Stanley Misler - US grants

An intriguing question in the physiology of the endocrine pancreas is how a rise in ambient glucose or amino acid concentration, a seemingly simple and ubiquitous stimulus which all cells are exposed to, results in the controlled pulsatile secretion of insulin by the B cell and inhibition of glucagon secretion by the A cell. The goal of this work is to study the role of single ion channels in the coupling of metabolite stimuli to insulin and glucagon secretion. Principally, we wish to examine whether stimulation by a metabolite results in B cell depolarization (and hence voltage-dependent Ca2+ influx) by inducing closure of an ATP sensitive K+ channel. To do this we shall examine the salient characteristics of this channel including ion selectivity, gating, single channel kinetics and pharmacology of this channel. These experiments will involve standard cell attached and excised patch recording, whole cell recording, cell attached patch recording with partially permeabilized cells and a novel "slow" whole cell technique. Second, we wish to examine whether intracellular release of Ca2+ in B cell, which may also be stimulated by metabolism, occurs through Ca2+ channels in endoplasmic reticulum (ER), by measuring Ca2+ channel activity of insulinoma ER fused into planar bilayer membranes. We also wish to determine whether this source of intracellular calcium contributes to a pool of Ca2+ which participates in triggering insulin exocytosis, using Ca2+-activated K+ channels in cell attached patches of partially permeabilized cells as an assay for local Ca2+ concentration. Third, we wish to begin exploring single channels underlying electrical activity in immunocytochemically identified A cells. Results of these experiments should contribute to our knowledge of stimulus-secretion coupling in the endocrine pancreas and pave the way for future investigations of membrane defects in diabetes mellitus.

A testable "metabolic hypothesis" for the postprandial induction of insulin secretion is that B cell metabolism of nutrient fuels, especially glucose, -(1)-> alteration of intracellular intermediates -(2)-> closure of metabolically regulated K+ channels _(3)-> cell depolarization -(4)-> action potential initiation and the opening of voltage dependent Ca2+ channels - (5)-> Ca2+ entry -(6)-> insulin granule exocytosis. Over the past five years important advances have been made in identifying "molecular players" in this hypothesis including an ATP1 sensitive K+ channel, whose closure depolarizes the cell; voltage dependent Ca2+, and K+ channels, which probably contribute to the action potential; and increases in cytosolic Ca2+ under conditions where B cells depolarize. We propose to further verify this hypothesis by investigating several dynamic links (4- 6) which connect cell depolarization to granule exocytosis by simultaneously applying several new "integrative" techniques to single normal adult human islet B cells harvested from cadaver donors. These techniques are "perforated patch" current or voltage clamp recording; microspectrofluometry, using Ca2+- sensitive dyes; and membrane capacitance measurements by "phase detection" techniques. First we shall examine the ionic channel currents underlying patterns of metabolite induced electrical activity, especially Ca2+ channels. Second, we shall correlate changes in cytosolic Ca2+ with patterns of electrical activity induced by nutrient fuel secretogogues. Third, we shall attempt to correlate patterns of electrical activity and Ca2+ entry with patterns of insulin granule exocytosis as reflected by changes in membrane capacitance. The last approach will require us to establish optimal conditions for insulin secretion using the "reverse hemolytic plaque assay" and to verify that capacitance changes seen reflect real-time incorporation of insulin granule membrane into the cell surface. The experiments should further our understanding of stimulus-secretion coupling in the normal human B cell and serve as a basis for studies on the pathophysiology of diminished insulin secretion in non-insulin dependent diabetes mellitus.

Volume regulation is a feature common to many vertebrate cells. When placed in hypotonic bathing solution, fluid transporting epithelial cells, peripheral blood cells and even glia and neurons initially swell, but then, over several minutes, shrink back to near their resting volumes. The regulatory volume decrease (RVD) is usually accompanied by the passive loss of intracellular K+ and anions including Cl-. Recently, using cell-attached patch clamp recording, we have investigated ion channel activity during cell swelling and RVD in clonal N1E115 neuroblastoma cells. The activity of a stretch-sensitive, non-selective cation channel (C+(SA)) increases shortly after the onset of osmotically induced cell swelling. Shortly thereafter, and roughly coincident with the onset of RVD, two types of voltage-dependent channels spontaneously open at the resting potential: (1) a delayed-rectifier type K+ channel, and (2) large conductance anion channel. We have hypothesized that the C+ (SA) channel may be a volume "sensor" mechanism, while the voltage- dependent K+ and anion channels may be potassium salt exit pathways during RVD. We now propose to test the latter hypothesis by combining videomicroscopic imaging of cell size with (a) single-channel recording and (b) the perforated patch variant of whole-cell recording to examine changes in membrane current, voltage and resistance during cell swelling and RVD in neuroblastoma under a variety of osmotic perturbations, and in the presence and absence of ion channel blockers which we shall demonstrate to be selective for a given channel. We shall focus on ascertaining (a) when and how many each ion channel type opens during RVD; (b) what forces and/or intracellular messengers actually gate each channel, as well as which ions permeate each channel when open; and (c) whether the net sum ion efflux through these channel during the RVD accounts for much or all of the salt loss from the cell. These results may help to elucidate the mechanism used by brain cells to limit swelling during hyponatremia, ischemia, insult by neurotoxic excitatory transmitters, and even cell growth.

DESCRIPTION: This application explores stimulation-secretion coupling in the beta islet cells of the pancreas. The investigator has hypothesized that a subpopulation of insulin granules are 'loosely co-localized' with Ca2+ channels in the plasmalemma, and these granules are released rapidly in response to local increases in Ca2+ concentration. The specific aims test four predictions of this co-localization model: 1) release should have a distinctive relationship to total Ca2+ entry that should be independent of the conditions initiating that entry; 2) widely-separated depolarizations should evoke insulin release with distinct latency periods; 3) sustained Ca2+ entry should deplete the co-localized pool of granules, resulting in a period of quiescence while that pool is replenished; and 4) conditions that increase the size of the co-localized pool should permit prolonged bouts of secretion. To evaluate these predictions, the investigator will use instrumentation capable of measuring these parameters of stimulation and secretion simultaneously in single beta cells.