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J F Worley

Publications and source records attributed to J F Worley.

At least 19 recordsLinked to original sources
PubMed2001

Linoleic acid both enhances activation and blocks Kv1.5 and Kv2.1 channels by two separate mechanisms.

Linoleic acid (LA) had two effects on human Kv1.5 and Kv2.1 channels expressed in Chinese hamster ovary cells: an increase in the speed of current activation process (EC(50) = 2.4 and 2.7 microM for Kv1.5 and Kv2.1, respectively) and current inhibition (IC(50) = 6.6 and 7.4 for Kv1.5 and Kv2.1, respectively). LA affected the activation kinetics via two processes: a leftward shift in the instantaneous activation curves and an increase in the rate of current rise. Current inhibition by LA was time dependent but voltage independent. Hill slopes for plots of current inhibition (3.5 and 3.9 for Kv1.5 and Kv2.1, respectively) vs. dose of LA suggested that cooperativity was involved in the mechanism of current inhibition. A similar analysis of the effects of LA on current activation did not reveal cooperative interactions. The effects of LA were mediated from the external side of the channels, since addition of 10 microM LA to the patch pipette solution was without effect. Additionally, the methyl ester of LA was effective at enhancing peak current and promoting channel activation for Kv1.5 and Kv2.1 without inducing significant current inhibition.

Animals↗
PubMed1998

Characterization of a Ca2+ release-activated nonselective cation current regulating membrane potential and [Ca2+]i oscillations in transgenically derived beta-cells.

Although stimulation of insulin secretion by glucose is regulated by coupled oscillations of membrane potential and intracellular Ca2+ ([Ca2+]i), the membrane events regulating these oscillations are incompletely understood. In the presence of glucose and tetraethylammonium, transgenically derived beta-cells (betaTC3-neo) exhibit coupled voltage and [Ca2+]i oscillations strikingly similar to those observed in normal islets in response to glucose. Using these cells as a model system, we investigated the membrane conductance underlying these oscillations. Alterations in delayed rectifier or Ca2+-activated K+ channels were excluded as a source of the conductance oscillations, as they are completely blocked by tetraethylammonium. ATP-sensitive K+ channels were also excluded, since the ATP-sensitive K+ channel blocker tolbutamide substituted for glucose in inducing [Ca2+]i oscillations. Thapsigargin, which depletes intracellular Ca2+ stores, and maitotoxin, an activator of nonselective cation channels, both converted the glucose-dependent [Ca2+]i oscillations into a sustained elevation. On the other hand, both SKF 96365, a blocker of Ca2+ store-operated channels, and external Na+ removal suppressed the glucose-stimulated [Ca2+]i oscillations. Maitotoxin activated a nonselective cation current in betaTC3 cells that was attenuated by removal of extracellular Na+ and by SKF 96365, in the same manner to a current activated in mouse beta-cells following depletion of intracellular Ca2+ stores. Currents similar to these are produced by the mammalian trp-related channels, a gene family that includes Ca2+ store-operated channels and inositol 1,4,5-trisphosphate-activated channels. We found several of the trp family genes were expressed in betaTC3 cells by reverse transcriptase polymerase chain reaction using specific primers, but by Northern blot analysis, mtrp-4 was the predominant message expressed. We conclude that a conductance underlying glucose-stimulated oscillations in beta-cells is provided by a Ca2+ store depletion-activated nonselective cation current, which is plausibly encoded by homologs of trp genes.

Animals↗
PubMed1996

Expression and function of pancreatic beta-cell delayed rectifier K+ channels. Role in stimulus-secretion coupling.

Voltage-dependent delayed rectifier K+ channels regulate aspects of both stimulus-secretion and excitation-contraction coupling, but assigning specific roles to these channels has proved problematic. Using transgenically derived insulinoma cells (betaTC3-neo) and beta-cells purified from rodent pancreatic islets of Langerhans, we studied the expression and role of delayed rectifiers in glucose-stimulated insulin secretion. Using reverse-transcription polymerase chain reaction methods to amplify all known candidate delayed rectifier transcripts, the expression of the K+ channel gene Kv2.1 in betaTC3-neo insulinoma cells and purified rodent pancreatic beta-cells was detected and confirmed by immunoblotting in the insulinoma cells. betaTC3-neo cells were also found to express a related K+ channel, Kv3.2. Whole-cell patch clamp demonstrated the presence of delayed rectifier K+ currents inhibited by tetraethylammonium (TEA) and 4-aminopyridine, with similar Kd values to that of Kv2.1, correlating delayed rectifier gene expression with the K+ currents. The effect of these blockers on intracellular Ca2+ concentration ([Ca2+]i) was studied with fura-2 microspectrofluorimetry and imaging techniques. In the absence of glucose, exposure to TEA (1-20 mM) had minimal effects on betaTC3-neo or rodent islet [Ca2+]i, but in the presence of glucose, TEA activated large amplitude [Ca2+]i oscillations. In the insulinoma cells the TEA-induced [Ca2+]i oscillations were driven by synchronous oscillations in membrane potential, resulting in a 4-fold potentiation of insulin secretion. Activation of specific delayed rectifier K+ channels can therefore suppress stimulus-secretion coupling by damping oscillations in membrane potential and [Ca2+]i and thereby regulate secretion. These studies implicate previously uncharacterized beta-cell delayed rectifier K+ channels in the regulation of membrane repolarization, [Ca2+]i, and insulin secretion.

4-Aminopyridine↗
PubMed1996

Activation of stimulus-secretion coupling in pancreatic beta-cells by specific products of glucose metabolism. Evidence for privileged signaling by glycolysis.

The energy requirements of most cells supplied with glucose are fulfilled by glycolytic and oxidative metabolism, yielding ATP. In pancreatic beta-cells, a rise in cytosolic ATP is also a critical signaling event, coupling closure of ATP-sensitive K+ channels (KATP) to insulin secretion via depolarization-driven increases in intracellular Ca2+ ([Ca2+]i). We report that glycolytic but not Krebs cycle metabolism of glucose is critically involved in this signaling process. While inhibitors of glycolysis suppressed glucose-stimulated insulin secretion, blockers of pyruvate transport or Krebs cycle enzymes were without effect. While pyruvate was metabolized in islets to the same extent as glucose, it produced no stimulation of insulin secretion and did not block KATP. A membrane-permeant analog, methyl pyruvate, however, produced a block of KATP, a sustained rise in [Ca2+]i, and an increase in insulin secretion 6-fold the magnitude of that induced by glucose. These results indicate that ATP derived from mitochondrial pyruvate metabolism does not substantially contribute to the regulation of KATP responses to a glucose challenge, supporting the notion of subcompartmentation of ATP within the beta-cell. Supranormal stimulation of the Krebs cycle by methyl pyruvate can, however, overwhelm intracellular partitioning of ATP and thereby drive insulin secretion.

Animals↗
PubMed1996

NIDDM is associated with loss of pancreatic beta-cell L-type Ca2+ channel activity.

Development of non-insulin-dependent diabetes mellitus (NIDDM) is associated with defects in glucose-stimulated insulin secretion. We have investigated Zucker diabetic fatty rats (ZDF), an animal model of NIDDM, and found that, compared with control islets, the expression of mRNA encoding C- and D-isoforms of alpha 1-subunits of beta-cell L-type voltage-dependent Ca2+ channels (VDCC) was significantly reduced in islets isolated from ZDF rats. This correlated with a substantial reduction of L-type Ca2+ currents (ICa) in ZDF beta-cells. Intracellular Ca2+ concentration responses in ZDF islets after glucose, KCI, or BAY K 8644 stimulation were markedly attenuated, whereas responses evoked by carbachol were unimpaired, consistent with a specific decrease in ICa in the diabetic islets. This reduction was accompanied by loss of pulsatile insulin secretion from ZDF islets treated with oscillatory increases of external glucose concentration. Our findings suggest that the attenuation of ICa in diabetic islets may contribute to the abnormal glucose-dependent insulin secretory responses associated with NIDDM and indicate that this defect is caused by decreased expression of genes encoding beta-cell VDCC alpha 1-subunits.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1994

Depletion of intracellular Ca2+ stores activates a maitotoxin-sensitive nonselective cationic current in beta-cells.

Glucose stimulation of beta-cell insulin secretion is initiated by membrane depolarization coupled with an elevation in intracellular Ca2+ concentration ([Ca2+]i). Both depolarization-dependent Ca2+ entry and intracellular Ca2+ store release contribute to the sugar-induced rise in [Ca2+]i. Here we show that maneuvers depleting intracellular Ca2+ stores induce membrane depolarization and a sustained nitrendipine-sensitive Ca2+ influx, whereas interventions promoting Ca2+ store refilling produce a hyperpolarization and inhibit Ca2+ influx. Both intracellular Ca2+ store depletion and maitotoxin activated a depolarizing nonselective cation current carried principally by Na+ in the physiological range of membrane potentials. The activation of such a current may form the paradigm by which excitable cells refill depleted intracellular Ca2+ stores by depolarization-driven opening of voltage-activated Ca2+ channels.

Animals↗
PubMed1994

Delayed rectifier K+ channel overexpression in transgenic islets and beta-cells associated with impaired glucose responsiveness.

Glucose stimulation of pancreatic beta-cell insulin secretion is closely coupled to alterations in ion channel conductances and intracellular Ca2+ ([Ca2+]i). To further examine this relationship after augmentation of voltage-dependent K+ channel expression, transgenic mice were produced which specifically overexpress a human insulinoma-derived, tetraethylammonium (TEA)-insensitive delayed rectifier K+ channel in their pancreatic beta-cells as shown by immunoblot of isolated islets and immunohistochemical analysis of pancreas sections. Whole-cell current recordings confirmed the presence of high amplitude TEA-resistant K+ currents in transgenic islet cells, whose expression correlated with hyperglycemia and hypoinsulinemia. Stable overexpression of the channel in insulinoma cells attenuated glucose-activated increases in [Ca2+]i and prevented the induction of TEA-dependent [Ca2+]i oscillations. These results, employing the first ion channel transgenic mouse, demonstrate the importance of membrane potential regulation in excitation-secretion coupling in the pancreatic beta-cell.

Animals↗
PubMed1994

Defective glucose-dependent endoplasmic reticulum Ca2+ sequestration in diabetic mouse islets of Langerhans.

Non-insulin-dependent diabetes mellitus (NIDDM) is a metabolic disease associated with abnormal insulin secretion, the underlying mechanisms of which are unknown. Glucose-dependent signal transduction pathways were investigated in pancreatic islets derived from the db/db mouse, an animal model of NIDDM. After stimulation with glucose (4-12 mM), the changes in intracellular Ca2+ concentration ([Ca2+]i) were different; unlike control islets, db/db islets lacked an initial reduction of [Ca2+]i and the subsequent [Ca2+]i oscillations following stimulation with 12 mM glucose. The severity of these defects in Ca2+ signaling correlated with the age-dependent development of hyperglycemia. Similarly defective glucose-induced Ca2+ signaling were reproduced in control islets by pre-exposure to thapsigargin, a selective inhibitor of endoplasmic reticulum (ER) Ca(2+)-ATPase. Estimation of ATPase activities from rates of ATP hydrolysis and by immunoblot hybridization with an antiserum directed against the sarco/endoplasmic reticulum Ca(2+)-ATPase both demonstrated that the ER Ca(2+)-ATPase was almost entirely absent from db/db islets. The effects of inhibition of ER Ca(2+)-ATPase on insulin secretion were also examined; a 4-day exposure of control islets to 1 microM thapsigargin resulted in basal and glucose-stimulated insulin secretion levels similar to those found in db/db islets. These results suggest that aberrant ER Ca2+ sequestration underlies the impaired glucose responses in the db/db mouse and may play a role in defective insulin secretion associated with NIDDM.

Animals↗
PubMed1994

Endoplasmic reticulum calcium store regulates membrane potential in mouse islet beta-cells.

Glucose stimulation of islet beta-cell insulin secretion is initiated by membrane depolarization and an elevation in intracellular free calcium concentration ([Ca2+]i) from a combination of influx through depolarization-activated Ca2+ channels and intracellular Ca2+ store release. Prevention of Ca2+ store refilling with thapsigargin produced a sustained depolarization, leading to enhanced Ca2+ influx and an elevation in [Ca2+]i in 12 mM glucose. Depletion of intracellular Ca2+ stores by external EGTA reduced [Ca2+]i and also caused a long-lasting depolarization. In single beta-cells, external EGTA activated an inward current, the voltage range and kinetic properties of which differed from those of voltage-dependent Ca2+ channels. A novel pathway thus exists in beta-cells by which depletion of endoplasmic reticulum Ca2+ stores results in the activation of an inward current that, by inducing depolarization, facilitates Ca2+ influx through voltage-gated Ca2+ channels. The physiological relevance of this pathway in the control of beta-cell function is indicated by the stimulation of insulin secretion by thapsigargin.

Animals↗
PubMed1994

Dependence on NADH produced during glycolysis for beta-cell glucose signaling.

An increase in cytosolic ATP following glucose metabolism by pancreatic beta-cells is the key signal initiating insulin secretion by causing blockade of ATP-dependent K+ channels (KATP). This induces membrane depolarization, leading to an elevation in cytosolic Ca2+ ([Ca2+]i) and insulin secretion. In this report we identify the critical metabolic step by which glucose initiates changes in beta-cell KATP channel activity, membrane potential, and [Ca2+]i. The signal stems from the glycolytic production of NADH during the oxidation of glyceraldehyde 3-phosphate, which is subsequently processed into ATP by mitochondria via the operation of discrete shuttle systems.

Adenosine Triphosphate↗
PubMed1994

Ion permeation, divalent ion block, and chemical modification of single sodium channels. Description by single- and double-occupancy rate-theory models.

Calcium ions, applied internally, externally, or symmetrically, have been used in conjunction with rate-theory modeling to explore the energy profile of the ion-conducting pore of sodium channels. The block, by extracellular and/or intracellular calcium, of sodium ion conduction through single, batrachotoxin-activated sodium channels from rat brain was studied in planar lipid bilayers. Extracellular calcium caused a reduction of inward current that was enhanced by hyperpolarization and a weaker block of outward current. Intracellular calcium reduced both outward and inward sodium current, with the block being weakly dependent on voltage and enhanced by depolarization. These results, together with the dependence of single-channel conductance on sodium concentration, and the effects of symmetrically applied calcium, were described using single- or double-occupancy, three-barrier, two-site (3B2S), or single-occupancy, 4B3S rate-theory models. There appear to be distinct outer and inner regions of the channel, easily accessed by external or internal calcium respectively, separated by a rate-limiting barrier to calcium permeation. Most of the data could be well fit by each of the models. Reducing the ion interaction energies sufficiently to allow a small but significant probability of two-ion occupancy in the 3B2S model yielded better overall fits than for either 3B2S or 4B3S models constrained to single occupancy. The outer ion-binding site of the model may represent a section of the pore in which sodium, calcium, and guanidinium toxins, such as saxitoxin or tetrodotoxin, compete. Under physiological conditions, with millimolar calcium externally, and high potassium internally, the model channels are occupied by calcium or potassium much of the time, causing a significant reduction in single-channel conductance from the value measured with sodium as the only cation species present. Sodium conductance and degree of block by external calcium are reduced by modification of single channels with the carboxyl reagent, trimethyloxonium (TMO) (Worley et al., 1986) Journal of General Physiology. 87:327-349). Elevations of only the outermost parts of the energy profiles for sodium and calcium were sufficient to account for the reductions in conductance and in efficacy of calcium block produced by TMO modification.

Animals↗
PubMed1994

Thapsigargin inhibits the glucose-induced decrease of intracellular Ca2+ in mouse islets of Langerhans.

Stimulation of pancreatic islets of Langerhans with glucose results in changes in intracellular Ca2+ concentration ([Ca2+]i). With the use of mouse islets loaded with fura 2, the earliest glucose-induced alteration of [Ca2+]i was a pronounced decline in [Ca2+]i. This effect (phase 0) was evident 1 min after increasing extracellular glucose from 2 to 12 mM and was sustained for 3-5 min. Phase 0 was also observed when glucose was increased from 5 to 12 mM, indicating that it was not an experimental artifact resulting from substrate depletion. The [Ca2+]i-lowering effect of glucose was mimicked by D-glyceraldehyde but not by 2-deoxyglucose, pyruvate, glyburide, or 30 mM extracellular KCl. Mannoheptulose inhibited phase 0, whereas diazoxide, sodium azide, calmidazolium, or increasing extracellular [Ca2+] to 10 mM were all without effect. After the elevation of islet [Ca2+]i with 5 microM glyburide, 12 mM glucose caused a considerable transient decrease in [Ca2+]i. Under similar conditions, 5 mM caffeine attenuated phase 0, whereas 1 microM thapsigargin, a specific inhibitor of the sarcoplasmic and endoplasmic reticulum family of Ca(2+)-adenosinetriphosphatases (SERCA), almost completely inhibited any glucose-induced reduction of [Ca2+]i. These observations suggest that glucose causes an elevation of beta-cell SERCA activity triggered by factors generated during the cytosolic stages of glycolysis.

Animals↗
PubMed1993

Amylin modulates beta-cell glucose sensing via effects on stimulus-secretion coupling.

The release of insulin from the pancreatic beta cell is dependent upon a complex interplay between stimulators and inhibitors. Recently, amylin, a peptide secreted by pancreatic beta cells, has been implicated in the development of type II (noninsulin dependent) diabetes through its modulation of the peripheral effects of insulin. However, the effect of amylin on insulin secretion from the beta cell has remained controversial. It is reported here that in single beta cells exhibiting normal glucose sensing, amylin causes membrane hyperpolarization, increases in net outward current, and reductions in insulin secretion. In contrast, in cells with abnormal glucose sensing (e.g., from db/db diabetic mice), amylin has no effect on electrical activity or secretion. Thus, amylin's effects on excitation-secretion coupling in the beta cell of the pancreas appear to be linked to the cell's capacity for normal glucose sensing.

Amyloid↗
PubMed1993

Voltage-dependent intracellular calcium release from mouse islets stimulated by glucose.

Glucose-activated beta-cell insulin secretion depends upon elevation of intracellular calcium concentration, [Ca2+]i, which is thought to arise from Ca2+ influx through voltage-dependent calcium channels. Using fura-2-loaded mouse islets, we demonstrate, in fact, that the major component of the glucose-activated [Ca2+]i rise represents voltage-dependent intracellular Ca2+ release. Furthermore, the Ca2+ release pool possesses a novel pharmacology in that it is caffeine-sensitive but ryanodine-insensitive. In the absence of external Ca2+, glucose still caused intracellular Ca2+ release, an effect blockable by tetrodotoxin. However, depolarization of the islet with KCl in low Ca(2+)-containing solutions induced intracellular Ca2+ release, which was resistant to tetrodotoxin. We conclude that glucose release of intracellular Ca2+ is dependent upon depolarization alone, possibly through increasing inositol 1,4,5-trisphosphate production.

Animals↗
PubMed1992

Lipid surface charge does not influence conductance or calcium block of single sodium channels in planar bilayers.

We have studied the effects of membrane surface charge on Na+ ion permeation and Ca2+ block in single, batrachotoxin-activated Na channels from rat brain, incorporated into planar lipid bilayers. In phospholipid membranes with no net charge (phosphatidylethanolamine, PE), at low divalent cation concentrations (approximately 100 microM Mg2+), the single channel current-voltage relation was linear and the single channel conductance saturated with increasing [Na+] and ionic strength, reaching a maximum (gamma max) of 31.8 pS, with an apparent dissociation constant (K0.5) of 40.5 mM. The data could be approximated by a rectangular hyperbola. In negatively charged bilayers (70% phosphatidylserine, PS; 30% PE) slightly larger conductances were observed at each concentration, but the hyperbolic form of the conductance-concentration relation was retained (gamma max = 32.9 pS and K0.5 = 31.5 mM) without any preferential increase in conductance at lower ionic strengths. Symmetrical application of Ca2+ caused a voltage-dependent block of the single channel current, with the block being greater at negative potentials. For any given voltage and [Na+] this block was identical in neutral and negatively charged membranes. These observations suggest that both the conduction pathway and the site(s) of Ca2+ block of the rat brain Na channel protein are electrostatically isolated from the negatively charged headgroups on the membrane lipids.

Animals↗
PubMed1992

Contractile agonists activate voltage-dependent calcium channels in airway smooth muscle cells.

To determine whether agents that cause contraction of airway smooth muscle affect sarcolemmal calcium channel activity, unitary calcium channel currents (using Ba2+ as the charge carrier) were recorded in on-cell configuration from acutely dissociated (dog, pig, and ferret) and cultured (human) airway smooth muscle cells. Addition of the contractile agonists methacholine or bradykinin increased the open-state probability of the large-conductance calcium channel 37.2- and 45-fold, respectively. The increase in open-state probability was not due to cellular depolarization because increases occurred in the absence of depolarization. Channel activation by the agonist was determined to result in the favoring of a long (16.5 +/- 5.0 ms) open lifetime for the channel, which was not observed under control conditions, in the absence of BAY K 8644. We also report the unitary calcium channel currents from a second, smaller conductance calcium channel. This channel was present in all cell types and had a mean conductance of 9.5 +/- 0.8 pS (80 mM Ba2+). Exposure of cells to agonist also resulted in an increase in the open-channel probability of the small-conductance calcium channel (10.4-fold), which did not result from cellular depolarization. These experiments demonstrate that the molecular pathways exist between contractile agonist receptors and sarcolemmal calcium channels in airway smooth muscle cells. Because membrane patches were not directly exposed to agonist, receptor-channel linkage probably occurs via a second messenger-coupling pathway.

Bradykinin↗
PubMed1991

Regulation of single calcium channels in cerebral arteries by voltage, serotonin, and dihydropyridines.

Unitary currents through Ca channels were measured from cell-attached patches on smooth muscle cells isolated from rabbit cerebral (basilar) arteries. Barium (80 and 10 mM) and calcium (80 and 10 mM) were used as the charge carriers. The dihydropyridine Ca channel agonist BAY R 5417 was used to increase open-state probability (Popen), with 500 nM BAY R 5417 increasing Popen 10-fold at 0 mV. Barium currents through single Ca channels were greater than calcium currents at any voltage, with single-channel conductances negative to -20 mV of 24.6 pS (80 mM barium), 15.1 pS (80 mM calcium), 17.2 pS (10 mM barium), and 5.8 pS (10 mM calcium). The single-channel Popen increased 2.7-fold per 5- to 7-mV membrane depolarization (negative to 0 mV) and was half maximal at +0.4 mV (80 mM calcium) and +13.5 mV (80 mM barium). Ca channels with calcium but not with barium as the charge carrier exhibited pronounced inactivation positive to -20 mV (half time, 112 ms at 0 mV). The dihydropyridine nimodipine (2 nM) inhibited average currents through Ca channels. The cerebral artery constrictor serotonin increased Popen of single Ca channels by as much as 200-fold without an effect on single-channel conductance. A second distinct amplitude of unitary currents was often observed, corresponding to a channel conductance of about one-half the more commonly observed level. The small-conductance-level channel was voltage dependent, did not inactivate over 0.5-s test pulses (with barium), and could be activated by serotonin.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗