Calcium currents in GH3 cultured pituitary cells under whole-cell voltage-clamp: inhibition by voltage-dependent potassium currents.

To isolate inward Ca2+ currents in GH3 rat pituitary cells, an inward Na+ current as well as two outward K+ currents, a transient voltage-dependent current (IKV) and a slowly rising Ca2+-activated current (IKCa), must be suppressed. Blockage of these outward currents, usually achieved by replacement of intracellular K+ with Cs+, reveals sustained inward currents. Selective blockage of either K+ current can be accomplished in the presence of intracellular K+ by use of quaternary ammonium ions. When IKCa and Na+ currents are blocked, the net current elicited by stepping the membrane potential (Vm) from -60 to 0 mV is inward first, becomes outward and peaks in 10-30 msec, and finally becomes inward again. Under this condition, in which both IKV and Ca2+ currents should be present throughout the duration of the voltage step, the Ca2+ current was not detected at the time of peak outward current. That is, plots of peak outward current vs. Vm are monotonic and are not modified by nisoldipine or low external Ca2+ as would be expected if Ca2+ currents were present. However, similar plots at times other than at peak current are not monotonic and are altered by nisoldipine or low Ca2+ (i.e., inward currents decrease and plots become monotonic). When K+ channels are first inactivated by holding Vm at -30 mV, a sustained Ca2+ current is always observed upon stepping Vm to 0 mV. Furthermore, substitution of Ba2+ for Ca2+ causes blockage of IKV and inhibition of this current results in inward Ba2+ currents with square wave kinetics. These data indicate that the Ca2+ current is completely inhibited at peak outward IKV and that Ca2+ conductance is progressively disinhibited as the transient K+ current declines due to channel inactivation. This suggests that in GH3 cells Ca2+ channels are regulated by IKV.     

Differential inhibition of potassium currents in rat ventricular myocytes by capsaicin.

OBJECTIVE: Capsaicin is a pungent irritant present in peppers of the Capsicum family. Its major target of action is believed to be sensory neurones. Capsaicin has also been shown to prolong cardiac action potential in atrial muscle, perhaps by local release of calcitonin gene related peptide which in turn enhances inward calcium currents. However, capsaicin has been shown to inhibit K+ current in neurones. Since such an action could contribute to action potential prolonging activity of capsaicin in heart, the aim of the study was to examine the effects of capsaicin on cardiac K+ currents. METHODS: Ionic currents and action potentials were examined in isolated adult rat ventricular myocytes using the whole cell variant of the patch clamp technique at 25 degrees C. RESULTS: Capsaicin (10 microM) increased the action potential duration (APD50) from 45 ms to 166 ms. This effect was associated with an inhibition of three distinct K+ currents. The decreasing rank order of potency was: transient outward K+ current (ITO, IC50 = 6.4 microM), a voltage dependent non-inactivating outward current (IK, IC = 11.5 microM), and the inward rectifier K+ current (IK1, IC50 = 46.9 microM). Capsaicin induced block of ITO was characterised by a decrease in the peak current amplitude and an increase in the rate of inactivation. The inactivation of ITO in the absence of capsaicin was well described by a single exponential [tau = 77 (SEM 2) ms at +40 mV, n = 10]. However, in the presence of 10 microM capsaicin inactivation was best described by the sum of two exponentials [tau FAST = 4.4(0.5) ms; tau SLOW = 92.4(3.0) ms, n = 10] with the fast component contributing 46(2)% of the total decay. A small but consistent hyperpolarising shift (approximately 3 mV) in the steady state voltage dependence of inactivation of ITO was induced by 10 microM capsaicin. Capsaicin had no effect on the rate of ITO recovery from inactivation (tau = 49 ms and 48 ms for control and drug respectively). The capsaicin analogue, resiniferatoxin, which as an irritant is up to 10(4)-fold more potent than capsaicin, had no effect on any of the K+ currents when present at concentrations of up to 10 microM. In contrast another capsaicin analogue, zingerone (30 microM) blocked ITO by 52(12)% and IK by 35%. CONCLUSIONS: Capsaicin produces a prolongation of the rat ventricular action potential, an effect which is associated with inhibition of potassium currents.     

Ethanol inhibition of insulin secretion by perifused rat islets

In vivo and in vitro effects of ethanol on the kinetics of insulin secretion in response to glucose and tolbutamide were studied in perifused rat islets. Phases I and II insulin response to 16.7 mM glucose was decreased 46% and 48%, respectively, in islets of rats given ethanol intragastrically 1 g/kg 1 h prior to sacrifice. Mean blood ethanol levels at the time of animal sacrifice were 19.4 mmol/l. The magnitude of insulin suppression was not significantly enhanced with higher ethanol doses, 2 or 3 g/kg, although mean blood ethanol levels increased to 25.9 and 60.3 mmol/l, respectively. Similarly, significant inhibition of both phases of insulin response to glucose occurred when ethanol 1 or 3 g/kg was given intraperitoneally instead of orally. Ethanol had no effect on insulin secretion when given orally 4 h instead of 1 h prior to islet isolation. Ethanol, 65 mmol/l, added directly to rat islets perifusate simultaneously with 16.7 mM glucose decreased both phases I and II insulin response nearly half; whereas addition of 21.7 instead of 65 mmol/l ethanol had no effect. Pre-treatment of islets with 21.7 or 65 mmol/l ethanol during 30 min basal islets perifusion period had no effect on subsequent insulin response to 16.7 mM glucose. Insulin response to 10 mM tolbutamide was decreased nearly 81% by the simultaneous presence of 65 mmol/l ethanol in islets perifusate.     

Calcium currents and fura-2 signals in fluorescence-activated cell sorted lactotrophs and somatotrophs of rat anterior pituitary

Optical and electrical recording techniques were applied to single primary pituitary cells to characterize the types of voltage-dependent calcium currents (ICa) and levels of intracellular calcium ([Ca2+]i). GH-containing somatotrophs and PRL-containing lactotrophs were isolated from adult female rats using fluorescence-activated cell-sorting techniques and were maintained in culture for 1-4 days. Whole cell patch-clamp recordings were made to analyze the ICa, and [Ca2+]i was measured with fura-2. Cell type was verified after each recording by indirect immunocytochemistry. GH and PRL cells could be divided into two groups: silent and spontaneously active. Silent cells had stable membrane potentials and stable levels of [Ca2+]i. Spontaneously active cells exhibited spontaneous action potentials and large fluctuations in [Ca2+]i. Two types of ICa were found: a low threshold, transient current which was insensitive to the dihydropyridine -Bay 5417 (the negative isomer of Bay K 8644), and a high threshold, sustained current which was enhanced by -Bay 5417. Both types of ICa were present in PRL and GH cells, but each cell type differed quantitatively in the proportion of each current type. While the GH cells had a more prominent, low threshold, transient ICa, the PRL cells had a more prominent, high threshold, sustained ICa. The enhancement of ICa by -Bay 5417 was greater in the PRL cells, which have a larger dihydropyridine-sensitive ICa. Parallel fura-2 measurements showed an increase in [Ca2+]i in response to 50 mM KCl and -Bay 5417 for both lactotrophs and somatotrophs.     

Calcium antagonists and insulin resistance

Calcium antagonists have become available for the treatment of hypertension and ischemic heart diseases. Large clinical trials have demonstrated that calcium antagonists prevent cardiovascular mortality and morbidity. Epidemiologic studies have shown insulin resistance is an independent predict factor for cardiovascular outcome. Cardiovascular diseases, including hypertension, diabetes mellitus, and hyperlipidemia are associated with insulin resistance. Insulin resistance is an initial step in atherosclerosis, leading to increase in the incidences of cardiovascular outcome. From a clinical perspective, it is important to select an appropriate intervention which is effective in improving insulin resistance in cardiovascular diseases. In this review, we would like to focus on the effects of calcium antagonists on insulin resistance (insulin sensitivity).     

Polyamines in rat adipocytes: their localization and their effects on the insulin receptor binding

Effects of polyamines on the insulin binding in isolated rat adipocytes were studied. In addition, the concentration of polyamines in adipose tissue was determined, and their localization revealed by fluorescence cytochemistry and immunocytochemistry. Spermine (0.1-10 mM) dose-dependently enhanced the insulin receptor binding; at a concentration of 5-10 mM spermine the insulin binding was enhanced by 100% above control values. Spermidine had a weaker effect than spermine whereas putrescine had no effect on insulin binding. Competition curves with unlabelled insulin indicated that spermine increased the insulin binding capacity without significantly affecting the binding affinity. Fluorescence and immunocytochemistry on adipose tissue localized the polyamines spermidine and/or spermine almost exclusively to the thin layer of adipocyte cytoplasm surrounding the lipid droplet. In addition, chemical analysis for polyamines in the adipocyte medium and in the cells indicated that a differential release of polyamines may have occurred from the cells. The possibility that polyamines may act as intracellular or intercellular (autocrine) regulators, modulating insulin binding, is discussed.     

Somatostatin-induced inhibition of insulin secretion by isolated pancreatic rat islets prepared by micro-dissection or collagenase digestion.

The effect of somatostatin on insulin secretion in response to a variety of stimuli was investigated in micro-dissected as well as collagenase isolated pancreatic rat islets. Somatostatin inhibited insulin secretion in both islet preparations in the presence of different initiators, but failed to affect the hormone output induced by theophylline, 1-methyl-3-isobutylxanthin (IBMX) or p-chloromercuriphenylsulfonic acid (CMBS). The inhibitory action was associated with decreased glucose metabolism by islets (measured as conversion of U-14C-glucose to 14CO2).     

Suppression by insulin treatment of glucose-induced inhibition of insulin release in non-insulin-dependent diabetics

Although restoration of normoglycemia in non-insulin-dependent diabetic subjects improves insulin release evoked by several secretagogues, conflicting data were reported concerning the effect of intensive insulin therapy on the first-phase response of the B-cell to an intravenous glucose challenge. In the present study, 14 non-insulin-dependent diabetics underwent an intravenous glucose test performed before and after 20 h of glycemic normalization. Before insulin treatment, glucose failed, as a rule, to provoke an early positive secretory response. On the contrary, a paradoxical inhibition of insulin release was observed in most patients. This phenomenon was reproducible when a second test was performed 120 min after the first one. The paradoxical inhibition was not observed any more after glycemic normalization. As judged from the paired difference (delta) between the early increment in insulin release before and after insulin treatment, normoglycemia resulted in an improved secretory response (delta greater than 5.0 microU/ml) in seven patients, whilst the first-phase response remained little affected (delta less than 3.0 microU/ml) in the other seven subjects. These findings suggest that an impaired first-phase response to glucose does not always represent an irreversible primary defect of the pancreatic B-cell in diabetic subjects.     

Time-dependent inhibition of insulin release: glucose-arginine interactions in the perfused rat pancreas

Summary The isolated perfused rat pancreas was stimulated sequentially with arginine or glucose to analyze the time-dependent modulation of insulin release. A 10-min perfusion with arginine (5.0 mmol/l) induced 75% inhibition of the insulin response to repeated arginine stimulation 10 min later. When glucose (8.3 mmol/l) was given as two pulses, inhibition of the second insulin response was less pronounced. The inhibitory effect generated by arginine also suppressed the insulin response to glucose (27.7 mmol/l), and this inhibitory effect persisted for over 80 min. Stimulation for 30 min with glucose (27.7 mmol/l) strongly potentiated the insulin responses to a pair of arginine stimuli given 20 min later. However, despite augmented secretion rates, the insulin response to the second arginine pulse was still inhibited by 75%. When insulin secretion was strongly amplified by two 10 min pulses of the synergistic mixture of arginine (5.0 mmol/l) and glucose (8.3 mmol/l), there was no inhibition of the second insulin response. If glucose (8.3 mmol/l) was present during the first arginine stimulation only, the response to the second arginine pulse was inhibited as in control experiments. However, when glucose was added to the second arginine pulse only, the inhibition generated by the first arginine pulse did not express itself, insulin release remaining similar to control. We conclude that: (1) short stimulations of the pancreas by arginine or glucose generate long-lasting inhibition of the insulin response to subsequent stimulations; (2) synergistic amplification of the insulin response by addition of glucose to arginine obliterates the inhibition; (3) glucose does not suppress the induction of inhibition, it blocks the expression of the inhibitory signal on insulin secretion; (4) these in vitro findings are in keeping with observations in normal and hyperglycaemic man.     

Calcium currents and arrhythmias: insights from molecular biology

Calcium channels are critical to normal cardiac function. They are involved in the generation and conduction of the action potential and in contraction. Three surface membrane channels have been identified. The L-type Ca channel is most abundant and is responsible for Ca entry into the cell that triggers contraction. T-type Ca channels are most prevalent in the conduction system and are probably involved in automaticity. A newly described TTX-sensitive calcium current may be important in "boosting" or enhancing conduction and contraction. The main intracellular Ca channel resides in the sarcoplasmic reticulum and is responsible for the release of the Ca that activates contraction. Oscillatory behavior of this channel influences the sarcolemmal membrane, causing delayed aftercontractions and arrhythmias such as those seen in digoxin toxicity. The on-going molecular characterization of these channels will enhance our knowledge of their normal function and dysfunction in disease states, leading to the development of new therapeutic agents to treat arrhythmias and contractile dysfunction.     

Pyridine nucleotides in pancreatic islets during inhibition of insulin release by exogenous insulin.

In vitro addition of rat insulin (200, 400 or 800 muU/ml) to collagenase-isolated pancreatic islets of adult rats diminished glucose (3 mg/ml)-induced insulin release which was correlated with a decrease of the ratio of total NADPH/NADP and inhibition of glucose oxidation via the pentose phosphate shunt (PPS). NADH and NAD levels were not affected. It is suggested that exogenous insulin diminishes the islet total NADPH/NADP ratio by a direct or indirect decrease in PPS activity. However, it is also conceivable that insulin decreases this ratio through another mechanism than PPS. It is possible that inhibition of insulin secretion by exogenous insulin is at least in part due to the decrease of the NADPH/NADP ratio.     

Proportional inhibition of glucose-induced insulin release by somatostatin

To test the hypothesis that somatostatin in a proportional inhibitor of glucose-induced insulin release, we examined the effect of somatostatin on the release of insulin stimulated either by endogenous signals (basal), by glucose infusion, or by glucose injection. Somatostatin infusions (1.7 μg/min × 30 min) produced a decrement of basal insulin output from the right lobe of the canine pancreas that was proportional to the initial rate of basal insulin secretion (r = −0.87, p < 0.001, n = 16) Glucose infusions of 1–6 mg/kg/min produced much higher rates of insulin output; again, the decrement of insulin output produced by somatostatin correlated with the initial rate of insulin secretion (r = −0.68, p < 0.01, n = 16). Rapid intravenous injection of either 2 or 20 g of glucose produced a wide range of acute insulin responses (AIR). Somatostatin produced a decrement that was proportional to the original magnitude of the AIR (r = −0.70, p < 0.005, n = 16). Thus, the absolute amount of inhibition produced by somatostatin increases, not decreases, with the magnitude of the stimulus. These data suggest that the inhibitory effect of somatostatin cannot be overcome by increasing the size of glucose stimulus. Thus, exogenous somatostatin, and presumably endogenous somatostatin as well, will produce an inhibitory effect at any physiologic level of glucose stimulation that the β-cell receives.     

Dose-dependent inhibition by ghrelin of insulin secretion in the mouse

Ghrelin is produced by stomach oxyntic cells and thought to be involved in the regulation of body weight and food intake. We demonstrate here that the peptide inhibits insulin secretion from overnight-incubated mouse islets in the presence of 8.3, 11.1, and 22.2 mmol/liter glucose. Ghrelin was most efficient at 1 nmol/liter and its effect disappeared by raising the dose more than 25 nmol/liter. Also, insulin secretion in the presence of high K(+) concentrations (20 mmol/liter) was inhibited by ghrelin. Furthermore, when administered iv to mice together with glucose (1 g/kg), ghrelin (50 nmol/kg) inhibited both the rapid 1-min insulin response (364 +/- 90 vs. 985 +/- 114 pmol/liter in controls, P < 0.001) and the area under the 50 min curve of insulin concentration (12.6 +/- 1.2 vs. 15.6 +/- 1.2 nmol/liter x 50 min; P = 0.046) without affecting the glucose disposal rate, insulin sensitivity or glucose effectiveness, i.e. glucose disposal independent from any dynamic change in insulin. The insulinostatic effect of ghrelin was inversely related to insulin sensitivity. In contrast, ghrelin had no influence at the lower dose of 5 nmol/kg and only slightly inhibited insulin secretion at the higher dose of 150 nmol/kg. These findings therefore show that ghrelin inhibits glucose-stimulated insulin secretion in the mouse. The effect is dependent on the dose and elicited on distal signaling steps in islet cells. The results suggest that the islet beta-cells are targets for ghrelin.     

Elevation of plasma tryptophan by insulin in rat

The concentration of tryptophan in rat plasma increases by 30–40% 2 hr after fasting animals receive insulin (2 U/kg) or consume a carbohydrate meal. These treatments decrease the concentrations of all other amino acids examined: Insulin administration also lowers plasma glucose levels. Large doses of glucagon (1 mg/kg) reduce plasma tryptophan concentrations. Lower doses are without a significant effect. The sources of plasma tryptophan responsible for its increase after insulin administration have not been identified.     

Monomethylated-adenines potentiate glucose-induced insulin production and secretion via inhibition of phosphodiesterase activity in rat pancreatic islets

Monomethyladenines have effects on DNA repair, G-protein-coupled receptor antagonism and autophagy. In islet ß-cells, 3-methyladenine (3-MA) has been implicated in DNA-repair and autophagy, but its mechanism of action is unclear. Here, the effect of monomethylated adenines was examined in rat islets. 3-MA, N6-methyladenine (N6-MA) and 9-methyladenine (9-MA), but not 1- or 7-monomethylated adenines, specifically potentiated glucose-induced insulin secretion (3-4 fold; p ≤ 0.05) and proinsulin biosynthesis (∼2-fold; p ≤ 0.05). Using 3-MA as a ‘model’ monomethyladenine, it was found that 3-MA augmented [cAMP]_i accumulation (2-3 fold; p ≤ 0.05) in islets within 5 minutes. The 3-, N6- and 9-MA also enhanced glucose-induced phosphorylation of the cAMP/protein kinase-A (PKA) substrate cAMP-response element binding protein (CREB). Treatment of islets with pertussis or cholera toxin indicated 3-MA mediated elevation of [cAMP]_i was not mediated via G-protein-coupled receptors. Also, 3-MA did not compete with 9-cyclopentyladenine (9-CPA) for adenylate cyclase inhibition, but did for the pan-inhibitor of phosphodiesterase (PDE), 3-isobutyl-1-methylxanthine (IBMX). Competitive inhibition experiments with PDE-isoform specific inhibitors suggested 3-MA to have a preference for PDE4 in islet ß-cells, but this was likely reflective of PDE4 being the most abundant PDE isoform in ß-cells. In vitro enzyme assays indicated that 3-, N6- and 9-MA were capable of inhibiting most PDE isoforms found in ß-cells. Thus, in addition to known inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3′K)/m Target of Rapamycin (mTOR) signaling, 3-MA also acts as a pan-phosphodiesterase inhibitor in pancreatic ß-cells to elevate [cAMP]_i and then potentiate glucose-induced insulin secretion and production in parallel.     

Monomethylated-adenines potentiate glucose-induced insulin production and secretion via inhibition of phosphodiesterase activity in rat pancreatic islets

Monomethyladenines have effects on DNA repair, G-protein-coupled receptor antagonism and autophagy. In islet ß-cells, 3-methyladenine (3-MA) has been implicated in DNA-repair and autophagy, but its mechanism of action is unclear. Here, the effect of monomethylated adenines was examined in rat islets. 3-MA, N6-methyladenine (N6-MA) and 9-methyladenine (9-MA), but not 1- or 7-monomethylated adenines, specifically potentiated glucose-induced insulin secretion (3-4 fold; p ≤ 0.05) and proinsulin biosynthesis (～2-fold; p ≤ 0.05). Using 3-MA as a ‘model’ monomethyladenine, it was found that 3-MA augmented [cAMP]_i accumulation (2-3 fold; p ≤ 0.05) in islets within 5 minutes. The 3-, N6- and 9-MA also enhanced glucose-induced phosphorylation of the cAMP/protein kinase-A (PKA) substrate cAMP-response element binding protein (CREB). Treatment of islets with pertussis or cholera toxin indicated 3-MA mediated elevation of [cAMP]_i was not mediated via G-protein-coupled receptors. Also, 3-MA did not compete with 9-cyclopentyladenine (9-CPA) for adenylate cyclase inhibition, but did for the pan-inhibitor of phosphodiesterase (PDE), 3-isobutyl-1-methylxanthine (IBMX). Competitive inhibition experiments with PDE-isoform specific inhibitors suggested 3-MA to have a preference for PDE4 in islet ß-cells, but this was likely reflective of PDE4 being the most abundant PDE isoform in ß-cells. In vitro enzyme assays indicated that 3-, N6- and 9-MA were capable of inhibiting most PDE isoforms found in ß-cells. Thus, in addition to known inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3′K)/m Target of Rapamycin (mTOR) signaling, 3-MA also acts as a pan-phosphodiesterase inhibitor in pancreatic ß-cells to elevate [cAMP]_i and then potentiate glucose-induced insulin secretion and production in parallel.     

Inhibition of K+ currents by homocysteine in rat ventricular myocytes

INTRODUCTION: Clinical evidence suggests that increased blood levels of homocysteine may be an independent risk factor for the development of cardiovascular disease, but the functional effects of this sulfhydryl amino acid on the myocardium are poorly understood. The present study was conducted to determine the direct effects of homocysteine on the electrophysiologic properties of the heart. METHODS AND RESULTS: Whole-cell voltage-clamp recordings were made in ventricular myocytes isolated from normal rat hearts to analyze the Ca2+-independent, transient outward K+ current (I(to)), a major repolarizing current in these cells. Maximum I(to) density (measured at +60 mV) was decreased approximately 47% from baseline in the presence of 500 microM homocysteine (P < 0.05), but the amount of block varied in a frequency- and voltage-dependent manner. Decreased I(to) density was not accompanied by significant changes in voltage- or time-dependent properties of the current, nor was it affected by pretreating myocytes with the protein kinase inhibitor staurosporine. Because a portion of total extracellular homocysteine is oxidized, we examined the response to homocystine, the oxidized form of homocysteine. In myocytes superfused with 500 microM homocystine, maximum I(to) density was decreased by approximately 40% from baseline (P < 0.05). In contrast, the thiolactone form of homocysteine did not alter I(to) amplitude. CONCLUSION: These data suggest that homocysteine and its oxidized form homocystine acutely inhibit I(to) channels in ventricular myocytes by mechanisms involving the free thiol or disulfide moieties of these compounds. High homocysteine or homocystine levels may contribute to abnormal repolarization and arrhythmogenic conditions in the intact heart.