Potassium-mediated stimulation of hepatic glycogenolysis

Increased extracellular potassium concentrations ([K+]o) stimulated transient increases in glucose release and 45Ca2+ washout in the perfused rat liver. Stimulated glucose release had a K0.5 of about 26 mM for [K+]o, was not desensitized by successive infusion intervals of increased [K+]o, was not affected by altering the direction of perfusion, was absolutely dependent on the presence of [Ca2+]o, and was blocked by 2 mM cobalt or 10 microM verapamil. The increase in 45Ca2+ washout resulting from increased [K+]o also was blocked by 2 mM cobalt or 10 microM verapamil. Inhibitors of vascular tone (nitroprusside, atriopeptin II), arachidonic acid metabolism (indomethacin, nordihydroguaiaretic acid), and alpha- or beta-adrenergic or muscarinic nerve stimulation/secretion (phentolamine, propranolol, atropine) were unable to inhibit the [K+]o-stimulated glucose release. ATP, ADP, and AMP concentrations in tissue freeze-clamped 2 min after the onset of infusion of 50 mM K+ were not significantly different from control tissue. Glucose release from freshly isolated suspensions or primary cultured monolayers of hepatocytes or from liver slices, all of which responded to glucagon or phenylephrine, did not respond to increased [K+]o. The results indicate that glycogenolysis stimulated by depolarizing gradients of K+ is dependent on an intact perfused vasculature and may be mediated by potential-sensitive Ca2+ channels present in the vascular endothelium of the liver.     

Nicotinic Autoreceptors Mediating Enhancement of Acetylcholine Release Become Operative in Conditions of "Impaired" Cholinergic Presynaptic Function

Abstract: The existence in the mammalian CNS of release-inhibiting muscarinic autoreceptors is well established. In contrast, few reports have focused on nicotinic autoreceptors mediating enhancement of acetylcholine (ACh) release. Moreover, it is unclear under what conditions the function of one type of autoreceptor prevails over that of the other. Rat cerebrocortex slices, prelabeled with [³H]choline, were stimulated electrically at 3 or 0.1 Hz. The release of [³H]ACh evoked at both frequencies was inhibited by oxotremorine, a muscarinic receptor agonist, and stimulated by atropine, a muscarinic antagonist. Nicotine, ineffective at 3 Hz, enhanced [³H]ACh release at 0.1 Hz; mecamylamine, a nicotinic antagonist, had no effect at 3 Hz but inhibited [³H]ACh release at 0.1 Hz. The cholinesterase inhibitor neostigmine decreased [³H]ACh release at 3 Hz but not at 0.1 Hz; in the presence of atropine, neostigmine potentiated [³H]ACh release, an effect blocked by mecamylamine. In synaptosomes depolarized with 15 mM KCI, ACh inhibited [³H]ACh release; this inhibition was reversed to an enhancement when the external [Ca²⁺] was lowered. The same occurred when, at 1.2 mM Ca²⁺, external [K⁺] was decreased. Oxotremorine still inhibited [³H]ACh release at 0.1 mM Ca²⁺. When muscarinic receptors were inactivated with atropine, the K⁺ (15 mM)-evoked release of [³H]ACh (at 0.1 mM Ca²⁺) was potently enhanced by ACh acting at nicotinic receptors (EC₅₀? 0.6 µM). In conclusion, synaptic ACh concentration does not seem to determine whether muscarinic or nicotinic autoreceptors are activated. Although muscarinic autoreceptors prevail under normal conditions, nicotinic autoreceptors appear to become responsive to endogenous ACh and to exogenous nicotinic agents under conditions mimicking impairment of ACh release. Our data may explain in part the reported efficacy of cholinesterase inhibitors (and nicotinic agonists) in Alzheimer's disease.     

Muscarinic receptor-mediated increase in cytoplasmic free Ca2+ in isolated bovine adrenal medullary cells. Effects of TMB-8 and phorbol ester TPA

The change in cytoplasmic free calcium, [Ca2+]i in isolated bovine adrenal medullary cells during stimulation by acetylcholine (ACh) in Ca2+-free incubation medium was measured using the fluorescent Ca2+ indicator quin2. ACh (1-100 microM) caused an increase in [Ca2+]i by mobilization of Ca2+ from the intracellular pool. Nicotine (10 microM) did not increase [Ca2+]i in the absence of extracellular Ca2+. Pretreatment of the cells with atropine (10 microM) completely inhibited ACh-induced increase in [Ca2+]i, whereas pretreatment with hexamethonium (100 microM) did not. The intracellular Ca2+ antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), inhibited ACh-induced increase in [Ca2+]i. The activator of protein kinase C 12-O-tetradecanoylphorbol-13-acetate (TPA), but not its 'inactive' analog 4 alpha-phorbol-12,13-didecanoate (PDD), also inhibited ACh-induced increase in [Ca2+]i. These findings suggest that in bovine adrenal medullary cells, stimulation of muscarinic ACh receptor causes an increase in [Ca2+]i by mobilizing Ca2+ from the intracellular pool and that protein kinase C is involved in 'termination' or 'down regulation' of this response.     

Evidence that isoproterenol-induced Ca2(+)-mobilization in rat parotid acinar cells is not mediated by activation of beta-adrenoceptors

The effects of isoproterenol (ISO), a beta-adrenoceptor agonist, on cytosolic free Ca2+ ([Ca2+]i) in rat parotid acinar cells were examined using the fluorescent Ca2(+)-indicator fura-2. At concentrations up to 1 mM, ISO caused a rapid increase in [Ca2+]i in a dose-dependent manner, while addition of 1 microM ISO, which evokes the maximum amylase secretion, had only a slight effect on [Ca2+]i. There was no such increase in [Ca2+]i with the addition (2 mM) of 8-bromo-cyclic AMP, a permeant cyclic AMP analogue. The alpha-adrenoceptor antagonist phentolamine blocked the ISO-induced [Ca2+]i increase better than the beta-adrenoceptor antagonist, propranol, and the muscarinic receptor antagonist, atropine. The IC50 value (the concentration which reduces the ISO-induced increase in [Ca2+]i by 50%) of phentolamine was estimated to be 7.6 nM, for propranolol 13.2 microM and for atropine 3.5 microM. The difference in potency between the three antagonists was similar to the difference in blocking the [Ca2+]i increase induced by phenylephrine, an alpha-adrenoceptor agonist. These results suggest that the Ca2(+)-mobilization in response to high concentrations of ISO results from an activation of alpha-adrenoceptors rather than beta-adrenoceptors.     

Effect of activation of muscarinic receptors on intracellular free calcium and secretion in bovine adrenal chromaffin cells

Stimulation of the nicotinic receptor of bovine chromaffin cells results in a rise in intracellular free calcium [( Ca2+]i) and subsequent release of catecholamine. This response is totally dependent on the presence of external Ca2+. Monitoring [Ca2+]i using quin-2 demonstrated a rise in [Ca2+]i in response to muscarinic agonists which was approximately 4-times less than that obtained in response to nicotinic stimulation. This atropine-sensitive [Ca2+]i rise occurred after a 10-s lag and was found to be independent of the external Ca2+, implying the existence of an intracellular Ca2+ source. Despite producing this [Ca2+]i rise low concentrations of the muscarinic agonist, methacholine (under 1 X 10(-3) M), failed to trigger secretion itself and did not effect the secretory response elicited by nicotine. Challenging the cells with higher methacholine concentrations (over 1 X 10(-3) M) resulted in the same [Ca2+]i rise, no secretion, but an inhibition of secretion due to nicotine. This latter response, however, was found to be atropine-insensitive and therefore non-muscarinic. The [Ca2+]i rise and secretion due to depolarization by 55 mM K+ were largely unaffected by prior addition 1 X 10(-2) M methacholine, inferring that high concentrations of methacholine inhibit nicotine-induced secretion by interacting with the nicotinic receptor. These results provide evidence consistent with the existence of an intracellular Ca2+ store mobilized by muscarinic receptor activation in bovine chromaffin cells and show that, despite causing a rise in [Ca2+]i, bovine chromaffin cell muscarinic stimulation does not effect secretion itself or secretion induced by either nicotine or high K+.     

Na+-K+ pump activation inhibits endothelium-dependent relaxation by activating the forward mode of Na+/Ca2+ exchanger in mouse aorta

The effect of Na+-K+ pump activation on endothelium-dependent relaxation (EDR) and on intracellular Ca2+ concentration ([Ca2+]i) was examined in mouse aorta and mouse aortic endothelial cells (MAECs). The Na+-K+ pump was activated by increasing extracellular K+ concentration ([K+]o) from 6 to 12 mM. In aortic rings, the Na+ ionophore monensin evoked EDR, and this EDR was inhibited by the Na+/Ca2+ exchanger (NCX; reverse mode) inhibitor KB-R7943. Monensin-induced Na+ loading or extracellular Na+ depletion (Na+ replaced by Li+) increased [Ca2+]i in MAECs, and this increase was inhibited by KB-R7943. Na+-K+ pump activation inhibited EDR and [Ca2+]i increase (K+-induced inhibition of EDR and [Ca2+]i increase). The Na+-K+ pump inhibitor ouabain inhibited K+-induced inhibition of EDR. Monensin (>0.1 microM) and the NCX (forward and reverse mode) inhibitors 2'4'-dichlorobenzamil (>10 microM) or Ni2+ (>100 microM) inhibited K+-induced inhibition of EDR and [Ca2+]i increase. KB-R7943 did not inhibit K+-induced inhibition at up to 10 microM but did at 30 microM. In current-clamped MAECs, an increase in [K+]o from 6 to 12 mM depolarized the membrane potential, which was inhibited by ouabain, Ni2+, or KB-R7943. In aortic rings, the concentration of cGMP was significantly increased by acetylcholine and decreased on increasing [K+]o from 6 to 12 mM. This decrease in cGMP was significantly inhibited by pretreating with ouabain (100 microM), Ni2+ (300 microM), or KB-R7943 (30 microM). These results suggest that activation of the forward mode of NCX after Na+-K+ pump activation inhibits Ca2+ mobilization in endothelial cells, thereby modulating vasomotor tone.     

Involvement of Ca2+ entry and inositol trisphosphate-induced internal Ca2+ mobilization in muscarinic receptor-mediated catecholamine release in dog adrenal chromaffin cells.

Catecholamine (CA) release from adrenal medulla evoked by muscarinic receptor stimulation has been studied using isolated perfused adrenal gland and cultured chromaffin cells from dogs. Muscarine and oxotremorine (1-100 microM), and bethanechol (0.1-1 mM) dose-dependently stimulated CA release. Muscarine-evoked CA release was antagonized with M1-antagonist, pirenzepine and, to a lesser extent, with atropine; and was reduced either by removal of extracellular Ca2+ or treatment with Ca2+ channel blockers. Muscarine caused an increase of 45Ca uptake and 22Na uptake. Tetrodotoxin (TTX) did not affect muscarine-evoked increase of 22Na uptake and CA release. Under the absence of extracellular Ca2+, muscarine stimulated a 45Ca efflux. Muscarine-induced CA release was attenuated by treating the cells with 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate-HCl (TMB-8) which blocks Ca2+ release from the intracellular store. A phospholipase C inhibitor, neomycin, markedly reduced muscarine-induced CA release but not nicotine- and high K(+)-evoked release. Cinnarizine, a Ca2+ channel blocker, attenuated muscarine-evoked but not caffeine-induced CA release and 45Ca efflux in the absence of extracellular Ca2+. Muscarine caused an increase in intracellular free Ca2+ concentration ([Ca2+]i) in the presence of extracellular Ca2+. It caused a similar increase, but to a lesser extent, in the absence of extracellular Ca2+. The increase of [Ca2+]i induced by muscarine without extracellular Ca2+ was reduced by neomycin and cinnarizine. Polymixin B and retinal, which reduced 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced CA release, had little effect on muscarine-induced CA release. Muscarine increased cellular Ins(1,4,5)P3 production, and atropine inhibited this increase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of Muscarinic Acetylcholine Receptors Increases Synaptosomal Free Calcium Concentration by Protein Kinase-Dependent Opening of L-Type Calcium Channels

In synaptosomes prepared from rat cerebral cortex, free cytosolic calcium concentration ([Ca2+]i) was measured using the fluorescent dye fura-2. Incubation of fura-2-loaded synaptosomes with carbachol increased [Ca2+]i in a dose-dependent manner (1-1,000 microM), with a maximum response of 22 +/- 2% at approximately 100 microM and an EC50 (calculated concentration producing 50% of the maximum response) of 30 microM. The effect of carbachol (100 microM) on [Ca2+]i was antagonised by atropine, but not by hexamethonium (10 microM). The calculated concentration of atropine needed for 50% inhibition (IC50) was 260 nM. The rise in [Ca2+]i produced by carbachol was reduced in the absence of extrasynaptosomal Ca2+ and effectively blocked by the L-type calcium channel blocker nifedipine (with an IC50 of 29 nM). The response to carbachol was reduced if the synaptosomes were preincubated with the protein kinase inhibitors H7 [1-(5-isoquinolinylsulfonyl)-2- methylpiperazine] (from 17% in the solvent control to 4%) and staurosporine (from 20% in the solvent control to 3%). These results show that stimulation of muscarinic acetylcholine receptors in synaptosomes increases [Ca2+]i by protein kinase-dependent activation of 1,4-dihydropyridine-sensitive calcium channels.     

Nicotonic and muscarinic components in acetylcholine stimulation of porcine adrenal medullary cells

Adrenal medullary chromaffin cells secrete catecholamines (CA) in response to cholinergic receptor activation by acetylcholine (ACh) released from splacnic nerve terminals. In cultured bovine chromaffin cells nicotinic receptors play a preponderant (> 90%) role in the control of CA release. By contrast, we found and report here that up to 40% of the ACh-evoked CA secretion from cultured porcine chromaffin cells can be associated with muscarinic receptor activation. The following results support our belief that in porcine adrenal medullary cells ACh (100 M) evoked CA secretion is mediated by both nicotinic and muscarinic cholinergic receptors. 1) Hexamethonium (100 M), a nicotinic receptor antagonist, inhibited ACh-induced CA secretion to ca. 40% of the control release and atropine (1 M), a muscarinic receptor antagonist, inhibited to ca. 60% of the control value. 2) We also found that ACh (100 M) evoked intracellular Ca2+ concentration ([Ca2+]i) rise was inhibited by these receptor antagonists to a different extent, and reversibly reduced by lowering the concentration of Ca2+ in the external medium ([Ca2+]o). This last maneuver ([Ca2+]o < 0.1 M) per se caused a marked reduction in the peak phase of the [Ca2+]i rise evoked by ACh (40% of the control response). Switching the external medium back to physiologic [Ca2+]o in the continued presence of ACh caused a partial recovery of the elevated [Ca2+]i. This [Ca2+]o-dependent [Ca2+]i rise was blocked by hexamethonium (100 M) but not by atropine (1 M). Conversely, the ACh-evoked [Ca2+]i rise in low external [Ca2+]o was blocked by atropine but not by hexamethonium. From these data we conclude that in porcine adrenal medullary cells an important fraction (ca. 0.4) of both ACh-induced CA secretion and peak [Ca2+]i rise is due to muscarinic receptor activation.     

Inhibition of endothelium-dependent vasorelaxation by extracellular K(+): a novel controlling signal for vascular contractility

The effects of extracellular K+ on endothelium-dependent relaxation (EDR) and on intracellular Ca2+ concentration ([Ca2+]i) were examined in mouse aorta, mouse aorta endothelial cells (MAEC), and human umbilical vein endothelial cells (HUVEC). In mouse aortic rings precontracted with prostaglandin F2alpha or norepinephrine, an increase in extracellular K+ concentration ([K+]o) from 6 to 12 mM inhibited EDR concentration dependently. In endothelial cells, an increase in [K+]o inhibited the agonist-induced [Ca2+]i increase concentration dependently. Similar to K+, Cs+ also inhibited EDR and the increase in [Ca2+]i concentration dependently. In current-clamped HUVEC, increasing [K+]o from 6 to 12 mM depolarized membrane potential from -32.8 +/- 2.7 to -8.6 +/- 4.9 mV (n = 8). In voltage-clamped HUVEC, depolarizing the holding potential from -50 to -25 mV decreased [Ca2+]i significantly from 0.95 +/- 0.03 to 0.88 +/- 0.03 microM (n = 11, P < 0.01) and further decreased [Ca2+]i to 0.47 +/- 0.04 microM by depolarizing the holding potential from -25 to 0 mV (n = 11, P < 0.001). Tetraethylammonium (1 mM) inhibited EDR and the ATP-induced [Ca2+]i increase in voltage-clamped MAEC. The intermediate-conductance Ca2+-activated K+ channel openers 1-ethyl-2-benzimidazolinone, chlorozoxazone, and zoxazolamine reversed the K+-induced inhibition of EDR and increase in [Ca2+]i. The K+-induced inhibition of EDR and increase in [Ca2+]i was abolished by the Na+-K+ pump inhibitor ouabain (10 microM). These results indicate that an increase of [K+]o in the physiological range (6-12 mM) inhibits [Ca2+]i increase in endothelial cells and diminishes EDR by depolarizing the membrane potential, decreasing K+ efflux, and activating the Na+-K+ pump, thereby modulating the release of endothelium-derived vasoactive factors from endothelial cells and vasomotor tone.     

Ca2+ dynamics in rat pancreatic AR-42J and AR-IP cells.

Spatiotemporal change of the cytosolic free Ca2+ concentration ([Ca2+]i) in response to a variety of secretagogues was examined in rat pancreatoma AR-42J and AR-IP cells by microspectroflurometry and digital imaging microscopy after loading with fura-2. In the presence of external Ca2+, carbachol, CCK-OP (cholecystokinin-octapeptide), gastrin, norepinephrine or high K+ evoked a large transient increase in [Ca2+]i in AR-42J cells which declined to a sustained level before slowly declining towards the resting level. In the absence of external Ca2+, a transient increase in [Ca2+]i were evoked by all the ligands except for high K+ stimulation, which declined rapidly towards the resting level. The [Ca2+]i increase caused by carbachol and high K+ treatment was inhibited by muscarinic receptor antagonist, atropine, and by L-type Ca2+ channel blocker, nifedipine, respectively. The transient [Ca2+]i increase induced by gastrin stimulation was not blocked by Ca2+ channel blocker, lanthanum. In the AR-IP cells, which are non-differentiated pancreatoma cell line, all stimulations including high K+ treatment have failed to evoke [Ca2+]i response. These intracellular Ca2+ mobilizations in response to ligands in AR-42J cells were displayed by digital imaging microscopy. From these results we conclude that AR-42J cells has an alpha-adrenergic receptor, in addition to muscarinic acetylcholine receptor, CCK-OP receptor, gastrin receptor and voltage dependent Ca2+ channel. In marked contrast, AR-IP cells have neither any hormone receptor for the above ligands nor voltage dependent Ca2+ channel.     

Calcium-Independent Release of Acetylcholine from Electric Organ Synaptosomes and Its Changes by Depolarization and Cholinergic Drugs

Chemiluminescent detection was applied to measure the continuous spontaneous Ca2+-independent liberation of acetylcholine (ACh) from Torpedo electric organ synaptosomes. Differentiation between the release of ACh and choline was achieved by inhibiting cholinesterases with phospholine, and a way to quantify the continuous release was devised. The method permitted measurements during short time intervals from minute amounts of tissue and without an accumulation of ACh in the medium. Synaptosomes continuously liberated small amounts of ACh during incubations in the presence of 3 mM K+ and in the absence of Ca2+. The spontaneous liberation of ACh was similar both quantitatively and qualitatively at pH values of 8.6 and 7.8. It was unaltered by MgCl2 (10.4 mM), 2-(4-phenylpiperidino)cyclohexanol (10 microM), ouabain (104 microM), atropine (10 microM), and valinomycin (102 nM). Carbamoylcholine brought about a decrease, which could be partially reversed by atropine. The Ca2+-independent output of ACh was increased considerably when the concentration of K+ ions was raised (eightfold at 103 and 35-fold at 203 mM K+). Carbamoylcholine (104 microM) blocked the increase in ACh release produced by high K+; this effect of carbamoylcholine was not reversed by atropine (10 microM). When Ca2+ was added to synaptosomes depolarized by a high concentration of K+, the amount of ACh released during the first 1-3 min after the addition of Ca2+ was at least 20 times higher than in the absence of Ca2+, but the release returned rapidly to predepolarization values. Similarly high values of ACh release could be achieved by adding Ca2+ plus the ionophore A23187 and even higher values by adding Ca2+ plus gramicidin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+ clearance mechanisms in neurohypophysial terminals of the rat

The importance of intracellular calcium ([Ca2+]i) in the release of vasopressin (AVP) and oxytocin from the central nervous system neurohypopyhysial nerve terminals has been well-documented. To date, there is no clear understanding of Ca2+ clearance mechanisms and their interplay with transmembrane Ca2+ entry, intracellular [Ca2+]i transients, cytoplasmic Ca2+ stores and hence the release of AVP at the level of a single nerve terminal. Here, we studied the mechanism of Ca2+ clearance in freshly isolated nerve terminals of the rat neurohypophysis using Fura-2 Ca2+ imaging and measured the release of AVP by radioimmuno assay. An increase in the K+ concentration in the perfusion solution from 5 to 50 mM caused a rapid increase in [Ca2+]i and AVP release. Returning K+ concentration to 5 mM led to rapid restoration of both responses to basal level. The K+-evoked [Ca2+]i and AVP increase was concentration-dependent, reliable, and remained of constant amplitude and time course upon successive applications. Extracellular Ca2+ removal completely abolished the K+-evoked responses. The recovery phase was not affected upon replacement of NaCl with sucrose or drugs known to act on intracellular Ca2+ stores such as thapsigargin, cyclopiazonic acid, caffeine or a combination of caffeine and ryanodine did not affect either resting or K+-evoked [Ca2+]i or AVP release. By contrast, the plasma membrane Ca2+ pump inhibitor, La3+, markedly slowed down the recovery phase. The mitochondrial respiration uncoupler, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), slightly but significantly increased the basal [Ca2+]i, and also slowed down the recovery phase of both [Ca2+]i and release responses. In conclusion, we show in nerve terminals that (i) Ca2+ extrusion through the Ca2+ pump in the plasma membrane plays a major role in the Ca2+ clearance mechanisms of (ii) Ca2+ uptake by mitochondria also contributes to the Ca2+ clearance and (iii) neither Na+/Ca2+ exchangers nor Ca2+ stores are involved in the Ca2+ clearance or in the maintenance of basal [Ca2+]i or release of AVP.     

Control of cytosolic free calcium by intracellular organelles in bovine adrenal glomerulosa cells. Effects of sodium and inositol 1,4,5-trisphosphate

The regulation of cytosolic free Ca2+ concentration ([Ca2+]c) by intracellular organelles was studied in permeabilized bovine adrenal glomerulosa cells. Two compartments, with distinct characteristics, were able to pump Ca2+. A first pool, sensitive to ruthenium red and presumably mitochondrial, required respiratory chain substrates to maintain [Ca2+]c around 700 nM. Ca2+ efflux from this compartment was activated by Na+ (ED50 = 5 mM). Inositol 1,4,5-trisphosphate (IP3) had no effect on this pool. A second nonmitochondrial pool required ATP to lower [Ca2+]c to about 200 nM and released Ca2+ transiently upon addition of IP3. When the two systems were allowed to work simultaneously, the nonmitochondrial pool regulated [Ca2+]c and IP3 released Ca2+ in a concentration-dependent manner (EC50 = 0.6 microM). Under these conditions the mitochondria seemed Ca2+ depleted. Upon repeated stimulations with IP3, a marked attenuation of the response was observed. This phenomenon was due to Ca2+ sequestration by a nonmitochondrial IP3-insensitive pool. Neither dantrolene (200 microM) nor 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (10 microM) were able to abolish IP3-induced Ca2+ release, though both compounds efficiently inhibited aldosterone production in intact cells stimulated with angiotensin II (10 nM) or K+ (12 mM). These results suggest that in permeabilized adrenal glomerulosa cells: the nonmitochondrial pool is responsible for buffering [Ca2+]c and for releasing Ca2+ in response to IP3; at resting [Ca2+]c levels, the mitochondria appear Ca2+ depleted; when [Ca2+]c rises above their set point, the mitochondria accumulate Ca2+ as a function of [Na+]c; 4) the mitochondria are not involved in the desensitization mechanism of the response to IP3.     

Inhibition of glucose-stimulated insulin release by alpha 2-adrenoceptor activation is parallelled by both a repolarization and a reduction in cytoplasmic free Ca2+ concentration

Effects of the alpha 2-adrenergic agonist clonidine on insulin release, membrane potential, and cytoplasmic free Ca2+ concentration ([Ca2+]i) were investigated using pancreatic beta-cells isolated from obese hyperglycemic mice. Addition of 2 microM clonidine promptly inhibited glucose-stimulated insulin release, an effect accompanied by a lowering in both membrane potential and [Ca2+]i. Within minutes, the effect on Ca2+ was partly reversed, [Ca2+]i attaining a new level, although still significantly lower than in the absence of agonist. This late increase in [Ca2+]i was inhibited by 50 microM D-600, a blocker of voltage-activated Ca2+ channels. The inhibitory effects of clonidine on membrane potential, [Ca2+]i, and insulin release were abolished by 5 microM of the alpha 2-adrenergic antagonist yohimbine. Depolarization with high K+ increased [Ca2+]i also in the presence of clonidine, conditions accompanied by only a minute release of insulin. Secretion was, however, partly restored by subsequent addition of 20 mM glucose. Addition of 5 mM Ca2+ transiently reversed the effects of clonidine on both membrane potential and [Ca2+]i. Although the clonidine-induced repolarization should be enough for closing the voltage-activated Ca2+ channels with a resulting decrease in [Ca2+]i, a direct interaction of the agonist with these channels cannot be excluded. The fact that it was possible to increase [Ca2+]i with only a minor effect on insulin release suggests that the inhibitory effect of clonidine not only is due to a reduction in [Ca2+]i, but also involves interference with some more distal step in the insulin secretory machinery.     

Caffeine inhibits the agonist-evoked cytosolic Ca2+ signal in mouse pancreatic acinar cells by blocking inositol trisphosphate production.

The inhibitory effects of caffeine on receptor-activated cytosolic Ca2+ signal generation in isolated mouse pancreatic acinar cells were investigated. Using the ability of caffeine to quench Indo-1 fluorescence we measured simultaneously the free intracellular Ca2+ concentration ([Ca2+]i) and the intracellular caffeine concentration ([caffeine]i). We also measured inositol 1,4,5-trisphosphate (InsP3) production with a radioreceptor assay. When caffeine was added to the extracellular solution during a sustained receptor-activated increase in [Ca2+]i, [caffeine]i rose to its steady level within a few seconds. This was accompanied by a decrease of [Ca2+]i, which started only after [caffeine]i had reached an apparent threshold concentration (about 2 mM in the case of 0.5 microM acetylcholine (ACh) stimulation). Above this [caffeine]i level there was a linear relationship between [caffeine]i and [Ca2+]i. Throughout the caffeine exposure [Ca2+]i remained at a steady low level. Following removal of caffeine from the bath, [caffeine]i decreased to zero within seconds. There was no significant increase in [Ca2+]i until [caffeine]i had been reduced to the threshold level (about 2 mM at 0.5 microM ACh). Caffeine inhibited Ca2+ signals evoked by ACh, cholecystokinin, and ATP and also inhibited signals generated in the absence of external Ca2+. Caffeine application had the same effect as removal of agonist allowing recovery from apparent desensitization. Caffeine inhibited the agonist-evoked production of InsP3 in a dose-dependent manner. Our results demonstrate the acute and reversible dose-dependent inhibition of agonist-evoked cytosolic Ca2+ signal generation due to rapid intracellular caffeine accumulation and washout. The inhibition can be explained by the reduction of agonist-evoked InsP3 production.     

Depolarization and Agonist-Stimulated Changes in Inositol 1,4,5-Trisphosphate and Inositol 1,3,4,5-Tetrakisphosphate Mass Accumulation in Rat Cerebral Cortex

Muscarinic receptor stimulation or depolarization with elevated extracellular K+ induced rapid and sustained increases in mass accumulations of myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and myo-inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] in cerebral cortex slices. Synergistic but transient responses of both inositol polyphosphate second messengers were observed when slices were stimulated with carbachol under depolarizing conditions; this synergy was observed as an increase in the maximal responsiveness, with no significant change in EC50 values for carbachol. Omission of buffer Ca2+ ([Ca2+]e 10-20 microM) reduced basal Ins(1,4,5)P3 and Ins(1,3,4,5)P4 concentrations; the relative stimulatory effects of muscarinic receptor stimulation were maintained, but the effects of depolarization were markedly attenuated under these conditions. A component of the response to depolarization appeared to be indirectly mediated by the release of acetylcholine, because the K(+)-evoked increase in Ins(1,3,4,5)P4 was enhanced by the cholinesterase inhibitor physostigmine, and was partially attenuated by atropine. An additive suppression by nitrendipine suggests that entry of Ca2+ through L-type Ca2+ channels may serve to accelerate phosphorylation of Ins(1,4,5)P3 by 3-kinase. Norepinephrine did not significantly increase Ins(1,4,5)P3 or Ins(1,3,4,5)P4 accumulation; however, in the presence of depolarizing K+, norepinephrine caused a dramatic increase in Ins(1,3,4,5)P4 mass accumulation. In contrast, the excitatory amino acid quisqualate caused significant increases in the mass accumulations of both inositol polyphosphates measured, with no further increase being observed under depolarizing conditions. The results are discussed with respect to the interactive effects of agonist and depolarization stimuli on inositol polyphosphate accumulation which might more accurately reflect the conditions pertaining in vivo.     

Calcium Dependency of Muscarinic and Nicotinic Agonist-Induced ATP and Catecholamine Secretion from Porcine Adrenal Chromaffin Cells

The secretion of catecholamines and ATP induced by cholinergic agonists and its dependence on extracellular Ca2+ were studied in cultured porcine adrenal chromaffin cells. Both nicotine and methacholine (a selective muscarinic agonist) induced secretion and increases in cytosolic free Ca2+ concentration ([Ca2+]in), although the activation of nicotinic receptors produced responses that were larger than those produced by activation of muscarinic receptors. The secretion and the increase in [Ca2+]in evoked by nicotine were completely dependent on extracellular Ca2+ and were blocked by prior depolarization of the cells with high extracellular K+ levels. In addition, nicotine induced significant 45Ca2+ influx. In contrast, the secretion and the increase in [Ca2+]in evoked by methacholine were partially dependent on extracellular Ca2+; methacholine also induced 45Ca2+ influx. Prior depolarization of the cells with high extracellular K+ levels did not block methacholine-induced secretion. In general, nicotinic responses were mediated by Ca2+ influx through voltage-dependent pathways. In contrast, muscarinic responses were dependent on both Ca2+ influx through an unknown mechanism that could not be inactivated by high K+ concentration-induced depolarization and presumably also intracellular Ca2+ mobilization.     

High external Ca2+ levels trigger membrane potential oscillations in mouse pancreatic beta-cells during blockade of K(ATP) channels.

Glucose depolarizes the pancreatic beta-cell and induces membrane potential oscillations, but the nature of the underlying oscillatory conductance remains unknown. We have now investigated the effects of the Ca2+ ionophore ionomycin and high external Ca2+ concentration ([Ca2+]o) on glucose-induced electrical activity and whole islet intracellular free Ca2+ concentration ([Ca2+]i), under conditions where the K(ATP) channel was blocked (100 microM tolbutamide or 4 microM glibenclamide). Raising [Ca2+]o to 10.2 or 12.8 mM, but not to 5.1 or 7.7 mM, turned continuous electrical activity into bursting activity. High [Ca2+]o (12.8 mM) regenerated a pattern of fast [Ca2+]i oscillations overshooting the levels recorded in tolbutamide. Ionomycin (10 microM) raised the [Ca2+]i and synergized with 5.1 mM Ca2+ to hyperpolarize the beta-cell membrane. The data indicate that a [Ca2+]i-sensitive and sulphonylurea-insensitive oscillatory conductance underlies the beta-cell bursting activity.     

Stimulus-secretion coupling in bovine parathyroid cells. Dissociation between secretion and net changes in cytosolic Ca2+

The relationship between the concentration of cytosolic free Ca2+ ([Ca2+]i) and secretion of parathyroid hormone (PTH) was investigated in isolated bovine parathyroid cells using the fluorescent Ca2+ indicator, quin 2. Increasing the concentration of extracellular Ca2+ from 0.5 to 2.0 mM caused a 3-fold increase in [Ca2+]i (from 183 +/- 4 to 568 +/- 21 nM) which was associated with a 2-4-fold decrease in secretion of PTH. Decreasing extracellular Ca2+ to about 1 microM caused a corresponding fall in [Ca2+]i to 60-90 nM. Extracellular Ca2+-induced changes in [Ca2+]i were not affected by omission of extracellular Na+. Depolarizing concentrations of K+ (30 mM) depressed [Ca2+]i at all concentrations of extracellular Ca examined, and this was associated with increased secretion of PTH. Ionomycin (0.1 or 1 microM) increased [Ca2+]i at extracellular Ca2+ concentrations of 0.5, 1.0, and 2.0 mM, but inhibited secretion of PTH only at Ca concentrations near the "Ca2+ set point" (1.25 microM). In contrast, dopamine, norepinephrine (10 microM each), and Li+ (20 mM) potentiated secretion of PTH without causing any detectable change in [Ca2+]i. The results obtained with these latter secretagogues provide evidence for a mechanism of secretion which is independent of net changes in [Ca2+]i. The phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) did not alter [Ca2+]i or secretion of PTH at low (0.5 mM) extracellular Ca2+ concentrations. At 2.0 mM extracellular Ca2+, however, TPA (20 nM or 1 microM) depressed [Ca2+]i and potentiated secretion of PTH. The addition of TPA prior to raising the extracellular Ca2+ concentration reduced the subsequent increase in [Ca2+]i. The results show that the effects of TPA on secretion in the parathyroid cell are not readily dissociated from changes in [Ca2+]i and suggest that some TPA-sensitive process, perhaps involving protein kinase C, may be involved in those mechanisms that regulate [Ca2+]i in response to changes in extracellular Ca2+.