William W. Douglas

How Do Neurones Secrete Peptides? Exocytosis and Its Consequences, Including “Synaptic Vesicle” Formation, in the Hypothalamo-Neurohypophyseal System

Publisher Summary This chapter addresses problem of how neurones secrete peptides by discussing the hypothalamo-neurohypophyseal system secreting the octapeptide hormones oxytocin and vasopressin (ADH). The secretory elements of this system are the archetypical “peptidergic” nerves, and more is known of their function than that of all other such neurones combined. It has long been suspected that these hypothalamo-neurohypophyseal cells possessing neuronal as well as glandular morphological characteristics generate impulses in response to osmotic or reflex synaptic activation and propagate them to their terminals in the neurohypophysis. The chapter also explores the following questions: (1) whether impulses provide the adequate stimulus for release of the posterior pituitary hormones, (2) if so, how these impulses act, and (3) what kind of secretory process is set in motion. The peptide-secreting nerves of the hypothalamo-neurohypophyseal tract possess the essential electrophysiological attributes of neurones: generation and propagation of impulses by means of the familiar regenerative sodium mechanism sensitive to tetrodotoxin (TTX). In neurohypophyseal terminals, granule membrane retrieved in this way is probably destroyed by lysosomes but a comparable mechanism in conventional neurones could permit recycling of synaptic vesicles.

Mast cell histamine secretion in response to somatostatin analogues: Structural considerations☆

Comparative studies of the activity of somatostatin and of several of its analogues in releasing histamine from rat mast cells suggest that the integrity of the positively charged amino terminus and of lysines at positions four and nine of somatostatin may be necessary to preserve its histamine releasing activity. D-Lys4 substitution reduced activity by 80% while D-Lys9 substitution increased it four fold. Replacement of the amino terminal Ala by Tyr or simultaneous removal of Ala1,Gly2 and the amino portion of the now terminal Cys3 inhibited the activity by about 95%. Finally, dihydrosomatostatin retained 33% activity while an analogue where both Cys sulfur atoms were permanently blocked by acetamidomethyl groups retained only about 13% activity. Using information from these studies and from the literature, two-dimensional and space-filling models approximating the conformation of somatostatin were constructed and compared with a plausible corresponding model of the hexameric form of 48/80, the most active congener of this classic mast cell secretagogue. By such modelling it was possible to show that the orientation of the cationic moieties in the two molecules could be similarly arranged thereby perhaps explaining the ability of these compounds to induce mast cell secretion.

Potassium-induced secretion of melanocytestimulating hormone from isolated pars intermedia cells signals participation of voltage-dependent calcium channels in stimulus-secretion coupling

Abstract The recent discovery of action potentials with a Ca component in pars intermedia cells raised the question whether voltage-dependent Ca 2+ influx participates in stimulus-secretion coupling in these cells. To test this, a study was made of the effects of a depolarizing concentration of K + on melanocyte-stimulating hormone (MSH) output from isolated pars intermedia cells of rats and mice obtained by tissue disaggregation and maintained in culture for periods ranging from 2 h to 10 days. The cells were placed in a column which was perfused thereby permitting continuous monitoring of MSH output. The cells secreted MSH promptly upon exposure to 50 m M K + and this effect was abolished when Ca 2+ was omitted or when Co 2+ was added to block Ca channels. It is concluded that activation of voltage-dependent Ca channels in pars intermedia cells allows influx of Ca ions that provides an adequate stimulus for MSH secretion. Furthermore, inhibitory effects of Ca 2+ lack and of Co 2+ addition on basal MSH output suggest the operation of such a Ca-dependent mechanism during spontaneous secretory activity. Secretory responses to high [K + ], although intense, were brief and waned rapidly during continuous exposure to K + . Experiments showing that Ca 2+ fails to elicit secretion when introduced with a delay of several minutes after exposure to high [K + ] indicate that the transience of the response is due to inactivation of voltage-dependent Ca channels. The brevity of the response of the isolated pars intermedia cells to high [K + ] may explain, at least in part, why stimulant effects of excess K + have not been seen previously, or in the present experiments in intact neurointermediate lobes of rats. When the much smaller, and seemingly more favorable, neurointermediate lobes of mice were used, K + elicited a Ca-dependent secretion of MSH comparable with that seen in the isolated cells. The demonstration that pars intermedia cells respond directly to a depolarizing stimulus by secreting MSH encourages the view that electrical activity in these cells is involved in the regulation of secretion.

Somatostatin-induced histamine secretion in mase cells. Characterization of the effect☆

Somatostatin, in concentrations up to 100 ng/ml, had no effect on mast cell secretion induced either by compound 48/80 or by the ionophore A23187. In higher concentration somatostatin induced mast cell secretion. Light and electron microscopic observations showed that the secretory response was identical with that induced by 48/80 and involved extrusion of secretory granules by exocytosis. As with 48/80, this response to somatostatin was inhibited by treating the mast cells with EDTA or EGTA or by exposing them briefly to A23187 in calcium-free media, all of which procedures seemingly deplete cellular calcium stores. Reintroduction of calcium (but not magnesium), restored secretory responsiveness. Somatostatin-induced secretion further resembled that induced by 48/80 or A23187 in its intensity, rapid time course, and dependence on albumin. Pretreatment of mast cells with dibutyryl cyclic AMP or 8-bromo cyclic AMP substantially reduced the secretory responses to both somatostatin and 48/80 but had little effect on the response to A23187. Somatostatin, like 48/80, lowered intracellular cyclic AMP levels in a time and dose-dependent fashion. Finally, as earlier found for 48/80, somatostatin attached to Sepharose columns retarded the passage of mast cells and elicited histamine release indicating an action at the cell surface. The stimulant action of somatostatin is thus very similar to that of the classic mast-cell secretagogue compound 48/80.

Pharmacological and ionic features of γ-aminobutyric acid receptors influencing electrical properties of melanotrophs isolated from the rat pars intermedia

Characteristics of the gamma-aminobutyric acid receptors on melanotrophs of the rat pars intermedia were studied by intracellular recording. Muscimol and 3-amino-1-propanesulfonic acid, but not baclofen or glycine, mimicked the depolarization and conductance increase produced by gamma-aminobutyric acid on the melanotrophs. These effects appeared to be due to an increase in chloride ion conductance since the null potentials for all three drugs were the same and were affected by changes in external or internal chloride ion concentration but not by changes in the concentrations of other ions present in the recording solution or by the addition of the calcium-channel blocker cobalt. Bicuculline abolished the effect of muscimol. Picrotoxin reduced the effect of gamma-aminobutyric acid; so too did furosemide. Muscimol mimicked the ability of gamma-aminobutyric acid to reduce the depolarization produced by excess potassium and this effect was also blocked by bicuculline. Rat melanotrophs thus appear to possess gamma-aminobutyric acid receptor-ionophore complexes similar to the classical sort found in neurons in the mammalian central nervous system. Furthermore, the parallels between the electrical responses observed and secretory effects previously noted, reinforce the view that electrical activity may participate in stimulus-secretion coupling in melanotrophs.

Effects of veratridine, tetrodotoxin and other drugs that alter electrical behaviour on secretion of melanocyte-stimulating hormone from melanotrophs of the pituitary pars intermedia

Since melanotrophs are electrically active and exhibit spontaneous Na spikes, a study was made of the effects, on melanotroph secretion, of drugs known to influence electrical properties. The output of melanocyte-stimulating hormone was measured from perifused neurointermediate lobes of mice or melanotrophs dispersed from such lobes of mice or rats. Veratridine (200 microM), which is known to increase Na permeability in a variety of cells, caused a large, although transient, increase in secretion from the melanotrophs that required extracellular Ca2+ and was blocked by the Na-channel blocker tetrodotoxin (1 microM). Tetraethylammonium (10 mM), which blocks K channels and thus prolongs the duration of the action potential in many cells, also stimulated secretion in the melanotrophs in a Ca-dependent manner. This response was not, however, blocked by tetrodotoxin, and is thus not attributable to prolongation of Na spikes in these cells. Moreover, tetrodotoxin did not inhibit basal secretion. The stimulant effect of veratridine on secretion in melanotrophs and its suppression by tetrodotoxin suggests that voltage-dependent Na channels can participate in the regulation of hormone output in these cells of the pituitary pars intermedia. However, the apparent lack of effect of tetrodotoxin on basal secretion suggests that the spontaneous Na spikes previously observed in these cells are not required for promoting the Ca influx which other evidence shows is important for basal secretion.

Effectiveness of GABAB antagonists in inhibiting baclofen‐induced reductions in cytosolic free Ca concentration in isolated melanotrophs of rat

1 The purpose of the present experiments was to assess the activities of GABAB receptor antagonists in mammalian isolated melanotrophs. 2 Cytosolic free Ca concentration ([Ca2+]i) in rat melanotrophs in primary culture was monitored with the fluorescent probe, fura‐2. 3 (−)‐Baclofen lowered [Ca2+]i in a concentration‐dependent manner with an EC50 of 0.96 μm. The reduction in [Ca2+]i produced by (−)‐baclofen at a maximally effective concentration (100 μm) was similar to that produced by the classic transmitter inhibitory to melanotroph secretion, dopamine, at a corresponding concentration (100 nm), or by perifusion with a nominally Ca‐free solution. 4 The GABAB receptor antagonists, 3‐aminopropyl(diethoxymethyl)phosphinic acid (CGP 35348), 2‐hydroxy saclofen, phaclofen and 4‐amino‐3‐(5‐methoxybenzo[b]furan‐2‐yl) butanoic acid (9H), had inhibitory effects on the reduction in [Ca2+]i produced by (−)‐baclofen (3 μm). Of the antagonists tested, CGP 35348 was the most potent with an IC50 of 60 μm, compared to 120 to 400 μm for the others. CGP 35348 acted competitively. 5 CGP 35348 alone had no effect on basal [Ca2+]i, or on the changes in [Ca2+]i produced by dopamine (10 nm) or the specific GABAA receptor agonist, muscimol (10 μm). 6 The evidence indicates that of the antagonists tested, CGP 35348 offers the greatest promise for pharmacological analysis of the functional significance of the GABAB receptors in melanotrophs.

Effects of BAY K 8644 and Other Dihydropyridines on Basal and Potassium-Evoked Output of MSH from Mouse Melanotrophs in vitro

Dihydropyridines that have been shown in studies on other tissues either to facilitate Ca influx (BAY K 8644) or depress it (nimodipine and nifedipine) were examined for their effects on the secretory activity of the melanotrophs. Isolated mouse neurointermediate lobes, cultured for a week or longer to allow the nerves to degenerate, were perifused and output of MSH activity was measured by bioassay. The Ca-channel agonist BAY K 8644 augmented secretion under basal conditions and potentiated secretion evoked by 30 mM K+, a submaximally depolarizing concentration. The stimulant effect on secretion under basal conditions persisted in the presence of tetrodotoxin (which blocks the action potentials, Na spikes, in the melanotrophs) but was lost when Ca2+ was omitted or nimodipine added. The results are considered to support the view that the prominent basal secretory activity in these cells, as well as that evoked by excess K+, involve the inward flux of Ca2+ through dihydropyridine-sensitive Ca channels. If the stimulant and inhibitory effects of the dihydropyridines on basal secretion are to be attributed to actions on voltage-regulated Ca channels, as the results from other tissues would suggest, then such channels in the melanotrophs are different from those previously described in other tissues.

Effects of BAY K 8644 on Ca-channel currents and electrical activity in mouse melanotrophs

The effects of BAY K 8644 on both evoked and spontaneous electrical activity were studied in mouse melanotrophs. The action potential in these cells consisted of both a Na and a Ca component. BAY K 8644 greatly increased the duration of the evoked action potential apparently by enhancing the Ca component since this prolongation remained when the Na component was blocked by tetrodotoxin but was diminished by the Ca-channel antagonists Co2+ or nimodipine. In addition to its effects on the evoked action potential, BAY K 8644 sometimes caused action potentials of long duration to occur in otherwise quiescent melanotrophs, even in the presence of tetrodotoxin. These action potentials appeared to be triggered by small, depolarizing prepotentials. Voltage-clamp experiments suggested the existence of 3 types of Ca-channel currents in these cells: one low-threshold current and two high-threshold currents, one of which rapidly inactivated and another which did not. BAY K 8644 caused a shift in the I-V relation for Ca-channel current to more negative values and reduced or abolished the hump in the initial part of the I-V curve produced by the low-threshold current suggesting that BAY K 8644 selectively affects high-threshold Ca current. The action of BAY K 8644 to initiate trains of long-lasting Ca spikes and thereby increase Ca influx, might explain its secretagogue effect on the unstimulated melanotroph.

Differential inhibitory effects of the arachidonic acid analog ETYA on rat mast cell exocytosis evoked by secretagogues utilizing cellular or extracellular calcium

ETYA (5,8,11,14-eicosatetraynoic acid; 50-100 microM), which inhibits both cyclo-oxygenase and lipoxidase, inhibited histamine release evoked by secretagogues dependent on extracellular calcium (antigen, dextran, and concanavalin A) but failed to inhibit secretion elicited by secretagogues capable of mobilizing calcium from intracellular sites (48/80, polymyxin B, protamine sulfate and poly-L-lysine). Responses to these latter secretagogues were inhibited only by higher concentrations of ETYA (100-200 microM) that were cytotoxic. Secretion evoked by the calcium ionophore A23187 (0.1 microgram/ml) was inhibited at much lower concentrations of ETYA (1-10 microM) but this inhibition could not be overcome by increasing the concentration of calcium. Responses to higher concentrations of ionophore were not inhibited by ETYA except in amounts affecting cell viability. Like ETYA, each of several fatty acids, including arachidonic acid, were inhibitory towards histamine release evoked by A23187 or 48/80. The results indicate that EYTA acts at some early stage of stimulus-secretion coupling rather than on the final, common, calcium-activated steps of exocytosis. Moreover, this action may be unrelated to inhibition of lipoxidase or cyclooxygenase.