Heteromultimeric Kv1.2-Kv1.5 channels underlie 4-aminopyridine-sensitive delayed rectifier K(+) current of rabbit vascular myocytes.

The molecular identity of vascular delayed rectifier K(+) channels (K(DR)) is poorly characterized. Inhibition by 4-aminopyridine (4-AP) of K(DR) of rabbit portal vein (RPV) myocytes was studied by patch clamp and compared with that of channels composed of Kv1.5 and/or Kv1.2 subunits cloned from the RPV and expressed in mammalian cells. 4-AP block of K(DR) was pulse-frequency dependent, required channel activation, and was associated with a positive shift in voltage dependence of activation. 4-AP caused a voltage-dependent reduction in mean open time of K(DR). Relief of 4-AP block of whole cell currents during washout required channel activation and was unaffected by voltage. Homotetrameric Kv1.5 channels did not exhibit the shift in voltage dependence of activation exhibited by the native channels. In contrast, Kv1.2 channels displayed a shift in voltage dependence of activation, and this characteristic was also evident during 4-AP treatment when Kv1.2 was coexpressed with Kv1.5 or coupled to Kv1.5 in a tandem construct to produce heterotetrameric [Kv1.5/Kv1.2](2) channels. K(DR) currents were not sensitive to charybdotoxin, which blocks homotetrameric Kv1.2 channels. The findings of this study (1) indicate that vascular K(DR) are inhibited by 4-AP via an open-state block mechanism and trapping of the drug within the pore on channel closure and (2) provide novel evidence based on a comparison of functional characteristics that indicate the dominant form of vascular K(DR) channel complex in RPV involves the heteromultimeric association of Kv1.2 and Kv1.5 subunits.

External nickel blocks human Kv1.5 channels stably expressed in CHO cells.

We have investigated the actions of Nickel (Ni(2+)) on a human cardiac potassium channel (hKv1.5), the main component of human atrial ultra-rapid delayed rectifier current, stably expressed in Chinese hamster ovary cell line using the whole-cell voltage-clamp technique. External Ni(2+) reversibly decreased the amplitude of the current in a concentration-dependent manner. The concentration for half-maximum inhibition of the current at +50 mV was 568 microm. The activation, deactivation, reactivation kinetics of the current were not affected by Ni(2+). Block was not voltage-dependent but frequency-dependent block was apparent. The extent of channel block during the first pulse increased when the duration of exposure to Ni(2+), prior to channel activation, was prolonged indicating that Ni(2+) interacted with hKv1.5 in the closed state. The percentage of current remaining in presence of Ni(2+) decreased steeply over the range of steady-state channel inactivation, consistent with an enhanced block with increased inactivation. This suggests that Ni(2+) preferentially blocks nonconducting hKv1.5 channels, either in the resting or inactivated state in a concentration-dependent manner. The data indicate that the mechanisms of hKv1.5 channel inhibition by Ni(2+) are distinct from those of other K(+) channels.

Effects of anorexinogen agents on cloned voltage-gated K(+) channel hKv1.5.

Appetite suppressants have been associated with primary pulmonary hypertension (PPH), inhibition of voltage-gated potassium channels, membrane depolarization, and calcium entry in pulmonary artery smooth muscle cells. In cells taken from pulmonary arteries of primary pulmonary hypertensive patients, voltage-gated potassium channels appear to be dysfunctional and in particular, reduced hKv1.5 gene transcription and hKv1.5 mRNA instability have been shown. We have compared the effects of anorexinogen agents on hKv1.5 channels stably expressed in mammalian cell line. We found that aminorex, phentermine, dexfenfluramine, sibutramine, and fluoxetine cause a dose-dependent inhibition of hKv1.5 current. Aminorex, phentermine, and dexfenfluramine had a K(D) of inhibition greater than to 300 microM and are not potent inhibitors of hKv1.5. Sibutramine and fluoxetine inhibited hKv1.5 current with lower K(D) values of 41 and 21 microM, respectively. Block by both drugs increased rapidly between -20 and +10 mV, coincident with channel opening and suggested an open channel block mechanism. This was confirmed by a slower deactivation time course resulting in a "crossover" phenomenon when tail currents recorded under control conditions and in the presence of either drug were superimposed. Single channel experiments demonstrated that open probability and open duration of hKv1.5 were decreased by fluoxetine and sibutramine. These results indicate that among the anorexinogen agents tested, sibutramine and fluoxetine are the most potent toward hKv1.5 channel, which they preferentially block in the open state. Nevertheless, their inhibitory effects do not correlate with their ability to produce PPH neither with their previously reported therapeutic plasma concentrations.

Characterization of mibefradil block of the human heart delayed rectifier hKv1.5.

The goal of this study was to analyze the effects of mibefradil on a human cardiac K(+) channel (hKv1.5) stably expressed in Chinese hamster ovary cells using the whole-cell configuration of the patch-clamp technique. Mibefradil inhibited in a concentration-dependent manner the hKv1.5 current with a K(D) value of 0.78 +/- 0.05 microM and a Hill coefficient of 0.97 +/- 0.06. Block induced by mibefradil was voltage dependent, consistent with a value of electrical distance of 0.13. The apparent association (k) and dissociation (l) rate constants measured at +50 mV were found to be 7.3 +/- 0.5 x 10(6) M(-1).s(-1) and 4.3 +/- 0.1 s(-1), respectively. Block increased rapidly between -20 and +10 mV, coincident with channel opening and suggested an open channel block mechanism, which was confirmed by a slower deactivation time course resulting in a "crossover" phenomenon when tail currents recorded under control conditions and in the presence of mibefradil were superimposed. Shifts toward negative potentials of the maximum conductance and the activation curve were observed, confirming the voltage dependence of block. Mibefradil induced a significant use-dependent block when trains of depolarization at frequencies between 0.02 and 2 Hz were applied. In the presence of mibefradil, recovery of inactivation was faster than under control conditions, suggesting that mibefradil might compete with the inactivation gate of hKv1.5. These results indicate that mibefradil blocks hKv1.5 channels in a concentration-, voltage-, time- and use-dependent manner and the concentrations needed to observe these effects are in the therapeutic range.

Identification, cloning and expression of rabbit vascular smooth muscle Kv1.5 and comparison with native delayed rectifier K+ current.

1. The molecular basis of voltage-gated, delayed rectifier K+ (KDR) channels in vascular smooth muscle cells is poorly defined. In this study we employed (i) an antibody against Kv1.5 and (ii) a cDNA clone encoding Kv1.5 derived from rabbit portal vein (RPV) to demonstrate Kv1.5 expression in RPV and to compare the properties of RPVKv1.5 expressed in mammalian cells with those of native RPV KDR current. 2. Expression of Kv1.5 channel protein in RPV was demonstrated by (i) immunocytolocalization of an antibody raised against a C-terminal epitope of mouse cardiac Kv1.5 in permeabilized, freshly isolated RPV smooth muscle cells and (ii) isolation of a cDNA clone encoding RPVKv1.5 by reverse transcription-polymerase chain reaction (RT-PCR) using mRNA derived from endothelium-denuded and adventitia-free RPV. 3. RPVKv1.5 cDNA was expressed in mammalian L cells and human embryonic kidney (HEK293) cells and the properties of the expressed channels compared with those of native KDR channels of freshly dispersed myocytes under identical conditions. 4. The kinetics and voltage dependence of activation of L cell-expressed RPVKv1.5 and native KDR current were identical, as were the kinetics of recovery from inactivation and single channel conductance. In contrast, there was little similarity between HEK293 cell-expressed RPVKv1.5 and native KDR current. 5. Inactivation occurred with the same voltage for half-maximal availability, but the kinetics and slope constant for the voltage dependence of inactivation for L cell-expressed RPVKv1.5 and the native current were different: slow time constants were 6.5 +/- 0.6 and 3.5 +/- 0.4 s and slope factors were 4.7 +/- 0.2 and 7.0 +/- 0.8 mV, respectively. 6. This study provides immunofluorescence and functional evidence that Kv1.5 alpha-subunits are a component of native KDR channels of vascular smooth muscle cells of RPV. However, the differences in kinetics and voltage sensitivity of inactivation between L cell- and HEK293 cell-expressed channels and native KDR channels provide functional evidence that vascular KDR current is not due to homomultimers of RPV Kv1.5 alone. The channel structure may be more complex, involving heteromultimers and modulatory Kvbeta-subunits, and/or native KDR current may have other components involving Kvalpha-subunits of other families.

Regulation of smooth muscle actin-myosin interaction and force by calponin.

Smooth muscle contraction is regulated primarily by the reversible phosphorylation of myosin triggered by an increase in sarcoplasmic free Ca2+ concentration ([Ca2+]i). Contraction can, however, be modulated by other signal transduction pathways, one of which involves the thin filament-associated protein calponin. The h1 (basic) isoform of calponin binds to actin with high affinity and is expressed specifically in smooth muscle at a molar ratio to actin of 1:7. Calponin inhibits (i) the actin-activated MgATPase activity of smooth muscle myosin (the cross-bridge cycling rate) via its interaction with actin, (ii) the movement of actin filaments over immobilized myosin in the in vitro motility assay, and (iii) force development or shortening velocity in permeabilized smooth muscle strips and single cells. These inhibitory effects of calponin can be alleviated by protein kinase C (PKC)-catalysed phosphorylation and restored following dephosphorylation by a type 2A phosphatase. Three physiological roles of calponin can be considered based on its in vitro functional properties: (i) maintenance of relaxation at resting [Ca2+]i, (ii) energy conservation during prolonged contractions, and (iii) Ca(2+)-independent contraction mediated by phosphorylation of calponin by PKC epsilon, a Ca(2+)-independent isoenzyme of PKC.

Angiotensin II activation of protein kinase C decreases delayed rectifier K+ current in rabbit vascular myocytes.

1. The effect of angiotension II (Ang) on delayed rectifier K+ current (IK(V)) was studied in isolated rabbit portal vein smooth muscle cells using standard whole-cell voltage clamp technique. The effect of 100 nM Ang on macroscopic, whole-cell IK(V) was assessed in myocytes dialysed with 10 mM BAPTA, 5 mM ATP and 1 mM GTP either at room temperature or at 30 degrees C. 2. Application of Ang caused a decline in IK(V) which was reversed upon washout of the drug. Tail current recorded after 250 ms pulses to +30 mV and repolarization to -40 mV was reduced from 3.9 +/- 0.7 to 2.5 +/- 0.5 pA pF-1 at 20 degrees C (n = 6) and from 4.5 +/- 0.5 to 3.13 +/- 0.4 pA pF-1 at 30 degrees C(n = 17). 3. Ang had no effect on outward current in the presence of an AT1 selective antagonist, losartan (1 microM), which alone had no direct effect on the amplitude of IK(V). Substitution of extracellular Ca2+ with Mg2+ in the presence of 10 microM intracellular BAPTA did not affect the suppression of IK(V) by Ang. 4. Ang induced a decrease in time constant for the rapid phase of inactivation of the macroscopic current (tau 1 reduced from 377 +/- 32 to 245 +/- 11 ms; tau 2 unchanged, n = 17). Neither the voltage dependence of activation nor inactivation were affected by Ang. 5. The inhibition of IK(V) by Ang was abolished by intracellular dialysis with the selective PKC inhibitors, calphostin C (1 microM) and chelerythrine (50 microM). These data provide strong evidence that the decline in IK(V) due to Ang treatment is due to PKC activation. 6. The pattern of expression of PKC isoforms was examined in rabbit portal vein using isoenzyme-specific antibodies: alpha, epsilon and zeta isoenzymes were detected, but beta, gamma, delta and eta isoenzymes were not. 7. The lack of requirement for Ca2+, as well as the sensitivity of the Ang response to chelerythrine, suggest the involvement of the Ca(2+)-independent PKC isoenzyme epsilon in the signal transduction pathway responsible for IK(V) inhibition by Ang.

Epsilon-isoenzyme of protein kinase C induces a Ca(2+)-independent contraction in vascular smooth muscle.

We provide here the first direct evidence for in situ functional specificity of protein kinase C (PKC)-epsilon as a regulator of smooth muscle contractility. PKC is known to cause a Ca(2+)-independent contraction of ferret aortic smooth muscle, and the expression of two Ca(2+)-independent PKC isoenzymes, epsilon and zeta, has been demonstrated in this tissue. To test directly the hypothesis that one of these isoenzymes regulates contractility, constitutively active forms of PKC-epsilon and PKC-zeta were applied to saponin-permeabilized single ferret aortic smooth muscle cells. PKC-zeta caused no significant force response, but PKC-epsilon induced contraction of a magnitude (105 +/- 8 micrograms) similar to that produced by phenylephrine (110 +/- 10 micrograms), a relatively selective alpha 1-adrenergic agonist that triggers a PKC-dependent contraction. The PKC-epsilon-induced contraction was reversed by the PKC pseudosubstrate inhibitory peptide, PKC19-31. The myosin light chain kinase inhibitor 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9) did not affect the force response of PKC-epsilon-activated cells, suggesting that PKC-epsilon may induce this contraction solely via thin filament disinhibition. In support of this conclusion, calponin and caldesmon were shown to be good in vitro substrates of PKC-epsilon but not of PKC-zeta.

Protein kinase C inhibits delayed rectifier K+ current in rabbit vascular smooth muscle cells.

The effect of protein kinase C (PKC) activation on 4-aminopyridine (4-AP)-sensitive delayed rectifier current (IdK) was studied in isolated rabbit portal vein smooth muscle cells by use of standard whole cell voltage clamp. The effects of the phorbol ester, 4 beta-phorbol 12,13-dibutyrate (PdBu, 100 nM) and diacylglycerol analogues, 1,2-dioctanoyl-sn-glycerol (1,2-diC8, 10 microM) and 1,3-dioctanoyl-sn-glycerol (1,3-diC8, 10 microM), on macroscopic whole cell IdK were assessed in myocytes dialyzed with 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and 5 mM ATP (20-22 degrees C). Activation of PKC by 1,2-diC8 or PdBu caused a decline in IdK that was reversed with washout of drug. 1,2-diC8 had no effect on outward current present after exposure to 4-AP (20 mM). The inactive analogue, 1,3-diC8, did not affect IdK, but subsequent exposure to the active analogue, 1,2-diC8, caused a marked depression of the current. The inhibition of IdK by 1,2-diC8 was significantly reduced by intracellular dialysis with the inhibitors of PKC, chelerythrine (50 microM) and calphostin C (1 microM). Substitution of extracellular Ca2+ with Mg2+ in the presence of 10 mM intracellular BAPTA did not affect the suppression of IdK by 1,2-diC8, indicating the involvement of a Ca(2+)-independent isoform of PKC. This study suggests a novel signal transduction mechanism for inhibition of 4-AP-sensitive IdK involving a phosphotransferase reaction catalyzed by PKC in vascular smooth muscle myocytes.

Effects of calponin on force generation by single smooth muscle cells.

Although the actin-binding and actomyosin adenosinetriphosphatase (ATPase) inhibitory properties of calponin are well documented in vitro, its function in the smooth muscle cell has not been elucidated. To address this question, we utilized the ferret aortic smooth muscle cell, which shows a protein kinase C-dependent contraction even at pCa (-log [Ca2+]) 9.0 in the absence of a change in myosin light chain phosphorylation. Force was recorded from single, briefly permeabilized cells stimulated via a Ca(2+)-independent pathway by either phenylephrine or the epsilon isoenzyme of protein kinase C. Treatment of stimulated cells with wild-type recombinant calponin reduced steady-state contractile force by 45-60%. When calponin application preceded protein kinase C epsilon treatment, contraction was completely suppressed. On the other hand, calponin phosphorylated at Ser175 or mutant calponin with a Ser175 --> Ala replacement had no effect on contractile force. A peptide corresponding to Leu166-Gly194 of calponin, which included an actin-binding domain but excluded the actomyosin ATPase inhibitory region, was synthesized. Treatment of aortic smooth muscle cells with this peptide triggered a concentration-dependent contraction, presumably by alleviating the inhibitory effect of endogenous calponin. A control peptide with a scrambled sequence of the same residues produced no detectable contractile response. Although other interpretations are possible, these results are consistent with the view that calponin participates in thin filament-mediated regulation of smooth muscle contraction and that it may be part of a Ca(2+)-independent pathway downstream of protein kinase C epsilon.

Regulation of 4-aminopyridine-sensitive, delayed rectifier K+ channels in vascular smooth muscle by phosphorylation.

Voltage-gated, delayed rectifier K+ current (KV) that is sensitive to 4-aminopyridine (4AP) block has been identified in all vascular smooth muscle tissues studied to date. These channels conduct outward, hyperpolarizing K+ current that influences resting membrane potential and contributes to repolarization of action potentials. Smooth muscle cells in most arterial resistance vessels regulate Ca2+ influx and contractile tone by low amplitude, tonic changes in membrane potential. Block of KV with 4-aminopyridine leads to contraction and an enhanced myogenic response to increased intravascular pressure. We investigated the modulation of KV currents in isolated, freshly dispersed smooth muscle cells from rabbit portal vein and coronary arteries in whole-cell voltage clamp experiments. Our findings indicate that KV channels are regulated by signal transduction mechanisms involving vasoactive agonists that activate cAMP-dependent protein kinase (PKA) or protein kinase C (PKC). In this paper, the properties and potential function of KV channels in vascular smooth muscle are reviewed. Further, the regulation and potential role of alterations in KV due to beta-adrenoceptor agonists, adenylyl cyclase and PKA, as well as angiotensin II, diacylglycerol, and PKC are discussed.

Protein kinase C mediation of Ca(2+)-independent contractions of vascular smooth muscle.

Tumour-promoting phorbol esters induce slow, sustained contractions of vascular smooth muscle, suggesting that protein kinase C (PKC) may play a role in the regulation of smooth muscle contractility. In some cases, e.g., ferret aortic smooth muscle, phorbol ester induced contractions occur without a change in [Ca2+]i or myosin phosphorylation. Direct evidence for the involvement of PKC came from the use of single saponin-permeabilized ferret aortic cells. A constitutively active catalytic fragment of PKC induced a slow, sustained contraction similar to that triggered by phenylephrine. Both responses were abolished by a peptide inhibitor of PKC. Contractions of similar magnitude occurred even when the [Ca2+] was reduced to close to zero, implicating a Ca(2+)-independent isoenzyme of PKC. Of the two Ca(2+)-independent PKC isoenzymes, epsilon and zeta, identified in ferret aorta, PKC epsilon is more likely to mediate the contractile response because (i) PKC epsilon, but not PKC zeta, is responsive to phorbol esters; (ii) upon stimulation with phenylephrine, PKC epsilon translocates from the sarcoplasm to the sarcolemma, whereas PKC zeta, translocates from a perinuclear localization to the interior of the nucleus; and (iii) when added to permeabilized single cells of the ferret aorta at pCa 9, PKC epsilon, but not PKC zeta, induced a contractile response similar to that induced by phenylephrine. A possible substrate of PKC epsilon is the smooth muscle specific, thin filament associated protein, calponin. Calponin is phosphorylated in intact smooth muscle strips in response to carbachol, endothelin-1, phorbol esters, or okadaic acid. Phosphorylation of calponin in vitro by PKC (a mixture of alpha, beta, and gamma isoenzymes) dramatically reduces its affinity for F-actin and alleviates its inhibition of the cross-bridge cycling rate. Calponin is phosphorylated in vitro by PKC epsilon but is a very poor substrate of PKC zeta. A signal transduction pathway is proposed to explain Ca(2+)-independent contraction of ferret aorta whereby extracellular signals trigger diacylglycerol production without a Ca2+ transient. The consequent activation of PKC epsilon would result in calponin phosphorylation, its release from the thin filaments, and alleviation of inhibition of cross-bridge cycling. Slow, sustained contraction then results from a slow rate of cross-bridge cycling because of the basal level of myosin light chain phosphorylation (approximately 0.1 mol Pi/mol light chain). We also suggest that signal transduction through PKC epsilon is a component of contractile responses triggered by agonists that activate phosphoinositide turnover; this may explain why smooth muscles often develop more force in response, e.g., to alpha 1-adrenergic agonists than to K+.

Identification of protein kinase C isoenzymes in smooth muscle: partial purification and characterization of chicken gizzard PKC zeta.

The pattern of expression of protein kinase C (PKC) isoenzymes was examined in chicken gizzard smooth muscle using isoenzyme-specific antibodies: alpha, delta, epsilon, eta, and zeta isoenzymes were detected. PKC alpha associated with the particulate fraction in the presence of Ca2+ and was extracted by divalent cation chelators. PKC delta required detergent treatment for extraction from the EDTA-EGTA-washed particulate fraction. PKC epsilon, eta, and zeta were recovered in the cytosolic fraction prepared in the presence of Ca2+. PKC zeta, which has been implicated in the regulation of gene expression in smooth muscle, was partially purified from chicken gizzard. Two peaks of PKC zeta-immunoreactive protein (M(r) 76 000) were eluted from the final column; only the second peak exhibited kinase activity. The specific activity of PKC zeta with peptide epsilon (a synthetic peptide based on the pseudosubstrate domain of PKC epsilon) as substrate was 2.1 mumol P(i).min-1.(mg PKC zeta)-1 and, with peptide zeta as substrate, was 1.2 mumol P(i).min-1.(mg PKC zeta)-1. Activity in each case was independent of Ca2+, phospholipid, and diacylglycerol. Lysine-rich histone III-S was a poor substrate for PKC zeta (specific activity, 0.1-0.3 mumol P(i).min-1.mg-1). Two proteins, calponin and caldesmon, which have been implicated in the regulation of smooth muscle contraction and are phosphorylated by cPKC (a mixture of alpha, beta, and gamma isoenzymes), were also poor substrates of PKC zeta (specific activities, 0.04 and 0.02 mumol P(i).min-1.mg-1, respectively). Chicken gizzard PKC zeta was insensitive to the PKC activator phorbol 12,13-dibutyrate or the PKC inhibitor chelerythrine. The properties of PKC zeta are, therefore, quite distinct from those of other well-characterized PKC isoenzymes.

Smooth muscle protein kinase C.

Protein kinase C (PKC) was first implicated in the regulation of smooth muscle contraction with the observation that phorbol esters induce slowly developing, sustained contractions. In some vascular smooth muscles, e.g., ferret aorta, phorbol ester induced contractions occur without an increase in sarcoplasmic free-Ca2+ concentration ([Ca]i) or myosin light chain phosphorylation. This response appears to be mediated by a Ca(2+)-independent isoenzyme of PKC (probably PKC epsilon), since saponin-permeabilized single ferret aortic smooth muscle cells, which retain receptor coupling, developed force in response to phenylephrine at low free [Ca2+] (pCa 7.0-8.6) and the constitutively active proteolytic fragment of PKC (PKM) elicited a contraction at pCa 7 comparable with the phenylephrine-induced contraction. Both contractions were reversed by a pseudo-substrate peptide inhibitor of PKC. These observations suggest a mechanism whereby alpha-adrenergic agonists may elicit a contractile response without a Ca2+ signal: alpha-adrenergic stimulation of phosphatidylcholine-specific phospholipase C or D (the latter in conjunction with phosphatidate phosphohydrolase) generates diacylglycerol. In the absence of an increase in [Ca2+]i, diacylglycerol specifically activates so-called novel PKCs, of which epsilon is the only isoenzyme known to be expressed in vascular smooth muscle. Recent evidence suggests that PKC may trigger a cascade of phosphorylation reactions, resulting in activation of mitogen-activated protein kinase and phosphorylation of the thin filament associated protein caldesmon. Alternatively, or additionally, PKC may directly phosphorylate calponin, another thin filament associated protein. These phosphorylations are predicted to alleviate inhibition of the cross-bridge cycling rate by these thin-filament proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.