Electrical activity of rat anterior pituitary cells in vitro

Regenerative responses were seen in most cells either after cessation of an inward current pulse or during an outward current pulse. Two cell groups were distinguished electrophysiologically. Type I cells showed action potentials with maximum rate of rise of 21.2 ± 9.0 V/sec (mean ± S.D., n= 19), while type II cells generated small graded depolarising responses with maximum rate of rise less than 3 V/s. The resting potentials of type I and II cells were 31.8± 14.9 mV (n = 19) and 41.7 ± 9.8 mV (n = 31), respectively. The steady-state current/voltage relationship was linear for both cell types when the membrane potential was more negative than — 60 mV. An outward rectification appeared when the membrane potential was more positive than —40 mV. The input resistance was smaller in type I cells (274±212 MΩ, n=19) than in type II cells (1 112 ± 456 MΩ, n=16). Even in Na-free solution regenerative responses were observed in most cells. When the Ca²+ concentration was increased tenfold to 24 mM, the maximum rate of rise of the off-response increased from 1.9 ± 0.8 V/s (n = 11) to 5.7 ± 2.12 V/s (n = 5). All-or-none action potentials could be evoked in this Ca²⁺ rich solution. Action potentials of similar maximum rate of rise could be evoked after replacing 24 mM Ca²⁺ with isomolar Sr²⁺. Prolonged action potentials were seen after substitution of Ca²⁺ for Ba²⁺. It is concluded that action potentials in most anterior pituitary cells have a Ca component, which in type I cells is additional to a Na component.     

Autocrine/paracrine modulation of calcium channels in bovine chromaffin cells

 Applying 10-s pulses of 10 mM Ba²⁺ to resting or K⁺-depolarized (70 mM) bovine adrenal chromaffin cells superfused with a nominal 0Ca²⁺ solution produced a large catecholamine secretory peak. In contrast, pulses of 10 mM Sr²⁺ or Ca²⁺ did not induce secretion from polarized resting cells, and induced smaller and narrower secretory peaks from depolarized cells; the areas of the secretory peaks from depolarized cells were 1.87, 3.06 and 27.4 nA s, respectively, for Ca²⁺, Sr²⁺ and Ba²⁺. Ca²⁺ channel currents in isolated cells or in cells surrounded by other unpatched cells (cell cluster) were studied with either the continuous-flow or the flow-stop method. When applied to an isolated cell, flow-stop reduced the amplitude of I _Ca by 19%, I _Sr by 31%, and I _Ba by 53%, compared with the current amplitude measured under continuous-flow conditions. This decrease in current amplitude was accompanied by a pronounced slowing down of current activation and could be largely relieved by applying strong depolarizing prepulses (facilitation). Under continuous-flow conditions, 10 μM exogenous ATP reduced (about 50%) I _Ca, I _Sr and I _Ba similarly. On the other hand, the use of Na⁺ as a charge carrier through Ca²⁺ channels, or intracellular dialysis with 1 mM BAPTA prevented the modulation of current by flow-stop. In cell clusters, activating secretion from unpatched cells, by either 10 mM Ba²⁺, 100 μM acetylcholine or 70 mM K⁺, caused a pronounced slowing down of current activation, as well as a decrease of its magnitude in the voltage-clamped cell immersed in the cluster. Such modulation of isolated cells was not observed. These data are compatible with the idea that the secretory activity of adrenal medullary chromaffin cells ”in situ” controls the activity of their Ca²⁺ channels through autocrine/paracrine mechanisms. Received: 29 June 1998 / Received after revision: 20 August 1998 / Accepted: 1 September 1998     

Actions of growth-hormone-releasing hormone on rat pituitary cells: intracellular calcium and ionic currents

Actions of growth-hormone-releasing hormone (GHRH) on single rat anterior pituitary cells were studied using indo-1 fluorescence to monitor changes in intracellular calcium, [Ca²⁺]_i, and perforated-patch recording to measure changes in membrane potential and ionic currents. GHRH elevated [Ca²⁺]_i in non-voltage-clamped cells by a mechanism that was dependent upon extracellular Na⁺ and Ca²⁺ and was blocked by the dihydropyridine Ca²⁺-channel blocker, nitrendipine. Resting cells had a fluctuating membrane potential whose a mean value depolarized by 9 mV in response to GHRH. The membrane-permeant cAMP analogue, 8-(4-chlorophenylthio)cAMP, mimicked the action of GHRH on membrane potential. Under voltage clamping, GHRH activated a small inward current (1–5 pA). Two types of response could be distinguished. The type I response had an inward current that was largest at more negative potentials (–90 mV), and the type II response had inward current that was larger at more positive potentials (–40 to –70 mV). Both types of response were reversible and blocked by removal of extracellular Na⁺. These results suggest that the rise in [Ca²⁺]_i produced by GHRH in non-voltage-clamped cells results from the activation via cAMP of a Na⁺-dependent conductance, which depolarizes the cell and increases the Ca²⁺ influx through voltage-gated Ca²⁺ channels.Dedicated in memory of the late Alexander P. Naumov.     

Contribution of BK channels to action potential repolarisation at minimal cytosolic Ca2+ concentration in chromaffin cells

BK channels modulate cell firing in excitable cells in a voltage-dependent manner regulated by fluctuations in free cytosolic Ca²⁺ during action potentials. Indeed, Ca²⁺-independent BK channel activity has ordinarily been considered not relevant for the physiological behaviour of excitable cells. We employed the patch-clamp technique and selective BK channel blockers to record K⁺ currents from bovine chromaffin cells at minimal intracellular (about 10 nM) and extracellular (free Ca²⁺) Ca²⁺ concentrations. Despite their low open probability under these conditions (V₅₀ of +146.8 mV), BK channels were responsible for more than 25% of the total K⁺ efflux during the first millisecond of a step depolarisation to +20 mV. Moreover, BK channels activated about 30% faster (τ = 0.55 ms) than the rest of available K⁺ channels. The other main source of fast voltage-dependent K⁺ efflux at such a low Ca²⁺ was a transient K⁺ (I_A-type) current activating with V ₅₀ = −14.2 mV. We also studied the activation of BK currents in response to action potential waveforms and their contribution to shaping action potentials both in the presence and the absence of extracellular Ca²⁺. Our results show that BK channels activate during action potentials and accelerate cell repolarisation even at minimal Ca²⁺ concentration, and suggest that they could do so also in the presence of extracellular Ca²⁺, before Ca²⁺ entering the cell facilitates their activity.     

Caffeine-induced oscillations of cytosolic Ca2+ in GH3 pituitary cells are not due to Ca2+ release from intracellular stores but to enhanced Ca2+ influx through voltage-gated Ca2+ channels

Caffeine, a well known facilitator of Ca²⁺-induced Ca²⁺ release, induced oscillations of cytosolic free Ca²⁺ ([Ca²⁺]_i) in GH₃ pituitary cells. These oscillations were dependent on the presence of extracellular Ca²⁺ and blocked by dihydropyridines, suggesting that they are due to Ca²⁺ entry through L-type Ca²⁺ channels, rather than to Ca²⁺ release from the intracellular Ca²⁺ stores. Emptying the stores by treatment with ionomycin or thapsigargin did not prevent the caffeine-induced [Ca²⁺]_i oscillations. Treatment with caffeine occluded phase 2 ([Ca²⁺]_i oscillations) of the action of thyrotropin-releasing hormone (TRH) without modifying phase 1 (Ca²⁺ release from the intracellular stores). Caffeine also inhibited the [Ca²⁺]_i increase induced by depolarization with high-K⁺ solutions (56% at 20 mM), suggesting direct inhibition of the Ca²⁺ entry through voltage-gated Ca²⁺ channels. We propose that the [Ca²⁺]_i increase induced by caffeine in GH₃ cells takes place by a mechanism similar to that of TRH, i.e. membrane depolarization that increases the firing frequency of action potentials. The increase of the electrical activity overcomes the direct inhibitory effect on voltage-gated Ca²⁺ channels with the result of increased Ca²⁺ entry and a rise in [Ca²⁺]_i. Consideration of this action cautions interpretation of previous experiments in which caffeine was assumed to increase [Ca²⁺]_i only by facilitating the release of Ca²⁺ from intracellular Ca²⁺ stores.     

Vasopressin responses in electrically coupled A7r5 cells

Changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and in membrane potential were monitored in single A7r5 smooth-muscle cells during spontaneous spiking and after arginine vasopressin stimulation. Spontaneous Ca²⁺ oscillations, which were associated with the occurrence of action potentials, occurred in about 90% of the confluent monolayers investigated. This spontaneous activity was synchronized amongst all the cells of the monolayer, indicating that the cells were electrically coupled. Arginine vasopressin stimulation produced a [Ca²⁺]_i rise that was about 5 times higher than the amplitude of the spontaneous Ca²⁺ oscillations and resulted in a subsequent cessation of spontaneous electrical activity and associated Ca²⁺ spiking, which persisted after [Ca²⁺]_i returned to baseline. Individual cells in the monolayer responded to arginine vasopressin with a different latency. Agonist-induced Ca²⁺ waves within one cell propagated much more slowly than spontaneous [Ca²⁺]_i rises. We conclude that agonist-induced [Ca²⁺]_i increases in an electrically coupled cell monolayer can be asynchronous.     

Action of stimulatory and inhibitory α-MSH secretagogues on spontaneous calcium oscillations in melanotrope cells of Xenopus laevis

The secretion of α-melanophore-stimulating hormone (α-MSH) from melanotrope cells in the pituitary gland of Xenopus laevis is regulated by various neural factors, both classical neurotransmitters and neuropeptides. The majority of these cells (80%) display spontaneous Ca²⁺ oscillations. In order to gain a better understanding of the external regulation of intracellular Ca²⁺ ([Ca²⁺]_i) in the melanotrope cell, we have examined the action of well known α-MSH secretagogues on the Ca²⁺ oscillations. It is shown that all secretagogues tested also control the oscillatory state of Xenopus melanotropes, that is, the secreto-inhibitors dopamine, isoguvacine (γ-aminobutyric acid, GABA_A agonist), baclofen (GABA_B agonist) and neuropeptide Y evoked a rapid quenching of the spontaneous Ca²⁺ oscillations, whereas the secreto-stimulant sauvagine, an amphibian peptide related to corticotropin releasing hormone, induced oscillatory activity in non-oscillating cells. Supporting argument is given for the idea that the regulation of Ca²⁺ oscillations is a focal point in the regulation of secretory activity of melanotrope cells. There was considerable heterogeneity among melanotrope cells in the threshold of their Ca²⁺ response to secretagogue treatment. This heterogeneity may be the basis for melanotrope cell recruitment observed during physiological adaptations of the animal to the light intensity of its background.     

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

The recent discovery of action potentials with a Ca component in pars intermedia cells raised the question whether voltage-dependent Ca²⁺ 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 mM K⁺ and this effect was abolished when Ca²⁺ was omitted or when Co²⁺ 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 Ca²⁺ lack and of Co²⁺ 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 Ca²⁺ 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.     

Electrophysiology of cultured parathyroid cells from the goat

The membrane potential and resistance of goat parathyroid (PT) cells in culture were 44.7±6.8 mV and 503±218 MΩ (mean ±S.D., n= 24) in 2.4 mM Ca²+ solution. The corresponding values in 0.8 mM Ca²+ solution were 29.2±12.2 mV and 290± 198 MΩ (n= 22). The current/voltage relation showed a pronounced outward rectification for membrane potentials more positive than -40 mV. Current injection produced graded, regenerative responses in half of the penetrated cells. Regenerative responses were also obtained in Na+-free solution, and these responses were reversibly inhibited by the Ca²+ blocker D-600. The results support the notion that PT cells possess voltage sensitive K+ and Ca²+ channels, and that exocytosis of stored PT hormone depends on influx of extracellular Ca²+ as in other secretory cells.     

Single Ca channel currents in mouse pancreatic B-cells

Barium currents flowing through single Ca²⁺ channels were recorded from outside-out patches isolated from mouse pancreatic B-cells. Only one type of Ca²⁺ channel was observed. In 110 mM Ba²⁺, the single channel conductance was 24 pS (at negative membrane potentials) and the current amplitude at 0 mV was–0.7 pA. Channel openings were activated by depolarisations more positive than –30 mV and showed little inactivation during 200 ms pulses. Open times were increased by BAY K 8644 an decreased by micromolar Cd²⁺. Channel activity was subject to rundown in excised patches and little activity remained after 10 min. These properties resemble those of L-type Ca²⁺ channels in other tissues. It is suggested that this Ca²⁺ channel participates in the generation of the B-cell action potential and mediates the increase in Ca²⁺ influx required for insulin secretion.     

Effects of a phorbol ester and diacyglycerols on secretion of mucin and arginine esterase by rat submandibular gland cells

The effects of a phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) and a diacylglyceride, 1-oleoyl-2-acetyl-glycerol (OAG) on the secretion of two major exocrine products by dispersed rat submandibular cells were investigated. TPA stimulated the release of acinar cell mucin and ductal cell protease (arginine esterase) in a dose- and time-dependent manner. Mucin secretion was also provoked by OAG, which, however, had no effect on arginine esterase release. The unsaturated diacylglycerol, 1,2-diolein, elicited a greater mucosecretory response than did OAG at the same concentration, while the saturated 1,2-distearin produced a smaller response.Mucin and enzyme secretion caused by TPA or OAG in the rat submandibular model was not inhibited by either of two putative antagonists, the antipsychotic drug, fluphenazine, and the antibiotic, polymyxin B.The involvement of extracellular Ca²⁺ in TPA-induced secretion was examined by comparing responses of cells maintained in normal or Ca²⁺-free medium, or in medium containing the ionophore A23187. Although extracellular Ca²⁺ was not an absolute requirement for a secretory response, the results indicate a synergistic relationship between TPA and Ca²⁺ in stimulating the release of both mucin and arginine esterase.These results suggest a role for the Ca²⁺-, phospholipid-dependent enzyme, protein kinase C in the secretory mechanism of mucous and serous cells in the submandibular gland. This is consistent with the proposal that receptor-mediated hydrolysis of membrane phosphoinositides is an initial event in stimulus-response coupling in exocrine cells.     

Calcium and barium permeation through calcium release-activated calcium (CRAC) channels

A Ca²⁺ current activated by store depletion has been described recently in several cell types and has been termed I _CRAC (for Ca²⁺ release-activated Ca²⁺ current). In this paper, the Ca²⁺ and Ba²⁺ permeability of CRAC channels is investigated in mast cells, rat basophilic leukaemia cells (RBL) and human T-lymphocytes (Jurkat). The selectivity of CRAC channels for Ca²⁺ over monovalent cations is identical in all three cell types and is at least as high as that of voltage-operated Ca²⁺ (VOC) channels in the various tissues tested. The amplitude of Ba²⁺ currents relative to Ca²⁺ currents (I _Ba/I _Ca) through CRAC channels was found to be strongly dependent on the membrane potential and was much smaller in Jurkat cells compared to mast and RBL cells. An anomalous mole-fraction behavior was observed at very negative membrane potentials in all three cell types when using different mixtures of external Ca²⁺ and Ba²⁺. In contrast to VOC channels, the anomalous mole-fraction effect was not observed at potentials positive to–20 mV.     

BK channels in intact clonal rat pituitary cells are activated by physiological elevations of the cytosolic Ca2+ concentration at the normal resting potential

Activation of large conductance Ca²⁺-activated K⁺ channels (BK channels) in intact clonal rat pituitary cells (GH₄ cells) was investigated using the cell-attached patch-clamp configuration. This method prevents loss of intracellular factors which might influence channel activity. BK channels are generally considered to be inactive at the resting membrane potential in excitable cells. However, at the resting potential (0 mV pipette potential), 40% of the cell-attached patches displayed spontaneously active BK channels, which remained active even at 20 mV hyperpolarization. The peptide thyroliberin (TRH) elevates the cytosolic Ca²⁺ concentration ([ Ca²⁺]_i) in GH cells by IP₃-induced release of Ca²⁺ from intracellular stores. This rise in [Ca²⁺]_i occurs concomitantly with membrane hyperpolarization. TRH stimulation caused activation of BK channels in nine out of 30 silent cell-attached patches, and caused enhanced channel activity in seven out of 29 cell-attached patches containing spontaneously active BK channels. The Ca²⁺ ionophore ionomycin activated silent BK channels in three out of 10 cell-attached patches, and increased the activity of spontaneously active BK channels in seven out of 16 cell-attached patches. The pipette potential was clamped to 0 mV in all these experiments. We conclude that the BK channels in GH₄ cells may be active at the resting membrane potential and more negative membrane potentials. The channels may also be activated further by physiological elevations of [Ca²⁺]_i in the same potential range. Our results point towards new possible physiological roles for the BK channels in GH₄ cells. This is in agreement with the emerging picture of BK channels highly sensitive to [Ca²⁺]_i in a wide variety of cell types.     

Activity-dependent intracellular Ca2+ transients in unmyelinated nerve fibres of the isolated adult rat vagus nerve

 Confocal laser scanning microscopy was used to follow changes in the free intracellular calcium concentration ([Ca²⁺]_i) in nerve fibres and adjacent Schwann cells in isolated rat vagus nerves. [Ca²⁺]_i was monitored by the Ca²⁺-sensitive fluorescent dyes Calcium Green-1 and Fura Red. Intracellular Ca²⁺ transients were observed during repetitive (1–50 Hz) supramaximal electrical stimulation or by bath application of ATP. Trains of action potentials were more effective at elongated, fibre-like structures of the vagus nerves, whereas ATP-induced Ca²⁺ transients were found predominantly in regions of Schwann cell bodies. Activity-induced Ca²⁺ signals were unaffected by pharmacological manipulation of intracellular Ca²⁺ stores, during long-lasting application of purinergic receptor agonists, or by substitution of extracellular Na⁺ with Li⁺. However, they were abolished in the presence of Ca²⁺-free bathing solution or after the blocking of Ca²⁺ channels with Cd²⁺. Ca²⁺ transients were also observed during Ca²⁺ action potentials. Such ”Ca²⁺ spikes” were elicited by electrical stimulation in the presence of a combination of tetrodotoxin and K⁺ channel blockers. These data suggest that voltage-dependent Ca²⁺ channels, activated during short trains of Na⁺ action potentials, produce an increase in intra-axonal [Ca²⁺] of rat vagus nerves. We did not find evidence for activity-dependent Ca²⁺ transients in the Schwann cells surrounding the unmyelinated axons. Received: 16 September 1997 / Received after revision: 25 November 1997 / Accepted: 27 November 1997     

Modulators of BK and SK channels alter electrical activity in vitro in single vasopressin neurons isolated from the rat supraoptic nucleus

Release of arginine vasopressin (AVP) from magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus (SON) is controlled by the electrical activities of the MNCs. Ca²⁺-activated K⁺ channels, such as the BK and SK channels, are K⁺-selective ion channels that are activated in response to increased intracellular calcium concentrations. Intrinsic affinities for Ca²⁺ permit these channels to exert a negative feedback effect on cellular excitability. In the present study, we used the whole-cell patch-clamp technique to examine the effects of BK or SK channel modulators on neuronal activity in single isolated rat SON MNCs that express an AVP-enhanced green fluorescent protein (eGFP) transgene. Application of BK or SK channel activators abolished the action potentials and induced hyperpolarization. In contrast, the number of action potentials was significantly increased after application of BK or SK channel blockers. Our results suggest that BK and SK channels in AVP neurons may play a role in the regulatory mechanisms of neural activity.     

Large and small conductance calcium-activated potassium channels in the GH3 anterior pituitary cell line

Single Ca²⁺-activated K⁺ channels were studied in membrane patches from the GH₃ anterior pituitary cell line. In excised inside-out patches exposed to symmetrical 150 mM KCl, two channel types with conductances in the ranges of 250–300 pS and 9–14 pS were routinely observed. The activity of the large conductance channel is enhanced by internal Ca²⁺ and by depolarization of the patch membrane. This channel contributes to the repolarization of Ca²⁺ action potentials but has a Ca²⁺ sensitivity at –50 mV that is too low for it to contribute to the resting membrane conductance. The small conductance channel is activated by much lower concentrations of Ca²⁺ at –50 mV, ad its open probability is not strongly voltage sensitive. In cell-attached patches from voltage-clamped cells, the small conductance channels were found to be active during slowly decaying Ca²⁺-activated K⁺ tails currents and during Ca²⁺-activated K⁺ currents stimulated by thyrotropin-releasing hormone induced elevations of cytosolic calcium. In cell-attached patches on unclamped cells, the small conductance channels were also active at negative membrane potentials when the frequency of spontaneously firing action potentials was high or during the slow afterhyperpolarization following single spontaneous action potentials of slightly prolonged duration. The small conductance channel may thus contribute to the regulation of membrane excitability.     

[Ca2+]i-induced augmentation of the inward rectifier potassium current (IK1) in canine and human ventricular myocardium

The inward rectifier K⁺ current (I_K1) plays an important role in terminal repolarization and stabilization of the resting potential in cardiac cells. Although I_K1 was shown to be sensitive to changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i), the nature of this Ca²⁺ sensitivity—in spite of its deep influence on action potential morphology—is controversial. Therefore, we aimed to investigate the effects of a nonadrenergic rise in [Ca²⁺]_i on the amplitude of I_K1 in canine and human ventricular myocardium and its consequences on cardiac repolarization. I_K1, defined as the current inhibited by 10 μM Ba²⁺, was significantly increased in isolated canine myocytes following a steady rise in [Ca²⁺]_i. Enhanced I_K1 was also observed when [Ca²⁺]_i was not buffered by ethylene glycol tetraacetic acid, and [Ca²⁺]_I transients were generated. This [Ca²⁺]_i-dependent augmentation of I_K1 was largely attenuated after inhibition of CaMKII by 1 μM KN-93. Elevation of [Ca²⁺]_o in multicellular canine and human ventricular preparations resulted in shortening of action potentials and acceleration of terminal repolarization. High [Ca²⁺]_o enhanced the action potential lengthening effect of the Ba²⁺-induced I_K1 blockade and attenuated the prolongation of action potentials following a 0.3-μM dofetilide-induced I_Kr blockade. Blockade of I_Ks by 0.5 μM HMR-1556 had no significant effect on APD₉₀ in either 2 mM or 4 mM [Ca²⁺]_o. It is concluded that high [Ca²⁺]_i leads to augmentation of the Ba²⁺-sensitive current in dogs and humans, regardless of the mechanism of the increase. This effect seems to be at least partially mediated by a CaMKII-dependent pathway and may provide an effective endogenous defense against cardiac arrhythmias induced by Ca²⁺ overload.     

Stimulatory actions of di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (di-8-ANEPPS), voltage-sensitive dye, on the BKCa channel in pituitary tumor (GH3) cells

Di-8-ANEPPS (4-{2-[6-(dibutylamino)-2-naphthalenyl]-ethenyl}-1-(3-sulfopropyl)pyridinium inner salt) has been used as a fast-response voltage-sensitive styrylpyridinium probe. However, little is known regarding the mechanism of di-8-ANEPPS actions on ion currents. In this study, the effects of this dye on ion currents were investigated in pituitary GH₃ cells. In whole-cell configuration, di-8-ANEPPS (10 μM) reversibly increased the amplitude of Ca²⁺-activated K⁺ current. In inside-out configuration, di-8-ANEPPS (10 μM) applied to the intracellular surface of the membrane caused no change in single-channel conductance; however, it did enhance the activity of large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels with an EC₅₀ value of 7.5 μM. This compound caused a left shift in the activation curve of BK_Ca channels with no change in the gating charge of these channels. A decrease in mean closed time of the channels was seen in the presence of this dye. In the cell-attached mode, di-8-ANEPPS applied on the extracellular side of the membrane also activated BK_Ca channels. However, neither voltage-gated K⁺ nor ether-à-go-go-related gene (erg)-mediated K⁺ currents in GH₃ cells were affected by di-8-APPNES. Under current-clamp configuration, di-8-ANEPPS (10 μM) decreased the firing of action potentials in GH₃ cells. In pancreatic βTC-6 cells, di-8-APPNES (10 μM) also increased BK_Ca-channel activity. Taken together, this study suggests that during the exposure to di-8-ANEPPS, the stimulatory effects on BK_Ca channels could be one of potential mechanisms through which it may affect cell excitability.     

Multisite regulation of insulin secretion by cAMP-increasing agonists: evidence that glucagon-like peptide 1 and glucagon act via distinct receptors

 The mechanisms by which glucagon-like peptide 1(7–36)amide (GLP-1[7–36]amide) potentiates insulin secretion were investigated by measurements of whole-cell K⁺ and Ca²⁺ currents, membrane potential, the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) and exocytosis in mouse pancreatic B-cells. GLP-1(7–36)amide (10 nM) stimulated glucose-induced (10 mM) electrical activity in intact pancreatic islets. The effect was manifested as a 34% increase in the duration of the bursts of action potentials and a corresponding 28% shortening of the silent intervals. GLP-1(7–36)amide had no effect on the electrical activity at subthreshold glucose con- centrations (≤6.5 mM). In cultured B-cells, GLP-1(7–36)amide produced a decrease of the whole-cell ATP-sensitive K⁺ (K_ATP) conductance remaining at 5 mM glucose by ≈30%. This effect was associated with membrane depolarization and the initiation of electrical activity. GLP-1(7–36)amide produced a protein-kinase-A- (PKA-) and glucose-dependent fourfold potentiation of Ca²⁺-induced exocytosis whilst only increasing the Ca²⁺ current marginally. The stimulatory action of GLP-1(7–36)amide on exocytosis was mimicked by the pancreatic hormone glucagon and exendin-4, a GLP-1 receptor agonist. Whereas the stimulatory action of GLP-1(7–36)amide could be antagonized by exendin-(9–39), this peptide did not interfere with the ability of glucagon to stimulate exocytosis. We suggest that GLP-1(7–36)amide and glucagon stimulate insulin secretion by binding to distinct receptors. The GLP-1(7–36)amide-induced stimulation of electrical activity and Ca²⁺ influx can account for (maximally) a doubling of insulin secretion. The remainder of its stimulatory action results from a cAMP/PKA-dependent potentiation of Ca²⁺-dependent exocytosis exerted at a stage distal to the elevation of [Ca²⁺]_i. Received: 3 March 1997 / Received after revision and accepted: 23 May 1997     

Toxin of the marine alga Prymnesium patelliferum enhances voltage dependent Ca2+-currents,elevates the cytosolic Ca2+-concentration and facilitates hormone release in clonal rat pituitary cells

The marine flagellate Prymnesium patelliferum produces toxins lethal to fish. The toxin extracted from the alga has haemolytic, cytotoxic and neurotoxic effects, but the action mechanisms of the toxin are not known in detail. We have examined the toxin effects on the voltage sensitive Ca²⁺-currents, the cytosolic Ca²⁺-level ([Ca²⁺]₁) and the prolactin release in clonal rat anterior pituitary GH₄C₁ cells, which possess T- and L-type Ca²⁺-channels. The trans-membrane Ca²⁺-current was recorded using whole-cell voltage clamp. After 5–15 min exposure to the algal toxin at a final concentration of 50000–100000 cells mL^-1, the Ca²⁺-currents through both the T- and L-channels showed a 2–3-fold enhancement. The voltage sensitivity of the Ca²⁺-currents was not affected by the algal toxin, and the toxin-induced currents were inhibited by 100 μM of the Ca²⁺-channel blocker D-600. In toxin-exposed cells microfluorometric measurements based on fura-2 revealed an increase of [Ca²⁺]₁ from 100–150 to 300–500 nM. This elevation was delayed and partially inhibited by 100 μM D-600. The algal toxin induced prolactin release in a dose-dependent manner, and this effect was inhibited by the Ca²⁺-channel blocker verapamil. We therefore conclude that the toxin of P. patelliferum affects the Ca²⁺ homeostasis of the pituitary cells by increasing the leak through voltage sensitive Ca²⁺-channels, resulting in increased [Ca²⁺]₁ and secretion of prolactin.