora erythrocephala) (Berridge & Patel, 1968; Berridge, 1970). However, we have recently shown that these compounds have opposite effects on the transepithelial potential (Berridge & Prince, 1971, 1972a). To reconcile these contradictory observations we suggested that 5-HT has two effects: one increases chloride movements and the other stimulates a potassium pump. These effects cause changes in the potential recorded across the apical membrane (Prince & Berridge, 1972). Cyclic AMP acts as an intracellular mediator in many other tissues (Sutherland & Robison, 1966; Robison, Butcher & Sutherland, 1968; Berridge & Prince, 19726). We have accumulated evidence supporting the hypothesis that cyclic AMP acts as an intracellular mediator for 5-HT in the salivary gland. The effect that cyclic AMP has on potential can be simulated by 5-HT when chloride is removed from the bathing medium and replaced by a less permeant anion such as isethionate. In addition, the cyclic AMP concentration increases during stimulation with 5-HT (Prince, Berridge & Rasmussen, 1972). Further, we suggested that the action of 5-HT on the potassium pump was mediated by cyclic AMP. In this paper we show that the action of 5-HT on chloride movements may be mediated by calcium. Brief reports of some of the work described in this paper have appeared elsewhere (Prince et al. 1972; Berridge & Prince, 1972 c).

2 368 W. T. PRINCE AND M. J. BERRIDGE METHODS The salivary glands were those from the adult blowfly, Calliphora erythrocephala (Oschman & Berridge, 1970). The technique for measurement of secretory rate was the same as that described previously (Berridge, 1970; Berridge & Prince, 1972a). Potential measurements were made by placing the salivary gland in a perspex tissuechamber consisting of three separate compartments (Berridge & Prince, 1971, 1972a). The closed end of the gland lay in one outer compartment (the perfusion bath) which was perfused with saline, and the open end lay in the other outer compartment (the saliva bath). The two outer chambers were separated by a chamber filled with liquid paraffin. The term 'transepithelial potential' used in this paper refers to the potential measured in the saliva bath with reference to the perfusion bath by electrodes connected to each outer compartment through agar bridges. Intracellular potential measurements were made using glass microelectrodes filled with 3 M potassium chloride. The term 'basal membrane potential' refers to the potential measured by an intracellular microelectrode with reference to the electrode connected to the perfusion bath. In the experiments shown in Fig. 3 secretory and potential measurements were performed on the same glands under identical conditions. For secretory measurements in such experiments the cut end of the gland was withdrawn from the saliva bath of the tissue chamber (described in Berridge & Prince, 1972 a) into the liquid paraffin. Drops of secretion were then collected and their diameters were measured. The physiological saline used in these experiments was the same as that used previously and had the following composition miyi/1: Na 155, K 20, Ca 2, Mg 2, Cl 156, phosphate 7, malate 2-7, glutamate 2-7, glucose, Phenol red < o-oi. In some experiments a Tris buffer ( mm) replaced the phosphate buffer to prevent precipitation by heavy metals. This tris-saline did not alter secretory or potential responses to either 5-HT or cyclic AMP. In experiments using calcium-free saline 5 mm EGTA (ethyleneglycol-bis-(/?-amino ethyl ether) iv,./v'-tetra-acetic acid) was sometimes added because difficulty was often experienced with calcium impurities in the chemicals used. When the calcium dose-response curve was obtained (Fig. 8) a series of calcium-egta buffers was used. In chloride-free saline sodium chloride was replaced by sodium isethionate, and magnesium and calcium were introduced as the sulphates. Measurements of chloride concentration in the bathing media and secreted fluid were made using the electrometric technique of Ramsay, Brown & Croghan (1955). RESULTS The effect of removing calcium on secretory rate In normal saline the application of IO^M 5-HT causes an increased rate of secretion that is maintained for up to 6 h (Berridge, 1970). However, when 5-HT was applied in the presence of 5 mm EGTA the secretory rate was not maintained (Fig. 1). Initially the rate of secretion increased to a value approaching the maximum secretory rate but after min began to decline, reaching a low rate after 60 min. Maximum secretion was restored by the addition of 2 mm calcium. Removal of

3 30 r- Role of Ca in secretion of salivary glands c 5-HT EGTA Minutes Fig. 1. The effect of io~* M 5-HT applied in the presence of 5 mm EGTA on the rate of fluid secretion by isolated salivary glands. 2 mm calcium replaced EGTA in the saline bathing the glands as indicated by the horizontal bars. 28i O u EGTA I Minutes \ / \ 5-HT Ca Fig. 2. The effect of 5 mm EGTA on the rate of fluid secretion. io" 8 M 5-HT and 2 mm calcium were added as indicated.

4 370 W. T. PRINCE AND M. J. BERRIDGE calcium again resulted in a decline in the secretory rate. This procedure could be, repeated a number of times. Thus, in the continued presence of 5-HT, secretion could be switched on and off simply by the addition and removal of 2 mm calcium. The decline in secretory rate is slower during the initial treatment with 5-HT in 5 mm EGTA than that seen subsequently after the addition of calcium for 15 min periods (Fig. 1). Rather surprisingly EGTA itself was found to increase the secretory rate (Fig. 2). When glands were pre-treated with EGTA before application of 5-HT it was found that there was a significant stimulation of secretion which declined after about 70 min (Fig. 2). This procedure reduced the effect of 5-HT applied subsequently. The onset of secretion stimulated by EGTA alone was substantially slower than that produced by EGTA added together with 5-HT (cf. Figs 1 and 2). As shown in Fig. 1, secretion could be restored to a maximum rate after treatment with EGTA simply by the addition of 2 mm calcium. The effect of removing calcium on the response of the transepithelial potential to 5-HT The transepithelial potential of resting glands is about + 4 mv. When salivary glands are stimulated with 5-HT in normal saline the transepithelial potential becomes about 16 mv negative to the resting potential and remains at this value for as long as 5-HT is perfused over the tissue (Berridge & Prince, 1971, 1972a). When the same experiment was performed in the absence of calcium (i.e. in the presence of 5 mm EGTA) the transepithelial potential first went negative as in normal control responses, but instead of remaining negative the potential went slowly positive and remained positive as long as calcium was absent from the bathing medium (Fig. 3). During the negative-to-positive phase of the responses some of the glands showed oscillations which stopped when the maximum positive value was reached. When calcium was added the potential rapidly returned to the negative value characteristic of the glands during normal responses to 5-HT. The average half-time of this latter response was 2-5 sec. Removal of calcium from the bathing medium in the absence of 5-HT had no effect on transepithelial potential. The secretory response of the glands used for the potential responses was also measured under identical conditions (Fig. 3) and was similar to that described in Fig. 1. Both the initial rate of rise and the subsequent decline in secretory rate during 5-HT stimulation in calcium-free conditions was slower than the corresponding changes in transepithelial potential (Fig. 3). Almost all of the transepithelial potential changes recorded in calcium-free solutions could be accounted for by changes in apical membrane potential. On addition of 5-HT in a calcium-free medium there was a small slow hyperpolarization (2-2 mv) of the basal membrane. This response was similar to that seen in normal saline and was very much smaller than the transepithelial potential change which was recorded simultaneously (Fig. 4). In the continued absence of calcium the hyperpolarization of the basal membrane declined until the potential had returned to the normal resting level (Fig. 4). On addition of calcium the hyperpolarization returned to the same peak value as was reached during the absence of calcium. Thus, the basal membrane response to 5-HT in a calcium-free medium differed from that in the presence of calcium in that the former was not maintained whereas the latter was.

5 50 Role of Ca in secretion of salivary glands frrr _o o OmV Time (min) Fig. 3. A comparison of the effect of 5-HT applied in the presence of 5 mm EGTA on the transepithelial potential (O) and rate of fluid secretion ( ) measured sequentially on the same salivary glands. 5-HT and EGTA were added at the first arrow. 5-HT and 2 mm calcium were added at the second arrow. Each experimental point is the average of experiments with eight different glands ( ± 2 S.E.). _44 L Time (min) EGTA 5-HT Fig. 4. The response of the transepithelial potential (top trace) and basal membrane potential (bottom trace) to io~* M 5-HT applied in the presence of 5 mm EGTA. At min EGTA was replaced by 2 mm calcium. That the basal membrane responds very little to 5-HT in a calcium-free medium indicates that the response of the transepithelial potential is accounted for mainly by changes in the apical membrane potential. The apical membrane potential could be measured directly by changing the reference point of an intracellular micro-

6 372 W. T. PRINCE AND M. J. BERRIDGE -r L Cyclic AMP Ca Time (min) 15 Fig. 5. The effect on the transepithelial potential of 2 mm calcium applied to a gland previously treated with cyclic AMP in the presence of 5 nun EGTA. electrode from the perfusion bath to the saliva bath (Prince & Berridge, 1972). When this was done it was found that when the lumen went negative on application of 5-HT this corresponded to depolarization of the apical membrane and when the lumen went positive in a calcium-free medium this corresponded to hyperpolarization of the apical membrane potential. Effect of removing calcium on the potential response to cyclic AMP In normal saline io~ 2 M cyclic AMP causes the transepithelial potential to go positive of the resting potential and remain positive as long as cyclic AMP is present (Berridge & Prince, 1971, 1972a). We found that removing calcium had no effect on this positivity although it did inhibit secretion (Prince et al. 1972). Positivity was maintained for as long as 40 min whilst the secretory rate diminished to % after 30 min. In an earlier paper we described how half of the glands treated with cyclic AMP in normal saline showed oscillations about a mean value that was positive of the resting potential during treatment with cyclic AMP in normal saline (Berridge & Prince, 1971). Few such oscillations were seen when glands were treated with cyclic AMP in a calcium-free saline. When oscillations were seen they occurred during the initial part of the response. However, if calcium was added during treatment of a gland with cyclic AMP in a calcium-free saline then oscillations were consistently produced (Fig. 5). If calcium was again removed then the oscillations would cease so that the oscillations could be turned on or off by the addition or removal of calcium. The effect of other divalent cations A similar protocol to that used for testing the effect of calcium removal and replacement was followed when testing the ability of different divalent cations to substitute for calcium in the secretory or potential responses to 5-HT. The glands were first treated with 5-HT in 5 mm EGTA until secretion had fallen to a resting level or the potential had stabilized at a value more positive than the resting potential.

7 Role of Ca in secretion of salivary glands Ca Sr Ca s HT Time (min).,. Sr, I I I 90 Time (min) Fig. 6. The effect of 5 mm strontium on the rate offluidsecretion (a) and transepithelial potential (b) of glands previously treated with 5-HT and EGTA. Strontium was applied for the duration of the open bar. 2 mm calcium was present as indicated by the shorter solid bars. In (a) 5-HT was present continuously, in (6) for the length of the top horizontal bar. 30 1x x" + \ I EGTA 2xlO- 3 \ f^ HT I xl0-5 2x- Minutes Fig. 7. (a) The effect of different calcium-egta buffers (containing free calcium concentrations as indicated) on the rate of fluid secretion. 5-HT was present continuously, (b) The effect of 5 x io" 8 M and 2 x io~* M calcium on the transepithelial potential of a gland treated with 5-HT in the absence of calcium. Such glands were then treated with a saline containing the cation to be tested. The results were compared with the effect of adding 2 mm calcium. Strontium and barium Both strontium and barium were found to be able to substitute completely for calcium in the secretory and potential responses to 5-HT (some of the experiments are shown in Fig. 6 a and b). Barium and strontium had no deleterious effect on the glands which remained fully responsive to calcium even after numerous treatments with these two divalent cations. 24 E X B

8 374 W. T. PRINCE AND M. J. BERRIDGE 0 r 50 " io- 4 Concentration (M) " Fig. 8. The ability of calcium ( ), barium (O) and strontium (0) to support 5-HT-induced secretion. Rate of fluid secretion is expressed as percentage of the maximal response. The effects of all three divalent cations were dose-dependent. Fig. 7(a) illustrates the effect of different calcium concentrations on the secretory rate. The effect of submaximal concentrations of calcium on the transepithelial potential is shown in Fig. 7(i); submaximal concentrations of barium and strontium produced similar changes. By comparing the secretory or potential effects of different concentrations of calcium, barium and strontium with the maximal response produced by calcium, dose response curves for each cation can be constructed. Fig. 8 shows the doseresponse curves obtained from secretory measurements. The curves from potential measurements were very similar to these, but they lay slightly to the left of those shown in Fig. 8. Manganese Manganese is also capable of replacing calcium but, unlike the effect of calcium, the effect of manganese is not readily reversible. After the addition of EGTA the secretory rate declined very slowly (Fig. ga). Similar results were obtained when the effect of manganese was tested on potential. When a gland previously treated with 5-HT and EGTA was exposed to 5 mm manganese for 30 sec the transepithelial potential went negative, as it did with calcium, but instead of immediately returning to a positive value when manganese was removed the potential remained negative for as long as 20 min. However, when a gland was treated with 0-5 mm manganese for the same period (20 sec) the potential went negative and then, when the gland was returned to an EGTA saline, recovered quickly to the potential seen before the addition of manganese (Fig. gb). The gland was responsive to subsequent treatment with calcium. However, the response to calcium after manganese was different from that before manganese treatment. In particular, the recovery of positivity in EGTA saline was much slower. In Fig. g(b) the half-time for the recovery from the calcium treatment before addition of 0*5 mm manganese is o-8 min whereas immediately after manganese the half-time is 1-2 min. After four such treatments with calcium the recovery time was similar to that seen before exposure to manganese. The slowly reversible effects of manganese on potential are also apparent in normal

9 Role of Ca in secretion of salivary glands i 20 g - + mv L Time (mm) 15 Fig. o. (a) The effect of 5 mm manganese (applied for the duration of the open bar) on the rate of secretion of glands treated with 5-HT and EGTA. (6) The effect of 0-5 min manganese on the transepithelial potential of a gland treated with s-ht and EGTA. Manganese was applied during the open bar, 2 mm calcium during the short solid bars. saline. In previous papers (Berridge & Prince, 1971, 1972 a) we have described how short treatments of salivary glands with 5-HT produced a biphasic potential response similar to those seen at the beginning of the trace in Fig.. However, when glands were exposed to manganese during one such brief treatment with 5-HT the potential went negative and stayed negative for some time. Only after 5 min did the potential begin to go positive and the gland become responsive to 5-HT once more. 34-3

10 376 W. T. PRINCE AND M. J. BERRIDGE -lor A A mv L 5-HT i i Time (min) 5-HT I I Fig. io. The effect of 5 mm manganese (open bar) on transepithelial responses to 5-HT (solid bars) in normal saline. 20 I Minutes - (.b) L An Ca I 5-HT Ca j I I I I I J I 15 Time (min) Fig. 11. (a) The effect of 1 mm lanthanum (open bar) on the rate of secretion. The glands were previously bathed in 5-HT and EGTA. 2 mm calcium (solid bar) was applied before and after exposure to lanthanum. (6) The effect of 5 mm lanthanum (open bar) on the transepithelial potential. The glands were treated initially with 5-HT and EGTA. a mm calcium was applied for the duration of the shorter solid bars. Ca

11 Role of Ca in secretion of salivary glands 377 c l/m c < r ( a ) «o o C / ^ ^ o /8 or 1 I I I I Chloride concentration (mm) Fig. 12. (a) The effect of changing the chloride concentration of the bathing medium on the rate of 5-HT-induced secretion. (6) The relationship between the chloride concentration (mm/1) in the saliva and that in the bathing medium. Lanthanum Lanthanum had no effect on the secretory rate of glands previously bathed in 5-HT and calcium-free saline, but it did reduce the subsequent effect of adding calcium (Fig. 11 a). After pre-treating the glands with lanthanum the secretory response to calcium was reduced by 68%. Lanthanum did have an effect on the transepithelial potential. When 5 mm lanthanum was applied to a gland previously bathed in 5-HT and EGTA the potential went slowly negative (Fig. 116). This response was much slower than that caused by adding 2 mm calcium before or after lanthanum treatment. When calcium was introduced during the treatment with lanthanum the effect normally seen on calcium addition was absent. In some preparations, as that illustrated in Fig. 11(6), the potential went slightly more negative and began to oscillate. When calcium and lanthanum were replaced by calcium-free saline then the potential returned to the level seen before lanthanum addition and the gland was once more responsive to 2 mm calcium. Magnesium Magnesium did not replace calcium and had no effect on the subsequent response of salivary glands to calcium. A comparison of the effects of 5-HT and cyclic AMP onfluidsecretion In a previous paper we suggested that 5-HT has two effects: one is to increase anion permeability and the other is to stimulate a potassium pump (Berridge & Prince, 1972ft; Prince & Berridge, 1972). Only the latter is directly mediated by cyclic AMP. The action of 5-HT on anion permeability appears to be unnecessary for a full stimulation of secretion because cyclic AMP alone can stimulate fluid secretion equally as well as 5-HT (Berridge, 1970). However, if the transepithelial potential is measured then the effects of cyclic AMP and 5-HT are opposite (Berridge & Prince, 1971). We investigated the possibility that the effects of 5-HT and cyclic AMP on secretion could be differentiated simply by limiting anion movement. The importance of chloride as the permeant anion was examined first.

12 378 W. T. PRINCE AND M. J. BERRTDGE D - [ : ] C.AMP mm chloride 50 mm chloride Fig. 13. The effect of 5-HT and cyclic AMP on the rate of fluid secretion by glands bathed in solutions containing 140 mm and 50 mm chloride. Each column represents the average secretory rate of 20 glands ( ± 2 S.E.). When chloride was progressively replaced with the less permeant anion isethionate, the ability of 5-HT to induce maximal rates of secretion declined sharply at a chloride concentration less than 50 mm (Fig. 12 a). However, even at the concentrations where chloride availability was limiting, chloride was the major anion appearing in the secretion (Fig. 126). The chloride concentration in the saliva declined only when the external chloride concentration was reduced to very low levels. The secretory rates produced by 5-HT and cyclic AMP were compared in glands bathed in normal saline containing 140 mm chloride and in a chloride-isethionate saline where chloride (50 mm) was just beginning to limit 5-HT-induced secretion (Fig. 12 a). In the high-chloride saline there was little difference between the rates in 5-HT and in cyclic AMP (Fig. 13). In the low-chloride saline, however, the average secretory rate in cyclic AMP was significantly less than in 5-HT. DISCUSSION Previously we proposed that 5-HT has two independent actions on the salivary gland (Berridge & Prince, 1971, 1972a, b, c; Prince & Berridge, 1972). One action was mediated by cyclic AMP and consisted of an increase in potassium transport across the apical plasma membrane. The other action of 5-HT was on chloride movement and occurred independently of cyclic AMP. From the results described in this paper we propose that the increase in chloride movement is mediated by calcium. Our current model for the control of secretion by salivary glands is depicted in Fig. 14. There are several lines of evidence implicating calcium as a second messenger in the action of 5-HT. Firstly, from tracer experiments we know that calcium influx into the cell is increased by 5-HT but not by cyclic AMP (Prince et al. 1972). Secondly, stimulation of fluid secretion by 5-HT is calcium-dependent. Thirdly, the chloridedependent depolarization of the apical membrane depends also on the presence of

13 Role of Ca in secretion of salivary glands HT Fig. 14. Diagram illustrating the proposed mode of action of 5-HT. The interaction of 5-HT with the receptor (stippled) leads to an increase in the entry of calcium and activation of adenyl cyclase (AC) to produce cyclic AMP (c. AMP). Cyclic AMP and calcium then act as intracellular intermediaries of 5-HT and initiate fluid secretion. Calcium increases anion movements possibly by increasing the permeability of the apical membrane to chloride. Cyclic AMP stimulates potassium transport across the apical membrane. In addition, various feedback mechanisms exist whereby one messenger can control the concentration of the other, i.e. calcium acts as a negative feedback on adenyl cyclase whereas cyclic AMP increases the release of calcium from an intracellular pool, probably mitochondria. calcium. 5-HT normally causes the lumen to go negative by inducing a large depolarization of the apical membrane (Berridge & Prince, 1972 a; Prince & Berridge, 1972). When calcium is absent from the bathing medium, 5-HT causes the lumen to go positive because the apical membrane becomes hyperpolarized. Hyperpolarization of the apical membrane is characteristic of the response to cyclic AMP in normal saline and to 5-HT if chloride in the bathing medium is replaced by the less permeant anion isethionate (Berridge & Prince, 1971, 1972a). In the experiments described in this paper hyperpolarization could be changed to depolarization simply by the addition of calcium (Figs. 3, 4). In other systems increasing the internal calcium concentration can cause changes in membrane permeability. In Aplysia and snail neurones calcium injected intracellularly increases potassium permeability (Meech, 1972). Changes in the intracellular calcium concentration will also effect changes in membrane permeability in the photoreceptors of Limulus (Lisman & Brown, 1972). In salivary glands a 5-HT-dependent influx of calcium may cause depolarization of the apical membrane. Previously we suggested that 5-HT increases the chloride permeability of the apical membrane (Prince & Berridge, 1972) and now we propose that this action is mediated by calcium. However, although this

14 380 W. T. PRINCE AND M. J. BERRIDGE explanation seems the more plausible on the evidence we have, we cannot, as yet, neglect the hypotheses that calcium might affect a pump controlling the entry of chloride into the cell across the basal membrane rather than affecting the apical membrane directly or that chloride might become linked to the potassium pump so that the pump at the apical membrane becomes electrically neutral. Calcium has long been recognized to play an essential role in both stimuluscontraction coupling in muscle (Triggle, 1971) and stimulus-secretion coupling in various secretory tissues including mammalian salivary glands (Douglas & Poisner, 1963). Recently, calcium has been implicated in the action of numerous hormones and cellular control mechanisms (Rasmussen, 1970, 1971; Berridge & Prince, 1972ft). The picture which is beginning to emerge is that the calcium may play a second messenger function equal in importance to that of cyclic AMP. In the case of salivary glands the chemical information resulting from a successful 5-HT-receptor interaction is transduced into two intracellular second messengers - cyclic AMP and calcium (Fig. 14). The subsequent actions of 5-HT are then mediated by the co-operative effort of both second messengers. Cyclic AMP stimulates potassium transport whereas calcium regulates the flow of anions. Any attempt to understand how cyclic AMP and calcium regulates cell function must take into account the possible existence of complex feedback relationships operating between these two intracellular mediators. For example, 5-HT induces a larger increase in the intracellular concentration of cyclic AMP in the absence than in the presence of calcium (Prince et al. 1972). This suggests that calcium may exert a negative feedback control on adenyl cyclase activity as described previously in heart (Namm, Mayer & Maltbie, 1968) and kidney (Nagata & Rasmussen, 1970). Indeed the stimulatory effect of EGTA on isolated salivary glands (Fig. 2) could be explained by an increase in cyclic AMP concentration resulting from removal of the inhibitory effect of calcium on adenyl cyclase. Such a mechanism may also account for the ability of chelating agents to stimulate smooth muscle (Triggle, 1971) and frog melanophores (Novales et al. 1962). Another important feedback relationship concerns the effect of cyclic AMP on the distribution of calcium between the cytosol and the various intracellular pools. The ability of cyclic AMP to increase the efflux of calcium from salivary glands may depend on this ability of cyclic AMP to stimulate the release of calcium from some intracellular pool such as the mitochondria (Prince et al. 1972). The release of calcium from intracellular storage sites could account for the brief appearance of normal secretory and potential responses when glands are first stimulated with 5-HT in a calcium-free saline (Figs. 1-3). 5-HT will stimulate adenyl cyclase to increase the intracellular level of cyclic AMP which in turn will stimulate the release of calcium from the intracellular calcium pool. Thus, the presence of both second messengers permits the cell to achieve its normal high rate of secretion and negative potential. However, when the intracellular pool of calcium is depleted and lost to the bathing medium, secretion fails and the potential goes positive. External calcium now becomes the limiting factor as shown by the sudden return to high secretory rates and the normal negative potential when calcium is returned. Subsequent removal of calcium in such depleted glands causes very rapid secretory and potential changes because the internal store of calcium is already depleted.

15 Role of Ca in secretion of salivary glands 381 Other feedback relationships have been described in various vertebrate tissues. For example, there is evidence that calcium may exert a negative feedback control on the enzyme phosphodiesterase which degrades cyclic AMP to 5'-AMP (Kakiuchi, Yamazaki & Teshima, 1972). Further, cyclic AMP may influence intracellular calcium homeostasis by altering the distribution of calcium between cytosol and intracellular pools such as the mitochondria or endoplasmic reticulum (Rasmussen, 1971). It is important to remember such feedback relationships when attempting to interpret the actions of hormones and cyclic AMP in normal and calcium-free solutions. One puzzling feature which may be answered by considering these feedback relationships concerns the ability of cyclic AMP to stimulate fluid secretion by salivary glands in the absence of the 5-HT-dependent increase in calcium influx (Fig. 14). Perhaps the ability of cyclic AMP to release calcium from intracellular pools enables the calcium concentration to increase sufficiently to raise anion permeability to a level necessary for near-normal rates of secretion. However, the problem of dragging anions through this relatively impermeable apical membrane results in the large increase in positivity characteristic of the action of cyclic AMP (Berridge & Prince, 1971, 1972 a, b). At this critical internal calcium concentration any further decrease in anion movement would be expected to inhibit the flow of potassium and hence the flow of water. This prediction was tested in the experiments outlined in Fig. 13. When salivary glands were stimulated with cyclic AMP in a medium where chloride was largely replaced with the impermeant anion isethionate, the secretory rate was considerably less than that observed during stimulation with 5-HT. Therefore, when chloride is just beginning to limit the 5-HT response, then the cyclic AMP response is much reduced. We interpret this result as a demonstration that the action of 5-HT on anion movement facilitates the movement of fluid across this epithelium. The fact that 5-HT activates two mediators may explain why a high dose of cyclic AMP has to be applied to glands in order to produce a response. This is often explained by saying that the membrane permeability to cyclic AMP is low and so high doses have to be applied externally in order to raise the internal cyclic AMP concentration sufficiently to induce a response. Another possibility, suggested by the work presented here, is that the cyclic AMP concentration must be raised above that produced by 5-HT to compensate for the lack of increase in intracellular calcium concentration and consequent anion movements. Cyclic AMP applied externally has to stimulate transport across a membrane where anion permeability is not increased as with 5-HT. If anion movements are reduced to a very low level by removing calcium from the bathing medium, fluid secretion declines even in the continued presence of 5-HT. During the period of calcium depletion it is possible to envisage the following conditions. (a) Soon after stimulation with 5-HT in calcium-free solution (5 mm EGTA) there will be a high level of cyclic AMP and calcium accounting for the initial high rate of secretion and negative potential as described earlier. (6) As the internal calcium concentration declines a condition will be reached where cyclic AMP remains high but calcium is at a critical level insufficient to

16 382 W. T. PRINCE AND M. J. BERRIDGE maintain the depolarization of the apical membrane but just sufficient to maintain secretion. Such a condition, which is analogous to that just described during the action of exogenous cyclic AMP, could explain why the potential becomes positive before secretion is reduced. This differential sensitivity to calcium may also explain why the dose-response curves for the potential responses are shifted to the left of those for secretion. (c) A further decrease in internal calcium reduces anion permeability to a very low level causing a subsequent reduction in secretion despite the higher than normal level of internal cyclic AMP (Prince et al. 1972). Although the rate of secretion declines drastically, the apical membrane remains hyperpolarized at a constant value (Fig. 3). This maintenance of hyperpolarization is probably the consequence of several contributory factors. Many enzymic systems require calcium as a co-factor, as exemplified by skeletal and heart muscle where activation of phosphorylase by cyclic AMP is calcium-dependent (Namm et al. 1968; Walsh, Perkins, Brostrom, Ho & Krebs, 1971). Similarly in salivary glands stimulation of the potassium pump by cyclic AMP may be calcium-dependent. Therefore, during calcium depletion the decrease in anion permeability may occur together with a decline in potassium pumping so that there will be little change in membrane potential. The decline in the basal membrane hyperpolarization which occurs during prolonged treatment with 5-HT in calcium-free solutions may be indicative of such a decrease in potassium transport. It was argued previously that the small hyperpolarization of the basal membrane produced by 5-HT might develop as an indirect effect of the increase in ion flux across the epithelium (Prince & Berridge, 1972). The existence of two intracellular mediators which appear to be linked through complex feedback relationships suggest one possible explanation for the oscillations in transepithelial potential seen in Fig. 5 and described in a previous paper (Berridge & Prince, 1971). Any interference in the feedback loops which control and integrate the activity of a complex system usually results in the appearance of oscillations. In the case of salivary glands, oscillations are always observed under conditions where cyclic AMP activates potassium transport and calcium is close to the threshold level necessary to completely depolarize the apical plasma membrane; for example: (a) during the application of submaximal doses of 5-HT (Berridge & Prince, 1971); (b) during treatment with cyclic AMP (the second condition discussed in the preceding paragraph) or sometimes with theophylline as well; (c) during the stimulation of glands with 5-HT in a calcium-free solution (Fig. 4) oscillations develop when the potential goes positive as the calcium level becomes limiting. Any oscillation in the calcium concentration at this critical level would be displayed as potential oscillations consisting of waves of depolarization and hyperpolarization as the concentration rises and falls. This suggestion that the potential oscillations reflect oscillations in internal calcium concentration are supported by the experiments described in Fig. 5, where oscillations are not seen in the absence of calcium, but consistently appear immediately after calcium is added. Another possibility is that these oscillations are independent of changes in calcium concentration but reflect instability of the apical plasma membrane produced by low calcium concentrations. Both strontium and barium are effective substitutes for calcium in both the secretory and potential responses. The effect of barium on potential was slightly slower than

17 Role of Ca in secretion of salivary glands 383 that of calcium or strontium in restoring negativity in a depleted gland. This temporal difference was not seen in secretory responses. The effect of manganese is more interesting and, as yet, is not satisfactorily explained. As seen in Fig. 9 and, 5 mm manganese was able to substitute for calcium in potential and secretory responses but once it had substituted for calcium the effect was not readily reversible; that is to say, the secretory and potential responses were maintained even though manganese and calcium were absent from the bathing medium. 0-5 mm manganese prolonged the recovery from a short exposure to 2 mm calcium in calcium-free saline. This result suggests a possible mode of action. Manganese may block the uptake processes for calcium inside the cells so that once calcium has entered a cell it remains free in the cytoplasm for longer than it normally would. Another possibility is that manganese may react with and substitute for calcium at calcium-binding sites but with a much greater affinity so that the effect is not so readily reversible. Lanthanum reduces the effect of 5-HT on both secretion and on potential (Fig. 11). There is some evidence to suppose that lanthanum can inhibit calcium fluxes across biological membranes (van Breemen & de Weer, 1970). If this is so in the salivary gland then the entry of calcium and consequently the effect of 5-HT will be reduced. In conclusion, cyclic AMP has long been established as an intracellular intermediary in hormone action and more recently in the action of neurotransmitters (McAfee, Schorderet & Greengard, 1971). Calcium too has been shown to serve many roles in cellular control. The results presented here suggest that both cyclic AMP and calcium play significant roles in cell activation during stimulation of salivary glands with 5-HT. Also there appear to be complex interactions between calcium and cyclic AMP within the cell. SUMMARY 1. The role of calcium in the potential and secretory responses of isolated salivary glands of Calliphora to 5-HT and cyclic AMP has been studied. 2. Secretion induced by 5-HT was reversibly inhibited by removal of calcium from the bathing medium. 3. The chloride-dependent depolarization of the apical membrane produced by 5-HT was calcium dependent whereas the potential response to cyclic AMP was little effected. 4. Strontium and barium effectively substituted for calcium. 5. Manganese replaced calcium at the onset of secretory and potential responses but these responses were maintained when manganese was removed. 6. Lanthanum did not substitute for calcium in secretory responses but did inhibit the secretory and potential responses to calcium in calcium-depleted glands. 7. The rate of secretion in a low-chloride medium produced by cyclic AMP was significantly lower than that induced by 5-HT, but there was little difference in normal saline. 8. A model for the mode of action of 5-HT is proposed in which calcium acts as an intracellular intermediary controlling chloride movements whilst cyclic AMP controls a potassium pump.

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