Sven Göpel

Fast exocytosis with few Ca(2+) channels in insulin-secreting mouse pancreatic B cells

The association of L-type Ca(2+) channels to the secretory granules and its functional significance to secretion was investigated in mouse pancreatic B cells. Nonstationary fluctuation analysis showed that the B cell is equipped with <500 alpha1(C) L-type Ca(2+) channels, corresponding to a Ca(2+) channel density of 0.9 channels per microm(2). Analysis of the kinetics of exocytosis during voltage-clamp depolarizations revealed an early component that reached a peak rate of 1.1 pFs(-1) (approximately 650 granules/s) 25 ms after onset of the pulse and is completed within approximately 100 ms. This component represents a subset of approximately 60 granules situated in the immediate vicinity of the L-type Ca(2+) channels, corresponding to approximately 10% of the readily releasable pool of granules. Experiments involving photorelease of caged Ca(2+) revealed that the rate of exocytosis was half-maximal at a cytoplasmic Ca(2+) concentration of 17 microM, and concentrations >25 microM are required to attain the rate of exocytosis observed during voltage-clamp depolarizations. The rapid component of exocytosis was not affected by inclusion of millimolar concentrations of the Ca(2+) buffer EGTA but abolished by addition of exogenous L(C753-893), the 140 amino acids of the cytoplasmic loop connecting the 2(nd) and 3(rd) transmembrane region of the alpha1(C) L-type Ca(2+) channel, which has been proposed to tether the Ca(2+) channels to the secretory granules. In keeping with the idea that secretion is determined by Ca(2+) influx through individual Ca(2+) channels, exocytosis triggered by brief (15 ms) depolarizations was enhanced 2.5-fold by the Ca(2+) channel agonist BayK8644 and 3.5-fold by elevating extracellular Ca(2+) from 2.6 to 10 mM. Recordings of single Ca(2+) channel activity revealed that patches predominantly contained no channels or many active channels. We propose that several Ca(2+) channels associate with a single granule thus forming a functional unit. This arrangement is important in a cell with few Ca(2+) channels as it ensures maximum usage of the Ca(2+) entering the cell while minimizing the influence of stochastic variations of the Ca(2+) channel activity.

Regulation of glucagon release in mouse -cells by KATP channels and inactivation of TTX-sensitive Na+ channels.

1 The perforated patch whole‐cell configuration of the patch‐clamp technique was applied to superficial glucagon‐secreting α‐cells in intact mouse pancreatic islets. 2 α‐cells were distinguished from the β‐ and δ‐cells by the presence of a large TTX‐blockable Na+ current, a TEA‐resistant transient K+ current sensitive to 4‐AP (A‐current) and the presence of two kinetically separable Ca2+ current components corresponding to low‐ (T‐type) and high‐threshold (L‐type) Ca2+ channels. 3 The T‐type Ca2+, Na+ and A‐currents were subject to steady‐state voltage‐dependent inactivation, which was half‐maximal at −45, −47 and −68 mV, respectively. 4 Pancreatic α‐cells were equipped with tolbutamide‐sensitive, ATP‐regulated K+ (KATP) channels. Addition of tolbutamide (0·1 mm) evoked a brief period of electrical activity followed by a depolarisation to a plateau of −30 mV with no regenerative electrical activity. 5 Glucagon secretion in the absence of glucose was strongly inhibited by TTX, nifedipine and tolbutamide. When diazoxide was added in the presence of 10 mm glucose, concentrations up to 2 μm stimulated glucagon secretion to the same extent as removal of glucose. 6 We conclude that electrical activity and secretion in the α‐cells is dependent on the generation of Na+‐dependent action potentials. Glucagon secretion depends on low activity of KATP channels to keep the membrane potential sufficiently negative to prevent voltage‐dependent inactivation of voltage‐gated membrane currents. Glucose may inhibit glucagon release by depolarising the α‐cell with resultant inactivation of the ion channels participating in action potential generation.

Voltage-gated and resting membrane currents recorded from B-cells in intact mouse pancreatic islets

1 The perforated patch whole‐cell configuration of the patch‐clamp technique was applied to superficial cells in intact pancreatic islets. Immunostaining in combination with confocal microscopy revealed that the superficial cells consisted of 35 % insulin‐secreting B‐cells and 65 % non‐B‐cells (A‐ and D‐cells). 2 Two types of cell, with distinct electrophysiological properties, could be functionally identified. One of these generated oscillatory electrical activity when the islet was exposed to 10 mm glucose and had the electrophysiological characteristics of isolated B‐cells maintained in tissue culture. 3 The Ca2+ current recorded from B‐cells in situ was 80 % larger than that of isolated B‐cells. It exhibited significant (70 %) inactivation during 100 ms depolarisations. The inactivation was voltage dependent and particularly prominent during depolarisations evoking the largest Ca2+ currents. 4 Voltage‐dependent K+ currents were observed during depolarisations to membrane potentials above −20 mV. These currents inactivated little during a 200 ms depolarisation and were unaffected by varying the holding potential between −90 and −30 mV. 5 The maximum resting conductance in the absence of glucose, which reflects the conductance of ATP‐regulated K+ (KATP) channels, amounted to ≈4 nS. Glucose produced a concentration‐dependent reduction of KATP channel conductance with half‐maximal inhibition observed with 5 mm glucose. 6 Combining voltage‐ and current‐clamp recording allowed the estimation of the gap junction conductance between different B‐cells. These experiments indicated that the input conductance of the B‐cell at stimulatory glucose concentrations (≈1 nS) is almost entirely accounted for by coupling to neighbouring B‐cells.

Patch-clamp characterisation of somatostatin-secreting δ-cells in intact mouse pancreatic islets

1 The perforated patch whole‐cell configuration of the patch‐clamp technique was applied to superficial cells in intact mouse pancreatic islets. 2 Three types of electrical activity were observed corresponding to α‐, β‐ and δ‐cells. The δ‐cells were electrically active in the presence of glucose but lacked the oscillatory pattern seen in the β‐cells. By contrast, the α‐cells were electrically silent at high glucose concentrations but action potentials could be elicited by removal of the sugar. 3 Both α‐ and β‐cells contained transient voltage‐activated K+ currents. In the δ‐cells, the K+ currents activated above −20 mV and were completely blocked by TEA (20 mm). The α‐cells differed from the δ‐cells in possessing a TEA‐resistant K+ current activating already at −40 mV. 4 Immunocytochemistry revealed the presence of Kv3.4 channels in δ‐cells and TEA‐resistant Kv4.3 channels in α‐cells. Thus the presence of a transient TEA‐resistant current can be used to functionally separate the δ‐ and α‐cells. 5 A TTX‐sensitive Na+ current developed in δ‐cells during depolarisations beyond −30 mV and reached a peak amplitude of 350 pA. Steady‐state inactivation of this current was half‐maximal at −28 mV. The δ‐cells were also equipped with a sustained Ca2+ current that activated above −30 mV and reached a peak of 60 pA when measured at 2·6 mm extracellular Ca2+. 6 A tolbutamide‐sensitive KATP channel conductance was observed in δ‐cells exposed to glucose‐free medium. Addition of tolbutamide (0·1 mm) depolarised the δ‐cell and evoked electrical activity. We propose that the KATP channels in δ‐cells serve the same function as in the β‐cell and couple an elevation of the blood glucose concentration to stimulation of hormone release.

Capacitance measurements of exocytosis in mouse pancreatic α-, β- and δ-cells within intact islets of Langerhans

Capacitance measurements of exocytosis were applied to functionally identified α‐, β‐ and δ‐cells in intact mouse pancreatic islets. The maximum rate of capacitance increase in β‐cells during a depolarization to 0 mV was equivalent to 14 granules s−1, <5% of that observed in isolated β‐cells. β‐Cell secretion exhibited bell‐shaped voltage dependence and peaked at +20 mV. At physiological membrane potentials (up to ∼−20 mV) the maximum rate of release was ∼4 granules s−1. Both exocytosis (measured by capacitance measurements) and insulin release (detected by radioimmunoassay) were strongly inhibited by the L‐type Ca2+ channel blocker nifedipine (25 μm) but only marginally (<20%) affected by the R‐type Ca2+ channel blocker SNX482 (100 nm). Exocytosis in the glucagon‐producing α‐cells peaked at +20 mV. The capacitance increases elicited by pulses to 0 mV exhibited biphasic kinetics and consisted of an initial transient (150 granules s−1) and a sustained late component (30 granules s−1). Whereas addition of the N‐type Ca2+ channel blocker ω‐conotoxin GVIA (0.1 μm) inhibited glucagon secretion measured in the presence of 1 mm glucose to the same extent as an elevation of glucose to 20 mm, the L‐type Ca2+ channel blocker nifedipine (25 μm) had no effect. Thus, glucagon release during hyperglycaemic conditions depends principally on Ca2+‐influx through N‐type rather than L‐type Ca2+ channels. Exocytosis in the somatostatin‐secreting δ‐cells likewise exhibited two kinetically separable phases of capacitance increase and consisted of an early rapid (600 granules s−1) component followed by a sustained slower (60 granules s−1) component. We conclude that (1) capacitance measurements in intact pancreatic islets are feasible; (2) exocytosis measured in β‐cells in situ is significantly slower than that of isolated cells; and (3) the different types of islet cells exhibit distinct exocytotic features.

CaM kinase II-dependent mobilization of secretory granules underlies acetylcholine-induced stimulation of exocytosis in mouse pancreatic B-cells

1 Measurements of cell capacitance were used to investigate the mechanisms by which acetylcholine (ACh) stimulates Ca2+‐induced exocytosis in single insulin‐secreting mouse pancreatic B‐cells. 2 ACh (250 μM) increased exocytotic responses elicited by voltage‐clamp depolarizations 2.3‐fold. This effect was mediated by activation of muscarinic receptors and dependent on elevation of the cytoplasmic Ca2+ concentration ([Ca2+]i) attributable to mobilization of Ca2+ from intracellular stores. The latter action involved interference with the buffering of [Ca2+]i and the time constant (τ) for the recovery of [Ca2+]i following a voltage‐clamp depolarization increased 5‐fold. As a result, Ca2+ was present at concentrations sufficient to promote the replenishment of the readily releasable pool of granules (RRP; > 0.2 μM) for much longer periods in the presence than in the absence of the agonist. 3 The effect of Ca2+ on exocytosis was mediated by activation of CaM kinase II, but not protein kinase C, and involved both an increased size of the RRP from 40 to 140 granules and a decrease in τ for the refilling of the RRP from 31 to 19 s. 4 Collectively, the effects of ACh on the RRP and τ result in a > 10‐fold stimulation of the rate at which granules are supplied for release.

Capacitance measurements of exocytosis in mouse pancreatic alpha-, beta- and delta-cells within intact islets of Langerhans.

Capacitance measurements of exocytosis were applied to functionally identified α‐, β‐ and δ‐cells in intact mouse pancreatic islets. The maximum rate of capacitance increase in β‐cells during a depolarization to 0 mV was equivalent to 14 granules s−1, <5% of that observed in isolated β‐cells. β‐Cell secretion exhibited bell‐shaped voltage dependence and peaked at +20 mV. At physiological membrane potentials (up to ∼−20 mV) the maximum rate of release was ∼4 granules s−1. Both exocytosis (measured by capacitance measurements) and insulin release (detected by radioimmunoassay) were strongly inhibited by the L‐type Ca2+ channel blocker nifedipine (25 μm) but only marginally (<20%) affected by the R‐type Ca2+ channel blocker SNX482 (100 nm). Exocytosis in the glucagon‐producing α‐cells peaked at +20 mV. The capacitance increases elicited by pulses to 0 mV exhibited biphasic kinetics and consisted of an initial transient (150 granules s−1) and a sustained late component (30 granules s−1). Whereas addition of the N‐type Ca2+ channel blocker ω‐conotoxin GVIA (0.1 μm) inhibited glucagon secretion measured in the presence of 1 mm glucose to the same extent as an elevation of glucose to 20 mm, the L‐type Ca2+ channel blocker nifedipine (25 μm) had no effect. Thus, glucagon release during hyperglycaemic conditions depends principally on Ca2+‐influx through N‐type rather than L‐type Ca2+ channels. Exocytosis in the somatostatin‐secreting δ‐cells likewise exhibited two kinetically separable phases of capacitance increase and consisted of an early rapid (600 granules s−1) component followed by a sustained slower (60 granules s−1) component. We conclude that (1) capacitance measurements in intact pancreatic islets are feasible; (2) exocytosis measured in β‐cells in situ is significantly slower than that of isolated cells; and (3) the different types of islet cells exhibit distinct exocytotic features.

Glucose-dependent regulation of rhythmic action potential firing in pancreatic β-cells by kATP-channel modulation

The regulation of a K+ current activating during oscillatory electrical activity (IK,slow) in an insulin‐releasing β‐cell was studied by applying the perforated patch whole‐cell technique to intact mouse pancreatic islets. The resting whole‐cell conductance in the presence of 10 mm glucose amounted to 1.3 nS, which rose by 50 % during a series of 26 simulated action potentials. Application of the KATP‐channel blocker tolbutamide produced uninterrupted action potential firing and reduced IK,slow by ≈50 %. Increasing glucose from 15 to 30 mm, which likewise converted oscillatory electrical activity into continuous action potential firing, reduced IK,slow by ≈30 % whilst not affecting the resting conductance. Action potential firing may culminate in opening of KATP channels by activation of ATP‐dependent Ca2+ pumping as suggested by the observation that the sarco‐endoplasmic reticulum Ca2+‐ATPase (SERCA) inhibitor thapsigargin (4 μm) inhibited IK,slow by 25 % and abolished bursting electrical activity. We conclude that oscillatory glucose‐induced electrical activity in the β‐cell involves the opening of KATP‐channel activity and that these channels, in addition to constituting the glucose‐regulated K+ conductance, also play a role in the graded response to supra‐threshold glucose concentrations.

Cellular function in multicellular system for hormone-secretion: electrophysiological aspect of studies on alpha-, beta- and delta-cells of the pancreatic islet

We review a neck method to explore the cellular functions in multicellular system by application of the perforated patch-clamp technique to intact pancreatic islet of Langerhans. Using this approach, the integrity of the islet is preserved and intercellular communication via gap junctions and paracrine processes are maintained. 13 using low-resistance patch electrodes, rapid current responses can be monitored wider voltage-clamp control. We have applied this methodology to answer questions not resolved by patch-clamp experiments on isolated single insulin-secreting, beta-cells. First, the role of a K+-current dependent on Ca2+-influx for the termination of burst of action potentials in beta-cells could be documented. Neither the current, nor the bursting pattern of electrical activity is preserved in isolated beta-cells. Second. the conductance of gap junctions (similar to1 nS) between beta-cells was determined. Third, electrical properties of glucagon-producing alpha- and somatostatin-secreting delta-cells and the different mechanisms for glucose-sensing in these cells could be explored. The findings emanating from these experiments may hake implications for neuroscience research such as the mechanism of oscillatory electrical activity in general anti processes involved in the glucose-sensing in some neurons, which response to changes of blood glucose concentration

Electrophysiology of pancreatic β-cells in intact mouse islets of Langerhans

When exposed to intermediate glucose concentrations (6-16 mol/l), pancreatic β-cells in intact islets generate bursts of action potentials (superimposed on depolarised plateaux) separated by repolarised electrically silent intervals. First described more than 40 years ago, these oscillations have continued to intrigue β-cell electrophysiologists. To date, most studies of β-cell ion channels have been performed on isolated cells maintained in tissue culture (that do not burst). Here we will review the electrophysiological properties of β-cells in intact, freshly isolated, mouse pancreatic islets. We will consider the role of ATP-regulated K⁺-channels (K(ATP)-channels), small-conductance Ca²⁺-activated K⁺-channels and voltage-gated Ca²⁺-channels in the generation of the bursts. Our data indicate that K(ATP)-channels not only constitute the glucose-regulated resting conductance in the β-cell but also provide a variable K⁺-conductance that influence the duration of the bursts of action potentials and the silent intervals. We show that inactivation of the voltage-gated Ca²⁺-current is negligible at voltages corresponding to the plateau potential and consequently unlikely to play a major role in the termination of the burst. Finally, we propose a model for glucose-induced β-cell electrical activity based on observations made in intact pancreatic islets.