The effect of adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA) activity on 4-aminopyridine (4-AP)-sensitive delayed rectifier current (IdK) in isolated rabbit portal vein smooth muscle cells was studied via whole cell voltage clamp (20-22 degrees C). A threefold increase in 4-AP-sensitive (5 mM) IdK was recorded after gaining cell access during dialysis with 5 mM intracellular ATP and internal Ca2+ buffered to a low level with 5 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Dialysis with the nonhydrolyzable ATP analogue 5'-adenylylimidodiphosphate (5 mM) or the specific peptide inhibitor of PKA (PKI; 10 microM) reduced current runup by 50 and 70%, respectively. Delayed dialysis with PKI reversed runup and inhibited IdK to below initial levels. Forskolin (1 microM) caused a reversible increase in IdK, which was inhibited by 4-AP (5 mM). Isoproterenol (1 microM) reversibly enhanced IdK; the increase was sensitive to propranolol (2 microM) and 4-AP (5 mM) and was prevented by dialysis with PKI (10 microM). IdK was enhanced over the entire voltage range of activation and associated with a negative shift in reversal potential of net whole cell current, consistent with hyperpolarization of resting membrane potential. The data provide the first evidence for a signal transduction mechanism involving beta-adrenoceptors, adenylate cyclase, and a phosphotransferase reaction mediated by PKA for the regulation of delayed rectifier K+ channels in vascular smooth muscle.

Cat ventricular myocytes loaded with [Ca2+]i‐ and pHi‐sensitive probes were used to examine the subcellular mechanism(s) of the Ang II‐induced positive inotropic effect. Ang II (1 μM) produced parallel increases in contraction and Ca2+ transient amplitudes and a slowly developing intracellular alkalisation. Maximal increases in contraction amplitude and Ca2+ transient amplitude were 163 ± 22 and 43 ± 8 %, respectively, and occurred between 5 and 7 min after Ang II administration, whereas pHi increase (0·06 ± 0·03 pH units) became significant only 15 min after the addition of Ang II. Furthermore, the inotropic effect of Ang II was preserved in the presence of Na+‐H+ exchanger blockade. These results indicate that the positive inotropic effect of Ang II is independent of changes in pHi. Similar increases in contractility produced by either elevating extracellular [Ca2+] or by Ang II application produced similar increases in peak systolic Ca2+ indicating that an increase in myofilament responsiveness to Ca2+ does not participate in the Ang II‐induced positive inotropic effect. Ang II significantly increased the L‐type Ca2+ current, as assessed by using the perforated patch‐clamp technique (peak current recorded at 0 mV: ‐1·88 ± 0·16 pA pF−1 in control vs. ‐3·03 ± 0·20 pA pF−1 after 6‐8 min of administration of Ang II to the bath solution). The positive inotropic effect of Ang II was not modified in the presence of either KB‐R7943, a specific blocker of the Na+‐Ca2+ exchanger, or ryanodine plus thapsigargin, used to block the sarcoplasmic reticulum function. The above results allow us to conclude that in the cat ventricle the Ang II‐induced positive inotropic effect is due to an increase in the intracellular Ca2+ transient, an enhancement of the L‐type Ca2+ current being the dominant mechanism underlying this increase.

Taken together, the results indicate that a low dose of angiotensin II induces release of endothelin, which, in autocrine/paracrine fashion activates the Na(+)/H(+) exchanger, increases [Na(+)](i) and changes E(NCX), promoting the influx of Ca(2+) that leads to a positive inotropic effect (PIE).

The perforated whole‐cell configuration of patch clamp and the pH fluorescent indicator SNARF were used to determine the electrogenicity of the Na+‐HCO3− cotransport in isolated rat ventricular myocytes. Switching from Hepes buffer to HCO3− buffer at constant extracellular pH (pHo) hyperpolarized the resting membrane potential (RMP) by 2.9 ± 0.4 mV (n= 9, P < 0.05). In the presence of HCO3−, the anion blocker SITS depolarized RMP by 2.6 ± 0.5 mV (n= 5, P < 0.05). No HCO3−‐induced hyperpolarization was observed in the absence of extracellular Na+. The duration of the action potential measured at 50 % of repolarization time (APD50) was 29.2 ± 6.1 % shorter in the presence of HCO3− than in its absence (n= 6, P < 0.05). Quasi‐steady‐state currents were evoked by voltage‐clamped ramps ranging from −130 to +30 mV, during 8 s. The development of a novel component of Na+‐dependent and Cl−‐independent steady‐state outward current was observed in the presence of HCO3−. The reversal potential (Erev) of the Na+‐HCO3− cotransport current (INa,Bic) was measured at four different levels of extracellular Na+. A HCO3−:Na+ ratio compatible with a stoichiometry of 2:1 was detected. INa,Bic was also studied in isolation in standard whole‐cell experiments. Under these conditions, INa,Bic reversed at −96.4 ± 1.9 mV (n= 5), being consistent with the influx of 2 HCO3− ions per Na+ ion through the Na+‐HCO3− cotransporter. In the presence of external HCO3−, after 10 min of depolarizing the membrane potential (Em) with 45 mm extracellular K+, a significant intracellular alkalinization was detected (0.09 ± 0.03 pH units; n= 5, P < 0.05). No changes in pHi were observed when the myocytes were pre‐treated with the anion blocker DIDS (0.001 ± 0.024 pH units; n= 5, n.s.), or when exposed to Na+‐free solutions (0.003 ± 0.037 pH units; n= 6, n.s.). The above results allow us to conclude that the cardiac Na+‐HCO3− cotransport is electrogenic and has an influence on RMP and APD of rat ventricular cells.

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Ca2+-Calmodulin kinase II (CaMKII) activation is deleterious in cardiac ischemia/reperfusion (I/R). Moreover, inhibition of CaMKII-dependent phosphorylations at the sarcoplasmic reticulum (SR) prevents CaMKII-induced I/R damage. However, the downstream targets of CaMKII at the SR level, responsible for this detrimental effect, remain unclear. In the present study we aimed to dissect the role of the two main substrates of CaMKII at the SR level, phospholamban (PLN) and ryanodine receptors (RyR2), in CaMKII-dependent I/R injury. In mouse hearts subjected to global I/R (45/120 min), phosphorylation of the primary CaMKII sites, S2814 on cardiac RyR2 and of T17 on PLN, significantly increased at the onset of reperfusion whereas PKA-dependent phosphorylation of RyR2 and PLN did not change. Similar results were obtained in vivo, in mice subjected to regional myocardial I/R (1/24 hrs). Knock-in mice with an inactivated serine 2814 phosphorylation site on RyR2 (S2814A), significantly improved post-ischemic mechanical recovery, reduced infarct size and decreased apoptosis. Conversely, knock-in mice, in which CaMKII site of RyR2 is constitutively activated (S2814D), significantly increased infarct size and exacerbated apoptosis. In S2814A and S2814D mice subjected to regional myocardial ischemia, infarct size was also decreased and increased respectively. Transgenic mice with double-mutant non-phosphorylatable PLN (S16A/T17A) in the PLN knockout background (PLNDM) also showed significantly increased post-ischemic cardiac damage. This effect cannot be attributed to PKA-dependent PLN phosphorylation and was not due to the enhanced L-type Ca2+ current, present in these mice. Our results reveal a major role for the phosphorylation of S2814 site on RyR2 in CaMKII-dependent I/R cardiac damage. In contrast, they showed that CaMKII-dependent increase in PLN phosphorylation during reperfusion opposes rather than contributes to I/R damage.

BACKGROUND AND PURPOSENa + /HCO3 -co-transport (NBC) regulates intracellular pH (pHi) in the heart. We have studied the electrogenic NBC isoform NBCe1 by examining the effect of functional antibodies to this protein. EXPERIMENTAL APPROACHWe generated two antibodies against putative extracellular loop domains 3 (a-L3) and 4 (a-L4) of NBCe1 which recognized NBCe1 on immunoblots and immunostaining experiments. pHi was monitored using epi-fluorescence measurements in cat ventricular myocytes. Transport activity of total NBC and of NBCe1 in isolation were evaluated after an ammonium ioninduced acidosis (expressed as H + flux, JH, in mmol·L -1 min -1 at pHi 6.8) and during membrane depolarization with high extracellular potassium (potassium pulse, expressed as DpHi) respectively. KEY RESULTSThe potassium pulse produced a pHi increase of 0.18 Ϯ 0.006 (n = 5), which was reduced by the a-L3 antibody (0.016 Ϯ 0.019). The a-L-3 also decreased JH by 50%. Surprisingly, during the potassium pulse, a-L4 induced a higher pHi increase than control,(0.25 Ϯ 0.018) whereas the recovery of pHi from acidosis was faster (JH was almost double the control value). In perforated-patch experiments, a-L3 prolonged and a-L4 shortened action potential duration, consistent with blockade and stimulation of NBCe1-carried anionic current respectively. CONCLUSIONS AND IMPLICATIONSBoth antibodies recognized NBCe1, but they had opposing effects on the function of this transporter, as the a-L3 was inhibitory and the a-L4 was excitatory. These antibodies could be valuable in studies on the pathophysiology of NBCe1 in cardiac tissue, opening a path for their potential clinical use.

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.