Nucleotide regulation of ATP sensitive potassium channels.
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Adenosine Triphosphate↗
Biomedical subjects
A Terzic
Publications and source records attributed to A Terzic.
Intracellular acidification and ADP enhance nicorandil induction of ATP sensitive potassium channel current in cardiomyocytes.
OBJECTIVE: Potassium channel openers target ATP sensitive potassium (KATP) channels, and might be beneficial in the treatment of ischaemic cardiac conditions. Ischaemia is associated with an increase in the concentrations of intracellular nucleoside diphosphates and protons, two regulators of KATP channels. Since it is largely unknown whether these factors modulate the action of potassium channel openers, the aim of this study was to define the effects of ADP and intracellular acidification on KATP channel activation by nicorandil, a potassium channel opener currently in clinical use. METHODS: To measure ionic currents, the whole cell patch clamp technique was employed in guinea pig single ventricular myocytes. Induction of KATP channel current by pinacidil, a potassium channel opener chemically unrelated to nicorandil, was used for comparison. RESULTS: Nicorandil (300 microM) and pinacidil (100 microM) induced a time independent K+ current, which was inhibited by glibenclamide (6 microM), a selective blocker of KATP channels. ADP in the pipette solution was required for the nicorandil induced activation of KATP channels when the pH of the pipette solution was 7.2. In the absence of ADP, lowering the pH of the pipette solution to 6.5 was necessary for nicorandil to induce KATP channel current. For pinacidil to induce KATP channel current neither the addition of intracellular ADP nor acidification was required. CONCLUSIONS: Nicorandil may activate KATP channel current more effectively in conditions associated with a change in intracellular proton and ADP concentrations such as cardiac ischaemia.
Action Potentials↗
HOE-234, a second generation K+ channel opener, antagonizes the ATP-dependent gating of cardiac ATP-sensitive K+ channels.
Effects of (3S,4R)-3-hydroxy-2,2-dimethyl-4-(2-oxo-1-pyrrolidinyl)-6- phenylsulfonylchroman hemihydrate (HOE-234), a novel dilator of bronchial and vascular smooth muscles, were examined on whole-cell and single channel currents in guinea pig cardiomyocytes. Under the whole-cell voltage-clamp condition, HOE-234 (0.6-100 microM) induced a time-independent K(+)-dominant current in atrial and ventricular myocytes. This current was inhibited by glyburide, a selective blocker of ATP-sensitive K+ (KATP) channels, suggesting that HOE-234 activates KATP channels. Neither intracellular acidification nor the absence of intracellular ADP inhibited the ability of HOE-234 to induce the current. In inside-out membrane patches in the presence of 200 microM ATP, HOE-234 (0.3-10 microM) increased concentration dependently (EC50 approximately 1 microM) the KATP channel activity. In the absence of HOE-234, half-inhibition of spontaneously operative KATP channels occurred at 25 microM of intracellular ATP (ATPi), whereas in the presence of HOE-234 (10 microM), 316 microM of ATPi was required for half-inhibition. In ATP-free internal solution, KATP channels appeared and then ran down. In the absence of ATPi, HOE-234 did not increase channel activity when channels were in a fully open or in the partially or completely run-down state. After run-down, the nucleoside diphosphate, UDP, restored KATP channel activity which was not further augmented by HOE-234. However, HOE-234 relieved at ATP- or ATP gamma S-mediated inhibition of the UDP-induced KATP channel activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenosine Triphosphate↗
Alpha 1-adrenoceptor and purinoceptor agonists modulate Na-H antiport in single cardiac cells.
We investigated the effects of an alpha 1-adrenoceptor (phenylephrine) and a purinoceptor agonist (ATP), both of which accelerate the phosphoinositide turnover, on the Na-H antiport activity of rat single cardiac cells using the pH-sensitive fluorescent indicator seminaphthorhodafluor-1 (SNARF-1). Both phenylephrine, in the presence of a beta-adrenoceptor blocker, and ATP enhanced the ability of the cell to regulate its intracellular pH (pHi) after an imposed acid load. This effect was observed in HCO3-free N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) and prevented by Na-H antiport inhibitors ethylisopropylamiloride (EIPA) or amiloride. Similar results were obtained when cells were bathed in an acidic extracellular medium. Hence, the alpha 1-adrenoceptor and purinoceptor agonists activate the Na-H antiport even when it is partially inhibited by extracellular protons. To further evaluate the effects of the two neurohormones, the rate of proton efflux was estimated as a function of the magnitude of the imposed acid load. The results indicate that the agonist-induced modulation of the Na-H antiport is caused by an acceleration of its exchange activity and by a shift of its dependence on pHi toward more alkaline pH values. The agonist-mediated stimulation of the antiport was also observed in partially depolarized cells and was not dependent on intracellular Ca. Phorbol 12-myristate 13-acetate was not able to reproduce the effects of the agonists on the Na-H antiport. Conversely, the inhibitors of protein kinase C did not prevent the activation of the antiport by the neurohormones. Thus our data suggest that neither a Ca-calmodulin-dependent kinase nor protein kinase C is responsible for the alpha 1-adrenoceptor- and purinoceptor-mediated stimulation of the antiport.
Acidosis↗
Cardiac alpha 1-adrenoceptors: an overview.
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Action Potentials↗
YM934, a novel K+ channel opener, activates ATP-sensitive K+ channels in cardiac myocytes.
2-(3,4-Dihydro-2,2-dimethyl-6-nitro-2H-1,4-benzoxazin-4-yl)pyridin e N-oxide (YM934) is a newly synthesized benzoxazin. The effects of YM934 on ATP-sensitive K+ (KATP) channels in guinea pig cardiac ventricular myocytes and in an insulin-secreting cell line, HIT T15 beta-cells, were examined using the gigaohm-seal patch-clamp techniques. Under the whole-cell clamp condition, YM934 induced in ventricular myocytes a time-independent, glibenclamide-sensitive K+ current in a concentration-dependent fashion (EC50 = approximately 3 microM). On formation of inside-out patches in ATP-free solution, the KATP channel current abruptly appeared and then ran down. YM934 was applied to inside-out patches before, during and after channel "run-down." Because nucleoside diphosphates, such as uridine diphosphate (UDP), can induce channel openings after complete run-down, the effects of YM934 on the UDP-induced channel openings were also examined. Before run-down, YM934 enhanced KATP channel activity by decreasing the sensitivity of channels to intracellular ATP. YM934 also enhanced the partially run-down channel, even in the absence of ATP. After run-down, YM934 had no effect but could enhance the UDP-induced KATP channel openings. These effects of YM934 on cardiac KATP channels were similar to those of pinacidil and lemakalim. In HIT T15 beta-cells, 100 microM YM934 was ineffective in both cell-attached and inside-out patch configurations, suggesting the tissue-specific nature of the action of this novel K+ channel opener.
Adenosine Triphosphate↗
Phenylephrine and ATP enhance an amiloride insensitive bicarbonate-dependent alkalinizing mechanism in rat single cardiomyocytes.
To expel the excess protons generated during a cellular acidification and to fully recover basal intracellular pH (pHi), cardiac cells rely on the amiloride-sensitive Na/H antiport. We report that rat single ventricular cardiomyocytes, loaded with the fluorescent pH indicator Snarf-1 and treated with inhibitors of the Na/H antiport, amiloride or its analogues, partially restored their pHi through a bicarbonate-dependent mechanism following an acidosis (imposed by the ammonia-pulse technique). In the presence of ethylisopropylamiloride (10 microM) or amiloride (1 mM) and 25 mM bicarbonate in the extracellular solution, the average time that cells needed to recover half of their pHi, following the removal of 20 mM NH4Cl, was 3.4 min, while the rate of proton efflux was calculated to be 2.0 mM/min. The stilbene derivative, 4-4'-di-isothiocyanostilbene-2,2'-disulphonate (DIDS 200 microM), a known blocker of anion transporters, inhibited this recovery. Both phenylephrine (100 microM, 3 microM propranolol present), an alpha 1-adrenoceptor agonist, and ATP (10 microM), a purinergic agonist, significantly enhanced the rate of proton efflux that was due to this HCO3-dependent alkalinizing mechanism. Phenylephrine and ATP also shortened by three-fold the time that a myocyte needed to recover half of its initial pHi. This bicarbonate-dependent alkalinizing mechanism could provide an additional means by which cardiac cells recover their pHi from acidosis, especially under conditions in which the Na/H antiport is inhibited. Furthermore, catecholamines and ATP, which are released under various pathophysiological conditions often associated with intracellular acidosis, could play an important role in the modulation of pHi under these conditions.
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
Cardiac alpha 1-adrenoceptors mediate positive inotropy via myofibrillar sensitization.
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Adrenergic alpha-Agonists↗
Alpha 1-adrenergic effects on intracellular pH and calcium and on myofilaments in single rat cardiac cells.
1. The cellular effects of alpha 1-adrenoceptor stimulation by phenylephrine were studied in the presence of propranolol in single cells isolated from the ventricles of rat hearts. 2. Phenylephrine (10-100 microM) induced a biphasic pattern of inotropism in these cells: a transient negative followed by a sustained positive inotropic effect as usually observed in cardiac tissues. 3. In Snarf-1-loaded cells, phenylephrine induced an alkalinization. This effect was reversible on wash-out and inhibited by prazosin, an alpha 1-adrenoceptor antagonist. 4. The alpha 1-adrenoceptor-mediated increase in intracellular pH (pHi) was 0.1 pH unit in HEPES buffer containing 4.4 mM-NaHCO3 and in Krebs buffer containing 25 mM-NaHCO3. 5. The alkalinization was blocked by the Na(+)-H+ antiport blocker, ethylisopropylamiloride (EIPA). 6. The recovery from an acidosis induced by a NH4Cl pre-pulse was accelerated by phenylephrine. The phenylephrine-induced alkalinization was attributed to activation of the Na(+)-H+ antiport. 7. Despite its ability to increase pHi, phenylephrine did not alter Ca2+ current amplitude and kinetics. 8. Ca2+ transients recorded in Indo-1-loaded cells were not augmented by phenylephrine. Diastolic calcium level was decreased. 9. In single skinned cells, the Ca2+ sensitivity of the contractile proteins was increased by a pre-treatment with phenylephrine even when the alpha 1-adrenoceptor-mediated alkalinizing effect had been prevented by EIPA. 10. These results lead us to propose that the alpha 1-adrenergic-induced positive inotropic response of heart muscle could result from an increased sensitivity of the myofilaments to Ca2+ ions. This alpha 1-adrenoceptor-mediated Ca2+ sensitization could result both from an intracellular alkalinization and from a direct effect on contractile proteins.
Amiloride↗
Opposite modulation of ouabain cardiotoxicity by hexamethyleneamiloride and phenylephrine.
To see whether the Na/H antiporter plays a role in digitalis cardiotoxicity, we investigated the influence of modulators of Na/H exchange on the toxic effects of ouabain in isolated, paced (0.4 Hz) rat left atria. Ouabain (1 mmol/l) caused a transient positive inotropic effect followed by toxic events, including a complete loss of developed force and a gradual increase in resting force. In the presence of hexamethyleneamiloride (3 and 10 mumol/l), an inhibitor of Na/H exchange, ouabain (1 mmol/l) caused a sustained positive inotropic effect without toxicity. By contrast, phenylephrine (100 mumol/l), an alpha-adrenoceptor agonist reported to stimulate the antiporter, hastened the development of ouabain's toxicity. Neither ouabain, at a subtoxic concentration (650 mumol/l), nor phenylephrine (100 mumol/l) affected diastolic force, but in their combined presence, a substantial contracture developed and twitch contractions disappeared. Phenylephrine (30 or 100 mumol/l) or adrenaline (30 mumol/l), in the presence of a beta-adrenoceptor antagonist, increased the intracellular pH by up to 0.15 pH unit, as measured using ion-selective microelectrodes in quiescent preparations. This effect on pHi was prevented by hexamethyleneamiloride (10 mumol/l). Consistent with phenylephrine's ability to stimulate Na+ influx via the Na/H antiporter, phenylephrine (100 mumol/l) increased intracellular Na+ activity by about 3 mmol/l in ouabain (650 mumol/l)-treated atria. These findings indicate that modulators of Na/H exchange affect the cardiotoxicity of digitalis glycosides and imply that the stimulation of myocardial alpha-adrenoceptors may aggravate digitalis toxicity.
Amiloride↗
On the mechanism of the positive inotropic action of the alpha adrenoceptor agonist, phenylephrine, in isolated rat left atria.
Alpha adrenoceptor agonists have been reported to increase contractile force and to stimulate Na+/H+ exchange in the heart. We studied the influence of hexamethylamiloride (HMA), a selective inhibitor of Na+/H+ exchange, on the positive inotropic action of phenylephrine in isolated, paced rat left atria (3 microM propranolol). HMA (10 microM) blocked the ouabain-induced contracture, an event dependent on Na+ uptake via the Na+/H+ exchanger. The same concentration of HMA prevented 50% of the positive inotropic effect of phenylephrine (10 microM), but had no effect on base-line developed force. HMA reduced the maximal effect (234 +/- 19 vs. 117 +/- 20% increase of base-line), but not the EC50 (4.4 +/- 1.0 vs. 3.6 +/- 2 microM) of phenylephrine. Phenylephrine (100 microM) caused both a leftward and upward shift of the Ca++ concentration-effect curve, but only a leftward shift, in the additional presence of HMA (3 microM). It is known that lithium, but not choline, will exchange for H+ via the Na+/H+ exchanger: phenylephrine's (100 microM) positive inotropic effect in choline-substituted solutions averaged 37% of that in lithium-substituted solutions. The positive inotropic effect of phenylephrine was amplified by ouabain (200 microM). These results are consistent with the hypothesis that alpha adrenoceptor agonists produce their positive inotropic effects, in part, via stimulation of Na+/H+ exchange. Such stimulation could cause an intracellular alkalinization and (in the presence of ouabain), elevated intracellular Na+.
Amiloride↗
Amiloride-sensitive actions of an alpha-adrenoceptor agonist and ouabain in rat atria.
The influence of amiloride, a known blocker of Na/H exchange, on the positive inotropic action of alpha-adrenoceptor stimulation was investigated in isolated rat left atria. Amiloride (300 microM) rapidly (within 10 min) and reversibly abolished the positive inotropic effect of phenylephrine (10 microM; 3 microM propranolol present). Lower concentrations of amiloride inhibited the increase in contractile force caused by phenylephrine in a concentration dependent manner (IC50 of 50 microM). At a concentration of 50 microM, amiloride caused a rightward and downward shift in the concentration-response curve to phenylephrine. Amiloride (10 to 300 microM) affected to only a small extent the increased contractile force in the presence of inotropic interventions known to increase Ca2+ influx via L-type calcium channels (Bay K 8644) and the Na/Ca exchanger (reduced extracellular Na+). To provide evidence that amiloride inhibits the Na/H antiporter intact atria, a contracture that depends on Na+ influx by the Na/H antiporter was examined. Amiloride fully relaxed the contracture induced by ouabain (1 mM) or potassium-free solutions in the identical concentration range over which amiloride inhibited the positive inotropic effect of phenylephrine. Phenylephrine increased the rate of development and the peak amplitude of the amiloride-sensitive contracture (ouabain-induced). The inhibitory action of amiloride on the positive inotropic response to phenylephrine may, in part, be the result of inhibition of the Na/H antiporter.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
Alpha-adrenergic regulation of action potentials in isolated rat cardiomyocytes.
Radioligand binding studies and studies in which inositol phosphate levels were assayed have provided substantial evidence for the existence of alpha 1-adrenoceptors in isolated rat ventricular myocytes, a pure cardiac preparation free of neuronal and vascular elements. Since correlational electrophysiological studies have not been carried out in this model, we investigated alpha-adrenergic effects on action potentials using intracellular microelectrodes to impale the myocytes. Epinephrine, in the presence of propranolol (10(-6) M), rapidly increased the action potential duration in a concentration-dependent manner (threshold concentration of 30 nM, EC50 of 100 nM). The maximal upstroke velocity and overshoot of Ca2+-mediated action potentials (15 mM K+) were also increased. Epinephrine did not significantly affect the resting membrane potential. The alpha 1-adrenoceptor antagonist, prazosin (10(-7) M) blocked epinephrine's effects. LiCl (10 mM), an inhibitor of inositol-phosphate phosphatases potentiated the effects of epinephrine (30 nM). The results suggest that the isolated rat cardiomyocyte is a suitable preparation for examining the ionic and molecular mechanisms of direct alpha-adrenergic effects on the cardiac membrane.
Action Potentials↗