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D L Kunze

Publications and source records attributed to D L Kunze.

At least 19 recordsLinked to original sources
PubMed1992

Localization and retention in vitro of fluorescently labeled aortic baroreceptor terminals on neurons from the nucleus tractus solitarius.

The anterograde fluorescent tracer DiA was used to visualize baroreceptor fibers and synaptic terminals both in living and fixed tissue. Baroreceptor fibers labeled with DiA terminated as a dense synaptic field in the medial nucleus tractus solitarius (NTS), making synaptic contact on the soma, as well as processes of neurons that they innervated. A similar distribution and morphology was observed in baroreceptor fibers and terminals labeled with horseradish peroxidase. DiA also identified baroreceptor terminals and the neurons receiving these synaptic contacts in vitro. NTS neurons were dissociated from their surrounding tissue and identified by attached baroreceptor terminals that retained the fluorescent dye. These results will enable us to study the electrophysiological properties of dispersed neurons that receive identified baroreceptor synaptic terminals.

Animals↗
PubMed1992

L-glutamate inhibition of an inward potassium current in neonatal neurons from the nucleus of the solitary tract.

Neurons isolated from the nucleus of the solitary tract (NTS) of 1- to 4-day-old rats were cultured for a study of the glutamatergic responses in this region of the medulla. Whole cell currents were examined under voltage clamp after 6-14 days in culture. An inwardly rectifying potassium current was identified in 107/174 cells. The presence of this K+ current diminished with time in culture from greater than 80% of the cells at day 6 to less than 30% of the cells after day 10. The current was inhibited by L-glutamate (IC50 = 10 microM).

Animals↗
PubMed1992

Anion and cation permeability of a large conductance anion channel in the T84 human colonic cell line.

A large conductance multi-state channel was identified and characterized in single channel recordings from cell-attached and excised patches of the human colonic tumor cell line, T84. The channel activity was dependent on the presence of both permeable cations and anions. In Na(+)-free symmetrical Cl- solutions or Cl(-)-free symmetrical Na+ solutions the channel was inactive. Addition of 5 mM NaCl (NaI or KCl) induced channel activity. The selectivity sequence obtained from the shift in reversal potential was I- (1.9) > Cl- (1) > Na+ (0.5) > K+ (0.3). SO2-(4), SCN- (thiocyanate) and NMDG+ were impermeant. Multiple subconductance states were identified at all voltages explored (+/- 90 mV). The minimum conductance encountered in symmetrical 100 mM NaCl was a 15 pS substrate, the maximum, 210 pS. The channel appeared to be composed of multiples of the 15 pS subunits which were reversibly blocked by the loop diuretic bumetanide (5 microM).

Anions↗
PubMed1992

G-protein-mediated regulation of a Ca(2+)-dependent K+ channel in cultured vascular endothelial cells.

The purpose of the present study was to determine the mechanism by which bradykinin activates the small conductance, inwardly rectifying, Ca(2+)-activated K+ channel (KCa) found in cultured bovine aortic endothelial cells. Channel activity was studied using the patch-clamp technique in whole-cell, cell-attached, inside-out and outside-out configurations. Channel conductance at potentials positive to 0 mV was 10 +/- 2 pS and at potentials negative to 0 mV 30 +/- 3 pS (n = 7) when examined in symmetrical K+ (150 mmol/l) solutions. The channel open probability (P(o)) was only weakly voltage dependent changing approximately 0.2 units over 160 mV. In contrast, raising the intracellular Ca2+ concentration from 100 nmol/l to 10 mumol/l at -60 mV produced a graded increase in channel P(o) from 0.15 to 0.96; the concentration required for half-maximum response (apparent K0.5) was 719 nmol/l. At a constant Ca2+ concentration, application of guanosine triphosphate (GTP) to the cytoplasmic surface of the patch increased channel P(o). This effect was dependent upon the simultaneous presence of both GTP and Mg2+, and was reversed by the subsequent application of the guanosine diphosphate (GDP) analogue, guanosine-5'-O-(2-thiodiphosphate) (GDP beta S). The hydrolysis-resistant GTP analogue, guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S), induced a long-lasting increase in channel P(o). In the presence of Mg(2+)-GTP, the apparent K0.5 for Ca2+ decreased from a control value of 722 nmol/l to 231 nmol/l. Addition of bradykinin to outside-out patches previously exposed to intracellular Mg(2+)-GTP further enhanced KCa activity, shifting the apparent K0.5 for Ca2+ from 228 nmol/l to 107 nmol/l.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Bradykinin-activated calcium influx pathway in bovine aortic endothelial cells.

Clusters of electrically coupled endothelial cells were used to characterize a bradykinin (BK)-activated Ca2+ influx pathway. Spatial voltage control of clusters containing three to eight cells, evaluated as the ratio of the voltage response in one cell to a voltage pulse in the most distant cell of the cluster, was 0.96 at a holding potential of 0 mV in normal saline bath and 0.88 in the presence of BK. BK activated an inward current that was carried by either Na+ or Ca2+ when the membrane potential was held at -60 mV. Current was activated within 3 s of application of BK and peaked within 1 min. With Ca2+ as the permeable extracellular ion the current was stable for 1-3 min and then declined over a period of 5-8 min in the continued presence of BK. However, when Na+ carried the current it was sustained over a 10-min test period. The reversal potential of the BK-activated current was near 0 mV, suggesting activation of a nonspecific cation channel(s). The inward current at -60 mV averaged 13 +/- 4.5 pA (n = 9)/cell in Ca2+ and 12.2 +/- 9.3 pA (n = 5)/cell in Na+. Both Na+ and Ca2+ currents were blocked by 200 microM lanthanum.

Animals↗
PubMed1992

Characterization of calcium currents in aortic baroreceptor neurons.

1. Calcium currents in identified rat aortic baroreceptors were characterized with the perforated patch whole-cell voltage-clamp technique. Aortic baroreceptors were distinguished from other neurons by the presence of a fluorescent tracer that was previously applied to the aortic depressor nerve. The diversity of calcium currents in unidentified neurons dissociated from neonatal rat nodose ganglia were also examined. 2. A population of aortic baroreceptors (63%, 7 of 11) possessed a low-threshold, also referred to as a T-type, calcium current. This current was typically less than 100 pA in 2 mM Ca [72.7 +/- 20.9 (SE) pA, n = 7], had a rapid activation and inactivation, and inactivated completely at conditioning voltages positive to -50 mV. 3. All aortic baroreceptors possessed high-threshold calcium currents that were activated at voltages positive to -30 mV, with typical maximum amplitudes of 600-1,000 pA (826 +/- 79 pA, n = 11). 4. The high-threshold current inactivated with three exponential rates of decay of tau = 10.7 +/- 2.2 ms, 138 +/- 14.6 ms, and a third tau greater than 3 s. It was not possible to separate the kinetic components of inactivation with conditioning voltages (voltage-dependent inactivation), activation thresholds, deactivation kinetics, or calcium-channel antagonists. 5. The voltage-dependent inactivation of high-threshold calcium currents began at voltages positive to -70 mV and became steeply voltage dependent between -60 and -10 mV. Unexpectedly, the three decay constants were present after all conditioning voltages. There were no conditioning voltages that excluded any component.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Characterization of the effects of a new Ca2+ channel activator, FPL 64176, in GH3 cells.

We examined the effects of the benzolpyrrole-type Ca2+ channel activator FPL 64176 on voltage-dependent L-type Ca2+ channels in rat anterior pituitary (GH3) cells. FPL 64176 increased K(+)-dependent Ca2+ influx into GH3 cells with an EC50 value of 1.2 x 10(-7) M but had no effect on the binding of [3H]PN200-110 to GH3 cell membranes at concentrations up to 10(-6) M. Whole-cell patch-clamp electrophysiology revealed that FPL 64176 (1 microM) increased L-type Ca2+ channel current amplitude and shifted the current-voltage relationship in the hyperpolarizing direction. Furthermore, Ca2+ channel current activation and deactivation were prolonged. Single-channel analysis showed that FPL 64176 increased both the probability of channel opening and the mean channel open time. Interestingly, the effect of FPL 64176 on channel open time was highly voltage dependent, with much longer openings being observed at more hyperpolarized potentials. We conclude that FPL 64176 represents a new class of L-type Ca2+ channel activator with a novel site and mechanism of action.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1991

Identification and dissociation of cardiovascular neurons from the medulla for patch clamp analysis.

This study describes a preparation that will enable us to study, using voltage clamp techniques, ionic currents from dissociated cardiovascular neurons that have retained their anatomical and functional identity of the intact animal. To identify dispersed preganglionic cardiac motoneurons various fluorescent dyes (rhodamine, fluorogold, microspheres, bizbenzimide and dextrans) were examined to determine which can be absorbed by preganglionic cardiac motorneuron nerve terminals (without surgical penetration of cardiac tissue), transported retrogradely to their soma in the medulla and retained during dissociation of the neurons. Rhodamine fulfilled these criteria. Dissociated preganglionic cardiac motorneurons had resting membrane potentials of -52.4 +/- 3 mV and input resistances of 236 +/- 71 M omega (mean +/- S.E.M., n = 10). Depolarizing voltage steps to -50 mV or above evoked a tetrodotoxin (TTX) sensitive inward sodium current followed by a biphasic outward current.

Animals↗
PubMed1991

Somatostatin inhibits insulin secretion by a G-protein-mediated decrease in Ca2+ entry through voltage-dependent Ca2+ channels in the beta cell.

We tested the hypothesis that somatostatin (SRIF) inhibits insulin secretion from an SV40 transformed hamster beta cell line (HIT cells) by an effect on the voltage-dependent Ca2+ channels and examined whether G-proteins were involved in the process. Ca2+ currents were recorded by the whole cell patch-clamp method, the free cytosolic calcium, [Ca2+]i, was monitored in HIT cells by fura-2, and cAMP and insulin secretion were measured by radioimmunoassay. SRIF decreased Ca2+ currents, [Ca2+]i, and basal insulin secretion in a dose-dependent manner over the range of 10(-12)-10(-7)M. The increase in [Ca2+]i and insulin secretion induced by either depolarization with K+ (15 mM) or by the Ca2+ channel agonist, Bay K 8644 (1 microM) was attenuated by SRIF in a dose-dependent manner over the same range of 10(-12)-10(-7) M. the half-maximal inhibitory concentrations (IC50) for SRIF inhibition of insulin secretion were 8.6 X 10(-12) M and 8.3 X 10(-11) M for K+ and Bay K 8644-stimulated secretion and 1 X 10(-10) M and 2.9 X 10(-10) M for the SRIF inhibition of the K+ and Bay K 8644-induced rise in [Ca2+]i, respectively. SRIF also attenuated the rise in [Ca2+]i induced by the cAMP-elevating agent, isobutylmethylxanthine (1 mM) in the presence of glucose. Bay K 8644, K+ and SRIF had no significant effects on cAMP levels and SRIF had no effects on adenylyl cyclase activity at concentrations lower than 1 microM. SRIF (100 nM) did not change K+ efflux (measured by 86Rb+) through ATP-sensitive K+ channels in HIT cells. SRIF (up to 1 microM) had no significant effect on membrane potential measured by bisoxonol fluorescence. Pretreatment of the HIT cells with pertussis toxin (0.1 microgram/ml) overnight abolished the effects of SRIF on Ca2+ currents, [Ca2+]i and insulin secretion implying a G-protein dependence in SRIF's actions. Thus, one mechanism by which SRIF decreases insulin secretion is by inhibiting Ca2+ influx through voltage-dependent Ca2+ channels, an action mediated through a pertussis toxin-sensitive G-protein.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1991

Sulfonylurea signal transduction.

In the pancreatic beta cells the proximal step in sulfonylurea signal transduction is the binding of these clinically important drugs to high-affinity receptors in the beta cell membrane. Using HIT cells as a model system, we have established an extremely close correlation between the affinity of binding of glyburide and its analog, iodoglyburide, and the activation of various steps in stimulus-secretion coupling--inhibition of 86Rb+ efflux, increase in [Ca2+]i resulting from gating of voltage-gated calcium channels by cell depolarization, and the exocytosis of insulin. Two different L-type channel cDNAs have been identified in an HIT cell library, one neuroendocrine in type and one more cardiac-like. A HIT cell membrane protein of Mr 140,000, which we believe to be the high-affinity sulfonylurea receptor, can be covalently linked to 5(125)-iodo-2-hydroxyglyburide by ultraviolet irradiation. The receptor has been solubilized and retains binding activity and the same rank order of displacement of the 5(125)-iodo-2-hydroxyglyburide as observed with the native receptor. The Mr 140,000 protein has been partially purified and the amino acid sequences of three proteolytic fragments have been used to design oligonucleotides to screen HIT cell cDNA libraries. Since the binding constant of glyburide or iodoglyburide is closely correlated with the ability of these compounds to inhibit the ATP-sensitive K+ channel, increase [Ca2+]i, and elicit insulin secretion, we have identified the Mr 140,000 protein as the sulfonylurea receptor. Expression of the cloned cDNA should allow us to test this hypothesis directly.

ATP-Binding Cassette Transporters↗
PubMed1990

Bradykinin-induced potassium current in cultured bovine aortic endothelial cells.

Bovine aortic endothelial cells (BAECs) respond to bradykinin with an increase in cytosolic-free Ca2+ concentration, [Ca2+]i, accompanied by an increase in surface membrane K+ permeability. In this study, electrophysiological measurement of K+ current was combined with 86Rb+ efflux measurements to characterize the K+ flux pathway in BAECs. Bradykinin- and Ca2(+)-activated K+ currents were identified and shown to be blocked by the alkylammonium compound, tetrabutylammonium chloride and by the scorpion toxin, noxiustoxin, but not by apamin or tetraethylammonium chloride. Whole-cell and single-channel current analysis suggest that the threshold for Ca2+ activation is in the range of 10 to 100 nM [Ca2+]i. The whole-cell current measurements show voltage sensitivity only at the membrane potentials more positive than 0 mV where significant current decay occurs during a sustained depolarizing pulse. Another K+ current present in control conditions, an inwardly rectifying K+ current, was blocked by Ba2+ and was not affected by noxiustoxin or tetrabutylammonium chloride. Efflux of 86Rb+ from BAEC monolayers was stimulated by both bradykinin and ionomycin. Stimulated efflux was blocked by tetrabutyl- and tetrapentyl-ammonium chloride and by noxiustoxin, but not by apamin or furosemide. Thus, 86Rb+ efflux stimulated by bradykinin and ionomycin has the same pharmacological sensitivity as the bradykinin- and Ca2(+)-activated membrane currents. The results confirm that bradykinin-stimulated 86Rb+ efflux occurs via Ca2(+)-activated K+ channels. The blocking agents identified may provide a means for interpreting the role of the Ca2(+)-activated K+ current in the response of BAECs to bradykinin.

Animals↗
PubMed1990

Multiple conductance levels of the dihydropyridine-sensitive calcium channel in GH3 cells.

Calcium channels in GH3 cells exhibit at least five conductance levels when examined in cell-attached or outside-out patches. These channels resemble the high threshold Ca2+ current in their range of activation and inactivation, and in their sensitivity to dihydropyridines (DHP). Mean open times for the five levels were brief (less than 1 msec) in control solutions but increased in the presence of BAY K 8644. In 100 mM Ba2+ and BAY K 8644, the five predominant slope conductances were 8-9, 12-13, 16-18, 23-24, and 28 pS. The present study is the first report of multiple levels of the DHP-sensitive Ca2+ channel occurring with high frequency in native membranes. The range of conductance levels that we observed encompasses the range of conductances found for two other different types of Ca2+ channels and indicates that unit conductance should be used with caution as a distinguishing characteristic for identification of different channel types.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1990

Excitatory amino acid receptors of guinea pig medial nucleus tractus solitarius neurons.

Neurons isolated from the medial subnuclei of nucleus tractus solitarius in adult guinea pigs were studied for responses to the excitatory amino acid glutamate and its analogues using the whole cell tight-seal voltage clamp technique. In 80% of the cells studied (n = 60) 100 microM glutamate produced inward currents at negative voltages. To further characterize the glutamate response, the agonists for three glutamate receptor subtypes, N-methyl-D-aspartate (NMDA), kainate, and quisqualate, were examined for their effects on membrane conductance. NMDA (25-250 microM) activated currents in 85% of the neurons tested (n = 30). NMDA currents were generally very small in amplitude. Of the neurons tested, 84% responded to kainate (10-30 microM, n = 19) and only 50% to quisqualate (25-50 microM, n = 26). The conductance activated by NMDA was outwardly rectifying. The conductance activated by kainate was voltage independent, while that activated by quisqualate showed varying degrees of outward rectification. Responses to NMDA were specifically antagonized by DL-2-amino-5-phosphonovaleric acid (AP-5, 50-100 microM). Kainate responses were blocked by kynurenate at concentrations (0.5-1.5 mM) ineffective on quisqualate-induced current. Glutamic acid diethyl ester (GDEE, 2-15 mM) was effective in reducing quisqualate responses at concentrations that had no effect on kainate responses. This characterization of the glutamate receptor subtypes and effective antagonists provides a basis for future determination of the specific receptor of glutamate responsible for mediation of the excitatory postsynaptic potentials produced by activation of the baroreceptor input.

Amino Acids↗
PubMed1990

Ion channels and insulin secretion.

We review the role of ion channels in regulating insulin secretion from pancreatic beta-cells. By controlling ion permeability, ion channels at the membrane play a major role in regulating both electrical activity and signal transduction in the beta-cell. A proximal step in the cascade of events required for stimulus-secretion coupling is the closure of ATP-sensitive K+ channels, resulting in cell depolarization. Of particular relevance is the finding that this channel is directly regulated by a metabolite of glucose, which is the primary insulin secretagogue. In addition, this channel, or a closely associated protein, contains the sulfonylurea-binding site. Another K+ channel, the Ca2(+)-activated K+ channel, may be involved in cell repolarization to create homeostasis. Voltage-dependent Ca2+ channels are activated by cell depolarization and regulate Ca2+ influx into the cell. By controlling cytosolic free-Ca2+ levels ([Ca2+]i), these channels play an important role in transducing the initial stimulus to the effector systems that modulate insulin secretion. The link between a rise in [Ca2+]i and the terminal event of exocytosis is the least-understood aspect of stimulus-secretion coupling. However, phosphorylation studies have identified substrate proteins that may correspond to those involved in smooth muscle contraction, suggesting an analogy in the processes of stimulus secretion and excitation contraction. The advent of new methodology, particularly the patch-clamp technique, has fostered a more detailed characterization of the beta-cell ion channels. Furthermore, biochemical and molecular approaches developed for the structural analysis of ion channels in other tissues can now be applied to the isolation and characterization of the beta-cell ion channels. This is of particular significance because there appear to be tissue-specific variations in the different types of ion channels. Given the importance of ion channels in cell physiology, a knowledge of the structure and properties of these channels in the beta-cell is required for understanding the abnormalities of insulin secretion that occur in non-insulin-dependent diabetes mellitus. Ultimately, these studies should also provide new therapeutic approaches to the treatment of this disease.

Animals↗
PubMed1989

Localization and mobility of omega-conotoxin-sensitive Ca2+ channels in hippocampal CA1 neurons.

Voltage-dependent Ca2+ channels (VDCCs) are modulators of synaptic plasticity, oscillatory behavior, and rhythmic firing in brain regions such as the hippocampus. The distribution and lateral mobility of VDCCs on CA1 hippocampal neurons have been determined with biologically active fluorescent and biotinylated derivatives of the selective probe omega-conotoxin in conjunction with circular dityndallism, digital fluorescence imaging, and photobleach recovery microscopy. On noninnervated cell bodies, VDCCs were found to be organized in multiple clusters, whereas after innervation the VDCCs were concentrated and immobilized at synaptic contact sites. On dendrites, VDCC distribution was punctate and was interrupted by extensive bare regions or abruptly terminated. More than 85% of the dendritic VDCCs were found to be immobile by fluorescence photobleach recovery. Thus, before synaptic contact, specific mechanisms target, segregate, and immobilize VDCCs to neuronal cell bodies and to specialized dendritic sites. Regulation of this distribution may be critical in determining the firing activity and integrative properties of hippocampal CA1 neurons.

Animals↗
PubMed1989

Catecholamine modulation of calcium currents in clonal pancreatic beta-cells.

The mechanisms by which norepinephrine and epinephrine activate alpha 2-adrenergic receptors and inhibit insulin release from the pancreatic beta-cell (19, 21, 23) are not yet clear but may involve modulation at several sites. Because intracellular calcium has been implicated in the secretory process, it has been suggested that catecholamines may inhibit secretion by blocking calcium influx, thus reducing the free cytosolic calcium concentration (23). The present study examines the effects of epinephrine, norepinephrine, and clonidine on calcium current in an SV40-transformed hamster beta-cell line (HIT cells). Under voltage-clamp conditions, calcium currents were reversibly inhibited by norepinephrine, epinephrine, and clonidine in the low nanomolar range. The effects were blocked by 1) the alpha 2-antagonist yohimbine, 2) preincubation of the cells with pertussis toxin (PTX), and 3) guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), the nonhydrolyzable GDP analogue that competitively inhibits the interaction of GTP with G proteins. In contrast, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) caused irreversible blockade by catecholamines. These effects could not be overcome by adenosine 3',5'-cyclic monophosphate (cAMP), suggesting that the adenylate cyclase pathway is not involved in the G protein coupling with the channels. These studies show that catecholamines inhibit calcium currents in beta-cells through an alpha 2-adrenoreceptor PTX-sensitive G protein pathway and could inhibit insulin secretion by this mechanism.

Adenylate Cyclase Toxin↗
PubMed1989

Characterization of voltage-dependent Ca2+ channels in beta-cell line.

Although there is compelling pharmacological evidence based on Ca2+-channel antagonist studies suggesting that the voltage-dependent Ca2+ channels regulate insulin release, no direct comparison with Ca2+ currents exists. This is particularly important because of the recent demonstration in other cell types of one and possibly two Ca2+ channels that are insensitive to Ca2+-channel antagonists, the dihydropyridines and the phenylalkylamines. Using an SV40-transformed pancreatic beta-cell line (HIT cells), we determined how voltage-dependent Ca2+ channels are involved in stimulus-secretion coupling. Ca2+ currents were measured with the tight-seal technique for whole-cell recording. The cytosolic free-Ca2+ concentration ([Ca2+]i) was followed with the fluorescent probe Fura 2, and the measurements were compared with insulin secretion stimulated by depolarizing the cells with K+. The Ca2+ current contained two components: a rapidly decaying current activated at -50 to -40 mV that decayed with a time constant of 25 ms and a very slowly decaying component activated at -40 mV. Both components were sensitive to the Ca2+-channel antagonist nimodipine. There is excellent agreement in the concentration of nimodipine that inhibited Ca2+ and the increase in [Ca2+]i in response to K+ depolarization (IC50 of 15 and 6 nM, respectively). Nimodipine inhibited insulin release over a similar dose-response range with an IC50 of 1.5 x 10(-9) M. These studies indicate that the increase in [Ca2+]i in response to beta-cell depolarization can be accounted for by the influx of this ion through a single class of dihydropyridine-sensitive Ca2+ channels in the cell membrane.

Animals↗