Modes of exocytosis and electrogenesis underlying canine biphasic insulin secretion.

Biphasic insulin secretion in response to glucose consists of a transient first phase followed by a progressive second phase. It is a well described feature of whole perfused pancreases as well as isolated pancreatic islets of Langerhans. Applying to single cell assays of exocytosis (capacitance monitoring and amperometry) to single canine Beta-cells we have examined the time courses of granule exocytosis in response to voltage-clamp depolarizations that mimic two modes of glucose-induced electrical activity, and then compared these to biphasic insulin secretion.

The isolated pancreatic islet as a micro-organ and its transplantation to cure diabetes: celebrating the legacy of Paul Lacy.

Over the past three decades the pancreatic islet of Langerhans has taken center stage as an endocrine micro-organ whose glucoregulatory function is highly explicable on the basis of the increasingly well understood activities of three highly interactive secretory cells. Islet dysfunction underlies both type 1 and type 2 diabetes mellitus (DM); its protection from immune attack and gluco-and lipo-toxicity may prevent the development of DM; and its replacement by non-surgical transplantation may be curative of DM. During a career marked by vision, focus and tenacity, Paul Lacy contributed substantially to the development of each of these concepts.

Unifying concepts in stimulus-secretion coupling in endocrine cells and some implications for therapeutics.

Stimulus-secretion coupling (SSC) in endocrine cells remains underappreciated as a subject for the study/teaching of general physiology. In the present article, we review key new electrophysiological, electrochemical, and fluorescence optical techniques for the study of exocytosis in single cells that have made this a fertile area for recent research. Based on findings using these techniques, we developed a model of SSC for adrenal chromaffin cells that blends features of Ca(2+) entry-dependent SSC (characteristic of neurons) with G protein receptor-coupled, Ca(2+) release-dependent, and second messenger-dependent SSC (characteristic of epithelial exocrine cells and nucleated blood cells).

Electrical activity and exocytotic correlates of biphasic insulin secretion from beta-cells of canine islets of Langerhans: contribution of tuning two modes of Ca2+ entry-dependent exocytosis to two modes of glucose-induced electrical activity.

Biphasic insulin secretion in response to glucose, consisting of a transient first phase followed by a progressive second phase, is well described in pancreatic islets. Using single canine beta-cells we have compared the time courses of electrical activity and insulin granule exocytosis to biphasic insulin secretion. Short trains of action potentials, similar those found during first phase insulin secretion, trigger phasic exocytosis from a small pool of insulin granules, likely an immediately releasable pool docked near voltage activated Ca(2+) channels.

Ion channels underlying stimulus-exocytosis coupling and its cell-to-cell heterogeneity in beta-cells of transplantable porcine islets of Langerhans.

Given the growing interest in porcine islets as model tissue for studying the pathogenesis of human diabetes mellitus and its treatment by transplantation, we investigated stimulus-exocytosis coupling in single porcine beta-cells using patch clamp electrophysiology, Ca2+ imaging, capacitance tracking and amperometry. We establish that porcine beta-cells display several features prominently seen in beta-cells from human islets of Langerhans. These include: (i) wide heterogeneity of electrical responsiveness to glucose; (ii) dependence of action potential activity on voltage-dependent Na(+) as well as high voltage activated Ca2+ current; (iii) heterogeneity of time course of depolarization-evoked insulin granule exocytosis; and (iv) the dependence of vigorous single cell electrical activity and insulin granule exocytosis on the presence of agents that enhance cytosolic cAMP concentration.

Phasic and tonic modes of depolarization-exocytosis coupling in beta-cells of porcine islets of Langerhans.

In response to depolarizations that open voltage dependent Ca2+ channels single porcine beta-cells display heterogeneous time courses of exocytosis. Some cells display phasic exocytosis that is triggered by individual or short burst of action potentials typically characteristic of glucose-induced electrical activity or brief voltage clamp depolarization. Other cells, singularly or additionally, display tonic exocytosis that (i) is triggered during prolonged (up to seconds-long) depolarizations to voltages (-30 to -20 mV), and (ii) coincides with rises in global cytosolic [Ca2+] >500 nM.

Electrophysiological and immunocytochemical evidence for P2X purinergic receptors in pancreatic beta cells.

Objectives: Glucose-induced insulin secretion from pancreatic beta cells is modulated by several hormones and transmitters, namely adenosine triphosphate (ATP) via purinergic receptors. Although P2Y receptors are well documented in beta cells, the presence of P2X receptors remains elusive. We present the first electrophysiological evidence for the presence of P2X receptors in single beta cells of different species.

Ca2+ controls slow NAD(P)H oscillations in glucose-stimulated mouse pancreatic islets.

Exposure of pancreatic islets of Langerhans to physiological concentrations of glucose leads to secretion of insulin in an oscillatory pattern. The oscillations in insulin secretion are associated with oscillations in cytosolic Ca(2+) concentration ([Ca(2+)](c)). Evidence suggests that the oscillations in [Ca(2+)](c) and secretion are driven by oscillations in metabolism, but it is unclear whether metabolic oscillations are intrinsic to metabolism or require Ca(2+) feedback.

Maintenance of stimulus-secretion coupling and single beta-cell function in cryopreserved-thawed human islets of Langerhans.

Studies of stimulus-secretion coupling in human beta-cells have been hampered by poor availability of tissue due to variability of the supply of cadaver pancreati and in the adequacy of enzymatic liberation of islets as well as by the shunting of isolates into transplant trials. Here we establish that aliquots of islets, several from high-quality but low-yield islet isolates (50,000-100,000 islets), cryopreserved and then thawed as needed, respond to glucose in a calcium- and metabolic-dependent fashion. Insulin secretion is modulated by blockers of voltage-dependent Na+ and Ca2+ channels, and paracrine hormones (glucagon and somatostatin) in manners indistinguishable from fresh tissue preparations.

alpha-Latrotoxin increases spontaneous and depolarization-evoked exocytosis from pancreatic islet beta-cells.

alpha-Latrotoxin (alpha-LT), a potent excitatory neurotoxin, increases spontaneous, as well as action potential-evoked, quantal release at nerve terminals and increases hormone release from excitable endocrine cells. We have investigated the effects of alpha-LT on single human, mouse and canine beta-cells. In isolated and combined measurements, alpha-LT, at nanomolar concentrations, induces: (i) rises in cytosolic Ca(2+), into the micromolar range, that are dependent on extracellular Ca(2+); (ii) large conductance non-selective cation channels; and (iii) Ca(2+)-dependent insulin granule exocytosis, measured as increases in membrane capacitance and quantal release of preloaded serotonin.

RyR2 and calpain-10 delineate a novel apoptosis pathway in pancreatic islets.

Cells are programmed to die when critical signaling and metabolic pathways are disrupted. Inhibiting the type 2 ryanodine receptor (RyR2) in human and mouse pancreatic beta-cells markedly increased apoptosis. This mode of programmed cell death was not associated with robust caspase-3 activation prompting a search for an alternative mechanism.

Ryanodine receptors in human pancreatic beta cells: localization and effects on insulin secretion.

It is clear that pancreatic beta-cell dysfunction, including basal hyperinsulinemia and reduced insulin release in response to glucose, is a key determinant of disease progression in type 2 diabetes, but the underlying molecular defects are not known. In diabetes, the expression and function of ryanodine receptor (RyR) Ca2+ release channels are reduced. The present studies were undertaken to define the subcellular location and role of RyR in the control of stimulated and basal insulin release from human pancreatic beta cells.

Increased islet apoptosis in Pdx1+/- mice.

Mice with 50% Pdx1, a homeobox gene critical for pancreatic development, had worsening glucose tolerance with age and reduced insulin release in response to glucose, KCl, and arginine from the perfused pancreas. Surprisingly, insulin secretion in perifusion or static incubation experiments in response to glucose and other secretagogues was similar in islets isolated from Pdx1(+/-) mice compared with Pdx1(+/+) littermate controls. Glucose sensing and islet Ca(2+) responses were also normal.

Nicotinic acid-adenine dinucleotide phosphate-sensitive calcium stores initiate insulin signaling in human beta cells.

Recent studies suggest a role for autocrine insulin signaling in beta cells, but the mechanism and function of insulin-stimulated Ca(2+) signals is uncharacterized. We examined Ca(2+)-dependent insulin signaling in human beta cells. Two hundred nanomolar insulin elevated [Ca(2+)](c) to 284 +/- 27 nM above baseline in approximately 30% of Fura-4F-loaded cells.

alpha-Latrotoxin alters spontaneous and depolarization-evoked quantal release from rat adrenal chromaffin cells: evidence for multiple modes of action.

alpha-Latrotoxin (alpha-LT) potently enhances both "spontaneous" and "depolarization-evoked" quantal secretion from neurons. Here we have used the patch-clamped rat adrenal chromaffin cell to examine simultaneously the effects of alpha-LT on membrane current or voltage, cytosolic Ca, and membrane capacitance, the latter used as an assay for exocytosis. In chromaffin cells exposed to toxin concentrations of >100 pM, the development of large conductance, Ca-permeable ion channels, accompanied by a rise in cytosolic Ca to levels near 1 microM, precedes the initiation of spontaneous exocytosis.

alpha-Latrotoxin-induced quantal release of catecholamines from rat adrenal chromaffin cells.

alpha-Latrotoxin (alpha-LT) potently enhances quantal release of neurotransmitter from nerve terminals. To develop the adrenal chromaffin cell as a 'model' system for the study of mechanisms of toxin action, we used amperometry to examine secretion of catecholamines and spectrofluorometry to measure cytosolic Ca. Several key features of toxin action emerged.

Characterization of a Ca2+-activated K+ current in insulin-secreting murine betaTC-3 cells.

1. The whole-cell perforated-patch recording mode was used to record a Ca2+-dependent K+ current (IK(Ca)) in mouse betaTC-3 insulin-secreting cells. 2.

An optimized approach to membrane capacitance estimation using dual-frequency excitation.

We present an optimized solution to the problem of membrane impedance estimation when a patch-clamped cell is stimulated by a dual-frequency, sinusoidal excitation. The complete data set of raw whole-cell current samples is typically reduced, via digital lock-in detection, to measurements of the complex cell model admittance at the two stimulus frequencies. We describe a statistical model of both data sets and demonstrate that the admittance data adequately represent the essential features obtained from the raw data.

Single-cell measurements of quantal secretion induced by alpha-latrotoxin from rat adrenal chromaffin cells: dependence on extracellular Ca2+.

alpha-Latrotoxin (alpha-LT), from black widow spider venom, is a potent enhancer of the spontaneous quantal release of neurotransmitter from a variety of nerve terminals and clonal neurosecretory cells. Using electrochemical amperometry and estimation of membrane impedance by phase detection, we present evidence that alpha-LT induces exocytosis of catecholamines from rat adrenal chromaffin cells beginning as rapidly as 30 s after close application of the toxin. This release is largely dependent on adequate levels of extracellular Ca2+ ([Ca2+]o).

Rapid fluctuations in transmitter release from single vesicles in bovine adrenal chromaffin cells.

Single-vesicle release of catecholamines from chromaffin cells can be detected in real time as current spikes by the electrochemical method of amperometry. About 70% of spikes are preceded by a small "foot," the trickle of transmitter out of the early fusion pore. In addition, 20-50% of foot signals exhibit rapid fluctuations that we interpret as flickering of the fusion pore.

Amperometric detection of quantal secretion from patch-clamped rat pancreatic beta-cells.

Serotonin (5-HT) is taken up in insulin granules and co-released with insulin on stimulation of pancreatic islet beta-cells. Based on these observations, we have used microcarbon fiber amperometry to examine secretogogue-induced 5-HT release from rat beta-cells preloaded for 4-16 h with 5-HT and then exposed to a bath solution containing 10 microM forskolin. In response to local application of KCl (60 mM) or tolbutamide (50-200 microM), we recorded barrages of amperometric events.

Voltage-dependent Na+ and Ca2+ currents in human pancreatic islet beta-cells: evidence for roles in the generation of action potentials and insulin secretion.

We describe three voltage-dependent inward currents in human pancreatic beta-cells. First, a rapidly inactivating Na+ current, blocked by tetrodotoxin (TTX) is seen upon brief depolarization to or beyond -40 mV. Second, a transient, low-voltage-activated (LVA), amiloride-blockable Ca2+ current is seen upon depolarization to or beyond -55 mV; it inactivates within less than 1s of sustained depolarization to -40 mV.

Coupling of exocytosis to depolarization in rat pancreatic islet beta-cells: effects of Ca2+, Sr2+ and Ba(2+)-containing extracellular solutions.

Using rat beta-cells we present evidence that Sr2+ and Ba2+, like Ca2+, support depolarization-induced increases in membrane capacitance which reflect insulin granule exocytosis. Even with identical total charge entry, Sr2+ and Ba2+ are 3-5 and 20-fold less effective than Ca2+ in supporting release. While exocytosis supported by Sr2+ is graded with cation entry and complete within 250ms of depolarization, exocytosis supported by Ba2+ begins abruptly after a threshold of charge entry and continues for many seconds.

Amperometric detection of stimulus-induced quantal release of catecholamines from cultured superior cervical ganglion neurons.

Amperometry has been used for real-time electrochemical detection of the quantal release of catecholamines and indolamines from secretory granules in chromaffin and mast cells. Using improved-sensitivity carbon fiber electrodes, we now report the detection of quantal catecholamine release at the surface of somas of neonatal superior cervical ganglion neurons that are studded with axon varicosities containing synaptic vesicles. Local application of a bath solution containing high K+ or black widow spider venom, each of which greatly enhances spontaneous quantal release of transmitter at synapses, evoked barrages of small-amplitude (2-20 pA), short-duration (0.

Pharmacology of volume regulation following hypotonicity-induced cell swelling in clonal N1E115 neuroblastoma cells.

When exposed to hypotonic solutions, clonal N1E115 neuroblastoma cells initially swell and later undergo a regulatory volume decrease (RVD). We studied the effects of a variety of transport inhibitors on the time course of cross-sectional area of N1E115 cells exposed to a solution of reduced osmolarity (pi = 186 mosm). Application to the bath of either: (i) blockers of net K efflux through K channels (e.