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PubMed1995

Characterization of the G protein coupling of SRIF and beta-adrenergic receptors to the maxi KCa channel in insulin-secreting cells.

Modulation of the Ca- and voltage-dependent K channel--KCa--by receptors coupled to the G proteins G(i)/G(o) and Gs has been studied in insulin-secreting cells using the patch clamp technique. In excised outside-out patches somatostatin (somatotropin-releasing inhibitory factor; SRIF) caused concentration-dependent inhibition of the KCa channel, an effect that was prevented by pertussis toxin (PTX). In inside-out patches, exogenous alpha subunits of either G(i)- or G(o)-type G proteins also inhibited the KCa channel (IC50 5.9 and 5.7 pM, respectively). These data indicate that SRIF suppresses KCa channel activity via a membrane-delimited pathway that involves the alpha subunits of PTX-sensitive G proteins G(i) and/or G(o). In outside-out patches, activation of Gs either by beta-agonists or with cholera toxin (CTX) increased KCa channel activity, consistent with a membrane-delimited stimulatory pathway linking the beta-adrenergic receptor to the KCa channel via Gs. In outside-out patches, channel inhibition by SRIF suppressed the stimulatory effect of beta-agonists but not that of CTX, while in inside-out patches CTX reversed channel inhibition induced by exogenous alpha i or alpha o. Taken together these data suggest that KCa channel activity is enhanced by activation of Gs and blocked by activated G(i) and/or G(o). Further, KCa channel stimulation by activated Gs may be "direct," while inhibition by G(i)/G alpha may involve deactivation of Gs. In inside-out patches KCa channel activity was reduced by an activator of protein kinase C (PKC) and enhanced by inhibitors of PKC, indicating that PKC also acts to inhibit the KCa channel via a membrane delimited pathway. In outside-out patches, chelerythrine, a membrane permeant inhibitor of PKC prevented the inhibitory effect of SRIF, and in inside-out patches PKC inhibitors prevented the inhibitory effect of exogenous alpha i or alpha o. These data indicate that PKC facilitates the inhibitory effect of the PTX-sensitive G proteins which are activated by coupling to SRIF receptors. To account for these results a mechanism is proposed whereby PKC may be involved in G(i)/G(o)-induced deactivation of Gs.

Calcium↗

PubMed1994

Characterization of the G protein coupling of a glucagon receptor to the KATP channel in insulin-secreting cells.

The G-protein-mediated coupling of a glucagon receptor to ATP-dependent K channels--KATP--has been studied in insulin-secreting cells using the patch clamp technique. In excised outside-out patches, KATP channel activity was inhibited by low concentrations of glucagon (IC50 = 2.4 nM); the inhibitory effect vanished at concentrations greater than 50 nM. In cell-attached patches, inhibition by bath-applied glucagon was seen most often, although stimulation was observed in a few cases. A dual action of the hormone is proposed to resolve these apparently divergent results. In excised inside-out patches, KATP channel activity was inhibited by addition of beta gamma subunits purified from either erythrocyte or retina (IC50 = 50 pM and 1 nM, respectively). Subsequent exposure of the patch to alpha i or alpha o reversed this effect. In excised inside-out patches, increasing Mg2+ in the bath stimulated the channel activity between 0 and 0.5 nM, but blocked it at higher concentrations (IC50 = 2.55 mM). In most cases (70%), GTP had a stimulatory effect at concentrations up to 100 microns. However, in three cases, similar GTP levels had clear inhibitory effects. In excised inside-out patches, cholera toxin (CTX) caused channel inhibition. Although the effect could not be reversed by removal of the toxin, the activity was restored by subsequent addition of purified alpha i or alpha o. These results are compatible with a model whereby channel inhibition by activated Gs-coupled receptors occurs, at least in part, via association of the beta gamma subunits of Gs with alpha i/alpha o subunits and deactivation of the alpha i/alpha o-dependent stimulatory pathway. On the basis of this hypothesis, a model is developed to describe the effects of G proteins on the KATP channel, as well as to account for the concentration-dependent stimulation and inhibition of KATP channel by Mg2+. An interpretation of the ability of glucagon to potentiate, but not initiate, insulin release is also given in terms of this model and the effects of ATP on KATP channels.

Adenosine Triphosphate↗

PubMed1995

Ca2+ channel activities in the limb bud of early embryonic chick.

Ca2+ channel activities were recorded in the limb bud of embryonic day 4 chick with Ca2+ sensitive fluorescence (Fura-2) measurements and patch clamp techniques. Rises in intracellular Ca2+ concentrations were evoked by depolarization with the application of 100 mM K+ and this Ca2+ response was abolished by removing extracellular Ca2+. The Ca2+ response was blocked by 10 microM nifedipine and enhanced by 5 microM Bay K 8644. Long-lasting inward currents were revealed by whole-cell patch clamp recordings from dissociated cells of the limb bud. The inward current was also blocked by 10 microM nifedipine. Our study suggested the presence of L-type Ca2+ channels in the limb bud cells.

Animals↗

PubMed1998

Endothelin-1 inhibits voltage-sensitive Ca2+ channels in cultured rat cerebellar granule neurones via the ET-A receptor.

In this study, we have investigated the effect of the vasoconstrictor peptide endothelin-1 (ET-1) on voltage-sensitive Ca2+ channels in rat cerebellar granule neurones using the patch-clamp technique. Using amphotericin B perforated-patch recording of whole-cell currents, the Ca2+ channel current was inhibited by 28.4+/-6.4% by 400 nM ET-1, but was unaffected when experiments were repeated using the whole-cell, ruptured-patch configuration. In cell-attached patches, 400 nM ET-1 inhibited unitary L-type Ca2+ channel currents (IBa) by 85+/-5%. ET-1 decreased the open probability (NPo) and the frequency of channel opening and increased the mean closed time of channels. No effects on the mean open time or the time constants for channel opening or closure were observed. L-type Ca2+ channel inhibition was dose dependent with an IC50 of 19 nM. The effect of ET-1 was prevented by the combined endothelin-A and -B receptor antagonist PD145065 (10 microM), indicating a receptor-mediated effect. The ET-A receptor antagonist BQ-123 (10 microM) prevented Ca2+ channel inhibition by ET-1, while the ET-B receptor agonist sarafotoxin 6c (500 nM) had no effect. The inhibition by ET-1 was not due to a change in the voltage of channel activation. Fura-2 Ca2+ imaging showed that no substantial rise in intracellular Ca2+ levels occurred during ET-1 application excluding a Ca2+-dependent inhibition of the channels. Thus in cultured rat cerebellar granule neurones, ET-1 inhibits L-type Ca2+ channels via activation of the ET-A receptor. Inhibition may be mediated by an as yet unidentified cytoplasmic second messenger.

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

PubMed2000

The effects of non-identifiability on testing for detailed balance in aggregated Markov models for ion-channel gating.

Aggregated Markov models are a widely used tool to model patch clamp data measured from single ion channels. These channels must obey the principle of detailed balance in thermodynamic equilibrium; otherwise, the channel is driven by an external source of energy. We investigate the power of a likelihood ratio test for detailed balance for a number of data points which is in the order of magnitude of patch clamp experiments. We show that for certain models with nearly equal dwell times, a test for detailed balance suffers from a loss of power to detect violations of detailed balance which is due to the non-identifiability of the transition rates for models with equal dwell times.

Computer Simulation↗

PubMed1997

GABA(A) receptor activation triggers a Cl- conductance increase and a K+ channel blockade in cerebellar granule cells.

GABA(A) receptor activation in cerebellar granule cells induced a complex physiological response, namely the activation of a Cl- conductance in concert with a blockade of the resting K+ outward conductance (by 71% as compared to controls). Both responses were mediated by the activation of GABA(A) receptors, since they were both mimicked by the GABA(A) receptor agonist muscimol and antagonized by picrotoxin and bicuculline. A substantial decrease of the mean open time of single, outwardly rectifying K+ channels was triggered by GABA as revealed from cell-attached recordings; this finding implies that an intracellular pathway links GABA(A) receptors and K+ channels. Furthermore, this action of GABA is mediated through the cytoplasm, as experiments with the cell-attached patch-clamp technique show. GABA induced a prominent membrane depolarization ranging from 10 to 25 mV as revealed by current-clamp recordings of gramicidin (or nystatin) permeabilized patches, thus selecting conditions not to perturb the physiological Cl- gradient across the cell. Our findings imply that the GABA-activated Cl- current depolarized the membrane as described for immature neurons. The blockade of the resting K+ channel conductance acts in concert and both mechanisms lead to this substantial depolarizing event.

Animals↗

PubMedDate not supplied

Extent of the selectivity filter conferred by the sixth transmembrane region in the CFTR chloride channel pore.

Point mutations within the pore region of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel have previously been shown to alter the selectivity of the channel between different anions, suggesting that part of the pore may form an anion 'selectivity filter'. However, the full extent of this selectivity filter region and the location of anion binding sites in the pore are currently unclear. As a result, comparisons between CFTR and other classes of Cl(-) channel of known structure are difficult. We compare here the effects of point mutations at each of eight consecutive amino acid residues (arginine 334-serine 341) in the crucial sixth transmembrane region (TM6) of CFTR. Anion selectivity was determined using patch-clamp recording from inside-out membrane patches excised from transiently transfected mammalian cell lines. The results suggest that selectivity is predominantly controlled by a single site involving adjacent residues phenylalanine 337 and threonine 338, and that the selectivity conferred by this 'filter' region is modified by anion binding to flanking sites involving the more extracellular arginine 334 and the more intracellular serine 341. Other residues within this part of the pore play only minor roles in controlling anion permeability and conductance. Our results support a model in which specific TM6 residues make important contributions to a single, localized anion selectivity filter in the CFTR pore, and also contribute to multiple anion binding sites both within and on either side of the filter region.

Animals↗

PubMed1996

Mechanism of action of K channel openers on skeletal muscle KATP channels. Interactions with nucleotides and protons.

The molecular mechanisms underlying the actions of K channel openers (KCOs) on KATP channels were studied with the patch clamp technique in excised inside-out patches from frog skeletal muscle fibers. Benzopyran KCOs (levcromakalim and SR 47063) opened channels partially blocked by ATP, ADP, or ATP gamma s, with and without Mg2+, but they had no effects in the absence of internal nucleotides, even after channel activity had significantly declined because of rundown. The effects of KCOs could therefore be attributed solely to a competitive interaction between KCOs and nucleotides, as confirmed by observations that ATP decreased the apparent affinity for KCOs and that, conversely, KCOs decreased ATP or ADP sensitivity. Protons antagonized the action of the non-benzopyran KCOs, pinacidil and aprikalim, by enhancing their dissociation rate. This effect resembled the effect of acidification on benzopyran KCOs (Forestier, C., Y. Depresle, and M. Vivaudou. FEBS Lett. 325:276-280, 1993), suggesting that, in spite of their structural diversity, KCOs could act through the same binding sites. Detailed analysis of the inhibitory effects of protons on channel activity induced by levcromakalim or SR 47063 revealed that, in the presence of 100 microM ATP, this effect developed steeply between pH 7 and 6 and was half maximal at pH 6.6. These results are in quantitative agreement with an allosteric model of the KATP channel possessing four protonation sites, two nucleotidic sites accessible preferentially to Mg(2+)-free nucleotides, and one benzopyran KCO site. The structural implications of this model are discussed.

Adenosine Diphosphate↗

PubMed1997

Adenosine triphosphate activates a noninactivating K+ current in adrenal cortical cells through nonhydrolytic binding.

Bovine adrenal zona fasciculata (AZF) cells express a noninactivating K+ current (IAC) that is inhibited by adrenocorticotropic hormone and angiotensin II at subnanomolar concentrations. Since IAC appears to set the membrane potential of AZF cells, these channels may function critically in coupling peptide receptors to membrane depolarization, Ca2+ entry, and cortisol secretion. IAC channel activity may be tightly linked to the metabolic state of the cell. In whole cell patch clamp recordings, MgATP applied intracellularly through the patch electrode at concentrations above 1 mM dramatically enhanced the expression of IAC K+ current. The maximum IAC current density varied from a low of 8.45 +/- 2.74 pA/pF (n = 17) to a high of 109.2 +/- 26. 3 pA/pF (n = 6) at pipette MgATP concentrations of 0.1 and 10 mM, respectively. In the presence of 5 mM MgATP, IAC K+ channels were tonically active over a wide range of membrane potentials, and voltage-dependent open probability increased by only approximately 30% between -40 and +40 mV. ATP (5 mM) in the absence of Mg2+ and the nonhydrolyzable ATP analog AMP-PNP (5 mM) were also effective at enhancing the expression of IAC, from a control value of 3.7 +/- 0.1 pA/pF (n = 3) to maximum values of 48.5 +/- 9.8 pA/pF (n = 11) and 67.3 +/- 23.2 pA/pF (n = 6), respectively. At the single channel level, the unitary IAC current amplitude did not vary with the ATP concentration or substitution with AMP-PNP. In addition to ATP and AMP-PNP, a number of other nucleotides including GTP, UTP, GDP, and UDP all increased the outwardly rectifying IAC current with an apparent order of effectiveness: MgATP > ATP = AMP-PNP > GTP = UTP > ADP >> GDP > AMP and ATP-gamma-S. Although ATP, GTP, and UTP all enhanced IAC amplitude with similar effectiveness, inhibition of IAC by ACTH (200 pM) occurred only in the presence of ATP. As little as 50 microM MgATP restored complete inhibition of IAC, which had been activated by 5 mM UTP. Although the opening of IAC channels may require only ATP binding, its inhibition by ACTH appears to involve a mechanism other than hydrolysis of this nucleotide. These findings describe a novel form of K+ channel modulation by which IAC channels are activated through the nonhydrolytic binding of ATP. Because they are activated rather than inhibited by ATP binding, IAC K+ channels may represent a distinctive new variety of K+ channel. The combined features of IAC channels that allow it to sense and respond to changing ATP levels and to set the resting potential of AZF cells, suggest a mechanism where membrane potential, Ca2+ entry, and cortisol secretion could be tightly coupled to the metabolic state of the cell through the activity of IAC K+ channels.

Adenosine Diphosphate↗

PubMed2005

Stretch-stimulated activity of large conductance calcium-activated potassium channels in human retinal pigment epithelial cells.

AIM: The aim of this study was to investigate the effect of membrane stretch on the activity of BK(Ca) channels and determine whether change in BK(Ca)-channel activity induced by membrane stretch can be altered in the presence of riluzole. METHODS: The effect and mechanisms of membrane stretch on the activity of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels in human retinal pigment epithelial (RPE) cells were investigated using the patch-clamp technique. RESULTS: In cell-attached patches, an increase in BK(Ca)-channel activity was induced by membrane stretch, which is mainly mediated by the increase in the number of long-lived openings. With inside-out patches, application of pipette suction did not modify single-channel conductance of BK(Ca) channels; however, it did enhance the channel activity. Membrane stretch-induced channel activity was found to be independent of the level of intracellular Ca(2+). Membrane stretch also potentiated riluzole-stimulated BK(Ca)-channel activity. CONCLUSIONS: BK(Ca) channel can be stimulated by membrane stretch in human RPE cells. During an increase in tensile strength of RPE, the BK(Ca) channel might exert a protective action against deleterious tension by promoting a hyperpolarizing outflow that forces the cell to rest.

Cell Line↗

PubMed2001

Differential transduction mechanisms underlying NaCl- and KCl-induced responses in mouse taste cells.

The transduction mechanism of salt-induced responses of mouse taste cells was investigated using the patch clamp and the local stimulation techniques under quasi-natural conditions. Apically applied NaCl induced a voltage-independent current, which was partially suppressed by amiloride and Cd2+. In contrast, apically applied 0.5 M KCl induced an inwardly rectifying current (KCl-induced Iir). The KCl-induced Iir was unaffected by amiloride. The Iir was suppressed not only by external Ba2+ and Cs+, but also by a Cl- channel blocker, niflumic acid. The Er of the KCl-induced response was independent of the apical ionic concentration, but rather was close to the equilibrium potential of Cl- (E(Cl)) at the basolateral membrane. The KCl-induced Iir displayed a fast run-down under the conditions of the conventional whole cell clamp method, but not under the perforated patch conditions. Immunohistochemical localization of an inwardly rectifying Cl- channel protein, ClC-2, was observed in taste bud cells of the fungiform papillae. It is concluded that the transduction mechanism of NaCl-induced responses is completely different from that of KCl-induced responses in mouse taste cells.

Amiloride↗

PubMed2004

Effects of the gap junction blocker glycyrrhetinic acid on gastrointestinal smooth muscle cells.

In the tunica muscularis of the gastrointestinal (GI) tract, gap junctions form low-resistance pathways between pacemaker cells known as interstitial cells of Cajal (ICCs) and between ICC and smooth muscle cells. Coupling via these junctions facilitates electrical slow-wave propagation and responses of smooth muscle to enteric motor nerves. Glycyrrhetinic acid (GA) has been shown to uncouple gap junctions, but previous studies have shown apparent nonspecific effects of GA in a variety of tissues. We tested the effects of GA using isometric force measurements, intracellular microelectrode recordings, the patch-clamp technique, and the spread of Lucifer yellow within cultured ICC networks. In murine small intestinal muscles, beta-GA (10 muM) decreased phasic contractions and depolarized resting membrane potential. Preincubation of GA inhibited the spread of Lucifer yellow, increased input resistance, and decreased cell capacitance in ICC networks, suggesting that GA uncoupled ICCs. In patch-clamp experiments of isolated jejunal myocytes, GA significantly decreased L-type Ca(2+) current in a dose-dependent manner without affecting the voltage dependence of this current. The IC(50) for Ca(2+) currents was 1.9 muM, which is lower than the concentrations used to block gap junctions. GA also significantly increased large-conductance Ca(2+)-activated K(+) currents but decreased net delayed rectifier K(+) currents, including 4-aminopyridine and tetraethylammonium-resistant currents. In conclusion, the reduction of phasic contractile activity of GI muscles by GA is likely a consequence of its inhibitory effects on gap junctions and voltage-dependent Ca(2+) currents. Membrane depolarization may be a consequence of uncoupling effects of GA on gap junctions between ICCs and smooth muscles and inhibition of K(+) conductances in smooth muscle cells.

Animals↗

PubMed1997

Modulation of ATP-sensitive K+ channels in rabbit ventricular myocytes by adenosine A1 receptor activation.

The objective of the present study was to characterize the role of adenosine in the regulation of ATP-sensitive K (KATP) channel activity in isolated rabbit ventricular myocytes using the patch-clamp technique. In an outside-out patch exposed to guanosine 5'-triphosphate and ATP at the intracellular surface, external adenosine stimulated KATP channel activity. In an inside-out patch exposed to external adenosine, ATP reduced KATP channel activity and guanosine 5'-triphosphate stimulated KATP channel activity. Guanosine 5'-O-(3-thiotriphosphate) resulted in a gradual increase of KATP channel activity even in the absence of adenosine. When myocytes were preincubated with pertussis toxin or 8-cyclopentyl-1,3-dipropylxanthine, adenosine A1 receptor activation failed to activate the KATP channel. Analysis of the open and closed time distributions showed that adenosine A1 receptor activation increased burst duration and decreased interburst duration. In a dose-response relationship for ATP, adenosine A1 receptor activation shifted the half-maximal inhibition of the KATP channel from 70 to 241 microM.

Adenosine↗

PubMed2001

Sodium channels in the apical membrane of human nasal epithelial cells.

OBJECTIVE: To study the electrophysiological properties of sodium channels in the apical membrane of human nasal epithelial cells. METHOD: Nasal epithelial cells of human inferior turbinate from patients with obstructive sleep apnea syndrome were cultured in serum free medium on collagen gel-coated membranes at an air-liquid interface and studied by a patch clamp technique. RESULTS: In cell-attached patches, a typical single channel current with a conductance of 21.09 pS and reversal potential of -50.96 were recorded. The permeability ratio PNa/PK was more than 5.80. In the presence of 10(-4) mmol/L amiloride in the pipette, the incidence of sodium channels decreased from 26.67% to 5.13%. This revealed that a population of channels were inhibited by amiloride at a dose of 10(-4) mmol/L. Ca2+ at dose of 10(-3) mmol/L did not influence the incidence of sodium channels. There was no obvious association between voltage and the open probability of the channels. CONCLUSIONS: Our results indicate that most Na+ channels in cell-attached patches of human nasal epithelial cells are amiloride-sensitive and Na+ selective. Only a few channels are amiloride-insensitive. The channels were not activated by extracellular Ca2+ and the open probability followed a voltage-independent manner.

Amiloride↗

PubMed1999

[Sodium channels in the apical membrane of human nasal epithelial cells].

OBJECTIVE: To study the physiological properties of sodium channels in the apical membrane of human nasal epithelial cells. METHODS: Serum-free cultured nasal epithelial cells of human inferior turbinate from obstructive sleep apnea syndrome patients on collagen gel-coated membranes at an air-liquid interface were studied by patch clamp technique. RESULTS: In cell-attached patches, a typical single channels current was recorded, their conductance was 21.09 pS, the reversal potential was -50.96, and 77.78% of them < -40 mV. Permeability ratio PNa/PK > 5.80. In the presence of 10(-4) mmol/L amiliride in the pipette, the incidence of sodium channels decreased from 26.67% to 5.13%. It revealed that a population of channels were inhibited by amiloride. Ca2+ did not influence the incidence of sodium channels(P > 0.05), there were no obviously association between voltage and the open probability of the channels. CONCLUSION: On Na+ channels in cell attached patches of human nasal epithelial cells, most of channels are amiloride-sensitive and selective Na+ over K+; The channels were not activated by extracellular Ca2+, but the open probability of them were voltage-independence.

Adult↗

PubMed1996

Tyrosine phosphorylation of the Kv1.3 potassium channel.

Kv1.3, a voltage-dependent potassium channel cloned from mammalian brain and T lymphocytes, contains multiple tyrosine residues that are putative targets for tyrosine kinases. We have examined the tyrosine phosphorylation of Kv1.3, expressed transiently in human embryonic kidney (or HEK) 293 cells, by endogenous and coexpressed tyrosine kinases. Tyrosine phosphorylation is measured by a strategy of immunoprecipitation followed by. Western blot analysis, using antibodies that specifically recognize Kv1.3 and phosphotyrosine. Coexpression of the constitutively active tyrosine kinase v-src, together with Kv1.3, causes a large increase in the tyrosine phosphorylation of the channel protein. This phosphorylation of Kv1.3 can be reversed by treatment with alkaline phosphatase before Western blot analysis. Coexpression with a receptor tyrosine kinase, the human epidermal growth factor receptor, also causes an increase in tyrosine phosphorylation of Kv1.3. The effects of endogenous tyrosine kinases were examined by treating Kv1.3-transfected cells with the specific membrane-permeant tyrosine phosphatase inhibitor pervanadate. Pervanadate treatment causes a time- and concentration-dependent increase in the tyrosine phosphorylation of Kv1.3. This increased tyrosine phosphorylation of Kv1.3 is accompanied by a time-dependent decrease in Kv1.3 current, measured by patch-clamp analysis with cell-attached membrane patches. The pervanadate-induced suppression of current and much of the channel tyrosine phosphorylation are eliminated by mutation of a specific tyrosine residue, at position 449 of Kv1.3, to phenylalanine. Thus, there is a continual phosphorylation and dephosphorylation of Kv1.3 by endogenous kinases and phosphatases, and perturbation of this constitutive phosphorylation/dephosphorylation cycle can profoundly influence channel activity.

Animals↗

PubMed1988

Ionic mechanisms of adenosine actions in pacemaker cells from rabbit heart.

1. Whole-cell and patch clamp techniques have been applied to cells isolated from the rabbit sino-atrial (S-A) node to study the ionic mechanism(s) of adenosine-induced slowing of cardiac pacemaker activity. 2. Viable spontaneously active cells were isolated from the central region of the S-A node of the rabbit heart by an enzymatic dispersion procedure similar to that reported by Giles & van Ginneken (1985) and van Ginneken & Giles (1988). In these spontaneously beating cells application of adenosine caused a dose-dependent slowing accompanied by a small hyperpolarization of the maximum diastolic potential. Relatively high doses of adenosine (greater than 20 microM) caused complete arrest, associated with a hyperpolarization of 12-15 mV. 3. In corresponding whole-cell voltage clamp experiments adenosine activated a time-independent potassium current, IK(ADO), which at -50 mV is approximately 30 pA in normal Tyrode solution and 50 pA in high [K+]o (20 mM) Tyrode solution. This current is similar to the one identified previously in guinea-pig atrium (Belardinelli & Isenberg, 1983a; Kurachi, Nakajima & Sugimoto, 1986). 4. Patch clamp recordings of the single-channel events underlying IK(ADO) showed that they have a conductance of approximately 25.0 +/- 1.9 pS. The whole-cell or macroscopic current, IK(ADO), and the adenosine-induced single-channel events exhibit strong inward-going rectification. 5. Adenosine in doses (10 microM) which significantly activate IK(ADO) failed to produce any measurable effect on the calcium current, ICa, in these isolated cardiac pacemaker cells. However, after ICa has been enhanced by the addition of isoprenaline, adenosine (1-10 microM) caused a significant inhibition: it reduced ICa back to approximately the control levels. 6. A similar 'indirect' effect of adenosine was observed on If, the slow time- and voltage-dependent inward current which is activated by hyperpolarizing these S-A node cells. Adenosine (10(-5) M) failed to influence the control or basal If; however, after If was enhanced by isoprenaline, adenosine markedly inhibited it. 7. These results provide explanations for both the direct and the indirect effects of adenosine in mammalian cardiac pacemaker tissue: activation of IK(ADO), and of a time-independent background potassium current and inhibition of ICa and If, respectively. Since it is known that there is significant adrenergic tone in the mammalian S-A node both the indirect and the direct effects of adenosine may be of physiological importance.

Adenosine↗

PubMed1992

Effects of diazoxide and glyburide on ATP-sensitive K+ channels from hypertrophied ventricular myocytes.

Hyperpolarizing vasodilators that specifically activate ATP-sensitive K+ currents (IK(ATP] in smooth muscle have been suggested as promising antihypertensive (if potentially arrhythmogenic and/or hyperglycemic) therapy. To date, however, the effects of agents presumed to influence these channels have not been characterized in hypertrophied cardiac muscle secondary to chronic hypertension. We used standard intracellular and patch clamp, single-channel recording techniques to study the effects of diazoxide, a presumed activator, as well as the sulfonylurea glyburide on IK(ATP) in cardiac muscle from control (WKY) and spontaneously hypertensive rats (SHR). Intracellular recordings were obtained from isolated left ventricles at 37 degrees C; unitary currents were recorded in excised, inside-out membrane patches with symmetrical transmembrane K+ at 21-23 degrees C. Diazoxide (5-100 microM) caused a decrease in action potential duration in both WKY and SHR ventricles. Glyburide (5-25 microM) produced dramatic dose-dependent increases in action potential duration approaching 100% in both groups. Action potential amplitude and resting membrane potential were unaffected by either agent. Before drug administration, unitary currents in hypertrophied myocytes exhibited a greater open state probability upon depolarization than those from control myocytes, although conductance, mean single-channel open time, and the number of channels per patch were not significantly different. Under patch clamp, both diazoxide (25 and 100 microM) and glyburide (50 microM) decreased IK(ATP) activity in cells from WKY and SHR in the absence of ATP. In both groups, the response reflected an overall decrease in open state probability. These data indicate that although IK(ATP) characteristics are altered in hypertension and myocardial hypertrophy, the effects of agents specific to this current are not significantly different in cells from SHR relative to control. On the other hand, the effects of diazoxide may be linked to temperature or to the metabolic state of the cell.

Adenosine Diphosphate↗