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R Y Pun

Publications and source records attributed to R Y Pun.

At least 19 recordsLinked to original sources
PubMed1998

Adenosine modulates hypoxia-induced responses in rat PC12 cells via the A2A receptor.

1. The present study was undertaken to determine the role of adenosine in mediating the cellular responses to hypoxia in rat phaeochromocytoma (PC12) cells, an oxygen-sensitive clonal cell line. 2. Reverse transcriptase polymerase chain reaction studies revealed that PC12 cells express adenosine deaminase (the first catalysing enzyme of adenosine degradation) and the A2A and A2B adenosine receptors, but not the A1 or A3 adenosine receptors. 3. Whole-cell current- and voltage-clamp experiments showed that adenosine attenuated the hypoxia-induced membrane depolarization. The hypoxia-induced suppression of the voltage-sensitive potassium current (IK(V)) was markedly reduced by adenosine. Furthermore, extracellularly applied adenosine increased the peak amplitudes of IK(V) in a concentration-dependent manner. This increase was blocked by pretreatment not only with a non-specific adenosine receptor antagonist, 8-phenyltheophylline (8-PT), but also with a selective A2A receptor antagonist, ZM241385. 4. Ca2+ imaging studies using fura-2 acetoxymethyl ester (fura-2 AM) revealed that the increase in intracellular free Ca2+ during hypoxic exposure was attenuated significantly by adenosine. Voltage-clamp studies showed that adenosine inhibited the voltage-dependent Ca2+ currents (ICa) in a concentration-dependent fashion. This inhibition was also abolished by both 8-PT and ZM241385. 5. The modulation of both IK(V) and ICa by adenosine was prevented by intracellular application of an inhibitor of protein kinase A (PKA), PKA inhibitor fragment (6-22) amide. In addition, the effect of adenosine on either IK(V) or ICa was absent in PKA-deficient PC12 cells. 6. These results indicate that the modulatory effects of adenosine on the hypoxia-induced membrane responses of PC12 cells are likely to be mediated via activation of the A2A receptor, and that the PKA pathway is required for these modulatory actions. We propose that this modulation serves to regulate membrane excitability in PC12 cells and possibly other oxygen-sensitive cells during hypoxia.

Adenosine↗
PubMed1997

Defective endothelium-dependent relaxation of vascular smooth muscle and endothelial cell Ca2+ signaling in mice lacking sarco(endo)plasmic reticulum Ca2+-ATPase isoform 3.

Sarco(endo)plasmic reticulum Ca2+ ATPase isoform 3 (SERCA3) is one of two Ca2+ pumps serving intracellular Ca2+ signaling pools in non-muscle tissues; however, unlike the ubiquitous SERCA2b, it exhibits a restricted cell-type distribution. Gene targeting was used to generate a mouse with a null mutation in the SERCA3 gene. Homozygous mutant mice were viable, fertile, and did not exhibit an overt disease phenotype. Because SERCA3 is expressed in arterial endothelial cells, aortic ring preparations were analyzed to determine whether it is involved in the regulation of vascular tone. Contraction-isometric force relations in response to phenylephrine or KCl, as well as relaxation produced by exposure to a nitric oxide donor, were similar in wild-type and null mutant aortas. Acetylcholine-induced endothelium-dependent relaxation of aortas after precontraction with phenylephrine was significantly reduced in homozygous mutants (61.3 +/- 5.6% in wild type, 35.4 +/- 7.3% in mutants). Ca2+ imaging of cultured aortic endothelial cells demonstrated that the acetylcholine-induced intracellular Ca2+ signal is sharply diminished in SERCA3-deficient cells and also indicated that replenishment of the acetylcholine-responsive Ca2+ stores is severely impaired. These results indicate that SERCA3 plays a critical role in endothelial cell Ca2+ signaling events involved in nitric oxide-mediated relaxation of vascular smooth muscle.

Acetylcholine↗
PubMed1997

Loss of protein kinase C inhibition in the beta-T594M variant of the amiloride-sensitive Na+ channel.

We previously reported the presence of a novel variant (beta-T594M) of the amiloride-sensitive Na+ channel (ASSC) in which the threonine residue at position 594 in the beta-subunit has been replaced by a methionine residue. Electrophysiological studies of the ASSC on Epstein-Barr virus (EBV)-transformed lymphocytes carrying this variant showed that the 8-(4-chlorophenylthio) adenosine 3':5'-cyclic monophosphate (8cpt-cAMP)-induced responses were enhanced when compared to wild-type EBV-transformed lymphocytes. Furthermore, in wild-type EBV-transformed cells, the 8cpt-cAMP-induced response was totally blocked by the phorbol ester, phorbol 12-myristate 13-acetate (PMA). This inhibitory effect of PMA was blocked by a protein kinase C inhibitor, chelerythrine. We now have identified individuals who are homozygous for this variant, and showed that PMA had no effect on the 8cpt-cAMP-induced responses in the EBV-transformed lymphocytes from such individuals. Cells heterozygous for this variant showed mixed responses to PMA, with the majority of cells partially inhibited by PMA. Our results demonstrate that an alteration in a single amino acid residue in the beta-subunit of the ASSC can lead to a total loss of inhibition to PMA, and establish the beta-subunit as having an important role in conferring a regulatory effect on the ASSC of lymphocytes.

Amiloride↗
PubMed1997

Norepinephrine-mediated calcium signaling is altered in vascular smooth muscle of diabetic rat.

We studied the influence of diabetes on norepinephrine (NE)-induced changes in intracellular free Ca2+ levels (receptor-mediated Ca2+ signaling) in single tail artery vascular smooth muscle (VSM) cells. VSM cells from 12-16 week streptozotocin-induced diabetic (SID) rats showed an increase in sensitivity to NE when compared to control VSM cells in that the concentration of NE needed to elicit half maximal response of the initial Ca2+ transient was reduced more than 4-fold though the maximal response attained was apparently reduced. In addition, the slope factor (steepness) of the dose-response relation was lowered 4-fold. Moreover, VSM cells of diabetic animals had a higher incidence of NE-induced Ca2+ oscillatory responses. The shift of the dose-response curve to the left, coupled with a higher incidence of oscillations, indicate that the noradrenergic receptor-mediated Ca2+ signaling pathways in tail artery VSM of diabetic rat may be altered.

Animals↗
PubMed1996

Persistence of the olfactory receptor current in a wide variety of extracellular environments.

We measured the current activated by cytoplasmic adenosine 3':5'-cyclic monophosphate (cAMP) in olfactory cilia from the frog Rana pipiens. The odorant-induced current in frog olfactory receptor neurons was also measured for comparison. In both cases, recordings were performed near the neuronal resting potential in a variety of extracellular bath solutions. 2. In Ca(2+)-free baths, cAMP activated an inward current in excised olfactory cilia that was carried entirely by cations. As extracellular Ca2+ was increased, the cationic current decreased while a second current, carried by C1-, increased. Total cAMP-activated current decreased with increasing extracellular CA2+. When external Na+ but not Ca2+ was eliminated, only the C1- component of the current persisted. When external Na+ and Ca2+ were both removed, there was no cAMP-activated current. 3. In receptor neurons, the total odorant-induced receptor current varied in a similar way with the extracellular ionic environment. Under conditions favoring the anionic receptor current, the response amplitude decreased and the latency increased. 4. It is known that olfactory receptor currents persist in a wide variety of extracellular environments. This persistence can be sufficiently explained by the balance between cationic and anionic currents demonstrated here.

Animals↗
PubMed1996

A novel variant of the beta-subunit of the amiloride-sensitive sodium channel in African Americans.

The amiloride-sensitive sodium channel is responsible for the rate-limiting step of sodium reabsorption in the distal renal tubule, and thus may play a key role in the maintenance of sodium balance and blood pressure. In this study, a genetic variant that results in a change of threonine to methionine at amino acid 594 (T594 M) in the carboxy-terminus of the beta-subunit of the amiloride-sensitive sodium channel has been identified. This variant was present in 6.1% of African-American subjects (N = 231) but was not seen in Caucasians (N = 192). Whole cell voltage clamp of B-lymphocytes from individuals with the T594 M variant showed similar basal membrane slope conductance, compared with the wild-type but increased response to cAMP analog.

Aldosterone↗
PubMed1994

Guanine nucleotides modulate steady-state inactivation of voltage-gated sodium channels in frog olfactory receptor neurons.

The voltage for half-inactivation (V1/2) of Na+ currents in frog olfactory receptor neurons (ORNs) under whole-cell voltage clamp showed a shift to more negative potentials with time. Inclusion of guanosine triphosphate (GTP) or its nonhydrolyzable analogue, guanosine-5'-O-3-thiotriphosphate (GTP-gamma-S), which activates G proteins, in the recording pipette, not only gave a more positive V1/2, but also reduced and delayed the negative shift observed in the absence of nucleotides. Guanosine-5'-O-2-thiodiphosphate (GDP-beta-S), a nonhydrolyzable analogue that prevents the binding of GTP to G proteins, did not affect the V1/2 significantly by itself but blocked the positive shift induced by GTP. Since the steady-state activation was not affected, our results indicate that a G protein or a G-protein-dependent process may be important in regulating the steady-state inactivation of Na+ channels in ORNs of the frog.

Animals↗
PubMed1994

G-protein activation mediates prepulse facilitation of Ca2+ channel currents in bovine chromaffin cells.

The effects of G-protein activation were investigated on tonic, large depolarization-induced Ca2+ channel facilitation in cultured bovine adrenal chromaffin cells. Under whole-cell voltage clamp, activation of G proteins by intracellular dialysis with 200 microM GTP-gamma S did not significantly affect prepulse facilitation or whole-cell Ba2+ current (IBa) density. In contrast, inactivation of G proteins by intracellular GDP-beta S or pertussis toxin (PTX) pretreatment completely abolished or markedly attenuated facilitation of IBa, respectively. GDP-beta S dialysis resulted in nearly a threefold increase in peak IBa density, whereas PTX pretreatment resulted in a 50% increase. Our results indicate that under control recording conditions (200 microM intracellular GTP), G proteins are tonically activated and suppress high-voltage-activated (HVA) Ca2+ channels in a voltage-dependent and voltage-independent manner. Local superfusion of chromaffin cells with normal bath solution produced a rapid and reversible increase (approximately 50%) in IBa amplitudes that also abolished prepulse facilitation. Together, these results demonstrate that tonic facilitation of HVA Ca2+ channels in bovine chromaffin cells involves the voltage-dependent relief of a G-protein-mediated suppression, imposed by chromaffin cell secretory products that feedback and activate G-protein-coupled autoreceptors.

Adenosine Triphosphate↗
PubMed1994

Angiotensin II suppresses Na+ currents in bovine adrenal chromaffin cells.

We investigated the effects of angiotensin II (ANG II) on the voltage-dependent Na+ channel currents (INa) recorded from bovine adrenal medullary chromaffin cells (BCCs) under whole-cell voltage clamp. Angiotensin II reversibly reduced the peak INa in a dose-dependent fashion. Inhibition was observed at a concentration of 1 nM (6.3 +/- 1.4%, mean +/- SEM) and reached a maximum at 1 microM (35 +/- 3.8%), with a half-maximal effect at 11.6 nM. The ANG II-induced inhibition resulted from a reduction in peak conductance (control, 7.2 +/- 0.7 nS; ANG II 4.3 +/- 0.5 nS; p < 0.01). Angiotensin II had no effect on the reversal potential or the decay time of INa. In addition, the V1/2 and k values, two parameters that describe the voltage dependence of INa for both steady-state activation and inactivation, were not affected by ANG II. The response to ANG II (1 microM) had a delay and attained maximum inhibition in 0.9 +/- 0.2 min (n = 10). Recovery from the effect was slow and took 3.5 +/- 0.8 min (n = 10) after the application of ANG II had been terminated. The inhibitory effects of ANG II were effectively blocked by a specific ANG II receptor antagonist. [Sar1, Val5, Ala8]ANG II. The present study demonstrates that ANG II inhibits voltage-dependent INa+ channel currents in BCCs via a specific receptor-coupled mechanism. The prolonged time course of the ANG II response indicates a possible involvement of second messenger(s) mediating this inhibition.

Adrenal Medulla↗
PubMed1993

Activation of serotonin1A receptors inhibits midbrain periaqueductal gray neurons of the rat.

The midbrain periaqueductal gray (PAG) is involved in a variety of functions including pain modulation, vocalization, autonomic control, fear and anxiety. This area contains serotonin receptors, particularly 5-HT1A that are known to play a role in the above functions. The goals of this study were to characterize the effects of 8-OH-DPAT, a selective 5-HT1A agonist, on the firing characteristics and membrane properties of PAG neurons. Both in vivo and in vitro preparations were used. The effects of 8-OH-DPAT on baseline activity of 91 neurons were tested in the in vivo preparation. In 50/91 cells, 8-OH-DPAT produced a decrease in the firing rate that ranged between 21 and 98% (mean +/- S.E.M. decrease of 49 +/- 1.9%). This inhibitory effect was dose dependent and could be blocked by spiperone. In 10/91 cells, 8-OH-DPAT produced an increase in the firing rate that ranged between 13 and 290%, with mean increase of 83 +/- 7.4%. The baseline firing rate of the remaining 31 cells was not affected by 8-OH-DPAT. In the PAG slice preparation, the effects of 8-OH-DPAT on synaptic and membrane properties of 17 PAG neurons were tested using whole-cell voltage clamp-recording procedures. In 14 cells, application of 8-OH-DPAT produced hyperpolarization that ranged between 6 and 21 mV, with mean of 8.4 +/- 2.0 mV. This hyperpolarization was associated with a decrease in membrane impedance that ranged between 8 and 45%, with mean decrease of 21.6 +/- 4.5%. The remaining three neurons did not respond to 8-OH-DPAT.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1992

Cyclic AMP-dependent phosphorylation modifies the gating properties of L-type Ca2+ channels in bovine adrenal chromaffin cells.

We investigated the effects of cAMP-dependent phosphorylation on the voltage- and time-dependent gating properties of Ca2+ channel currents recorded from bovine adrenal chromaffin cells under whole-cell voltage clamp. Extracellular perfusion with the membrane-permeant activator of cAMP-dependent protein kinase, 8-bromo(8-Br)-cAMP (1 mM), caused a 49%, 29%, and 21% increase in Ca2+ current (ICa) amplitudes evoked by voltage steps to 0, +10, and +20 mV respectively (mean values from eight cells, p less than or equal to 0.05). Analysis of voltage-dependent steady-state activation (m infinity) curves revealed a 0.70 +/- 0.27 charge increase in the activation-gate valency (zm) following 8-Br-cAMP perfusion. Similar responses were observed when Ba2+ was the charge carrier, where zm was increased by 1.33 +/- 0.34 charges (n = 8). The membrane potential for half activation (V1/2) was also significantly shifted 6 mV more negative for IBa (mean, n = 8). The time course for IBa (and ICa) activation was well described by second-order m2 kinetics. The derived time constant for activation (tau m) was voltage-dependent, and the tau m/V relation shifted negatively after 8-Br-cAMP treatment. Ca2+ channel gating rates were derived from the tau m and m infinity 2 values according to a Hodgkin-Huxley type m2 activation process. The forward rate (alpha m) for channel activation was increased by 8-Br-cAMP at membrane potentials greater than or equal to 0 mV, and the backward rate (beta m) decreased at potentials less than or equal to + 10 mV. Time-dependent inactivation of ICa consisted of a slowly decaying component (tau h approximately 300 ms) and a "non-inactivating" steady-state component. The currents contributed by the two inactivation processes displayed different voltage dependences, the effects of 8-Br-cAMP being exclusively on the slowly inactivating L-type component.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1991

Single cell assay of exocytosis from adrenal chromaffin cells using "perforated patch recording".

We have combined "perforated patch recording" with phase detection to examine depolarization-induced changes in membrane capacitance (delta Cm) in bovine adrenal chromaffin cells. With this technique, voltage dependent Ca2+ currents and resultant delta Cm's often show little rundown over 1-2 hours even when the free Ca2+ concentration of the pipette is in the millimolar range. By limiting washout of cytosolic components and by maintaining more intact cytosolic Ca2+ buffering, this approach should facilitate the study of stimulus-exocytosis coupling evoked by physiological stimuli which involve cell metabolism and/or membrane receptor triggered second messenger cascades.

Adrenal Medulla↗
PubMed1991

Somatic sodium channels of frog olfactory receptor neurones are inactivated at rest.

Membrane excitability of acutely isolated olfactory receptor neurones (ORNs) of the grass frog (R. pipiens) was studied with the use of the whole-cell "tight-seal" patch recording technique. ORNs of the frog had a mean resting membrane potential of -52 mV, a mean input resistance of 1-2 G omega, and a mean capacitance of 4.5 pF. In the majority of cells examined (over 70%), short duration (several milliseconds) action potentials were elicited at the end of a hyperpolarising pulse (off-spike) or following hyperpolarization of the membrane potential by injection of current. Under voltage-clamp conditions, a fast inward current followed by an outward current could be evoked upon depolarisation of the membrane. The fast inward current decayed with a time constant of 1-2 ms, with an e-fold decrease per 52 mV increase in voltage, and was blocked by the selective voltage-dependent sodium channel blocker tetrodotoxin (0.5-1 microM). Steady-state inactivation studies revealed that the mean voltage for half-inactivation (V1/2) was -82 mV (range -72 to -98 mV), which indicates that the voltage-dependent Na+ channels in the cell body or soma of frog ORNs are not available for conducting currents at the resting membrane potential. This finding raises the possibility that voltage-dependent Na+ channels may not play a significant role in sensory transduction at the soma. Our results indicate that ORNs of the frog are very efficient in transducing signals towards the brain since currents generated at the cilia will be directed towards depolarising the axons.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Long-term monitoring of depolarization-induced exocytosis from adrenal medullary chromaffin cells and pancreatic islet B cells using "perforated patch recording".

(1) Membrane capacitance measurements using perforated patch recording offer the possibility of studying the process of depolarization-secretion coupling (DSC) in single endocrine cells with unprecedented time resolution and stability. (2) Early results with catecholamine-secreting adrenal chromaffin cells and insulin-secreting pancreatic B cells support longstanding ideas that the Ca(2+)-dependent processes underlying DSC are fundamentally similar to those of nerve terminals. (3) Future experiments using these approaches should prove useful in sorting out those effects of humoral substances that have a predominant effect on excitability and Ca2+ entry from those that affect the secretory process itself.

Adrenal Medulla↗
PubMed1990

Cultured rat olfactory neurons are excitable and respond to odors.

Newborn rat nasal tissues containing olfactory epithelium were dissociated and maintained in a monolayer cell culture. Neurons were present, as determined by immunostaining with antibodies to 4 neuron-specific proteins: neuron-specific enolase, microtubule-associated protein 2, tau protein and synaptophysin. Immunostained neurons had a distinctive morphology resembling olfactory neurons. By patch-clamp analysis, these cells were electrically active. Responses of some neurons to physiological concentrations of an odorant mixture identified them as olfactory receptor cells.

Action Potentials↗
PubMed1990

Thyrotropin-releasing hormone has profound presynaptic action on cultured spinal cord neurons.

Thyrotropin-releasing hormone (TRH) receptors are widely distributed throughout the nervous system. In particular, both the dorsal and the ventral horn (VH) neurons contain a rich distribution of TRH receptors, and TRH application to these sites has profound physiological effects. Currently the mechanism of action of TRH is not known. We examined the effect of TRH on ventral horn neurons using intracellular and patch-clamp techniques. Our results indicate that TRH application profoundly increases the firing rate of VH cells by decreasing membrane conductance. More importantly, TRH causes a significant increase in frequency and amplitude of postsynaptic potentials. Under voltage-clamp condition, TRH reduces holding current and causes a significant increase in the rate of occurrence and the amplitude of excitatory postsynaptic currents (EPSCs), an effect that lasts for more than 5 minutes. This effect of TRH is not observed in cultured neurons pretreated with tetanus toxin. TRH also fails to alter the characteristics of the EPSCs when it is applied to a region of the cell that is sparsely innervated. These results provide strong evidence that presynaptic mechanisms have a significant role in the excitatory effect of TRH on the VH neurons. Because there is evidence that trophic factors are released from presynaptic terminals, by increasing synaptic activity, TRH can have a trophic influence on the spinal cord neurons. In addition, because there are a significant number of TRH containing neurons within the spinal cord, it is likely that TRH has a major role in information processing within the spinal cord.

Animals↗
PubMed1990

A rapidly inactivating Ca2(+)-dependent K+ current in pheochromocytoma cells (PC12) of the rat.

The membrane electrical properties of undifferentiated pheochromocytoma cells of the rat (PC12) were studied using both current- and voltage-clamp techniques with the use of low-resistance blunt-tipped micropipettes (patch electrodes). In the presence of tetrodotoxin (TTX, 2-3 microM), a spike-like wave form with a prominent after-hyperpolarization (AHP) was recorded following brief (less than 10 ms) depolarizing current pulses. The inorganic divalent cations, Cd2+ (0.5 mM), Mn2+ (4 mM), and 0 mM Ca2+/4 mM Mg2+ solution prolonged the duration, attenuated the AHP, slowed the rate of repolarization, and slightly enhanced the amplitude of this wave form. A rapidly inactivating outward current was recorded in over 70% of the cells under voltage-clamp conditions. This transient current was elicited at about -30 mV, and was blocked by tetraethylammonium (5 mM), inorganic divalent cations (Cd2+, 0.5 mM; Mn2+, 4 mM; Ba2+, 3 mM), and removal of Ca2+ (0 mM Ca2+/4 mM Mg2+) from the local perfusion medium. In addition, 4-aminopyridine (5 mM), which blocks the transient outward K+ current IA in a variety of excitable cells, did not have any appreciable effect on this rapidly inactivating current. Moreover, it was possible to elicit the current at a holding potential of -40 mV. The reversal potential of this current was -90 mV, and shifted positively when extracellular K+ concentrations were elevated. It is concluded that PC12 cells have a rapidly inactivating Ca2(+)-dependent K+ current. A possible explanation for the transient nature of this current may be the presence of an effective intracellular Ca2+ buffering (uptake or extrusion) system.

Adrenal Gland Neoplasms↗