Mechanisms of Inactivation of Neurohypophysial Hormone Release

The Neusohyprphysis :Stsuc.tuse. Function upid Coritsol, Psogrc.ss in Bruin Reseurch, Vol. 60. edited iqi B.A. Cross mid G . Leng 01983 Elsevies Science Puhlishrss 8.V .

Mechanisms of Inactivation of Neurohypophy sial Hormone Release F.D. SHAW, R.E.J. DYBALL* and J.J. NORDMANN] Deppartment of Anatomy. King’s College London, Strand. London WCZR 2LS ( U . K . ) arid ‘Insritu! National de Recherche Medicale. Rue Camille Sain!-Snens, 33077 BnsdrLiux Cedex (Fsuncr)

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INTRODUCTION ltmctivation clf hormone release :the problem The biosynthesis of the neurohypophysial hormones, followed by packaging into granules and transport to neurosecretory swellings and terminals (see Morris et al., 1978), provides a large pool of stored product from which hormone secreted into the blood is derived. The mechanisms which regulate the release of available hormone are fundamental to the physiology of the magnocellular neurosecretory system, and are probably of relevance to peptidergic neurones in general, The application in vivo of appropriate stimuli to release hormone is associated with an increase in the firing rates of magnocellular neurones (see Poulain and Wakerley, 1982). A direct relationship between supply and demand for neurohypophysial hormones might reasonably be inferred, but this is demonstrably not the case. As long ago as 1960, Sachs and his colleagues discovered that, in dogs subjected to haemorrhage, an increased blood concentration of vasopressin is not maintained beyond the first few minutes of the stimulus (Weinstein et al., 1960). Later experiments showed that a much reduced secretory response is elicited by a haemorrhagic stimulus applied 1 h after an initial stimulus of only 10 min duration (Sachs et al., 1967). Hormone release from isolated neurohypophyses subjected to a prolonged depolarizing stimulus in vitro has also been shown to decline at a rate greater than can be accounted for by depletion of the total hormone store (Sachs et al., 1967 ; Sachs and Haller, I968 ;Thorn, 1966). These results were originally interpreted as evidence for the existence of two separate hormone pools, one of which was “readily releasable” and constituted 5-20 % of the total store. It was postulated that this pool was constituted by the free cytoplasmic hormone then thought to exist (Ginsburg, 1968; Thorn, 1966), or alternatively by those granules situated close to the release site (Sachs et al., 1967; Sachs and Haller, 1968). However, it has since been demonstrated that stimulation in vitro can release 30 96 or more of the stored hormone (Muller et al., 1975; Nordmann, 1976). It was accordingly suggested that the “readily releasable pool” might represent those granules present in the secretory terminals, which account for some 30 % of the total granule population in the unstimulated neurohypophysis

* Author for correspondence at present address: Department of Anatomy, Downing Street, Cambridge, CB2 3DY. U.K.

306 (Nordmann, 1976, 1977). The decrease (inactivation) of hormone release during prolonged stimulation in vitro was attributed to a decline in membrane calcium conductance, similar to that first described in the squid giant axon (Baker et al., 1973b). The existence of such a mechanism has been demonstrated in anumber of cell types (e.g. D'Amore and Shepro, 1977 ; Foreman and Garland, 1974; Maddrell and Gee, 1974), and is thought, for instance, to limit catecholamine release by adrenal medullary cells (Baker and Rink, 1975). However, many questions remain. Is the calcium channel inactivation hypothesis a sufficient explanation for all the observed features of the inactivation of hormone release? Is the mechanism exclusively depolarization-dependent, as suggested by the earlier work ? Does an exactly similar mechanism operate when hormone release is elicited by the transient depolarization which follows the arrival of action potentials at the secretory terminals, rather than by the maintained depolarization employed in previous experiments ? What is the physiological significance of inactivation of release, and can the same mechanisms be demonstrated in vivo? The present experiments were thus undertaken to re-examine the calcium entry hypothesis, and to extend the observations to electrical stimulation in vitro and to the intact system in vivo. The characteristics of hormone release inactivation will be shown to be rather more complex than previously supposed. EXPERIMENTAL APPROACHES Most of the experiments described employed an isolated neural lobe preparation maintained in vitro, which permits manipulation of the ionic environment and the precise determination of the time course of secretory response. Hormone release was evoked either by raising extracelMar potassium, which is known to cause maintained depolarization (Nordmann et al., 1982), or by application of electrical stimulus pulses. The pulses were delivered at 10Hz, a frequency similar to those recorded from the cell bodies of magnocellular neurones in rats subjected to haemorrhage or dehydration (see Poulain and Wakerley, 1982). Hormone release was stimulated in vivo by the administration of a 2 % solution of NaCl in place of drinking water (Jones and Pickering, 1969) and the neurohypophyses were subsequently removed and incubated in vitro. The bicarbonate-buffered incubation medium was maintained at 37" C, constantly bubbled with 95 % 0, and 5 % CO,, and had the following basic composition : NaCl 150 mM ;KHCO, 5.6mM; MgC1, 1 .0 mM; CaCI, 2.2 mM, and glucose 10mM. During potassium stimulation, KC1 was increased to 56mM and NaCl reduced to l00mM; in other experiments using a reduced sodium concentration, choline chloride was added for osmotic compensation. Tissue to be electrically stimulated was impaled upon one of a pair of platinum electrodes immersed in the incubation medium (Dutton and Dyball, 1979). The medium was changed every 15 min, and the collected samples assayed by the rat milk-ejection tnethod (Bisset et al., 1967). Experimental decay constants for hormone release inactivation were calculated together with their correlation coefficients, as described by Nordmann (1976). Significance levels quoted in the text all refer to Student's r-test. RESULTS Inactivation and reactivation in vitro :potussium stimulation Stimulation for 2 h with 56 mM potassium in the presence of 100 mM sodium resulted in progressive inactivation of hormone release from isolated neurohypophyses (Fig. la), similar

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to the observations of Miiller et al. ( 1975)and Nordmann ( 1975, 1976). The inactivation could be described by a single exponential with a decay constant ( k ) of 0.012 min-' (Y = 0.96). If calcium was omitted from the incubation medium or if 0.1 mM D600 was present, hormone release failed to increase over basal levels during stimulation, again in accordance with previous results (Dreifuss et al., 1973). Inactivation cannot be attributed to a non-specific decline in viability of the tissue, as it is shown below that neurohypophyses first stimulated after as long as 5 h in vitro exhibit normal responsiveness. 100 Na

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Fig. I .a: hormone released from the isolated rat neurohypophysis during depolarization with a raised (56mM) K f concentration added at time 0 and a sodium concentration of 100mM. In each case hormone release is shown as milk-ejection activity (MEA) expressed in terms of mU of synthetic oxytocin. In this and the following figures, the horizontal dotted line indicates basal release (control,e---. ;experiments in which calcium was omitted for the first 60 min, M). b: similar experiments camed out in the absence of sodium. Both experiments show that, in the absence of calcium, inactivation was less severe and imply that calcium is important for inactivation.

To investigate the calcium-dependence of inactivation, a second group of neurohypophyses, incubated in parallel with the above experiment, were stimulated for 1 h in the absence of calcium, after which time calcium was reintroduced and the subsequent hormone release determined over a further 1 h (Fig. I a). The release during the first sample period after calcium addition was similar to the corresponding value when calcium was present throughout, suggesting that inactivation had occurred by a calcium-independent mechanism (Nordmann, 1976). However, the following samples all contained significantly more hormone (P< 0.01) than the relevant controls. It would thus appear that prolonged depolarization in the absence of calcium provokes only a limited degree of inactivation, and that a major component is calcium-dependent. The delayed increase in secretion may represent the time required for calcium to infiltrate the extracellular space, and also to reach the appropriate concentration since extracellular binding sites would also demand reoccupation. A similar pair of experiments was performed in the absence of extracellular sodium (Fig. Ib). When calcium was present throughout the period of stimulation, initial hormone release was much greater than in I O O m M sodium, in accord with previous accounts of

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antagonism between extracellular sodium and calcium in secretory systems (Kelly, 1965; Dreifuss et al., 1971). The subsequent inactivation proceeded more rapidly than in I O O m M sodium, with k = 0.02 I min-’ (r = 0.98) rather than at arate similar to that previously reported and cited as evidence for calcium channel inactivation (Nordmann, 1976). When calcium was introduced after 1 h of stimulation, release was higher than the corresponding controls throughout the following I h period (Fig. Ib). This occurred despite the small increase in secretion during the initial 1 h stimulation without added calcium, due presumably to the remaining extracellular calcium, as chelaters were not employed. Hormone release inactivation therefore showed a major dependence on extracellular calcium, both in the presence and absence of sodium, and a relatively minor dependence upon depolarization. These results led to the consideration of recovery from inactivation, referrcd to here as “reactivation”. Different groups of neurohypophyses were potassium-stimulated for 1 h (in the presence of sodium), returned to a normal medium for different time intervals, and then exposed once again to the stimulus. The results are expressed in Fig. 2a, in terms of the percentage of the initial release achieved by each neurohypophysis. Hormone release had significantly inactivated (P< 0.0 1) by the final 15 min period of the initial stimulation, and fell

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Fig. 2.a: hormone released (expressed as % initial MEA rclease) at diffcrent intervals after a I h potassium stimulus in the presence of 2.2 mM calcium, which ended at time 0. Reactivation occurs only slowly and is not complete after 3 h. b : similar experiments in which the initial stimulation was carried out in the absence ofcalcium. Inactivation was less severe and had recovered to values not significantly below control within 2 h. This implies the existence of at least two mechanisms of inactivation: a potential-dependent one which reactivates quickly and a calcium-dependent onc which does not. (Error bars arc added in a since the values were derived by comparing initial and subsequent release. They were not added in b since there was no initial release, and the release which followed the second stimulus was compared with that from a separate control series of neurohypophyaes.)

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to the basal levels by 30 min after its termination. Restimulation after intervals of 1 and 2 h evoked an increase in hormone output, but only to levels significantly lower (P< 0.01) than that from the same neurohypophyses at the end of the initial stimulation period. After a 3 h interval some recovery of secretory capacity was observed, but secretion nevertheless achieved only 54 % of its initial rate. It is unlikely that any damage was caused by the initial 1 h stimulus as the rate of loss of the cytoplasmic enzyme lactate dehydrogenase was unaltered throughout the incubation period, and as electron microscopy failed to reveal any structural alterations. A potassium stimulus therefore exerts chronic effects on subsequent hormone release. The calcium-dependence of reactivation was investigated by stimulating for 1 h in the absence of calcium, followed by an interval and restimulation in its presence (Fig. 2b). The releases evoked by restimulation were compared with those for control neurohypophyses, treated identically except that they were not depolarized during the initial I h. As can be appreciated by comparison of Fig. 2a and b, recovery following stimulation was much more rapid in the absence of calcium. After a 1 h interval, release was 63 % of the control, but was nevertheless significantly less ( P < 0.01), while after a 2 h interval release was not significantly different from the control. These results presumably represent the characteristics of the depolarization-dependent, calcium-independent process, and it would thus appear that the calcium-dependent process is responsible for long-term inactivation.

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Fig. 3.a: hornionercleased (expressed asinFig. I)duringa2 hperiodofelectrical stimulation at IOHz withasodium ;experiments in which calcium was omitted for the first concentration of 100 niM, begun at time 0 (control,60 inin,-). With electrical stimulation, omission of calcium did not appcar to impair thc degrce of inactivation (but lowered external sodium may have enhanced it). b : the effects of manipulation of calcium and sodium concentration on inactivation. Significant inactivation occurred only in those experiments with lowered cxternal sodium concentration.

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Inactivution and reactivation :in vitro electrical s t i ~ ~ l a t j o n Hormone release during 2 h of electrical stimulation is shown in Fig. 3a. It should be noted that the neurohypophyses were incubated in a medium containing l O O m M sodium (see below). Stimulated release could be abolished by the omission of calcium, and by the presence of 0.1 mM D600 or low7g/ml tetrodotoxin, this last suggesting that propagation of sodium-dependent action potentials to the neurosecretory terminals was involved. The rate of secretion was initially about one-third of that obtained with potassium stimulation in the presence of lOOmM sodium, but inactivation actually proceeded more rapidly, with k = 0.020min-' (r = 0.99). This result prompted investigation of the effects of external sodium and calcium on electrically stimulated release (Fig. 3b). The rapid inactivation during electrical stimulation in 100mM sodium was initially interpreted as arising from activation of a calcium-mediated potassium permeability, as identified in both invertebrate and vertebrate neurones (see Meech, 1976), due to the increased calcium influx during stimulation. Such an increase in potassium permeability would reduce the duration of the action potential, and hence the rate of calcium entry and of hormone release. However, this explanation was rendered unlikely by the finding that significant inactivation occurs after 1 h of stimulation in a medium containing 100 mM sodium and only 1.5 mM calcium, despite the much reduced initial release (Fig. 3b). Further, prolonged stimulation in media containing 150 mM sodium did not result in inactivation after 1 h, whether a normal or a raised (4.95 mM) calcium concentration was employed, despite the large initial calcium influx which must have occurred in the latter case (Fig. 3b). These results suggest that inactivation caused by electrical stimulation at this frequency is a quite specific effect of lowering the sodium concentration of the medium, regardless of the calcium concentration or the ratio between the two ions. The effect of adding calcium after I h of stimulation in its absence (Fig. 3a) is consistent with the hypothesis of a sodium-dependent, calcium-independent effect. Addition of calcium resulted in an increased rate of secretion, but only to values almost identical with the corresponding controls. Reactivation following electrical stimulation in I O O m M sodium is shown in Fig. 4. After an initial stimulus of only 30 min, and an interval of 30 min, restimulated release was significantly reduced (P< O.Ol), amounting to only 52 % of the initial rate. More surprisingly, release of only about 30 % of the initial rate was observed after intervals of 1 and 4 h (Fig. 4a). Hormone release from control neurohypophyses which were stimulated after incubation in vitro for an equivalent time to the experimental group subjected to a 4 h interval, was not significantly below the initial release rate and was significantly higher ( P < 0.0 1) that the restimulated release at that time (Fig. 4a). Further, there was no increase in lactate dehydrogenase release, and no ultrastructural changes were observed at that time, so it may be presumed that the tissue was fully functional. Long-term effects were also seen after only 15 min initial electrical stimulation, although the results were more variable and the inactivation not so profound (Fig. 4b). By contrast, neurohypophyses stimulated for 30 min in the absence of calcium, followed by an interval of 30 min and restimulation in its presence, released hormone equivalent to 8 I % of the control level, and not significantly different from it. Reactivation after a relatively short initial electrical stimulus thus apparently displays a calcium-dependence similar to that of potassium stimulation. However, it remains possible that the sodium-dependent effect also contributes to long-term inactivation ; more experiments are required to determine whether further inactivation occurs after intervals of 1 4 h, as was observed following an initial 30 min stimulus in the presence of calcium (Fig. 4a).

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Fig. 4.a: hormone release (expressed as in Fig. 2) at different intervals after 30 min electrical stimulation at lOHz, which ended at time 0. Restimulation after an interval of I h released less hormone than after an interval of 30 min and there was no recovery within the 4h incubation period; control release at this time confirmed the viability of the tissue. b: similar experiments after only 15 min initial stimulation.

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Fig. 5.a: the influence of 1 h potassium stimulation ending at time 0 (-) on a subsequent electrical stimulation (w) after an interval of 1 h ; also shown are the equivalent values for control electrical stimulation (*---*). b: the ending , at time 0 on a subsequent potassium stimulation (-). influence of 30 min electrical stimulation ( M ) Also shown are the equivalent values for a control potassium stimulation (0--4). Both initial stimuli impaired the effectiveness of the second stimulus, so that potassium and electrical stimulation clearly interact.

3 12 Interaction between potassium and electrical stimulation

The results outlined above suggest that there are three apparently separate processes governing inactivation, dependent upon depolarization, extracellular calcium and extracellular sodium. As the characteristics of inactivation and reactivation associated with either potassium or electrical stimulation were generally similar, the effect of one upon the other was investigated. Neurohypophyses were stimulated with raised potassium for 1 h or electrically for 30 min, followed by an interval of 1 h and application of the alternative stimulus (Fig. Sa, b). Initial potassium stimulation significantly ( P < 0.05) reduced the hormone release evoked by a subsequent electrical stimulus, to 30 % of the control values (Fig. 5a). Tissue initially stimulated with electrical pulses also released significantly less (P< 0.05)in response to a subsequent potassium stimulus than the appropriate controls, amounting to 33 % of the latter (Fig. Sb). Potassium and electrical stimulation thus interact, 1 h of the former being approximately equivalent to 30 min of the latter in terms of their subsequent effects on reactivation (see also Figs. 2a and 4a). It should nevertheless be noted that hormone release evoked by 30 min electrical stimulation is only about 20% that evoked by 1 h potassium stimulation. The results encourage the view that similar processes are responsible for the inactivation which follows both stimuli. Further, electrical stimulation followed after an interval by potassium constitutes an in vitro equivalent of the experiments of Sachs et al. (1967), who found that neurohypophyses taken from dogs subjected to a haemon-hagic stimulus released much less hormone when stimulated with high potassium than did controls. This in turn suggests that the process occurring in vivo is similar to that observed in vitro. Inacfivution in vivo and reactivation in vitro

The possibility of inactivation in vivo was here investigated using the chronic stimulus of substituting 2 % NaCl for drinking water, which is known to release both oxytocin and vasopressin (Jones and Pickering, 1969). After only 24 h of such treatment, neurohypophyses incubated in vitro and electrically stimulated released substantially less than controls, the

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Fig. 6. Hormone release during 2 h electrical stimulation (beginning at time 0) from control glands (-) and neurohypophyses from rats that had becn given 2 % NaCl to drink instead of water for 24 h (M). Inset are the values for the relative reduction in gland content and hormone release after 24 h of such treatment. Clearly drinking 2 % NaCl (in vivo) reduced the subsequent release from isolated glands in vitro and the relative reduction of release was significantly greater ( P < 0.01) than that of gland content. This implies that inactivation of release also occurs in vivo.

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initial release reaching only 48 % of the control level (Fig. 6). It is unlikely that the decrease was due entirely to depletion, as the treated neurohypophyses contained 76 % of the hormone content of controls, so the proportional decrease in release was significantly greater (P< 0.01) than that of neurohypophysial content. It is thus likely that prolonged stimulation in vivo causes inactivation by mechanisms similar to those described above, although a far higher proportion of the gland content can ultimately be released in vivo than in vitro. This might be because the rate of hormone release in vivo, which in the present experiments amounted to approximately 2 mUlh for the 24 h (ignoring the effects of resynthesis), is sufficiently low to minimize inactivation. In addition, the characteristic discharge patterns of magnocellular neurones (Dutton and Dyball, 1979; Bicknell et a]., 1981) might be adapted to optimize release relative to the extent of inactivation caused. DISCUSSION Is there a readily releasuble pool ? The results presented here offer no support for the hypothesis of a readily releasable pool, at least in its original forms (see above). In the case of potassium stimulation, inactivation is demonstrable both in the presence of l O O m M sodium and in its absence, although far less hormone was released in the former case. Further, previous authors have shown that a second increase in secretion may be evoked by application of an appropriate stimulus, such as exposure to barium or lanthanum (Nordmann, 1976), or sodium withdrawal (when inactivation has proceeded in its presence: Miiller et al., 1975; Nordmann, 1976). The alkaloid veratridine, which opens sodium channels permanently (Ohta et a]. , 1973), can also stimulate a second release (Dyball and Nordmann, 1977). Inactivation of release has also been demonstrated here with electrical stimulation, although 2 h of such stimulation in 100 mM sodium releases only about 25 5% of that released by potassium stimulation in the absence of sodium. Further, inactivation associated with electrical stimulation depends not on the magnitude of hormone release, but on the sodium concentration of the incubation medium. It must be concluded that electrically evoked hormone release is normally not limited by availability of secretory product, although depletion may partially account for the declining release seen during potassium stimulation in the absence of sodium. The actual “readily releasable pool”, that is the granules available for release by sufficiently powerful stimuli, is therefore likely to represent at least the 30 o/o contained in the neurosecretory terminals. During prolonged stimulation, more hormone may also become available by migration of granules into the terminals from the axonal swellings. It should nevertheless be emphasized that a proportion of the granules within the terminals may only be accessible to more potent stimuli : for instance, the associated large calcium influx might penetrate deeper into the terminals, and recruit granules unavailable to weaker stimuli. Viewed in this way, the size of the release pool depends upon the particular stimulus employed. Evidence that exhaustion of this pool cannot account for release inactivation is provided by the findings for electrical stimulation in different sodium and calcium concentrations mentioned above. In addition, electrical stimulation causes inactivation of release in response to a subsequent potassium stimulation, which is proportionately greater than can be accounted for by the hormone released by the electrical stimulus itself. This in turn suggests that the mechanism of hormone release inactivation resides in a stage prior to the recruitment and exocytosis of the neurosecretory granules.

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Possible mechanisms of hormone release inactivation Since the original work of Douglas and Poisner (1 964a, b), evidence has accumulated that an increase of the intracellular ionized calcium concentration is responsible for initiation of secretion (see Nordmann, this volume). It is extremely unlikely that inactivation is due to failure of the secretory mechanism, in view of evidence already discussed, such as the reactivation of potassium-stimulated hormone release by veratridine. Inactivation of hormone release might therefore occur because of limitation of the availability of calcium at exocytotic sites; this availability is influenced by the rate of calcium influx across the neurosecretory terminal membrane and release inactivation may thus reflect the properties of the membrane calcium permeability. It remains to incorporate the depolarization-dependent, calcium-dependent and sodium-dependent elements of hormone release inactivation into a model of the mechanism involved. If the properties of inactivation described here are all attributable to the “late” calcium channel (Baker et al., 1973a, b ; Nordmann, 1976), several modifications to these previous concepts of its operation are necessary. Baker et al. (1973b) demonstrated a potential-dependent inactivation of calcium conductance in the squid axon, and showed that recovery from previous stimulation was extremely slow by the standards of other membrane ion channels, being half-complete 3-4 min after 2-3 min of potassium depolarization. This bears some resemblance to the depolarization-dependent component of hormone release inactivation demonstrated here with potassium stimulation. However, an additional component of release inactivation was calcium-dependent, and it is of considerable interest that inactivation of calcium channels dependent upon the entry of calcium has been demonstrated in a number of cell types (e.g. Tillotson, 1979; Brehm et al., 1980). Further, Brown et al. (1981) have reported both calcium- and potential-dependent inactivation of calcium currents in Helix neurones. The sodium-dependent component of hormone release inactivation is more difficult to incorporate into this model. One possibility is suggested by the work of Birks and Cohen (1968a, b), who showed that transmitter release at the neuromuscular junction is enhanced by an increase in intracellular sodium. It was postulated that sodium might displace calcium from an intracellular binding site, and thus exert the opposite effect on calcium entry to the extracellular antagonism observed in the same preparation (Kelly, 1965). Thus in the neurohypophysis, lowered extracellular sodium would allow increased access of calcium to its channels and hence increased hormone secretion, whereas lowered intracellular sodium would inhibit intracellular liberation of calcium, and hence decrease secretion. As no sodium-dependent effects were observed with potassium stimulation, it must be postulated that potassium stimulation did not alter intracellular sodium. Certainly, the potential-dependent sodium conductance would probably inactivate almost immediately. In the case of electrical stimulation, potential-dependent gating of sodium, potassium and calcium would occur with every pulse, causing activation of the sodium pump (Ritchie and Straub, 1957). It must be assumed that the activity of the pump would reduce intracellular sodium, and that this would occur more readily in a medium containing only 100 mM sodium, thus accounting for the sodium-dependent component of inactivation seen with electrical stimulation. However, the extremely long time course of calcium-dependent release inactivation is difficult to reconcile with the rapid recovery of calcium conductance seen in systems investigated electrophysiologically (e.g. Brown et al., 198 1). An alternative scheme, involving the sodium-calcium exchange mechanism known to be present in neurohypophysial membranes (Nordmann and Zyzek, 1982) is therefore presented as a further working hypothesis. If three or more sodium ions are exchanged for a single calcium ion without accompanying cations, the

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direction of exchange would vary with membrane potential, such that calcium flux would be directed predominantly outward at negative (resting) potentials, but would become predominantly inward at more positive (depolarized) potentials. A similar mechanism is thought to be responsible for tonic contraction in heart muscle (Horackova and Vassort, 1979). In this scheme, hormone release during potassium stimulation would be maintained by calcium influx in exchange for intracellular sodium, the release declining as the electrochemical gradient for outward movement of sodium decreases. This would also account for the enhanced release and increased rate of inactivation seen when extracellular sodium is reduced from 100 mM to zero. In the case of electrical stimulation, depletion of intracellular sodium would proceed as described for the first model. It can be shown that relatively small changes in the intracellular sodium concentration would lead to a large decrease in intracellular calcium at a membrane potential around the resting level. Thus, inactivation induced by electrical stimulation could be due to failure of stimulus-induced calcium entry to increase the intracellular concentration by an increment great enough to cause hormone release. It is certainly feasible that alterations in the intracellular sodium concentration could persist following stimulation, as a facilitation of transmission in crayfish and crab motoneurones, attributable to sodium accumulation, remains undiminished after 2 h (Atwood et al., 1975). Both the above schemes explain the apparently disproportionate effect of electrical stimulation upon inactivation relative to the amount of hormone it releases, as intracellular sodium may thereby be altered independently of calcium influx. The involvement of three processes in the mechanism of hormone release inactivation has thus been demonstrated, and their interaction provides evidence that all act upon the membrane calcium permeability. Similarprocesses, the nature of which could not have been envisaged by Sachs and his colleagues when the pioneering work on this subject was performed, are likely to operate during both acute and chronic stimulation of the magnocellular neurosecretory system in vivo. The precise mechanism by which the effects of calcium are exerted, together with the nature of the influence of the sodium concentration on inactivation, remain matters of conjecture.

SUMMARY During prolonged stimulation, the rate of secretion from neurohypophyses stimulated in vivo or in vitro is not maintained at its original level, but declines at a rate higher than can be accounted for by depletion of the total hormone store. It has previously been suggested that this inactivation of hormone release is due either to depletion of a “readily releasable pool” of hormone, or to a potential-dependent reduction of membrane calcium permeability. In the present experiments, the mechanism of inactivation was further investigated using both potassium and electrical stimulation in vitro, and the stimulus of 2 % saline drinking in vivo. It has been found that inactivation of release elicited by potassium stimulation is dependent not only on membrane potential, but also on calcium entry. Recovery from the potential-dependent component is much faster than from the calcium-dependent one. Calciumdependent inactivation of hormone release can also be demonstrated with electrical stimulation, which additionally reveals a third component of inactivation dependent on external sodium. “Ex vivo” experiments, involving incubation of neurohypophyses from animals previously given 2 % NaCl rather than drinking water, suggest that similar mechanisms serve to limit hormone release in vivo.

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It is proposed that all three components of inactivation exert their effects upon membrane calcium permeability, and that it is the magnitude of intracellular calcium entry during stimulation, rather than decreased availability of hormone or fatigue of the exocytotic mechanism, which is responsible for inactivation of release. ACKNOWLEDGEMENTS

F.D. Shaw was in receipt of an MRC Studentship while much of this work was performed. We gratefully acknowledge the participation of P.G. Roe in the experiments shown in Fig. 6.

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