The perforated patch whole‐cell configuration of the patch‐clamp technique was applied to superficial glucagon‐secreting α‐cells in intact mouse pancreatic islets. 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. 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. 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. 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. 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.

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). 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. 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. 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. 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. 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.

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 Ca 2+ channel blocker nifedipine (25 µM) but only marginally (<20%) affected by the Rtype Ca 2+ 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 Ca 2+ 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 Ca 2+ channel blocker nifedipine (25 µM) had no effect. Thus, glucagon release during hyperglycaemic conditions depends principally on Ca 2+ -influx through N-type rather than L-type Ca 2+ channels. Exocytosis in the somatostatinsecreting 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.

We have applied the perforated patch whole-cell technique to β cells within intact pancreatic islets to identify the current underlying the glucose-induced rhythmic firing of action potentials. Trains of depolarizations (to simulate glucose-induced electrical activity) resulted in the gradual (time constant: 2.3 s) development of a small (<0.8 nS) K+ conductance. The current was dependent on Ca2+ influx but unaffected by apamin and charybdotoxin, two blockers of Ca2+-activated K+ channels, and was insensitive to tolbutamide (a blocker of ATP-regulated K+ channels) but partially (>60%) blocked by high (10–20 mM) concentrations of tetraethylammonium. Upon cessation of electrical stimulation, the current deactivated exponentially with a time constant of 6.5 s. This is similar to the interval between two successive bursts of action potentials. We propose that this Ca2+-activated K+ current plays an important role in the generation of oscillatory electrical activity in the β cell.

The sequence of biochemical events involved in mechanical stress-induced signaling in osteoblastic cells remains unclear. Runx2, a transcription factor involved in the control of osteoblast differentiation, has been identified as a target of mechanical stress-induced signaling in osteoblastic cells. In this study, uniaxial sinusoidal stretching (15% strain, 115% peak-to-peak, at 1/12 Hz) stimulated the differentiation of osteoblast-like MC3T3-E1 cells and rat primary osteoblastic cells by activating Runx2. We examined the involvement of diverse mitogen-activated protein kinase (MAPK) pathways in the activation of Runx2 during mechanical stress. Mechanical stress increased alkaline phosphatase activity, a marker of osteoblast differentiation, increased the expression of the osteoblast-specific extracellular matrix (ECM) protein osteocalcin, and induced Runx2 activation, along with increased osterix expression. Furthermore, activation of ERK1/2 and p38 MAPKs increased significantly. U0126, a selective inhibitor of ERK1/2, completely blocked Runx2 activation during periods of mechanical stress, but the p38 MAPK-selective inhibitor SB203580 did not alter nuclear phosphorylation of Runx2. Small interfering RNA (siRNA) targeting Rous sarcoma kinase (RAS), an upstream regulator of both ERK1/2 and p38 MAPKs, inhibited stretch-induced ERK1/2 activation, but not mechanically induced p38 MAPK activity. Furthermore, mechanically induced Runx2 activation was inhibited by Ras depletion, using siRNA. These findings indicate that mechanical stress regulates Runx2 activation and favors osteoblast differentiation through the activation of MAPK signal transduction pathways and Ras/Raf-dependent ERK1/2 activation, independent of p38 MAPK signaling.

The perforated patch whole‐cell configuration of the patch‐clamp technique was applied to superficial cells in intact mouse pancreatic islets. 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. 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. 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. 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+. 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.

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