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.     

Cyclic AMP raises cytoplasmic calcium in pancreatic alpha 2-cells by mobilizing calcium incorporated in response to glucose

The cytoplasmic Ca2+ concentration ([Ca2+]i) was monitored in individual guinea-pig pancreatic alpha 2-cells exposed to modulators of glucagon release. Addition of the stimulatory amino acid arginine resulted in a sustained increase in [Ca2+]i, whereas the inhibitor glucose had the opposite effect. Epinephrine, the beta-adrenergic agonist isoproterenol, the adenylate cyclase activator forskolin and 8-bromo-cAMP transiently raised [Ca2+]i provided that the cells had been pretreated with glucose. However, simultaneous presence of glucose was not required and the effect occurred even in the absence of extracellular Ca2+. Carbachol, the alpha 2-adrenergic agonist clonidine and the sulfonylurea tolbutamide lacked effects on [Ca2+]i. In addition to providing support for the concept that glucagon release is positively modulated by [Ca2+]i, the results demonstrate that cAMP raises [Ca2+]i in the alpha 2-cells by mobilizing calcium incorporated in response to glucose.     

Beta-adrenergic receptor stimulation induces inositol trisphosphate production and Ca2+ mobilization in rat parotid acinar cells

In dispersed rat parotid gland acinar cells, the beta-adrenergic agonist (-)-isoproterenol, but not its stereoisomer (+)-isoproterenol, induced a transient 1.6-fold (at maximum stimulation, 2 x 10(-4) M) increase in cytosolic free calcium ([Ca2+]i) within 9 s, which returned to resting levels (approximately 190 nM) by 60 s. This [Ca2+]i response was not altered by chelating extracellular Ca2+ with [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) and could be completely blocked by the beta-adrenergic antagonists propranolol (beta 1 + beta 2) and ICI 118,551 (beta 2) but not by atenolol (beta 1). The muscarinic-cholinergic agonist carbachol (at maximum stimulation, 10(-5) M) induced a 3-4-fold elevation in [Ca2+]i within 6 s, which slowly returned to resting levels by 8-10 min. The peak carbachol [Ca2+]i response was not substantially altered by the addition of EGTA to the extracellular medium. However, if the cells were first stimulated with isoproterenol in the EGTA-containing medium, the peak carbachol response was decreased approximately 54%. When carbachol was added to cells in the presence of high extracellular calcium, at the isoproterenol-stimulated [Ca2+]i peak, the resulting [Ca2+]i level was equal to that achieved when carbachol was either added alone or added after propranolol and isoproterenol. 8-Bromo-cyclic AMP induced a [Ca2+]i response similar to that elicited by isoproterenol, which was not additive to that by carbachol. Carbachol induced a approximately 3.5-fold increase in inositol trisphosphate (IP3) production in parotid cells within 30 s. 8-Bromo-cAMP, N6,O2'-dioctanoyl-cAMP, and isoproterenol consistently induced a significant stimulation in IP3 production. The half-maximal concentration of isoproterenol required for [Ca2+]i mobilization and IP3 production was comparable (approximately 10(-5) M). Isoproterenol-induced IP3 formation was blocked by propranolol. The data show that in rat parotid acinar cells, beta-adrenergic stimulation results in IP3 formation and mobilization of a carbachol-sensitive intracellular Ca2+ pool by a mechanism involving cAMP. This demonstrates an interaction between the cAMP and phosphoinositide second messenger systems in these cells.     

Actions of two native GnRHs and protein kinase C modulators on goldfish pituitary cells. Studies on intracellular calcium levels and gonadotropin release.

Previous results indicate that the two native gonadotropin (GtH)-releasing hormones of the goldfish, sGnRH and cGnRHII, stimulate GtH secretion in an extracellular Ca2+ ([Ca2+]o) dependent manner. In the present study, sGnRH, cGnRHII, KCI and the protein kinase C (PKC) activators TPA and DiC8, stimulated increases in intracellular Ca2+ ([Ca2+]i) levels in goldfish pituitary cells. Testing in Ca(2+)-deficient medium abolished the [Ca2+]i responses to cGnRHII, TPA and KCI and attenuated responses to sGnRH and DiC8. These results are the first to demonstrate that in teleost pituitary cells both native GnRHs stimulate increases in [Ca2+]i levels via [Ca2+]o entry. sGnRH- and DiC8-stimulated increases in [Ca2+]i also appear to be partially due to mobilization of Ca2+ from intracellular stores. Other results are consistent with a role for PKC in mediating GnRH action especially extracellular Ca2+ entry. Firstly, the PKC inhibitor staurosporine decreased GnRH- and TPA-induced [Ca2+]i responses. Secondly, incubation with Ca(2+)-deficient medium attenuated TPA- and DiC8-stimulated GtH release. Thirdly, GtH release responses to PKC activators were enhanced and reduced by an agonist and an antagonist of Ca2+ channel function, respectively. However, differences in the sensitivity of DiC8- and TPA-elicited responses to manipulations of [Ca2+]o entry indicate that these two PKC activators may have different actions in the goldfish pituitary. A difference in action of the two GnRHs on mobilization of Ca2+ from intracellular stores is also indicated.     

The effect of [Ca2+] and [H+] on the functional recovery of rat brain synaptosomes from anoxic insult in vitro.

The energy status (as measured by the ATP/ADP ratio), oxidative metabolism (14CO2 output) and neurotransmitter synthesis ( [14C]acetylcholine production) by rat brain synaptosomes utilizing [U-14C]glucose has been studied. The ability of anoxia in vitro to permanently alter these parameters was investigated with reference to external [Ca2+] and [H+]. It has previously been shown that anoxic damage to synaptosomal preparations is only apparent when their metabolism is stimulated by veratridine [Harvey, Booth & Clark (1982) Biochem. J. 206, 433-439]. It is concluded that low [Ca2+] ameliorates, and high [H+] exacerbates, the damage sustained by veratridine-stimulated anoxic synaptosomes. The combined effects of low pH, anoxia and veratridine stimulation on synaptosomal metabolism most closely approximated to the irreversible damage to brain metabolism observed during acute hypoxia in vivo [Booth, Harvey & Clark (1983) J. Neurochem. 40, 106-110]. Suitably treated synaptosomal preparations may therefore be usefully employed as models to study impaired neurotransmitter synthesis in vivo.     

Elevation of cytosolic calcium precedes anoxic gene expression in maize suspension-cultured cells.

Based on pharmacological evidence, we previously proposed that intracellular Ca2+ mediates the perception of O2 deprivation in maize seedlings. Herein, using fluorescence imaging and photometry of Ca2+ in maize suspension-cultured cells, the proposal was further investigated. Two complementary approaches were taken: (1) real time analysis of anoxia-induced changes in cytosolic Ca2+ concentration ([Ca]i) and (2) experimental manipulation of [Ca]i and then assay of the resultant anoxia-specific responses. O2 depletion caused an immediate increase in [Ca2+]i, and this was reversible within a few seconds of reoxygenation. The [Ca]i elevation proceeded independent of extracellular Ca2+. The kinetics of the Ca2+ response showed that it occurred much earlier than any detectable changes in gene expression. Ruthenium red blocked the anoxic [Ca]i elevation and also the induction of adh1 (encoding alcohol dehydrogenase) and sh1 (encoding sucrose synthase) mRNA. Ca2+, when added along with ruthenium red, prevented the effects of the antagonist on the anoxic responses. Verapamil and bepridil failed to block the [Ca]i rise induced by anoxia and were equally ineffective on anoxic gene expression. Caffeine induced an elevation of [Ca]i as well as ADH activity under normoxia. The data provide direct evidence for [Ca]i elevation in maize cells as a result of anoxia-induced mobilization of Ca2+ from intracellular stores. Furthermore, any manipulation that modified the [Ca]i rise brought about a parallel change in the expression of two anoxia-inducible genes. Thus, these results corroborate our proposal that [Ca]i is a physiological transducer of anoxia signals in plants.     

Anoxic survival of the isolated cerebellum of the turtle Pseudemis scripta elegans

Intact turtle brain provides a useful model for the study of anoxia and potential survival strategies, since this tissue maintains transmembrane ion gradients and ATP levels during prolonged anoxia and recovers functional activity afterwards. Since isolated tissues offer experimental advantages, the present study sought to determine effects of anoxia on the isolated turtle cerebellum and to define relationships between anoxia survival and glucose supply. In normoxia, the extracellular potassium ([K+]o) activity and evoked potentials were maintained with 5 mM glucose, but 20 mM glucose was required to maintain adenosine triphosphate (ATP) levels and prevent significant increases in [K+]o during anoxia. Inhibition of glycolysis by iodoacetic acid (IAA) during anoxia provoked large increases in [K+]o at all glucose levels. These results demonstrate the usefulness of the isolated turtle cerebellum for studies of anoxic survival since this tissue can maintain ATP levels and [K+]o during prolonged anoxia with 20 mM glucose in the artificial cerebrospinal fluid medium. They also suggest the presence of a Pasteur effect at least during the transition to a hypometabolic state.     

Enzyme-substrate interactions in the hydrolysis of peptides by cathepsins B and H from rat liver.

Free Ca2+ in the cytosol ([Ca2+]i) of individual rat ventricle cells injected with aequorin was measured under anoxia. In glucose-free medium myocytes spontaneously shortened after about 60 min, although [Ca2+]i was still at or near resting levels. However, within minutes a net inward movement of Ca2+ across the sarcolemma developed and [Ca2+]i began to rise. Provided oxygen was readmitted before [Ca2+]i exceeded 2-3 microM, cells were able to restore [Ca2+]i to resting levels through caffeine-sensitive sequestration of Ca2+ in the sarcoplasmic reticulum. We suggest that Ca2+-independent shortening of anoxic cardiomyocytes reflects onset of rigor which triggers loss of [Ca2+]i homoeostasis.     

Na^＋／Ca^2＋交换抑制剂在大鼠海马缺氧损伤中的作用

本文以大鼠离体海马脑片和分散培养的海马神经元为标本,分别采用微电极记录技术和激光扫描共聚焦显微镜动态监测单个神经元［Ｃａ２＋］ｉ的方法,研究Ｎａ＋／Ｃａ２＋交换抑制剂Ｂｅｎｚａｍｉｌ对缺氧后海马脑片损伤以及海马神经元［Ｃａ２＋］ｉ变化的影响。结果显示,预先用Ｂｅｎｚａｍｉｌ（５０μｍｏｌ）灌流的海马脑片缺氧后ＰＶ持续时间较对照组显著延长,提示其可延缓海马不可逆缺氧损伤的发生;共聚焦测［Ｃａ２＋］ｉ实验进一步发现,急性缺氧可诱导海马神经元［Ｃａ２＋］ｉ迅速升高,而Ｂｅｎｚａｍｉｌ（２０μｍｏｌ）能显著抑制缺氧引起的［Ｃａ２＋］ｉ升高。上述结果表明,抑制神经元Ｎａ＋／Ｃａ２＋交换活动可提高海马脑片抗缺氧能力,其作用机制可能与抑制缺氧所致神经元［Ｃａ２＋］ｉ升高有关,由此推测Ｎａ＋／Ｃａ２＋交换体参于大鼠海马脑区缺氧损伤,它可能是导致缺氧后神经元［Ｃａ２＋］ｉ升高的重要途径之一     

Glucose sensing of individual pancreatic beta-cells involves transitions between steady-state and oscillatory cytoplasmic Ca2+.

Glucose stimulation of individual pancreatic beta-cells is associated with a rise of the cytoplasmic Ca2+ concentration ([Ca2+]i) manifested either as large amplitude oscillations (0.2-0.5/min) or as a sustained increase. Determinants for the transitions between the basal and the two stimulated states have now been studied using dual-wavelength fluorometric measurements on individual ob/ob mouse beta-cells loaded with the Ca2+ indicator Fura-2. The transition from the basal state to large amplitude oscillations was induced by raising the glucose concentration to 7 mM or above. The frequencies and shapes of the [Ca2+]i cycles remained largely unaffected when raising glucose as high as 40 mM. However, in some cells the oscillatory pattern was transformed into a sustained increase of [Ca2+]i at high glucose concentrations. Although the peak values for the oscillations exceeded the steady-state increase, the time average [Ca2+]i was higher during the latter phase. Both types of glucose-induced transitions were facilitated by the presence of 1-100 nM glucagon. Protein kinase C activation by 10 nM of the phorbol ester TPA resulted in a transformation of the glucose-induced oscillations into a sustained increase of [Ca2+]i but the levels reached were considerably lower than obtained with glucose alone. It is concluded that the glucose sensing of the individual beta-cell is based on sudden transitions between steady-state and oscillating cytoplasmic Ca2+. It is these transitions rather than alterations of the oscillatory characteristics which determine the average [Ca2+]i regulating insulin release.     

Relationship between cytosolic Ca2+ concentration and amylase release in rat parotid acinar cells following muscarinic stimulation.

Carbachol (CCh), a muscarinic-cholinergic agonist, increased both cytosolic free calcium concentration ([Ca2+]i) and amylase release in rat parotid acinar cells or acini in a dose-dependent manner. Treatment of acinar cells with the intracellular Ca2+ antagonist, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), or the intracellular Ca2+ chelator, 1,2-bis(O-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid (BAPTA), strongly attenuated the increases in [Ca2+]i evoked by CCh, but amylase release from acini was not significantly suppressed by the treatment with TMB-8 or BAPTA. Low concentrations (0.02-0.5 microM) of ionomycin, a Ca2+ ionophore, caused increases in [Ca2+]i comparable to those induced by CCh, but the same concentrations had only a little effect on amylase release. The protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulated amylase release in quantities similar to those induced by CCh, although TPA alone did not cause any change in [Ca2+]i. Combined addition of TPA and ionomycin potentiated only modestly amylase release stimulated by TPA alone. Staurosporine, a protein kinase C-inhibitor, similarly inhibited both the CCh- and TPA-induced amylase release. These results suggest that an increase in [Ca2+]i elicited by CCh does not play an essential role for inducing amylase release in rat parotid acini. Amylase release by muscarinic stimulation may be mediated mainly by activation of protein kinase C.     

Cytoplasmic Ca2+ in platelets is controlled by cyclic AMP: antagonism between stimulators and inhibitors of adenylate cyclase

Activation of platelets by thrombin rapidly increases cytoplasmic free calcium, [Ca2+]i, measured by Quin -2, and induces secretion. Stimulators of adenylate cyclase (i.e. PGI2, PGD2, forskolin) suppressed or reversed the increase of [Ca2+]i. Inhibitors of adenylate cyclase (i.e. epinephrine, ADP), added before or after thrombin, counteracted PGI2, PGD2 and forskolin and thereby increased [Ca2+]i and restored secretion. Responses to epinephrine (via alpha-2 adrenoreceptors) and ADP were independent of extracellular Ca2+, but required maintained occupancy of thrombin receptors and intact cAMP-phosphodiesterase activity. These results indicate that cAMP serves as an inhibitory second-messenger that antagonizes the mobilization of Ca2+, an activator second-messenger.     

Single pancreatic beta-cells from normal rats exhibit an initial decrease and subsequent increase in cytosolic free Ca2+ in response to glucose.

Since it was reported that glucose stimulation initially lowers as well as subsequently raises the cytosolic free calcium concentration [( Ca2+]i) in pancreatic islet cells from hyperglycemic ob/ob mice, it has been argued whether the lowering of [Ca2+]i is physiological or artifactual. In the present study, [Ca2+]i in single pancreatic beta-cells from normal rats was measured by Fura-2 microfluorometry. Following elevation of the glucose concentration from 2.8 mM (basal) to 16.7 mM, a bimodal change in [Ca2+]i, an initial decrease and subsequent increase, was demonstrated. When the basal glucose concentration was raised to 5.6 mM, the stimulation with 16.7 mM glucose also induced the decrease in [Ca2+]i in the majority of the cells, though the amplitude of the decrease was reduced. An elevation of the glucose concentration from 2.8 to 5.6 mM induced the decrease in [Ca2+]i but not usually the increase in [Ca2+]i. Removal of extracellular Ca2+ eliminated the increase in [Ca2+]i without affecting the decrease in [Ca2+]i. Thus, the decrease and increase in [Ca2+]i were clearly dissociated under certain conditions. In contrast, mannoheptulose (an inhibitor of glucose metabolism) inhibited both the decrease and increase in [Ca2+]i. These results demonstrate that the glucose-induced bimodal change in [Ca2+]i is a physiological response of islet beta-cells, and that the decrease and increase in [Ca2+]i are generated by mutually-independent mechanisms which are operated through glucose metabolism by islet beta-cells.     

Cytoplasmic Ca2+ during differentiation of 3T3-L1 adipocytes. Effect of insulin and relation to glucose transport

The cytoplasmic concentration of ionized Ca2+ [( Ca2+]i) was determined in 3T3-L1 cells during their differentiation from fibroblasts to adipocytes, suspended and loaded with the fluorescent Ca2+ indicators quin2 or indo-1. In undifferentiated fibroblasts, as well as in differentiated adipocytes up to day 9, [Ca2+]i was steady around 170 nM, and it increased significantly only in old adipocytes (day 12). During differentiation, stimulation of glucose uptake by insulin increased from a few percent to severalfold. Stimulation of uptake was already apparent after 10 min of addition of the hormone, and 10 nM insulin produced maximal stimulation in 30 min. Insulin (10(-6) M) added to quin2- or indo-1-loaded, suspended adipocytes had no detectable effect on [Ca2+]i for at least 10 min. In contrast, addition of the general anesthetic halothane increased [Ca2+]i from 172 to 251 nM in 3 min. In EGTA solution, the Ca2+ ionophore ionomycin elicited release of Ca2+ from intracellular stores that resulted in a transient increase in [Ca2+]i. A smaller but measurable Ca2+ release from intracellular stores (increasing [Ca2+]i by 20 nM) resulted upon addition of 20 micrograms/ml phosphatidic acid. In contrast, insulin did not produce any detectable release of Ca2+ from intracellular stores. Incubation of 3T3-L1 adipocytes with insulin in the presence of EGTA (the latter in excess over the Ca2+ concentration of the medium) did not prevent the stimulation of hexose uptake by the hormone, indicating that extracellular Ca2+ does not play a role in the insulin response. Furthermore, incubation of cells with quin2/AM in EGTA medium during exposure to insulin did not prevent stimulation of hexose uptake. Under these conditions it is demonstrated that intracellular quin2 suffices to chelate cytoplasmic Ca2+ even if releasable Ca2+ from intracellular stores were to pour into the cytoplasm. Thus, quin2 effectively lowers [Ca2+]i without impairing insulin action. It is concluded that insulin does not produce changes in [Ca2+]i and that chelating intracellular Ca2+ does not prevent stimulation of hexose uptake by insulin. These results suggest that it is unlikely that changes in [Ca2+]i may play a role in the transduction of information in insulin stimulation of glucose uptake in 3T3-L1 adipocytes.     

Metabolic responses of perfused rat livers to alpha- and beta-adrenergic agonists, glucagon and cyclic AMP.

1. The mechanism of action of glucagon and epinephrine was studied in perfused rat livers. Hormone-induced transitions from one metabolic steady state to another were followed in a non-recirculating perfusion system. Glucose and lactate production rates, oxygen uptake and K+ redistribution were measured. 2. Glucagon (3 nM), cyclic AMP (0.2 mM) and epinephrine (0.5 muM) had similar effects on K+ concentrations in the perfusate. Glycogenolysis responded more rapidly and O2 uptake was enhanced to a larger extent with epinephrine than with the other agents. alpha- and beta-receptor responses were differentiated by the use of phenylephrine (0.5 muM), isoproterenol (0.5 muM) and adrenergic blocking agents (phentolamine and beta-blocker Ro 3-4787 at 0.1 mM). 3. alpha-receptors mediated an activation of glucose production that was very rapid and was paralleled by a transient decrease of K+ concentrations in the effluent from the liver, lactate production rose gradually. Respiration was also enhanced, but fell again as lactate production increased. 4. beta-receptor stimulation was followed by an increase of glucose production that was less drastic and was paralleled by a K+ release, lactate production and respiration were only slightly enhanced. beta stimulation and glucagon both resulted in an inhibition of the alpha-adrenergic effect on lactate release and simultaneously increased O2 uptake. 5. We concluded that in perfused rat livers alpha- as well as beta-adrenergic receptor stimulation resulted in an activation of glycogenolysis, possibly by two different mechanisms.     

Effects of protein kinase C activation on the regulation of the stimulus-secretion coupling in pancreatic beta-cells.

Effects of protein kinase C (PKC) activation on the insulin-secretory process were investigated, by using beta-cell-rich suspensions obtained from pancreatic islets of obese-hyperglycaemic mice. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), which is known to activate PKC directly, the muscarinic-receptor agonist carbamoylcholine and high glucose concentration enhanced the phosphorylation of a specific 80 kDa PKC substrate in the beta-cells. At a non-stimulatory glucose concentration, 10 nM-TPA increased insulin release, although there were no changes in either the cytoplasmic free Ca2+ concentration ([Ca2+]i) or membrane potential, as measured with the fluorescent indicators quin-2 and bisoxonol respectively. At a stimulatory glucose concentration TPA caused a lowering in [Ca2+]i, whereas membrane potential was unaffected. Despite the decrease in [Ca2+]i, there was a large stimulation of insulin release. Addition of TPA lowered [Ca2+]i also in beta-cells stimulated by tolbutamide or high K+, although to a lesser extent than in those stimulated by glucose. There was no effect of TPA on either Ca2+ buffering or the ability of Ins(1,4,5)P3 to release Ca2+ in permeabilized beta-cells. However, the phorbol ester inhibited the rise in [Ca2+]i in response to carbamoylcholine, which stimulates the formation of InsP3, in intact beta-cells. Down-regulation of PKC influenced neither glucose-induced insulin release nor the increase in [Ca2+]i. Hence, although PKC activation is of no major importance in glucose-stimulated insulin release, this enzyme can serve as a modulator of the glucose-induced insulin-secretory response. Such a modulation involves mechanisms promoting both amplification of the secretory response and lowering of [Ca2+]i.     

Dual effect of glucose on cytoplasmic free Ca2+ concentration and insulin release reflects the beta-cell being deprived of fuel

Influence of basal glucose concentration on the response evoked by subsequent stimulation with the sugar, was evaluated by investigating changes in free cytoplasmic Ca2+ concentration, [Ca2+]i, and insulin release, using beta-cells isolated from obese hyperglycemic mice. When increasing the glucose concentration from 0 to either 11 or 20 mM, there was a transient decrease in both [Ca2+]i and insulin release. The decrease was followed by a pronounced increase in both of the parameters. When increasing the basal glucose concentration, the initial decrease gradually disappeared, being abolished already at 5 mM of the sugar and the subsequent increase appeared more rapidly. It is suggested that the observed decrease in [Ca2+]i and thereby insulin release reflects a phenomenon associated with fuel deprived beta-cells.     

Effects of In Vitro Anoxia and Low pH on Acetylcholine Release by Rat Brain Synaptosomes

Acetylcholine and choline release from rat brain synaptosomes have been measured using a chemiluminescent technique under a variety of conditions set up to mimic anoxic insult, including conditions of low pH (6.2) and the presence of lactate plus pyruvate as substrate. Lactate plus pyruvate as substrate consistently gave higher respiration rates than glucose alone, but with either substrate (glucose or lactate plus pyruvate) the omission of Ca2+ caused an increase in respiration whereas a low pH caused a decreased respiration. Acetylcholine release under control conditions (glucose, pH 7.4) was Ca2+-dependent, stimulated by high K+ concentrations, and decreased significantly during anoxia but recovered fully after a period of postanoxic oxygenation. Low pH (6.2) suppressed K+ stimulation of acetylcholine release, and after a period of anoxia at low pH the recovery of acetylcholine release was only partial. With lactate plus pyruvate as substrate, the effects of anoxia and/or low pH on acetylcholine release and its subsequent recovery were exacerbated. Choline release from synaptosomes, however, was not affected by anoxic/ionic conditions in the same way as acetylcholine release. At low pH (6.2) there was a marked reduction in choline release both under aerobic and anoxic conditions. These results suggest that acetylcholine release per se from the nerve is very sensitive to anoxic insult and that the low pH occurring during anoxia may be an important contributory factor.     

Glucose-stimulated efflux of indo-1 from pancreatic beta-cells is reduced by probenecid

Indo-1 loaded pancreatic beta-cells, isolated from obese hyperglycaemic mice, were studied with respect to cytoplasmic free Ca2+ concentration ([Ca2+]i), efflux of indicator and insulin release. In the absence of glucose there was a continuous efflux of indo-1 which increased upon stimulation with 20 mM of the sugar. The anion exchange inhibitor probenecid reduced both basal efflux of indo-1 and prevented that promoted by glucose. Measurements of [Ca2+]i and insulin release revealed similar results as previously reported with quin-2 and fura-2. Furthermore, probenecid did not influence the [Ca2+]i responses. It is thus possible to reduce efflux of indo-1 probenecid and thereby improve the measurements of [Ca2+]i in pancreatic beta-cells.     

On the Mechanism of Ouabain-Induced Release of Acetylcholine from Synaptosomes

Ouabain (5 x 10(-8)-5 x 10(-4) M) was confirmed to cause a dose-dependent increase in [3H]acetylcholine ([3H]ACh) release, cytosolic free Ca2+ concentration ([Ca2+]i), and 22Na+ uptake in cerebrocortical synaptosomes of rats in the presence of extracellular Ca2+. Ouabain also caused a dose-dependent decrease in membrane potential. In a low-Na+ (10 mM) medium, ouabain failed to increase [3H]ACh release and [Ca2+]i. Tetrodotoxin (10(-6) M) had no effect on the ouabain-induced increase in both [3H]ACh release and [Ca2+]i but abolished the increase in 22Na+ uptake and partially inhibited the depolarizing effect. Verapamil (10(-6)-5 x 10(-4) M) inhibited the ouabain-induced increase in both [3H]ACh release and [Ca2+]i in a dose-dependent manner. Removal of extracellular Ca2+ abolished the effect of ouabain on [Ca2+]i but not on [3H]ACh release and 22Na+ uptake, regardless of the presence or absence of EGTA. In the absence of extracellular Ca2+, 10 mM Mg2+ blocked ouabain-induced [3H]ACh release, which was resistant to verapamil. These results suggest that ouabain can increase ACh release from synaptosomes without the preceding increases in intracellular Ca2+ and/or Na+ content. It seems likely that the removal of extracellular Ca2+ unmasks mechanisms of ouabain action different from those operating in the presence of Ca2+.