Potassium-induced secretion of melanocyte-stimulating hormone from the melanotrophs of the neurointermediate lobe of the lizard Anolis carolinensis.

Secretion of melanocyte-stimulating hormone (MSH) from the melanotrophs of the neurointermediate lobe (NIL) of the lizard Anolis carolinensis was studied to investigate the role of membrane potential and extracellular calcium ions in the control of secretion in this species. MSH secretion was monitored from perifused NILs which had been in organ culture for 7-14 days prior to experiment to allow the nerve terminals present in the tissue to degenerate. Elevation of the K(+) concentration in the perifusate induced a marked increase in MSH secretion. Perifusion of the cultured NILs with a nominally Ca-free solution did not reduce basal MSH secretion but blocked K-induced secretion. Moreover, nimodipine, an antagonist of voltage-gated Ca(2+) channels, inhibited K-induced secretion, whereas BAY K 8644, a Ca(2+) channel agonist, potentiated it; but neither drug affected basal secretion. High ¿K(+) perifusate also stimulated MSH secretion from freshly excised (acute) NILs. Furthermore, in these preparations nominally Ca-free solution reduced basal secretion by 40% in addition to blocking K-induced secretion. As in the cultured NILs, nimodipine blocked and BAY K 8644 potentiated K-induced secretion in the acute NILs while not affecting basal secretion. The results from the cultured NILs are consistent with the hypothesis that stimulated MSH secretion from the melanotrophs of the anole is dependent upon Ca(2+) influx through voltage-gated calcium channels. The qualitatively similar results obtained from the acute NILs in response to high ¿K(+), nimodipine, and BAY K 8644 suggest that, for the most part, what is being observed are the direct effects of these substances on the melanotrophs. Basal secretion of MSH in cultured NILs is significantly less than that in the acute preparations. The calcium-sensitive fraction of basal secretion present in acute NILs may require the presence of nerve terminals.

Dopamine (D2) or gamma-aminobutyric acid (GABAB) receptor activation hyperpolarizes rat melanotrophs and pertussis toxin blocks these responses and the accompanying fall in [Ca2+]i.

The effects, on membrane potential, of dopamine (DA) and gamma-aminobutyric acid (GABA), transmitters present in the secreto-inhibitor innervation to the melanotrophs, were monitored in primary cultures of rat melanotrophs with bis-oxonol. DA and GABA, acting through D2 and GABAB receptors, hyperpolarized the melanotrophs. Hyperpolarization was not suppressed by tetrodotoxin but was prevented by pertussis toxin and may thus be due to a G protein mediated mechanism. Pertussis toxin also blocked the effects mediated by the two receptors to reduce intracellular free Ca2+ ([Ca2+]i).

Cytosolic Ca2+ in melanotrophs: pharmacological insights into regulatory influences of electrical activity and ion channels.

The concentration of intracellular free Ca2+ ([Ca2+]i) was measured in melanotrophs, the characteristic endocrine cells of the pars intermedia of the rat pituitary gland, using the fluorescent Ca indicator fura-2. The resting [Ca2+]i was 211 +/- 8 nM and was little affected by tetrodotoxin (TTX; 5 or 10 microM), which inhibits the spontaneous action potentials that occur in these cells. Removal of extracellular Ca2+ (by chelation with EGTA) or addition of the Ca channel blocker nimodipine (1 microM) produced a rapid fall in [Ca2+]i, which occurred whether TTX was present or not. Excess K+ (60 mM), veratridine (10 or 100 microM) and BAY K 8644 (1 microM) each caused a rapid rise in [Ca2+]i, which was blocked or truncated by EGTA or nimodipine. TTX blocked or truncated the increases in [Ca2+]i induced by veratridine, but not those induced by either excess K+ or BAY K 8644. The results show that manipulations that increase or decrease hormone output increase or decrease [Ca2+]i. Furthermore, the resting [Ca2+]i appears to depend importantly on Ca influx, since it is rapidly and markedly reduced by removal of extracellular Ca2+ or addition of a Ca channel blocker.

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.

Electrical stimulation of neurointermediate lobes of mice elicits calcium-dependent output of melanocyte-stimulating hormone.

Current pulses applied to isolated neurointermediate lobes of mice increased output of melanocyte-stimulating hormone. This response was dependent on extracellular calcium over a wide range of stimulus intensities and thus appears to be a true secretory response from the melanotrophs. Since substantial responses persisted in the presence of tetrodotoxin, much of the effect seems to be independent of Na spiking.

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.

Pharmacological and ionic features of gamma-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.

Electrical activity in adenohypophyseal cells and effects of hypophyseotropic substances.

Normal endocrine cells isolated from adenohypophyses of the rat, lizard, and fish, and maintained in short-term culture, produce action potentials spontaneously or in response to depolarizing current pulses or stimulant hypophyseotropic substances. Thyrotropin-releasing hormone elicited action potentials in a fraction of a mixed population of rat pars distalis cells. Dopamine, which inhibits basal secretion from rat melanotrophs and teleostean prolactin cells, inhibited action potential discharge in both these cell types. 5-Hydroxytryptamine, which stimulates secretion from lizard pars intermedia cells, stimulated action potentials in these cells. gamma-Aminobutyric acid, recently found in nerves to the rat pars intermedia and shown to have both stimulant and inhibitory effects on secretion, depolarized rat melanotrophs. The effect involved an increase in Cl conductance, which could be blocked by bicuculline and potentiated by diazepam. A Ca component that indicates inward Ca flux was present in the action potentials in each of the cell types examined, although its contribution varied with species and gland region. Electrical activity may therefore participate in secretory control in adenohypophyseal cells. Attention is drawn to some unexpected electrical responses to agents that are likely to be used in attempts to examine the correlation between the electrical events and secretion.

Secretagogue effect of barium on output of melanocyte-stimulating hormone from pars intermedia of the mouse pituitary.

Ba ions caused an intense and prolonged discharge of melanocyte-stimulating hormone (MSH) from perifused neurointermediate lobes of mouse pituitaries and dispersed pars intermedia cells. The effect persisted in chronically cultured lobes or cells. It did not require Ca, but, like the Ca-dependent response to excess K, was blocked by cyanide combined with glucose lack. The secretagogue effect of Ba was blocked or prevented by Co or by excess Ca, both of which can reduce inward Ba currents through Ca channels. Prior exposure to excess K partially reduced the secretagogue effect of Ba, suggesting that depolarization caused some inactivation of Ba current. In contrast to Ba, excess K elicited secretion that was transient, and prior exposure of preparations to excess K (in the absence of Ca) profoundly suppressed the secretagogue effect of Ca. The evidence is consistent with the view that inward Ca current rapidly inactivates in these cells. It is concluded that Ba ions have a potent and persistent direct secretagogue effect on the melanotrophs that may reflect, in part, their ability to penetrate Ca channels more easily than Ca ions. The strong secretagogue effects of Ba on melanotrophs may be of considerable utility in studies on MSH secretion since a physiological secretagogue has yet to be discovered. Moreover, since the responses of melanotrophs (and other endocrine cells) to Ba can be distinguished from those of various other secretory cells and neurones, it is suggested that Ba may provide a tool for characterizing the distinctive membrane properties of the Ba-responsive endocrine cells.

GABA acts directly on cells of pituitary pars intermedia to alter hormone output.

Recent immunohistochemical evidence from the rat, indicating that gamma-aminobutyric acid (GABA)-containing fibres of central nervous origin project to the pars intermedia of the pituitary1,2, prompts inquiry into the function of this innervation. There is electrophysiological evidence that GABA acts directly on melanotrophs isolated from rat, through bicuculline-blockable receptors, to increase Cl- conductance and thereby drive the membrane potential towards the Cl- equilibrium potential in these cells, resulting in depolarization at rest or reduction of the depolarization caused by excess K+ (ref. 3). As voltage-dependent Ca channels can participate in the regulation of secretion in these cells4, we have now examined the effect of GABA on hormone output and find that it first stimulates and then inhibits spontaneous secretion of melanocyte-stimulating hormone (MSH) and inhibits K+-evoked secretion. Moreover, our pharmacological evidence suggests that similar receptors are involved in the secretory and the electrophysiological responses. A function of the GABAergic innervation may therefore be to regulate hormone output by acting directly on the melanotrophs, and this regulation may be affected by the changes in electrical properties induced by GABA.

Slowing effects of dopamine and calcium-channel blockers on frequency of sodium spikes in rat pars intermedia cells.

1. Spontaneous discharge of action potentials (Na spikes) in cells isolated from rat pars intermedia was slowed or arrested by Co(2+), Ni(2+) or Mn(2+), which block voltage-dependent Ca channels in these cells. The amplitude of persisting spikes was undiminished. The effects resembled those of dopamine.2. Action potential frequency decreased when the Ca(2+) concentration was lowered to 0.1 mM and increased when the Ca(2+) concentration was raised from this level to 1 mM or 2 mM or when Ba(2+) (2 mM) was introduced. These effects, together with those of Co(2+), Ni(2+) and Mn(2+), are consistent with the possibility that Ca(2+) participates in the regulation of spike discharge.3. Verapamil, methoxyverapamil (D600), and nifedipine reduced the amplitude of the individual Na spikes in concentrations that had little effect on voltage-dependent Ca channels. Action potential frequency was comparatively little affected by these drugs.4. K(+) (15 mM) stimulated action potential frequency and this effect too was suppressed by dopamine or Co(2+).5. The effect which dopamine had of slowing spontaneous discharge, like the inhibitory effect on secretion, was blocked by metoclopramide. But otherwise the mechanism is unclear: dopamine blocked voltage-dependent Ca channels in some cells but not in most others.6. The effects of K(+) and Ba(2+) of eliciting spikes, the suppression of Na-spike discharge by Co(2+) and related Ca-channel blocking cations, and the unspecific effects of the organic ;Ca channel blockers', all have implications for the use of these substances as tools to analyse stimulus-secretion coupling.

Calcium component to action potentials in rat pars intermedia cells.

1. The ionic dependence of the action potential of rat pars intermedia cells was investigated by using intracellular recording techniques. 2. In the presence of tetrodotoxin (TTX, 5 x 10(-6)M), the action potentials evoked by passing depolarizing current through the recording electrode were abolished, confirming that they are mainly dependent on Na; however, when tetraethylammonium (TEA, 10 mM) was added to the TTX-containing solution the imposed depolarizations triggered all-or-none regenerative potentials indicative of involvement of another ion. 3. There TTX-insensitive regenerative potentials persisted when the cells were perifused with Na-free solution but were severely reduced or abolished by Ca-free solution. This suggests that the ion producing these potentials is Ca. 4. These Ca action potentials were suppressed by Ni, Co and Mn in concentrations that did not suppress the "Na spikes' recorded in the absence of TTX and TEA. 5. Sr and Ba could substitute for Ca in maintaining the action potentials recorded in the presence of TTX. These ions also prolonged the duration of these action potentials. 6. The demonstration of a Ca component to the predominantly Na-dependent action potentials of pars intermedia cells heightens the possibility that these action potentials participate in the regulation of secretion.

Action potentials in gland cells of rat pituitary pars intermedia: inhibition by dopamine, an inhibitor of MSH secretion.

1. Cells were dissociated from rat pituitary pars intermedia, maintained in culture, and subjected to electrophysiological study.2. Recorded membrane potentials varied widely (range - 18 to about - 80 mV). They were relatively insensitive to changes in external Na but were rapidly and reversibly lowered by excess K.3. Action potentials were elicited by passing current through intracellular recording electrodes. They were reversibly blocked by tetrodotoxin (TTX, 2 x 10(-6)M) or by removal of Na from the recording solution and thus appeared to be Na spikes. Cells yielding action potentials in response to depolarization had relatively high membrane potentials (about - 65 mV) which may be representative of the true resting membrane potential of pars intermedia cells.4. Spontaneous action potentials were recorded extracellularly from nearly all the many isolated pars intermedia cells studied with a microsuction electrode. Their amplitude was reduced by TTX (0.1-2 x 10(-6)M). Electron microscopic examination of cells producing action potentials showed them to be hormone-containing parenchymal (gland) cells.5. Dopamine (10(-6)M), a presumed physiological inhibitor of secretion of melanocyte stimulating hormone (MSH) from pars intermedia cells, decreased the frequency of spontaneous action potentials but not their amplitude. Similar effects were seen with noradrenaline (10(-6)M), another inhibitor of MSH secretion, whereas isoprenaline and 5-hydroxytryptamine (5-HT), which do not inhibit MSH secretion, had no effect.6. Action potentials may be involved in stimulus-secretion coupling in pars intermedia cells.