ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

The mechanism responsible for the regulation of cardiac function by endogenous nitric oxide (NO) remains unclear. In this investigation, O2consumption by freshly isolated myocardial muscle segments from the left ventricular free wall of canine hearts was quantified by a Clark-type O2electrode at 37 degrees C. S-nitroso-N-acetylpenicillamine (SNAP, 9+/-3% to 50+/-8%), bradykinin (BK, 14+/-3% to 30+/-5%), or carbachol (CCh, 15+/-4% to 29+/-4%) significantly attenuated tissue O2consumption at doses of 10 sup -7 to 10 sup -4 mol/L (mean+/-SE, P<.05). The effects of BK and CCh, but not SNAP, were blocked by 10 sup -4 mol/L NG-nitro-L-arginine, consistent with both BK and CCh stimulating NO biosynthesis and with SNAP decomposing to release NO, respectively. Similar doses of 8-Br-cGMP caused a respiratory inhibition, but to a lesser extent (9+/-2% to 14+/-6%). A mitochondrial uncoupler, 2,4-dinitrophenol (at 1 mmol/L), blocked the effects of 8-Br-cGMP, but not those of SNAP, BK, or CCh, suggesting that the major site of action of NO is on mitochondrial electron transport. Myocardial muscle from dogs with pacing-induced heart failure had a basal O2consumption rate of 251+/-21 nmol centered dot min sup -1 centered dot g sup -1, which was 54% higher than the rate seen in muscle from normal healthy canine hearts. The inhibitory effects of BK and CCh on O2consumption were not observed in failing cardiac tissue, but SNAP showed an unaltered inhibitory effect. Therefore, our results indicate that NO released from microvascular endothelium by BK, stimulation of muscarinic receptors, and perhaps flow velocity may play an important physiological role in the control of cardiac mitochondrial respiration, and the loss of this regulatory function may contribute to the development of heart failure.(Circ Res. 1996;79:381-387.)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

We have previously demonstrated that at least four isoforms of protein kinase C (PKC; alpha, delta, epsilon, and zeta) are expressed in neonatal rat ventricular myocytes and that development is associated with a decline in their expression. The mechanism(s) regulating PKC isoform expression in ventricular myocytes is completely unknown. The developmental decline in PKC expression occurs, in large part, during the first 2 weeks of postnatal life, while thyroid hormone levels are known to be progressively increasing. Accordingly, this study examined the influence of thyroid hormone on PKC isoform expression to determine whether thyroid hormone can be implicated as a potential physiological regulator of PKC gene expression during normal cardiac development. Hypothyroidism was induced in adult rats by surgical thyroidectomy; thyroid status was manipulated in cultured neonatal ventricular myocytes by growth in serum-free medium with varying triiodothyronine (T3) levels. In each case, hypothyroidism was verified by a 10- to 50-fold increase in steady state mRNA for beta-myosin heavy chain. In hypothyroid adult ventricular myocardium, there was a selective 60% increase in the expression of PKC epsilon protein that corresponded to an increase in maximally stimulated PKC enzyme activity with PKC epsilon substrate peptide (epsilonpep) but not with histone as substrate. Northern blot analysis revealed a 70% increase in PKC epsilon mRNA, indicating that the regulatory effects of thyroid hormone are mediated, at least in part, at the message level. In neonatal ventricular myocytes, there was a T3-dependent reduction in immunoreactivity for both PKC alpha and PKC epsilon that was associated with significant reductions in both histone- and epsilonpep-kinase activities. The concentration of T3that half-maximally repressed PKC alpha and PKC epsilon expression was approximate equal 0.5 nmol/L. Thyroid hormone had no effect on PKC delta and PKC zeta expression in neonatal or adult ventricular myocytes. PKC isoform expression in cardiac fibroblasts was not influenced by variations in the thyroid hormone concentration during culture. These results provide evidence that thyroid hormone specifically represses PKC alpha and PKC epsilon in the neonatal heart and PKC epsilon in the adult heart. Thyroid hormone-induced changes in PKC may play an important permissive role in the modulation of autonomic responsiveness in ventricular cardiomyocytes.(Circ Res. 1996;79:388-398).

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

Activation of both ATP-sensitive K sup + (KATP) channels and the enzyme protein kinase C (PKC) has been associated with the cardioprotective response of ischemic preconditioning. We recently showed that at low cytoplasmic ATP (<or=to50 micro mol/L), PKC inhibits KATPchannel activity. This finding is surprising, as both KATPchannels and PKC are activated during preconditioning. However, PKC also altered ATP binding to the channel, changing the Hill coefficient from approximate equals 2 to approximate equals 1. This apparent change in stoichiometry would lead to a PKC-induced activation of KATPchannels at more physiological (millimolar) levels of ATP. The aim of the present study was to determine whether PKC activates cardiac KATPchannels at millimolar levels of ATP. The effects of PKC on single KATPchannels were studied at millimolar internal ATP levels using excised inside-out membrane patches from rabbit ventricular myocytes. Application of purified constitutively active PKC (20 nmol/L) to the intracellular surface of the patches produced an approximately threefold increase in the channel open probability. The specific PKC inhibitor peptide PKC(19-31) prevented this increase. Heat-inactivated PKC had no effect on KATPchannel properties. KATPchannel activity spontaneously returned to control levels after washout of PKC. This spontaneous reversal did not occur in the presence of 5 nmol/L okadaic acid, suggesting that the reversal of PKC's action is dependent on activity of a membrane-associated type 2A protein phosphatase (PP2A). In the presence of exogenous PP2A (7.5 nmol/L), PKC had no effect. We conclude that the PKC-induced increase in KATPchannel activity at millimolar ATP results from a crossing of the ATP concentration-response curves for inhibition of the phosphorylated and nonphosphorylated forms of the channel. This identifies a mechanism by which PKC activates KATPchannels at near physiological levels of ATP and thus could link these two components in a signaling pathway that induces ischemic preconditioning.(Circ Res. 1996;79:399-406.)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

The delay of infarct size development by ischemic preconditioning involves the activation of protein kinase C in rats and rabbits. In dogs the role of protein kinase C in ischemic preconditioning is controversial. We investigated whether or not the activation of protein kinase C is a prerequisite for ischemic preconditioning in swine. Swine were used, since they are large mammals and since infarct development in this species, due to the lack of an innate collateral circulation, is similar to that in humans. In 20 enflurane-anesthetized swine, the proximal left anterior descending coronary artery was cannulated and perfused from an extracorporeal circuit. The impact of continuous intracoronary infusion of 10 sup -7 mol/L staurosporine, a potent protein kinase C inhibitor, on global and regional myocardial function (sonomicrometry), subendocardial blood flow (ENDO, microspheres), and infarct size (IS, triphenyltetrazolium chloride staining after 120 minutes of reperfusion) was analyzed. Staurosporine (10 sup -7 mol/L) abolished the 1.6-fold increase in coronary arterial resistance in response to 10 sup -6 mol/L IC 4 beta-phorbol 12-myristate 13-acetate, a potent protein kinase C activator. In the presence of staurosporine, 90 minutes of low-flow ischemia at an ENDO of 0.05+/-0.04 (mean+/-SD) mL centered dot min sup -1 centered dot g sup -1 resulted in an IS of 12.5+/-8.6% (n=10) of the area at risk. Also, in the presence of staurosporine, ischemic preconditioning by a cycle of 10 minutes of low-flow ischemia followed by 15 minutes reperfusion before the 90 minutes sustained ischemic period (ENDO, 0.05+/-0.03 mL centered dot min sup -1 centered dot g sup -1) reduced IS to 3.3+/-3.4% (n=10, P<.05). The protein kinase C inhibitor staurosporine does not prevent ischemic preconditioning in swine.(Circ Res. 1996;79:407-414.)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

PD 81,723 (PD) acts allosterically to increase agonist binding to A sub 1 adenosine receptors and to enhance functional A sub 1 receptor-mediated responses in the heart and other tissues. To determine if PD lowers the threshold for ischemic preconditioning (PC), pentobarbital-anesthetized dogs were subjected to 60 minutes of left anterior descending coronary artery (LAD) occlusion and 3 hours of reperfusion. Ischemic PC was produced by either 2.5 or 5 minutes of LAD occlusion 10 minutes before the 60-minute occlusion. PD (100 micro gram/kg total dose, 5 to 50 micro mol/L in coronary arterial blood) or vehicle was infused intracoronarily for 17.5 minutes before the 60-minute occlusion period in non-PC dogs or in dogs preconditioned with 2.5 minutes of ischemia. Myocardial infarct size was determined by triphenyltetrazolium staining and expressed as a percentage of the area at risk. Compared with the control group (26.3+/-3.6%, mean+/-SEM), infarct size was not significantly affected by 2.5 minutes of PC alone (23.4+/-4.2%) or by PD alone (26.5+/-1.7%) but was decreased by PD+PC (14.6+/-1.7%, P<.05) or by a longer period (5 minutes) of PC alone (12.5+/-3.3%). The intravenous administration of the selective antagonist of A1adenosine receptors, 8-cyclopentyl-1,3-dipropylxanthine (1 mg/kg), or the ATP-sensitive K sup + channel blocker, glibenclamide (0.3 mg/kg), for 15 minutes before PD+PC blocked the protection (23.6+/-2.3% or 25.9+/-3.3%, respectively). None of the compounds studied affected systemic hemodynamics, collateral blood flow, or AAR. To determine which subtypes of canine adenosine receptors were affected by 10 micro mol/L PD, radioligand binding studies were conducted using membranes derived from COS-7 cells expressing recombinant canine receptors and agonist radioligands. PD enhanced the binding of [sup 125 I]N sup 6-4-amino-3-iodobenzyladenosine (sup 125 I-ABA) to A1receptors by increasing the t1/2 for dissociation by 2.18-fold, but PD had no effect on the dissociation kinetics of125I-ABA from A3receptors or [sup 125 I]-[2-(4-amino-3-iodo-phenyl)ethylamino] adenosine from A2Areceptors. Glibenclamide at concentrations up to 10 micro mol/L had no effect on the binding of radioligands to recombinant canine A1, A2A, or A3receptors. These data suggest that PD reduces the amount of time required for ischemia to produce preconditioning by enhancing adenosine binding to its A1receptor. Glibenclamide prevents the protection afforded by A1receptor activation by a mechanism not involving adenosine receptor blockade.(Circ Res. 1996;79:415-423.)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

6 hours to develop, lasts for at least 60 hours after its appearance (with the most effective protection present at 24 hours and 3 days), and disappears within 6 days after the preconditioning ischemia, a time course that is consistent with the synthesis and degradation of cardioprotective proteins. In view of its sustained duration, this endogenous cardioprotective mechanism is of potential clinical importance.(Circ Res. 1996;79:424-434.)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

Prior transient episodes of ischemia (``ischemic preconditioning'') reduce lactate accumulation and attenuate acidosis during a subsequent prolonged ischemic insult. The mechanisms responsible for attenuated glycolytic catabolite accumulation have not been established but may include earlier exhaustion of glycogen stores, slowed glycogenolysis before complete glycogen depletion, and/or inhibition of glycolysis. Simultaneous repeated measures of myocardial glycogen and the rates of glycolysis, glycogenolysis, glucose utilization, and glycolytic ATP production were obtained during total ischemia by13C nuclear magnetic resonance spectroscopy in control and ischemia-preconditioned isolated rat hearts. Both [sup 13 C]glycolytic and [sup 13 C]glycogenolytic rates were significantly lower during total ischemia in preconditioned compared with control hearts (0.77+/-0.04 versus 1.06+/-0.06 micro mol/min per gram wet weight [P<.01] for glycolysis and 0.15+/-0.07 versus 0.78+/-0.12 micro mol/min per gram wet weight [P<.001] for glycogenolysis, respectively, at 2.5 minutes of ischemia). Slowed glycolysis was present even during the early minutes of ischemia, when significant amounts of available [sup 13 C]glycogen were still present. Importantly, the reduction in the rate of glycogenolysis was larger and out of proportion to the reduction in glycolysis and occurred despite an increase in glucose utilization in preconditioned hearts (2.23+/-0.15 versus 1.5+/-0.10 micro mol/min per gram wet weight at 1.25 minutes, P<.01). During early ischemia, conversion of glycogen phosphorylase to the a or ``active'' form was less in preconditioned than in control hearts (29.1+/-2.6% versus 41.2+/-9.8%, respectively; P<.05). Taken together, these findings demonstrate that ischemic preconditioning significantly depresses glycolytic catabolite accumulation during sustained ischemia not by more severe glycolytic inhibition or exhaustion of glycogen stores but by depressed glycogenolysis from the onset of ischemia.(Circ Res. 1996;79:435-446.)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

The decrease in Ca sup 2+ responsiveness of myofilaments in stunned myocardium implies that there may be structural changes in proteins composing the contractile machinery. To elucidate the lesion in stunned myocardium, isolated guinea pig hearts were subjected to global ischemia at 37 degrees C and reperfused. SDS-PAGE revealed that the contents of desmin, alpha-actinin, and spectrin decreased in the myofibrillar fraction isolated from hearts reperfused after 60-minute ischemia compared with nonischemic control hearts. To examine the change of cytoskeletal proteins in stunned myocardium, immunohistochemical studies with antibodies against these proteins were performed after 15 minutes of ischemia. In stunned myocardium, the staining was largely intact, but there were some lesions where desmin was not stained and alpha-actinin and spectrin were only weakly identified. The percentage of normally stained areas in the myocardium (percent stained area), quantified by image processing, was significantly lower in stunned myocardium (79.6+/-3.6%, mean+/-SEM) than in nonischemic control myocardium (96.5+/-0.7%). Percent recovery of developed pressure significantly correlated with percent stained area (r=.82, P<.001). In hearts subjected to 15-minute ischemia but not reperfused, or in hearts reperfused with Ca2+-free solution after 15-minute ischemia, staining by the antibodies remained intact, suggesting that the change of the cytoskeletal proteins is mediated by Ca2+ overload during reperfusion. In hearts treated with the protease inhibitor leupeptin (50 micro mol/L) or calpain inhibitor I (100 micro mol/L), both developed pressure and staining were well preserved. These results indicate that contractile dysfunction in stunned myocardium has a strong correlation with the disappearance of cytoskeletal proteins that may be mediated by a Ca2+-dependent intracellular protease activated during reperfusion. The disruption of cytoskeletal proteins is a possible mechanism for stunning, although it may be a secondary effect of protease activation.(Circ Res. 1996;79:447-454.)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

During myocardial ischemia, a local release of noradrenaline coincides with an increased density of beta-adrenergic receptors. The functional activity of these receptors, however, is mainly determined by their state of phosphorylation. The beta-adrenergic receptor kinase (beta ARK) specifically phosphorylates and thereby inactivates beta-adrenergic receptors after stimulation by receptor agonists, facilitating the binding of the inhibitor protein beta-arrestion to the receptors. beta ARK activation involves a translocation of the enzyme to the membrane. In the present study, we investigated the density and the functional activity of beta-adrenergic receptors, the enzymatic activity of beta ARK in membranes and cytosol, the mRNA levels of beta ARK-1, and the expression of beta-arrestin during stop-flow and low-flow ischemia in the isolated perfused rat heart. After 60 minutes of stop-flow ischemia, beta-adrenergic receptor density was upregulated, but beta-agonist-mediated adenylate cyclase activity was blunted. Simultaneously, beta ARK activity in the particulate fraction was significantly induced. The increase in beta ARK activity was reversible after inhibition of ischemia-evoked noradrenaline release by desipramine. Also, exposure to externally given noradrenaline increased beta ARK activity in the particulate fraction. Cytosolic beta ARK activity remained largely unchanged during stop-flow or low-flow ischemia. The steady state concentration of beta ARK-1 mRNA increased after 20 minutes of stop-flow ischemia and then returned to baseline values after another 20 minutes. Cardiac ischemia did not alter beta-arrestin levels. During myocardial ischemia, an increase in the number of beta-adrenergic receptors is paralleled by increased membrane activity of the receptor kinase beta ARK. This increased membrane activity may contribute to enhanced receptor phosphorylation and inactivation.(Circ Res. 1996;79:455-460.)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID