ATP-sensitive K+ channel-dependent regulation of glucagon release and electrical activity by glucose in wild-type and SUR1-/- mouse alpha-cells

Patch-clamp recordings and glucagon release measurements were combined to determine the role of plasma membrane ATP-sensitive K+ channels (KATP channels) in the control of glucagon secretion from mouse pancreatic alpha-cells. In wild-type mouse islets, glucose produced a concentration-dependent (half-maximal inhibitory concentration [IC50]=2.5 mmol/l) reduction of glucagon release. Maximum inhibition (approximately 50%) was attained at glucose concentrations >5 mmol/l. The sulfonylureas tolbutamide (100 micromol/l) and glibenclamide (100 nmol/l) inhibited glucagon secretion to the same extent as a maximally inhibitory concentration of glucose. In mice lacking functional KATP channels (SUR1-/-), glucagon secretion in the absence of glucose was lower than that observed in wild-type islets and both glucose (0-20 mmol/l) and the sulfonylureas failed to inhibit glucagon secretion. Membrane potential recordings revealed that alpha-cells generate action potentials in the absence of glucose. Addition of glucose depolarized the alpha-cell by approximately 7 mV and reduced spike height by 30% Application of tolbutamide likewise depolarized the alpha-cell (approximately 17 mV) and reduced action potential amplitude (43%). Whereas insulin secretion increased monotonically with increasing external K+ concentrations (threshold 25 mmol/l), glucagon secretion was paradoxically suppressed at intermediate concentrations (5.6-15 mmol/l), and stimulation was first detectable at >25 mmol/l K+. In alpha-cells isolated from SUR1-/- mice, both tolbutamide and glucose failed to produce membrane depolarization. These effects correlated with the presence of a small (0.13 nS) sulfonylurea-sensitive conductance in wild-type but not in SUR1-/- alpha-cells. Recordings of the free cytoplasmic Ca2+ concentration ([Ca2+]i) revealed that, whereas glucose lowered [Ca2+]i to the same extent as application of tolbutamide, the Na+ channel blocker tetrodotoxin, or the Ca2+ channel blocker Co2+ in wild-type alpha-cells, the sugar was far less effective on [Ca2+]i in SUR1-/- alpha-cells. We conclude that the KATP channel is involved in the control of glucagon secretion by regulating the membrane potential in the alpha-cell in a way reminiscent of that previously documented in insulin-releasing beta-cells. However, because alpha-cells possess a different complement of voltage-gated ion channels involved in action potential generation than the beta-cell, moderate membrane depolarization in alpha-cells is associated with reduced rather than increased electrical activity and secretion.     

Palmitate stimulation of glucagon secretion in mouse pancreatic alpha-cells results from activation of L-type calcium channels and elevation of cytoplasmic calcium

We have investigated the short-term effects of the saturated free fatty acid (FFA) palmitate on pancreatic alpha-cells. Palmitate (0.5 or 1 mmol/l bound to fatty acid-free albumin) stimulated glucagon secretion from intact mouse islets 1.5- to 2-fold when added in the presence of 1-15 mmol/l glucose. Palmitate remained stimulatory in islets depolarized with 30 mmol/l extracellular K(+) or exposed to forskolin, but it did not remain stimulatory after treatment with isradipine or triacsin C. The stimulatory action of palmitate on secretion correlated with a 3.5-fold elevation of intracellular free Ca(2+) when applied in the presence of 15 mmol/l glucose, a 40% stimulation of exocytosis (measured as increases in cell capacitance), and a 25% increase in whole-cell Ca(2+) current. The latter effect was abolished by isradipine, suggesting that palmitate selectively modulates l-type Ca(2+) channels. The effect of palmitate on exocytosis was not mediated by palmitoyl-CoA, and intracellular application of this FFA metabolite decreased rather than enhanced Ca(2+)-induced exocytosis. The stimulatory effects of palmitate on glucagon secretion were paralleled by a approximately 50% inhibition of somatostatin release. We conclude that palmitate increases alpha-cell exocytosis principally by enhanced Ca(2+) entry via l-type Ca(2+) channels and, possibly, relief from paracrine inhibition by somatostatin released by neighboring delta-cells.     

An altered repolarizing potassium current in rat cardiac myocytes after subtotal nephrectomy

Renal failure in humans is associated with electrocardiographic changes including altered QT interval dispersion, which suggests that cardiac myocyte repolarization is abnormal and which appears to correlate with cardiac prognosis. In this study, cardiac myocyte repolarizing currents have been studied in isolated cells from rats 8 wk after subtotal nephrectomy (SNx), using sham-operated animals as controls. In addition, monophasic cardiac action potentials were recorded from the epicardial surface of the left ventricle (LV) apex, LV base, and the right ventricle of isolated perfused hearts paced at 320/min. SNx was associated with cardiac hypertrophy and histologic evidence of myocardial fibrosis, but SNx rats were not hypertensive. Repolarizing K(+) currents were measured using whole-cell patch-clamp, and 4-aminopyridine (4-AP)-sensitive transient outward (I(to)) and 4-AP-insensitive sustained outward (I(so)) components were quantified. After SNx, I(to) was increased by two to threefold at voltages from -30 to +60 mV and showed increased heterogeneity. For example, at 0 mV voltage clamp pulse, the median I(to) was increased from 3.23 pA/pF in control myocytes (interquartile range 3.20 pA/pF, n = 24) to 5.86 pA/pF in SNx myocytes (interquartile range 7.32 pA/pF, n = 21, P: < 0.005). The kinetics of inactivation of I(to) were altered after SNx with slowing both of the onset and the recovery from inactivation. The mean time constant of inactivation at +30 mV after SNx was 14.2 +/- 1.6 ms (n = 20) compared with control values of 9.8 +/- 0.6 ms (n = 23, P: < 0.05). Neither I(so) nor inward rectifier K(+) currents were altered after SNx. The action potential duration (APD(50)) at the left ventricular base was approximately 20% shorter (P: < 0.02) in hearts from SNx rats compared with controls. 4-AP (2 mM) prolonged the APD(50) in all regions in hearts from both SNx and control rats and abolished the APD(50) shortening in SNx. These results indicate that abnormalities of the cardiac transient outward K(+) current contribute to alterations in the cardiac action potential in renal failure and warrant further investigation because they may contribute to altered repolarization and arrythmogenesis.     

In vitro electrophysiologic effects of morphine in rabbit ventricular myocytes

BACKGROUND: Morphine is widely used in patients undergoing surgical operations and is also reported to mediate cardioprotection of preconditioning. The current study determined effects of morphine at therapeutic to pharmacologic concentrations on cardiac action potential, L-type Ca2+ current (ICa.L), delayed rectifier K+ current (IK), and inward rectifier K+ current (IK1) in isolated rabbit ventricular myocytes. METHODS: Ventricular myocytes were enzymatically isolated from rabbit hearts. Action potential and membrane currents were recorded in current and voltage clamp modes. RESULTS: Morphine at concentrations from 0.01 to 1 microM significantly prolonged cardiac action potential, and at 0.1 and 1 microM slightly but significantly hyperpolarized the resting membrane potential. In addition, morphine at 0.1 microM significantly augmented ICa.L (at +10 mV) from 5.9 +/- 1.9 to 7.3 +/- 1.7 pA/pF (by 23%; P < 0.05 vs. control) and increased IK1 (at -60 mV) from 2.8 +/- 1.0 to 3.5 +/- 0.9 pA/pF (by 27%; P < 0.05 vs. control). Five microM naltrindole (a selective delta-opioid receptor antagonist) or 5 microM norbinaltorphimine (a selective kappa-opioid receptor antagonist) prevented the increase in ICa.L induced by morphine, but 5 microM CTOP (a selective mu-opioid receptor antagonist) did not. The three types of opioid antagonists did not affect the augmentation of IK1 by morphine. Morphine had no effect on IK. CONCLUSIONS: These results indicate that morphine prolongs action potential duration by increasing ICa.L, an effect mediated by delta- and kappa-opioid receptors. It also hyperpolarizes cardiac resting membrane potential by increasing IK1, which is not mediated by opioid receptors.     

In Vitro Electrophysiologic Effects of Morphine in Rabbit Ventricular Myocytes

Background: Morphine is widely used in patients undergoing surgical operations and is also reported to mediate cardioprotection of preconditioning. The current study determined effects of morphine at therapeutic to pharmacologic concentrations on cardiac action potential, L-type Ca2+ current (ICa.L), delayed rectifier K+ current (IK), and inward rectifier K+ current (IK1) in isolated rabbit ventricular myocytes.

Methods: Ventricular myocytes were enzymatically isolated from rabbit hearts. Action potential and membrane currents were recorded in current and voltage clamp modes.

Results: Morphine at concentrations from 0.01 to 1 [mu]m significantly prolonged cardiac action potential, and at 0.1 and 1 [mu]m slightly but significantly hyperpolarized the resting membrane potential. In addition, morphine at 0.1 [mu]m significantly augmented ICa.L (at +10 mV) from 5.9 +/- 1.9 to 7.3 +/- 1.7 pA/pF (by 23%; P < 0.05 vs. control) and increased IK1 (at -60 mV) from 2.8 +/- 1.0 to 3.5 +/- 0.9 pA/pF (by 27%; P < 0.05 vs. control). Five [mu]m naltrindole (a selective [delta]-opioid receptor antagonist) or 5 [mu]m norbinaltorphimine (a selective [kappa]-opioid receptor antagonist) prevented the increase in ICa.L induced by morphine, but 5 [mu]m CTOP (a selective [mu]-opioid receptor antagonist) did not. The three types of opioid antagonists did not affect the augmentation of IK1 by morphine. Morphine had no effect on IK.     

Inward calcium currents in cultured and freshly isolated detrusor muscle cells: evidence of a T-type calcium current

PURPOSE: We carefully examined the possible routes of Ca2+ influx, and determined whether cultured cells retain Ca2+ channels and whether the culturing process changes their properties. MATERIALS AND METHODS: Inward currents were measured under voltage clamp in freshly isolated cells and myocytes from confluent cell cultures of detrusor smooth muscle. RESULTS: In guinea pig and human cells mean peak inward current density plus or minus standard deviation decreased significantly in cell culture (2.0 +/- 0.9 versus 4.5 +/- 2.2 pA.pF.(-1)) but there was no species variation. In primary cultured and passaged guinea pig cells an inward current was identified as L-type Ca2+ current. In freshly isolated cells another component to the inward current was identified that was insensitive to 20 micromol. l(-1) verapamil and 20 to 50 micromol. l(-1) cadmium chloride but abolished by 100 micromol. l(-1) nickel chloride and identified as T-type Ca2+ current. In addition, total inward current was greater at a holding potential of -100 than -40 mV., also indicating a component of current activated at negative voltage. Steady state activation and inactivation curves of the net inward current were also compatible with a single component in cultured cells but a dual component in freshly isolated cells. The action potential was completely abolished in cultured cells by L-type Ca2+ channel blockers but incompletely so in freshly isolated cells. Outward current depended strongly on previous inward current, suggesting a predominant Ca2+ dependent outward current. CONCLUSIONS: In freshly isolated guinea pig cells T and L-type Ca2+ current is present but T-type current is absent in confluent cultures.     

静止压力对血管平滑肌细胞容积调节性氯电流的影响

目的 观察不同静止压力对血管平滑肌细胞容积调节性氯电流(ICl,vol)的影响.方法 将胚胎鼠血管平滑肌细胞株A10细胞分别在正常大气压下、100、200 mm Hg(1 mm Hg=0.133 kPa)的静止压力下培养,流式细胞仪测试样品的细胞周期与凋亡率,膜片钳全细胞记录技术观察细胞ICl、vol的变化.结果 100 mm Hg静止压力下培养的A10细胞基础电流在+100 mV和-100 mV分别为(32.08±3.22)、(-12.50±1.34)pA/pF,低渗激活的电流在+100 mV和-100 mV分别为(77.91±8.38)、(-39.39±5.72)pA/pF,较对照组(20.49±2.30)、(-9.14±1.35)和(42.34±4.34)、(-24.02±2.71)pA/pF显著增强(P<0.05);当压力达200 mm Hg时,在+100 mV和-100 mV电流密度分别为(12.16±1.29)、(-5.80±0.87)pA/pF(Ib),(28.29±5.20)、(-14.33±2.49)pA/pF(Ihypo),较对照组显著减弱(P<0.05).结论 容积调节性氯通道在静止压力诱导细胞增殖与凋亡过程中发挥了重要作用.     

Glucose or insulin, but not zinc ions, inhibit glucagon secretion from mouse pancreatic alpha-cells

The mechanisms by which hypoglycemia stimulates glucagon release are still poorly understood. In particular, the relative importance of direct metabolic coupling versus paracrine regulation by beta-cell secretory products is unresolved. Here, we compare the responses to glucose of 1) alpha-cells within the intact mouse islet, 2) dissociated alpha-cells, and 3) clonal alphaTC1-9 cells. Free cytosolic concentrations of ATP ([ATP](c)) or Ca(2+) ([Ca(2+)](c)) were imaged using alpha-cell-targeted firefly luciferase or a green fluorescent protein-based Ca(2+) probe ("pericam"), respectively. Consistent with a direct effect of glucose on alpha-cell oxidative metabolism, an increase in glucose concentration (from 0 or 3 mmol/l to 20 mmol/l) increased [ATP](c) by 7-9% in alpha-cells within the intact islet and by approximately 4% in alphaTC1-9 cells. Moreover, glucose also dose-dependently decreased the frequency of [Ca(2+)](c) oscillations in both dissociated alpha-cells and alphaTC1-9 cells. Although the effects of glucose were mimicked by exogenous insulin, they were preserved when insulin signaling was blocked with wortmannin. Addition of ZnCl(2) slightly increased the frequency of [Ca(2+)](c) oscillations but failed to affect glucagon release from either islets or alphaTC1-9 cells under most conditions. We conclude that glucose and insulin, but not Zn(2+) ions, independently suppress glucagon secretion in the mouse.     

Effects of the anesthetic gases xenon, halothane, and isoflurane on calcium and potassium currents in human atrial cardiomyocytes

BACKGROUND: Negative inotropic and proarrhythmic side effects on the heart are well known for the volatile anesthetics halothane and isoflurane but not for the noble gas xenon. We investigated the effects of halothane, isoflurane, and xenon on calcium and potassium currents in human atrial myocytes to elucidate the cellular and molecular basis of their cardiac actions. METHODS: Atrial myocytes were prepared from the right auricles obtained from patients undergoing heart surgery. Ion currents were measured with the whole cell patch clamp technique during superfusion of the cells with solutions that contained halothane, isoflurane, or xenon at concentrations corresponding to their respective minimum alveolar concentration (MAC); gas concentrations were determined with the head space-gas chromatography/mass spectrometry/selected ion monitoring method. RESULTS: L-type calcium currents were significantly depressed by 31.9 +/- 4.1%, from -1.8 +/- 0.3 to -1.2 +/- 0.4 picoampere (pA)/picofarad (pF) (n = 4; P < 0.05) at 1 MAC halothane and by 21.7 +/- 9.2%, from -1.6 +/- 0.7 to -1.2 +/- 0.6 pA/pF (n = 7; P < 0.05) at 1 MAC isoflurane, but not affected by 70% xenon (1 MAC). Inwardly rectifying potassium currents were not influenced by any anesthetic. Halothane (1 MAC) significantly inhibited the transient as well as the sustained part of voltage-gated potassium outward currents, by 19.4 +/- 6.7%, from 6.7 +/- 2.1 to 5.4 +/- 1.6 pA/pF (n = 8; P < 0.05), and by 8.6 +/- 4.8%, from 5.5 +/- 1.7 to 5.0 +/- 1.5 pA/pF (n = 8; P < 0.05), respectively. Transient K+ outward currents were even more inhibited, by 25.8 +/- 4.8%, from 9.8 +/- 3.1 to 7.3 +/- 2.1 pA/pF (n = 5; P < 0.05) at 1 MAC isoflurane, whereas xenon evoked only a slight (albeit significant) inhibition, by 6.1 +/- 3.7%, from 8.2 +/- 6.0 to 7.7 +/- 5.8 pA/pF (n = 10; P < 0.05). Isoflurane and xenon did not affect sustained potassium currents. All effects of the anesthetics were fully reversible after washout. CONCLUSIONS: Halothane and isoflurane exhibited considerable inhibitory effects on voltage-gated cardiac Ca2+ and K+ currents important for the duration of action potentials and the repolarization. Xenon, in contrast, did not affect Ca2+ currents and only slightly inhibited transient K+ outward currents, in line with the almost absent cardiac side effects of the noble gas.     

新福林增强人心房肌细胞肿胀激活的氯离子电流

目的 通过全细胞膜片钳技术研究α1-肾上腺素受体激动剂新福林对人心房肌细胞肿胀激活的氯离子电流(ICl.swell)的调节作用.方法 术中取冠脉搭桥患者右心耳组织,酶解分离出心房肌细胞.全细胞膜片钳技术记录氯离子电流.等渗、低渗台氏液灌流;分别加入新福林、氯离子电流阻滞剂4,4'-二异硫氰基芪-2,2'-二磺酸(DIDS)、α1受体阻断剂哌唑嗪,观察电流变化.结果 低渗台氏液灌流心房肌细胞,可记录到ICl.swell.用含有100μmol/L新福林的低渗台氏液灌流,ICl.swell电流强度增加.在-90 mV,电流强度由(-1.11±0.55)pA/pF增加到(-1.56±0.69)pA/pF(P＜0.05,n=7);+40mV,由(3.24±2.04)pA/pF增加到(4.64±2.61)pA/pF(P＜0.01,n=7).这种效应为浓度依赖性,加入DIDS或者用等渗灌流液灌流,该效应被逆转.加入哌唑嗪1 μmol/L可以使新福林的量效关系曲线右移.结论 在人心房肌细胞新福林可以增强ICl.swell,这和在其他动物得到的结果不同.该现象是首次发现,表明新福林对ICl.swell的调节具有种属特异性.     

Characterization of the ion channel currents in single myocytes of the guinea pig prostate

PURPOSE: We characterized membrane ionic currents underlying the action potential in single myocytes freshly isolated from the stroma of the guinea pig prostate. MATERIAL AND METHODS: Whole cell and single channel currents were recorded in single stromal smooth muscle cells using standard patch clamp techniques. RESULTS:: A rapidly activating, nifedipine (1 microM) sensitive Ca current was recorded in CsCl (130 mM) filled myocytes at potentials positive to -50 mV This current was half maximally activated at -22 mV and half maximally inactivated at -53 mV. In KCl (130 mM) filled myocytes membrane depolarization evoked a complex set of K selective outward currents, consisting of a rapidly activating transient outward current (IKto) followed by a more slowly developing transient outward current (IP2), which decayed to a steady state current (ISS). Tetraethylammonium (1 mM), a blocker of large conductance, Ca activated K channels, substantially blocked IP2 and ISS. Initial IKto was half maximally activated at -5 mV, half maximally inactivated at -65 mV and blocked by 4-aminopyridine (IC50 0.8 mM). IP2 and ISS were decreased by ryanodine (10 microM) or cyclopiazonic acid (10 microM) and increased by caffeine (1 mM), suggesting that Ca release from internal stores participates in the activation of these large conductance, Ca activated K channel currents. CONCLUSIONS: We speculate that membrane currents characterized in stromal myocytes underlie the generation of simple action potentials triggered during the slow wave recorded in the intact guinea pig prostate and pharmacological manipulation of IKto and IP2 may well provide a selective avenue of modulating stromal excitability and muscle tone.     

Effects of ketamine on voltage-dependent Ca2+ current in single smooth muscle cells from rabbit portal vein

The effects of ketamine on membrane potentials and voltage-dependent Ca2+ current were studied in dispersed single smooth muscle cells from rabbit portal vein. The resting membrane potential (-56.2 +/- 1.5mV) was not affected by ketamine (10(-5)-10(-3)M). The duration and the amplitude of the action potential evoked by intracellular stimulation were significantly decreased by ketamine in the concentrations of 10(-4)M and 10(-3)M. Voltage-gated Ca2+ current in single smooth muscle cells was apparently decreased by ketamine at concentrations of between 10(-5)M to 10(-3)M. The activation threshold of Ca2+ current (approx. -30mV) was also decreased by ketamine. Therefore, these results suggest that inhibition of the contractile response by ketamine in vascular smooth muscle from rabbit portal vein may be attributable to the inhibition of the Ca2+ current.     

Potassium permeable channels in primary cultures of rabbit cortical collecting tubule.

Rabbit cortical collecting tubule (RCCT) primary cultures, were grown on permeable, collagen supports with 1.5 microM aldosterone. Single K+ permeable channels in principal cell apical membranes were examined. At applied patch pipette potential (Vapp) from -60 to +60 mV (cell interior with respect to pipette interior), outward currents (cell to pipette) with a unitary conductance of 8 to 10 pS were seen in cell-attached (N = 31) and excised inside-out (N = 15) patches. At resting membrane potential (Vapp = 0 mV), mean open probability (Po = 0.85 +/- 0.16) decreased by 50% with 0.75 mM luminal BaCl2 exposure. In cell-attached patches, a second type of outward current was seen only at extreme depolarization, Vapp greater than +80 mV (N = 9). Usually in the closed state (Po less than 0.0005) at no applied potential, Po for this 150 pS channel increased dramatically with depolarization and/or raising cytoplasmic Ca2+. With a calculated K+ equilibrium potential of -84 mV, excised patch reversal potentials were less than -50 mV for both the above channel types, indicating high selectivity for K+ over Na+. In cultures grown without aldosterone low conductance K+ channels were rarely observed, while mineralocorticoid status did not appear to affect high conductance K+ channel frequency. Finally, a 30 pS cation channel was found to be nonselective for K+ over Na+, and insensitive to voltage, intracellular Ca2+ or luminal Ba2+. We conclude that: 1) Principal cell apical membranes from aldosterone-stimulated, RCCT primary cultures contain (a) low conductance, Ba(2+)-inhibitable and (b) high conductance, Ca2+/voltage-dependent K+ channels; and c) nonselective cation channels. 2) The low conductance K+ channel may play an important physiologic role in native RCCT mineralocorticoid-controlled K+ secretion, while the latter two channels' functions are unknown, although similar channels have been suggested to play a role in cell volume regulation.     

Potentiation of GABA(A) currents after mechanical injury of cortical neurons

Numerous studies have implicated glutamate receptors, glutamate neurotoxicity, and hyperexcitation in the pathobiology of traumatic brain injury, yet much less is known about the effects of neurotrauma on inhibitory GABA channels of the brain. Using an in vitro cell injury model, we tested whether mild stretch injury altered the GABA(A) currents of cultured rat cortical neurons. The application of 1-100 microM GABA to single pyramidal neurons voltage clamped to -60 mV activated an inward current that reversed near 0 mV in solutions containing symmetrical [Cl-]. This current was inhibited by bicuculline, consistent with mediation by GABA(A) receptor channels. In injured neurons, 50 microM GABA elicited a peak current density of 41.2 +/- 2.6 pA/pF (n = 82), which was significantly larger than in uninjured control neurons, 20.2 +/- 1.7 pA/pF (n = 69, p < 0.01). The GABA(A) currents of injured neurons did not differ from those of control neurons in their sensitivity to GABA or their reversal potentials, suggesting that GABA current potentiation did not result from changes in the agonist affinity or ionic selectivity of the channels. GABA current potentiation was prevented by injuring neurons in the presence of the NMDA antagonist APV, or the CaMKII inhibitor KN93. These results thus suggest that NMDA receptor activation following neuronal injury may potentiate GABA(A) channels through the activation of CaMKII. The increase in GABA(A) receptor function observed following injury could potentially contribute to dysfunctional synaptic function and information processing as well as unconsciousness and coma following human brain trauma.     

Calcium currents in interstitial cells from the guinea-pig bladder

OBJECTIVE: To determine whether there are inward currents in interstitial cells (IC) isolated from the guinea-pig detrusor and if so, to characterise them using the patch-clamp technique and pharmacological agents. MATERIALS AND METHODS: Using the whole-cell patch-clamp technique, inward currents were studied in IC enzymatically isolated from the detrusor of the guinea-pig bladder. Currents were evoked by stepping positively from a holding potential of - 80 mV. RESULTS: Outward K+ currents were blocked by Cs+ internal solution to reveal inward currents, which activated at voltages more positive than - 50 mV, peaked at 0 mV, reversed near + 50 mV and were half-maximally activated at - 27 mV. The inward currents showed voltage-dependent inactivation and were half-maximally inactivated at - 36 mV. Fitting the activation and inactivation data with a Boltzmann function revealed a window current between - 40 mV and + 20 mV. The decay of the current evoked at 0 mV could be fitted with a single exponential with a mean time-constant of 88 ms. Replacing external Ca2+ with Ba2+ significantly increased this to 344 ms. The current amplitude was augmented by Ba2+, and by Bay K 8644. Inward currents were significantly reduced by 1 microm nifedipine, across the voltage range, but the blockade was more effective on the current evoked at 0 mV than that evoked by a step to - 20 mV, perhaps indicating voltage-dependence of the action of nifedipine or another component of inward current. Increasing the concentration of the drug to 10 microm caused no further significant reduction either at 0 mV or at -20 mV. However, in the presence of 1 microm nifedipine the latter current was significantly reduced by 100 microm Ni2+. Both currents were significantly reduced in Ca2+-free solution. CONCLUSIONS: IC from the guinea-pig detrusor possess inward currents with typical characteristics of L-type Ca2+ current. They also have a component of inward Ca2+ current, which was resistant to nifedipine, but sensitive to Ni2+. Further work is needed to characterise the latter conductance.     

Vesicular inhibitory amino acid transporter is present in glucagon-containing secretory granules in alphaTC6 cells,mouse clonal alpha-cells,and alpha-cells of islets of Langerhans

Islets of Langerhans contain gamma-aminobutyrate (GABA) and may use it as an intercellular transmitter. In beta-cells, GABA is stored in synaptic-like microvesicles and secreted through Ca(2+)-dependent exocytosis. Vesicular inhibitory amino acid transporter (VIAAT), which is responsible for the storage of GABA and glycine in neuronal synaptic vesicles, is believed to be responsible for the storage and secretion of GABA in beta-cells. However, a recent study by Chessler et al. indicated that VIAAT is expressed in the mantle region of islets. In the present study, we investigated the precise localization of VIAAT in rat islets of Langerhans and clonal islet cells and found that it is present in alpha-cells, a minor population of F-cells and alphaTC6 cells, and clonal alpha-cells but not in beta-cells, delta-cells, or MIN6 m9-cells (clonal beta-cells). Combined biochemical, immunohistochemical, and electronmicroscopical evidence indicated that VIAAT is specifically localized with glucagon-containing secretory granules in alpha-cells. ATP-dependent uptake of radiolabeled GABA, which is energetically coupled with a vacuolar proton pump, was detected in digitonin-permeabilized alphaTC6 cells as well as in MIN6 m9 cells. These results demonstrate that functional neuronal VIAAT is present in glucagon-containing secretory granules in alpha-cells and suggest that the ATP-dependent GABA transporter in beta-cells is at least immunologically distinct from VIAAT. Because glucagon-containing secretory granules also contain vesicular glutamate transporter and store L-glutamate, as demonstrated by Hayashi et al., the present results suggest more complex features of the GABAergic phenotype of islets than previously supposed.     

The effects of volatile anesthetics on L- and T-type calcium channel currents in canine cardiac Purkinje cells.

The effects of halothane (0.45 and 0.9 mM, equivalent to 0.7 and 1.5%, respectively), isoflurane (0.54 and 1.23 mM, equivalent to 0.9 and 2.0%, respectively) and enflurane (0.65 and 1.48 mM, equivalent to 1.2 and 2.5%, respectively) on macroscopic L- and T-type Ca2+ channel currents were compared in single canine cardiac Purkinje cells using the whole-cell voltage-clamp technique. Cells were dialyzed with pipette solution containing CsCl and superfused with an external solution containing 10 mM BaCl2 and tetraethylammonium chloride. The long-lasting (L) and transient (T)-type Ca2+ channel currents were measured by depolarizing the membrane from different holding potentials (HPs). Voltage steps from an HP of either -80 or -70 mV elicited a low threshold, rapidly inactivating inward current at -40 to -30 mV, which maximally activated at -14 +/- 0.9 mV. This current was reduced by Ni2+ (100 microM) but not by nifedipine (1 microM), therefore resembling T-type Ca2+ channel current. In contrast, depolarizing steps from an HP of -40 mV elicited a sustained inward current that maximally activated at +4.1 +/- 0.8 mV and was nifedipine-sensitive, showing the characteristics of an L-type Ca2+ channel current. Halothane, isoflurane, and enflurane produced a concentration-dependent suppression of total Ca2+ channel current in every cell studied. Separation of Ca2+ channel types showed that both L- and T-type Ca2+ channel currents were depressed to a similar extent by anesthetic administration. These agents reduced peak L- and T-type current elicited at each pulse potential but did not shift the current-voltage (I-V) relationship for either T- or L-type current activation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulus-secretion coupling in beta-cells of transplantable human islets of Langerhans. Evidence for a critical role for Ca2+ entry.

With human islets isolated for transplantation, we examined the applicability to humans of a metabolic fuel hypothesis of glucose transduction and a Ca2+ hypothesis of depolarization-secretion coupling, both previously proposed for rodent islet beta-cells. We report that several features of human beta-cell physiology are well accounted for by these hypotheses. With whole-islet perifusion, we demonstrated that insulin secretion induced by glucose, tolbutamide, or elevated K+ is dependent on extracellular Ca2+. Insulin release induced by these secretagogues is enhanced by the dihydropyridine Ca2+ channel agonist BAYk8644 and depressed by the dihydropyridine Ca(2+)-channel antagonist nifedipine. All of the aforementioned secretagogues provoke increases in cytosolic free Ca2+, which are dependent on extracellular Ca2+ and are altered by the dihydropyridine drugs. Individual beta-cells in the islet display diminished resting membrane conductance, graded depolarization, and complex electrical patterns, including bursts of action potentials in response to stimulatory concentrations of glucose or tolbutamide. Individual islet beta-cells display voltage-dependent Ca2+ currents that are activated at membrane potentials traversed during the excursion of the action potential. In most cells, the Ca2+ currents are enhanced by BAYk8644 and depressed by nifedipine at concentrations that have parallel effects on secretagogue-induced increases in cytosolic Ca2+ and insulin secretion. These survey studies should provide the basis for more detailed investigations of the relationship of voltage-dependent ionic currents to electrical activity patterns and of electrical activity patterns to granule exocytosis in single human beta-cells.     

Glucose induces opposite intracellular Ca2+ concentration oscillatory patterns in identified alpha- and beta-cells within intact human islets of Langerhans

Homeostasis of blood glucose is mainly regulated by the coordinated secretion of glucagon and insulin from alpha- and beta-cells within the islets of Langerhans. The release of both hormones is Ca(2+) dependent. In the current study, we used confocal microscopy and immunocytochemistry to unequivocally characterize the glucose-induced Ca(2+) signals in alpha- and beta-cells within intact human islets. Extracellular glucose stimulation induced an opposite response in these two cell types. Although the intracellular Ca(2+) concentration ([Ca(2+)](i)) in beta-cells remained stable at low glucose concentrations, alpha-cells exhibited an oscillatory [Ca(2+)](i) response. Conversely, the elevation of extracellular glucose elicited an oscillatory [Ca(2+)](i) pattern in beta-cells but inhibited low-glucose-induced [Ca(2+)](i) signals in alpha-cells. These Ca(2+) signals were synchronic among beta-cells grouped in clusters within the islet, although they were not coordinated among the whole beta-cell population. The response of alpha-cells was totally asynchronic. Therefore, both the alpha- and beta-cell populations within human islets did not work as a syncitium in response to glucose. A deeper knowledge of alpha- and beta-cell behavior within intact human islets is important to better understand the physiology of the human endocrine pancreas and may be useful to select high-quality islets for transplantation.