Acute estradiol application increases inward and decreases outward whole-cell currents of neurons in rat hypothalamic ventromedial nucleus.

Acute estradiol (E2) can potentiate the excitatory responses of hypothalamic ventromedial nucleus (VMN) neurons to neurotransmitters. To investigate the mechanism(s) underlying the potentiation, the whole-cell patch voltage clamp technique was used to study VMN neurons in hypothalamic slices prepared from female juvenile (3-5 weeks) rats. A voltage step and/or ramp was applied every 5 min to evoke whole-cell currents before, during and after a treatment with E2 (10 nM), corticosterone (10 nM) or vehicle for up to 20 min. Acute E2 increased inward currents in 38% of neurons tested. Their average peak inward current amplitudes started to increase within 5 min and reached the maximum of 163% of pretreatment level (Pre) at 20 min of treatment before recovering toward Pre. These increases are significantly greater than the Pre and corresponding vehicle controls and non-responsive neurons. Outward currents were decreased significantly by E2 in 27% of E2-treated cells, down to 60% of Pre levels. E2 also appeared to affect the kinetics of the inward and outward currents of estrogen-responsive neurons. Whenever observed, the effects of acute E2 were reversible after a 5- to 10-min washing. Probability analysis indicates that E2 affected the inward and the outward currents independently. The E2 effects are specific in that they were not produced by similar treatment with vehicle or corticosterone. Pharmacological characterizations using ion replacement and channel blockers showed that the inward currents were mediated practically all by Na(+) and the outward currents mainly by K(+). Thus, acute E2 can enhance inward Na(+) and attenuate outward K(+) currents. Since both effects will lead to an increase in neuronal excitability, they may explain our previous observation that E2 potentiates the excitation of VMN neurons.

Metabolic regulations and biological functions of phospholipids in Escherichia coli.

Extensive genetic and biochemical studies in the last two decades have elucidated almost completely the framework of synthesis and turnover of quantitatively major phospholipids in E. coli. The knowledge thus accumulated has allowed to formulate a novel working model that assumes sophisticated regulatory mechanisms in E. coli to achieve the optimal phospholipid composition and content in the membranes. E. coli also appears to possess the ability to adapt phospholipid synthesis to various cellular conditions. Understanding of the functional aspects of E. coli phospholipids is now advancing significantly and it will soon be able to explain many of the hitherto unclear cell's activities on the molecular basis. Phosphatidylglycerol is believed to play the central role both in metabolism and functions of phospholipids in E. coli. The results obtained with E. coli should undoubtedly be helpful in the study of more complicated phospholipid metabolism and functions in higher organisms.

Acute oxidative stress modulates secretion and repetitive Ca2+ spiking in rat exocrine pancreas.

The effects of the oxidant tert-butylhydroperoxide (t-buOOH) on carbachol-stimulated pancreatic secretion in the vascularly perfused rat pancreas have been studied in parallel with [Ca2+]i signalling and amylase output in perifused rat pancreatic acinar cells. Perfusion of the pancreas with t-buOOH (0.1-1 mM) caused a rapid and irreversible inhibition of carbachol-stimulated (3x10-7 M) amylase and fluid secretion. Pre-perfusion of the pancreas with vitamin C and dithiothreitol or a cocktail of GSH and GSH-precursor amino acids provided only marginal protection against the deleterious effects of t-buOOH, even though GSH levels were elevated significantly. In perifused pancreatic acini, repetitive [Ca2+]i spikes evoked by carbachol (3x10-7 M) were sustained for 40 min. t-buOOH (1 mM) acutely increased the amplitude and duration of Ca2+ spikes, then attenuated Ca2+ spiking and subsequently caused a marked and sustained rise in [Ca2+]i. t-buOOH-induced alterations in carbachol-stimulated [Ca2+]i signalling and amylase release in perifused pancreatic acini were prevented by vitamin C. Although vitamin C restored impaired Ca2+ signalling and maintained amylase output in pancreatic acini, it seems likely that oxidative stress inhibits fluid secretion irreversibly in the intact pancreas, resulting in a loss of amylase output. Thus, perturbations in [Ca2+]i signalling may not fully explain the secretory block caused by oxidative stress in acute pancreatitis.

Identification of alpha- and beta-cells in intact isolated islets of Langerhans by their characteristic cytoplasmic Ca2+ concentration dynamics and immunocytochemical staining.

Ratiometric images of cytoplasmic Ca2+ concentration ([Ca2+]c) in individual cells were recorded simultaneously with a confocal ultraviolet-laser microscope in the Indo-1-loaded islets isolated from mice. After changes in [Ca2+]c in response to glucose or amino acids were recorded, the islet was fixed, permeabilized, and stained by the indirect immunofluorescence method against insulin or glucagon in situ; the individual cells were then identified in the focal plain identical to that used for the [Ca2+]c imaging. Almost all cells identified as insulin-positive (beta-cells) by their distinct immunofluorescence responded to the increase in glucose concentration from 3 to 11 mmol/l with an increase in [Ca2+]c. Major populations of cells (approximately 65%) identified as glucagon-positive (alpha-cells) responded to the addition of arginine (5-10 mmol/l) to 3 mmol/l glucose solution with an increase in [Ca2+]c. About half of the alpha-cells (47.6%) responded to the addition of alanine (5-10 mmol/l) to 3 mmol/l glucose solution with an increase in [Ca2+]c. In contrast, <13% of beta-cells responded to the addition of alanine (5-10 mmol/l) or arginine (5-10 mmol/l) to 3 mol/l glucose with an increase in [Ca2+]c. More than one-fourth of alpha-cells responded with an increase in [Ca2+]c when glucose concentration in perifusion solution was reduced from 11 to 0 mmol/l. These results indicate that [Ca2+]c changes in islet cells stimulated by glucose or amino acid were characteristic of the cell species, at least in the alpha- and beta-cell. This technique provides a useful tool to investigate not only the intracellular signal transduction but also the intercellular signal transmission in the intact islet.

Indications from Mn-quenching of Fura-2 fluorescence in melanotrophs that dopamine and baclofen close Ca channels that are spontaneously open but not those opened by high [K+]O; and that Cd preferentially blocks the latter.

In unstimulated melanotrophs, which secrete spontaneously, Mn caused a progressive quenching of Fura-2 fluorescence (F360) which was: (a) unaffected by tetrodotoxin to suppress spontaneous Na-action potentials; (b) slowed by lowering temperature to 23 degrees C; and (c) arrested by the Ca channel blocker, Ni. Mn quenching slowed on lowering [K+]O from 5 to 2 mM to hyperpolarize (indicating Mn entry through voltage-dependent channels) and accelerated on raising [K+]O to 50 or 100 mM to strongly depolarize (indicating recruitment of high threshold channels). The secreto-inhibitor, dopamine, arrested spontaneous Mn quenching and so, too, did the GABAB agonist, baclofen; and these effects like those of the two agonists on secretion and [Ca2+]i were blocked by the specific D2 and GABAB antagonists, sulpiride and CGP 35348, respectively, and were lost following exposure to pertussis toxin. By contrast, neither dopamine nor baclofen prevented Mn quenching in response to high K, although this was arrested by Ni. A second Ca channel blocker, Cd, in concentrations that inhibited the response to high K, failed to inhibit spontaneous entry of Mn. This preferential effect offers an explanation for observations made with Cd that have been interpreted as contrary to the notion of Ca-regulated secretion in the melanotroph. The results we have obtained are interpreted to mean that in the melanotroph secreting spontaneously some voltage-dependent Ca channels are in the open state; that this open state is not dependent on any Na spiking activity; and that these channels are preferentially closed by dopamine and baclofen which are without effect on Ca channels opened by strongly depolarizing concentrations of K.

Spontaneous cytosolic calcium pulsing detected in Xenopus melanotrophs: modulation by secreto-inhibitory and stimulant ligands.

The purpose of the experiments was to examine the behavior of cytosolic Ca2+ ([Ca2+]i) in individual pituitary melanotrophs and how that is affected by physiological ligands that inhibit or stimulate melanotroph secretion. Melanotrophs were dispersed from neurointermediate lobes of Xenopus laevis adapted to a black background, and [Ca2+]i was measured with fura-2. In basal (unstimulated) conditions, repetitive transient elevations in [Ca2+]i, not hitherto observed in any melanotrophs, were detected in 73% of the cells. These cytosolic Ca pulses occurred at fairly regular intervals (1-10 min) and lasted from a half to several minutes, during which [Ca2+]i rose several-fold. Pulsing was promptly and reversibly arrested by the secreto-inhibitory transmitters, dopamine, neuropeptide-Y (NPY), and gamma-aminobutyric acid (GABA), and also by quinpirole, muscimol, and baclofen. Pertussis toxin eliminated the responses to dopamine, NPY, and GABAB receptor activation, but spared responses to GABAA receptor activation. The responses to the physiological inhibitors and to the Ca channel blocker Ni were close to all or nothing; a mere doubling of an ineffective concentration commonly sufficed to arrest pulsing. Submaximal responses, seen over a narrow concentration range, involved a slowing of the pulsing. Cells not pulsing spontaneously were responsive to dopamine, GABA, and NPY and pulsed in response to the secretagogues CRF and TRH. They are suggested to be melanotrophs not actively secreting. The behavior of [Ca2+]i parallels secretory activity and strengthens the view that spontaneous secretion is driven by spontaneous influx of Ca ions and that secreto-inhibitory transmitters act by suppressing this influx and lowering [Ca2+]i. Cytosolic Ca pulsing may provide an efficient means of sustaining a high rate of spontaneous secretion.

Calcium channels in rat melanotrophs are permeable to manganese, cobalt, cadmium, and lanthanum, but not to nickel: evidence provided by fluorescence changes in fura-2-loaded cells.

Cobalt (Co), nickel (Ni), manganese (Mn), cadmium (Cd), and lanthanum (La) are commonly used as calcium (Ca) channel blockers, but some of them, besides reducing Ca entry, also traverse Ca channels and can exert effects intracellularly that confound interpretation of functional responses. Because of this and our need to use Ca channel blockers in an ongoing analysis of Ca channel activity in the regulation of the cytosolic free Ca concentration ([Ca2+]i) and secretion in melanotrophs, we assessed whether the cations mentioned enter these cells. This was done by incorporating the fluorescence for changes that would signal the presence of the cations in the cytosol. In cell-free solution, where the probe and cations can interact freely, Mn, Co, and Ni all quench fluorescence, whereas Cd and La act in a Ca-like manner. When tested on fura-2-loaded melanotrophs in basal (unstimulated) conditions, Mn, Co, and Cd each yielded corresponding signals, thereby showing that they had penetrated the cells. By contrast, Ni caused no quenching of fluorescence even in melanotrophs exposed to 100 mM K+ to recruit additional Ca channels. Ni, therefore, did not penetrate the cells. However, as expected, Ni quenched fluorescence when given artificial access to the cytoplasm by ionomycin. Ni blocked spontaneous entry of Mn, Co, and Cd. It also lowered [Ca2+]i in unstimulated melanotrophs, consistent with blockade of spontaneous Ca entry. Like Ni, La lowered basal [Ca2+]i in unstimulated melanotrophs without penetrating the cells; however, unlike Ni, it penetrated when the melanotrophs were exposed to high potassium. We conclude that Ni is the most specific of the Ca channel blockers tested and that results obtained with Mn, Co, Cd, and La must be interpreted with reserve.

Spontaneous cytosolic calcium pulses in Xenopus melanotrophs are due to calcium influx during phasic increases in the calcium permeability of the cell membrane.

We recently discovered that melanotrophs of Xenopus laevis exhibit spontaneous pulse-like rises in cytosolic free calcium ([Ca2+]i) and that this cytosolic Ca pulsing is inhibited by the secreto-inhibitory transmitters dopamine, gamma-aminobutyric acid, and neuropeptide-Y and stimulated by the secretagogues CRF and TRH. Here we provide evidence for the factors responsible for the individual cytosolic Ca pulse and the repetitive behavior. Isolated melanotrophs of Xenopus were loaded with fura-2, and fluorescence was recorded from perifused single cells to measure [Ca2+]i and assess the patency of divalent cation channels by Mn quenching of fluorescence. Cytosolic Ca pulsing was arrested by omission of Ca and by the Ca channel blockers Ni and Co, but was unaffected by tetrodotoxin. Mn (0.3 mM) caused phasic quenching, each "quench" being synchronous with the rising phase of a cytosolic Ca pulse. Quenching was blocked by Ni, but was unaffected by tetrodotoxin. When introduced during the course of an individual Ca pulse, Ni aborted the pulse and [Ca2+]i collapsed. Extracellular K+ (10 mM) or K channel block with tetraethylammonium stimulated Ca pulsing. Threshold concentrations of Ni slowed Ca pulsing without reducing the amplitude of the individual pulses. The overshoots in quenching and Ca pulsing observed after exposure to Ni, dopamine, or baclofen are interpreted as off responses. It is concluded that each cytosolic Ca pulse is attributable, probably quantitatively, to Ca influx during a spontaneous increase in Ca permeability independent of Na-mediated action potentials, and that the pacemaker for the repetitive phenomenon is voltage sensitive and may involve Ca currents active around basal membrane potential.

Inhibition of voltage-dependent calcium channels by prostaglandin E2 in rat melanotrophs.

The effects of PGE2 on voltage-dependent Ca2+ channel currents were studied in dissociated rat melanotrophs by the whole-cell configuration of the patch-clamp technique. In about 90% of melanotrophs examined, PGE2 reversibly inhibited voltage-dependent Ba2+ currents elicited by voltage steps from a holding potential of -80 to 0 mV, with an ED50 of 68 nM. The maximum inhibition of Ba2+ currents by 1 microM PGE2 (35.3%) was comparable with that by the maximally effective concentration (100 nM) of dopamine. The EP1/EP3 PGE (EP) agonists, 17PT-PGE2 and sulprostone, and the EP2/EP3 agonist, misoprostol, mimicked the inhibition by PGE2, whereas the selective EP2 agonist, butaprostol, had little effect. The inhibition by PGE2 was partially, but significantly, reduced by the selective EP1 antagonist, SC-51322. The magnitude of the PGE2-induced inhibition of Ba2+ currents was greatly reduced by pretreatment with pertussis toxin, or by a depolarizing prepulse, to +80 mV, lasting for 50 msec. Although four distinct types (N-, P/Q-, L-, and R-types) of high-threshold Ba2+ currents were observed, PGE2 (1 microM) caused significant inhibition of only P/Q- and L-type currents, which were 17.3 and 10.1%, respectively, of the total Ba2+ currents. These results suggest that PGE2 inhibits P/Q- and L-type Ca2+ channels of rat melanotrophs via EP1 and EP3 receptors, which are coupled to pertussis toxin-sensitive G proteins, and produces both voltage-sensitive and -insensitive inhibition of Ca2+ channels.

Melanotrophs of Xenopus laevis do respond directly to neuropeptide-Y as evidenced by reductions in secretion and cytosolic calcium pulsing in isolated cells.

Neuropeptide-Y (NPY) is present, along with dopamine and gamma-aminobutyric acid, in the neurons innervating the intermediate lobe of the pituitary gland of Xenopus laevis, and all three neurotransmitters have been shown to inhibit melanotroph secretion from isolated neurointermediate lobes. However, unlike dopamine and gamma-aminobutyric acid, NPY has been reported to be without inhibitory effect on secretion from dispersions of intermediate lobe cells. Moreover, binding studies have been taken as indicating that Xenopus melanotrophs lack NPY receptors, although such receptors appear to be present on folliculostellate cells. For these reasons, NPY has been considered to act indirectly on Xenopus melanotrophs; the putative intermediary is supposed to be the folliculo-stellate cell. However, the present experiments show that NPY does strongly inhibit melanotroph secretion from cells dispersed from Xenopus intermediate lobes. In addition, they demonstrate that NPY acts directly on individual Xenopus melanotrophs (immunohistochemically identified and under conditions that preclude any interaction between cells) to inhibit the intermittent rises in cytosolic free Ca (cytosolic Ca pulsing). From these observations, we conclude that NPY does act directly on melanotrophs of Xenopus.

Pituitary adenylate cyclase-activating polypeptide causes rapid Ca2+ release from intracellular stores and long lasting Ca2+ influx mediated by Na+ influx-dependent membrane depolarization in bovine adrenal chromaffin cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been reported to increase intracellular Ca2+ concentrations ([Ca2+]i) and catecholamine release in adrenal chromaffin cells. We measured [Ca2+]i with fura-2 and recorded ion currents and membrane potentials with the whole cell configuration of the patch-clamp technique to elucidate the mechanism of PACAP-induced [Ca2+]i increase in bovine adrenal chromaffin cells. PACAP caused [Ca2+]i to increase due to Ca2+ release and Ca2+ influx, and this was accompanied by membrane depolarization and inward currents. The Ca2+ release was suppressed by ryanodine, an inhibitor of caffeine-sensitive Ca2+ stores, but was unaffected by cinnarizine, an inhibitor of inositol trisphosphate-induced Ca2+ release. Ca2+ influx and inward currents were both inhibited by replacement of extracellular Na+, and Ca2+ influx was inhibited by nicardipine, an L-type Ca2+ channel blocker, or by staurosporine, a protein kinase C (PKC) inhibitor, but was unaffected by a combination of omega- conotoxin-GVIA, omega-agatoxin-IVA, and omega-conotoxin- MVIIC, blockers of N-, P-, and Q-type Ca2+ channels. Moreover, 1-oleoyl-2-acetyl-sn-glycerol, a PKC activator, induced inward currents and Ca2+ influx. These results indicate that PACAP causes both Ca2+ release, mainly from caffeine-sensitive Ca2+ stores, and Ca2+ influx via L-type Ca2+ channels activated by membrane depolarization that depends on PKC-mediated Na+ influx.

Pituitary adenylate cyclase-activating polypeptide potentiation of Ca2+ entry via protein kinase C and A pathways in melanotrophs of the pituitary pars intermedia of rats.

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been reported to stimulate melanotroph secretion, and PACAP-like immunoreactivity and expression of PACAP type I receptor messenger RNA have been identified in the pituitary pars intermedia (PI). The present study showed that PACAP messenger RNA is also expressed in the PI. To examine the mechanism of PACAP action in the PI, cytosolic Ca2+ concentrations ([Ca2+]i) and ionic currents were measured in acutely dissociated rat melanotrophs. In about 40% of the melanotrophs studied, PACAP induced an increase in [Ca2+]i, which was suppressed by extracellular Ca2+ removal; extracellular Na+ replacement; the blocker of L-type Ca2+ channels, nicardipine; or the secreto-inhibitory neurotransmitter, dopamine. The PACAP-induced [Ca2+]i increase was mimicked by activators of protein kinase A (PKA) and protein kinase C (PKC), Sp-diastereomer of cAMP and 1-oleoyl-2-acetyl-sn-glycerol, and was reduced by inhibitors of PKA and PKC, Rp-diastereomer of cAMP and staurosporine. Patch-clamp analysis revealed that PACAP caused inward currents with a reversal potential of -0.8 mV and facilitated voltage-dependent Ba2+ currents. It further revealed that PACAP-induced inward currents were mimicked by 1-oleoyl-2-acetyl-sn-glycerol and inhibited by staurosporine, and that Sp-diastereomer of cAMP facilitated Ba2+ currents. These results suggest that PACAP potentiates Ca2+ entry mechanisms of rat melanotrophs by activation of nonselective cation channels via PKC and facilitation of voltage-dependent Ca2+ channels via PKA.

Water deprivation increases the expression of pituitary adenylate cyclase-activating polypeptide gene in the rat subfornical organ.

The effect of water deprivation on the expression of pituitary adenylate cyclase-activating polypeptide (PACAP) was examined in the rat subfornical organ (SFO), using a combination of immunohistochemistry and in situ hybridization histochemistry. In the euhydrated condition, PACAP-immunoreactivity (PACAP-IR) and the expression of PACAP gene was observed in the SFO. Water deprivation for 24 h and 48 h caused a significant increase in PACAP gene transcripts in the SFO, compared with euhydrated animals. Additionally, water deprivation for 48 h caused an increase in PACAP-IR. This increase of PACAP-IR was demonstrated in both nerve fibers and cell bodies. High correlation was found between the localization of PACAP-IR cell bodies and PACAP messenger RNA synthesizing cell bodies in the peripheral part of the SFO. These results suggest that PACAP in the SFO may play a role in the humoral and neural changes associated with the regulation of body fluid balance after water deprivation.

Centrally administered adrenomedullin increases plasma oxytocin level with induction of c-fos messenger ribonucleic acid in the paraventricular and supraoptic nuclei of the rat.

The effects of intracerebroventricular (i.c.v.) administration of adrenomedullin (AM) on plasma oxytocin (OXT), c-Fos protein (Fos), and c-fos messenger RNA (mRNA) in the paraventricular (PVN) and supraoptic nuclei (SON) of the rat were investigated using RIA for OXT, immunohistochemistry for Fos, and in situ hybridization histochemistry for c-Fos mRNA. Central administration of AM caused a significant increase in the plasma OXT level. Intracerebroventricular administration of AM caused a marked induction of Fos-like immunoreactivity (LI) in the PVN and in the dorsal parts of the SON. In the PVN and SON, OXT-LI cells predominantly exhibited nuclear Fos-LI in comparison with arginine vasopressin-LI cells. In situ hybridization histochemistry revealed that the induction of c-fos mRNA in the PVN and SON was increased in a dose-related manner 30 min after i.c.v. administration of AM. This induction was reduced by pretreatment with the AM receptor antagonist, human AM-(22-52)-NH2. These results suggest that central AM is responsible for activating the neurosecretory cells in the PVN and SON via selective AM receptors, and that AM stimulates the secretion of OXT by activating hypothalamic OXT-producing cells.

Active increase in cardiolipin synthesis in the stationary growth phase and its physiological significance in Escherichia coli.

Activity of the Escherichia coli cardiolipin synthase, encoded by cls, increased about 10-fold in the stationary growth phase, while other committed-step enzymes in phospholipid biosynthesis rather decreased. A null cls mutant lost viability to 10(-4) of the wild-type cells during the prolonged incubation for 5 days. Cardiolipin was most stable among membrane phospholipids during the incubation. Accordingly, cardiolipin should play a role in survival of the cell and E. coli employs a sophisticated way to form cardiolipin according to need even under non-growing conditions.

Loss of phosphatidylserine synthesis results in aberrant solute sequestration and vacuolar morphology in Saccharomyces cerevisiae.

Null cho1 mutants of Saccharomyces cerevisiae are incapable of phosphatidyl-serine synthesis. They were more susceptible than wild-type strains to 100 mM CaCl2, 3 mM ZnCl2 or 1 mM MnCl2, but not to MgCl2 nor KCl. They were also susceptible to high concentrations of basic amino acids, L-lysine and L-arginine, and to an L-lysine analog, S-2-aminoethyl-L-cysteine. Their vacuolar pools of amino acids, especially those of basic ones, were decreased. Pigmentation of cho1 ade2 double mutants was obscured and vacuoles of cho1 mutants were considerably fragmented. These indicate that phosphatidylserine plays vital roles in normal vacuolar function and morphogenesis.

Studies on pituitary melanotrophs reveal the novel GABAB antagonist CGP 35-348 to be the first such compound effective on endocrine cells.

One obstacle to understanding the action and physiological significance of the responsiveness of various endocrine cells to gamma-aminobutyric acid (GABA) has been that previously available substances, all active as GABAB antagonists in the nervous system, are ineffective on endocrine cells. The introduction of a potent new member of this class, CGP 35-348, of very different chemical structure, encouraged us to examine its effect on endocrine cells. For this purpose, we studied melanotroph secretion from pituitary neurointermediate lobes. We found that CGP 35-348, in contrast to previously available members of this class, suppressed completely, in rat and toad, secretory responses to baclofen, the classic GABAB agonist. Analysis, in toad, showed CGP 35-348 did not affect responses to GABAA agonists (muscimol; isoguvacine), dopamine, or neuropeptide Y. When tested against GABA, the physiological ligand present in the innervation of melanotrophs (along with dopamine and neuropeptide Y), CGP 35-348 completely suppressed the secretory response, which, in toad, is purely inhibitory and unaffected by bicuculline, the specific GABAA antagonist. In addition, CGP 35-348 unmasked a stimulant effect that bicuculline blocked. In CGP 35-348, we thus have a new tool with which to analyse responses to GABA and their physiological involvement in endocrine cells.

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 microM. The reduction in [Ca2+]i produced by (-)-baclofen at a maximally effective concentration (100 microM) 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 microM). Of the antagonists tested, CGP 35348 was the most potent with an IC50 of 60 microM, compared to 120 to 400 microM 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 microM). 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.

Heterogeneous increases of cytoplasmic calcium: distinct effects on down-regulation of cell surface sodium channels and sodium channel subunit mRNA levels.

1. Long-term (> or = 12 h) treatment of cultured bovine adrenal chromaffin cells with A23187 (a Ca(2+) ionophore) or thapsigargin (TG) [an inhibitor of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)] caused a time- and concentration-dependent reduction of cell surface [(3)H]-saxitoxin (STX) binding capacity, but did not change the K:(D:) value. In A23187- or TG-treated cells, veratridine-induced (22)Na(+) influx was reduced (with no change in veratridine EC(50) value) while it was enhanced by alpha-scorpion venom, beta-scorpion venom, or Ptychodiscus brevis toxin-3, like in nontreated cells. 2. The A23187- or TG-induced decrease of [(3)H]-STX binding was diminished by BAPTA-AM. EGTA also inhibited the decreasing effect of A23187. A23187 caused a rapid, monophasic and persistent increase in intracellular concentration of Ca(2+) ([Ca(2+)](i)) to a greater extent than that observed with TG. 2,5-Di-(t-butyl)-1,4-benzohydroquinone (DBHQ) (an inhibitor of SERCA) produced only a rapid monophasic increase in [Ca(2+)](i), without any effect on [(3)H]-STX binding. 3. Reduction in [(3)H]-STX binding capacity induced by A23187 or TG was attenuated by Gö6976 (an inhibitor of conventional protein kinase C) or calpastatin peptide (an inhibitor of calpain). When the internalization rate of cell surface Na(+) channels was measured in the presence of brefeldin A (an inhibitor of vesicular exit from the trans-Golgi network), A23187 or TG accelerated the reduction of [(3)H]-STX binding capacity. 4. Six hours treatment with A23187 lowered Na(+) channel alpha- and beta(1)-subunit mRNA levels, whereas TG had no effect. 5. These results suggest that elevation of [Ca(2+)](i) caused by A23187, TG or DBHQ exerted differential effects on down-regulation of cell surface functional Na(+) channels and Na(+) channel subunit mRNA levels.

Stimulatory effects of vanadate on amylase release from isolated rat pancreatic acini.

The effects of vanadate on exocrine pancreatic function were examined in isolated rat pancreatic acini. Vanadate caused a concentration-dependent stimulation of amylase release above a concentration of 1 mM. Co-incubation of vanadate with vasoactive intestinal polypeptide, 8-bromoadenosine 3':5'-cyclic monophosphate, and the Ca2+ ionophore A23187 produced a synergistic pattern of amylase release, whereas co-incubation with cholecystokinin octapeptide (CCK-8), carbamylcholine, and 12-O-tetradecanoylphorbol 13-acetate produced an additive effect. Vanadate alone had no influence on acinar cyclic AMP content, Ca2+ efflux, or intracellular Ca2+ concentration. However, preincubation with vanadate prevented the plateau phase of CCK-8-induced Ca2+ transient increase from returning to baseline. Moreover, depletion of the intracellular Ca2+ pool by pretreatment of acini with CCK-8 in Ca2+-free medium (plus ethyleneglycol bis[beta-aminoethylether]-N,N'-tetraacetic acid) had no effect on subsequent stimulation by vanadate, although it abolished the response to both CCK-8 and carbamylcholine stimulation. The protein kinase C (PKC) inhibitors staurosporine and calphostin C significantly inhibited vanadate-stimulated amylase release, whereas the protein tyrosine kinase inhibitor genistein had no inhibitory effect. Moreover, vanadate caused a significant translocation of PKC from cytosol to membrane fraction in pancreatic acinar cells. This translocation was inhibited significantly by staurosporine and calphostin C but not by genistein. These results suggest that vanadate acts directly on pancreatic acini and stimulates amylase release by activating PKC without an effect on Ca2+ mobilization, cyclic AMP, or protein tyrosine kinase.