Morphine preconditioning confers cardioprotection in doxorubicin-induced failing rat hearts via ERK/GSK-3ß pathway independent of PI3K/Akt.

Preconditioning against myocardial ischemia-reperfusion (I/R) injury can be suppressed in some pathological conditions. This study was designed to investigate whether morphine preconditioning (MPC) exerts cardioprotection in doxorubicin (DOX)-induced heart failure in rats and the mechanisms involved. Phosphatidylinositol-3 kinase/protein kinase B (PI3K/Akt), extracellular signal-regulated kinase (ERK) and glycogen synthase kinase (GSK)-3ß pathways were examined. Normal and DOX-induced failing rat hearts were subjected to I/R injury using a Langendorff perfusion system with or without MPC or ischemic preconditioning (IPC). The PI3K inhibitor (wortmannin) or ERK inhibitor (PD98059) was infused before MPC. In normal hearts, both MPC and IPC significantly reduced infarct size and the rise in lactate dehydrogenase (LDH) level caused by I/R injury. Pretreatment with wortmannin or PD98059 abrogated the protective effects of MPC and suppressed the phosphorylation of Akt, ERK and GSK-3ß. In failing rat hearts, however, MPC retained its cardioprotection while IPC did not. This protective effect was abolished by PD98059 but not wortmannin. MPC increased the level of p-ERK rather than p-Akt. The phosphorylation of GSK-3ß induced by MPC was reversed by PD98059 only. IPC did not elevate the expression of p-ERK, p-Akt and p-GSK-3ß in failing rat hearts. We conclude that MPC is cardioprotective in rats with DOX-induced heart failure while IPC is not. The effect of MPC appears to be mediated via the ERK/GSK-3ß pathway independent of PI3K/Akt.

The function of calcineurin and ERK1/2 signal in the antihypertrophic effects of kappa-opioid receptor stimulation on myocardial hypertrophy induced by isoprenaline.

The aim of the present study, performed in an in vitro model of cardiac hypertrophy, was to examine the possible function of calcineurin and ERK1/2 in the inhibitory effects of kappa-opioid receptor stimulation on Ca2+ transients and myocardial hypertrophy induced by beta1-adrenoceptor stimulation. We determined the effects of trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamid methanesulfonate salt (U50,488H), a selective kappa-opioid receptor agonist, on the enhancement of spontaneous Ca2+ transients and the induction of hypertrophy by isoprenaline, a beta-adrenoceptor agonist, in cultured neonatal ventricular myocytes. Total protein content, [3H]leucine incorporation and cell size were used as indices of hypertrophy; calcineurin activity and phospho-ERK1/2 level were determined by immunoblotting. Isoprenaline (10 micromol x L(-1)) increased all the three indices of hypertrophy, Ca2+ transients, calcineurin activity and the level of phospho-ERK1/2. The effects of isoprenaline were abolished by 1 micromol x L(-1) U50,488H in the absence but not in the presence of nor-binaltorphimine, a kappa-opioid receptor antagonist. The inhibitory effects of U50,488H were reproduced by cyclosporine-A, an inhibitor of calcineurin, U0126, the inhibitor of ERK1/2 and verapamil, a L-type Ca2+ channel antagonist. In addition, suppression of calcineurin activity by cyclosporine-A was associated with modest suppression of ERK1/2 phosphorylation. Meanwhile, suppression of ERK1/2 phosphorylation by U0126 was associated with modest suppression of calcineurin activity. In conclusion, the inhibitory effects of kappa-opioid receptor stimulation involved calcineurin and ERK1/2, and the two signaling pathways showed interaction in the mechanism of antihypertrophic effects afforded by kappa-opioid receptor stimulation.

Polyol pathway impairs the function of SERCA and RyR in ischemic-reperfused rat hearts by increasing oxidative modifications of these proteins.

A number of studies have shown that the polyol pathway, consisting of aldose reductase (AR) and sorbitol dehydrogenase (SDH), contributes to ischemia-reperfusion (I/R)-induced myocardial infarction due to depletion of ATP. In this report we show that the polyol pathway in I/R heart also contributes to the impairment of sacro/endoplasmic reticulum Ca(2+)-ATPase (SERCA) and ryanodine receptor (RyR), two key players in Ca(2+) signaling that regulate cardiac contraction. Rat hearts were isolated and retrogradely perfused with either Krebs' buffer containing 1 microM AR inhibitor, zopolrestat, or 200 nM SDH inhibitor, CP-170,711, and challenged by 30 min of regional ischemia and 45 min of reperfusion. We found that post-ischemic contractile function of the isolated perfused hearts was improved by pharmacological inhibition of the polyol pathway. I/R-induced contractile dysfunction is most likely due to impairment in Ca(2+) signaling and the activities of SERCA and RyR. All these abnormalities were significantly ameliorated by treatment with ARI or SDI. We showed that the polyol pathway activities increase the level of peroxynitrite, which enhances the tyrosine nitration of SERCA and irreversibly modifies it to form SERCAC674-SO(3)H. This leads to reduced level of S-glutathiolated SERCA, contributing to its inactivation. The polyol pathway activities also deplete the level of GSH, leading to decreased active RyR, the S-glutathiolated RyR. Thus, in I/R heart, inhibition of polyol pathway improved the function of SERCA and RyR by protecting them from irreversible oxidation.

Intrathecal morphine preconditioning induces cardioprotection via activation of delta, kappa, and mu opioid receptors in rats.

BACKGROUND: Small doses of intrathecal morphine provide cardioprotection similar to that conferred by IV morphine. However, the extent of intrathecal morphine preconditioning (IT-MPC) relative to that resulting from ischemic preconditioning (IPC) is unknown. Further, it is uncertain whether IT-MPC is mediated by opioid receptor dependent pathways. In this study, we compared the extent of cardioprotection conferred by IT-MPC with IPC and investigated the role of opioid receptors in this effect. METHODS: Eighty anesthetized, open-chest, male Sprague-Dawley rats were assigned to 1 of 13 groups (n = 6-7) after successful intrathecal catheter placement. Rats in the IPC group were subjected to three 5-min cycles of myocardial ischemia (induced by occlusion of the left main coronary artery) interspersed with 5 min of reperfusion. After IPC, myocardial ischemia and reperfusion injury was induced by 30 min of left main coronary artery occlusion followed by 2 h of reperfusion. In the IT-MPC groups, the rats were given 3 consecutive 5 min intrathecal morphine infusions (0.03, 0.3, 3.0, or 30.0 microg/kg, respectively) interspersed with 5 min infusion-free periods, before myocardial ischemia reperfusion injury. In 2 other groups either 300microg/kg of IV morphine or 10 microL of intrathecal normal saline were given. The selective delta, kappa, and mu opioid receptor antagonists naltrindole, nor-binaltorphimine, and D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), respectively, were given to groups of animals receiving IT-MPC to evaluate the relative role of the specific opioid receptor subtypes in IT-MPC preconditioning. Myocardial infarct size (IS), as a percentage of the area at risk (AAR), was determined by 2,3,5-triphenyltetrazolium staining. RESULTS: Intrathecal morphine 0.3 to 30 microg/kg reduced myocardial IS compared with intrathecal normal saline control animals. The IS/AAR were 33% +/- 10% (0.3 microg/kg), 29% +/- 10% (3 microg/kg) and 29% +/- 16% (30 mug/kg), versus 53% +/- 8% for the control group (P < 0.01). The reduction in IS/AAR with IT-MPC was similar to that produced by IV morphine (33% +/- 6%, P = 0.84) and IPC (22% +/- 4%, P = 0.41). Myocardial preconditioning due to IT-MPC was attenuated by co-administration of any one of the opioid receptor antagonists (IT-MPC + naltrindole 50% +/- 9%, IT-MPC + nor binaltorphimine 43% +/- 6%, IT-MPC + CTOP 53% +/- 9%, P = 0.14). CONCLUSIONS: IT-MPC produced comparable cardioprotection to myocardial IPC and IV morphine. Myocardial preconditioning from intrathecal morphine seems to involve delta, kappa, and mu opioid receptors.

Protein kinase C mediates the effects of delta-opioid receptor stimulation on survival and apoptosis in neonatal cardiomyocytes cultured in serum-deprived condition.

The aims of the present study were to determine whether Delta opioid receptor (delta-OR) stimulation improved the survival of cardiomyocytes cultured in serum-deprived conditions, which impaired their growth. [D-Ala2, D-Leu5]-enkephalin (DADLE), a selective delta-OR agonist, at a concentration range of 0.1 micromol/L to 10 micromol/L for 48 h increased the viability of the cardiomyocyte under serum deprivation conditions. DADLE (0.1 micromol/L) also decreased the early cell apoptosis rate and the expression of Caspase-3. The effects of 0.1 micromol/L DADLE were abolished by 10 micromol x L(-1) naltrindole, a selective delta-OR antagonist, or by blockade of protein kinase C (PKC) with its blockers, 10 micromol x L(-1) GF109203X or 1 micromol/L staurosporine. Furthermore, 0.1 micromol x L(-1) DADLE increased the expression of PKC, an effect abrogated by 10 micromol x L(-1) naltrindole. The observations indicate that delta-OR stimulation improves the viability and reduces the apoptosis via PKC pathway in neonatal cardiomyocytes cultured in serum deprived conditions.

Polyol pathway mediates iron-induced oxidative injury in ischemic-reperfused rat heart.

Recent studies have shown that the polyol pathway is involved in ischemia-reperfusion (I/R)-induced myocardial infarction, but the mechanism is unclear. We previously found that lack of aldose reductase (AR), the first enzyme of the polyol pathway, attenuated the increase in transferrin (Tf) level in I/R brain, suggesting that AR contributes to iron-catalyzed free radical-induced damage. We therefore investigated if this mechanism occurs in I/R hearts. We found that inhibition of AR or sorbitol dehydrogenase (SDH), the second enzyme of the polyol pathway, both attenuated the I/R-mediated increases in HIF-1alpha, Tf, TfR, and intracellular iron content and reduced the I/R-induced infarct area of the heart. Further, administration of niacin, which replenishes NAD+, the cofactor for SDH, also normalized TfR and HIF-1alpha levels in I/R hearts. These results suggest that during I/R polyol pathway activity increases the cytosolic NADH/NAD+ ratio. This activates HIF-1alpha that induces the expression of TfR, which in turn increases Tf uptake and iron accumulation and exacerbates oxidative damage that increases the lipid peroxidation. This was confirmed by the fact that administration of the iron chelator deferoxamine attenuated the I/R-induced myocardial infarction.

kappa-Opioid receptor stimulation inhibits augmentation of Ca(2+) transient and hypertrophy induced by isoprenaline in neonatal rat ventricular myocytes - Role of CaMKIIdelta(B).

We aimed to further define the pathway mediating the inhibitory effects of kappa-opioid receptor stimulation on Ca(2+) transients and hypertrophic responses to beta(1)-adrenoceptor stimulation. We determined the effects of trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamid methanesulfonate salt (U50,488H), a selective kappa-opioid receptor agonist, on the enhancement of spontaneous Ca(2+) transients and the induction of hypertrophy by isoprenaline, a beta-adrenoceptor agonist, in cultured neonatal ventricular myocytes. The results were compared with those found with KN93, a selective Ca(2+)/calmodulin-dependent kinase (CaMKII) inhibitor, propranolol, a beta-adrenoceptor antagonist, and verapamil, a L-type Ca(2+) channel antagonist. Hypertrophy of cardiomyocytes was characterized by increases in (i) total protein content; (ii) cell size; and (iii) [(3)H]leucine incorporation. 10 micromol/l isoprenaline increased all three parameters. We also determined the expression of nuclear CaMKIIdelta in response to U50,488H in the presence or absence of isoprenaline. To determine whether the effects of U50,488H were receptor-mediated, its effects were also measured following blockade of the kappa-opioid receptor with nor-binaltorphimine. kappa-Opioid receptor stimulation suppressed the stimulatory effect of isoprenaline on Ca(2+) transients and cardiac hypertrophy, as did KN93, propranalol and verapamil. U50,488H also suppressed the expression of nuclear CaMKIIdelta(B) in the presence, but not in the absence of isoprenaline. These results suggest that the inhibitory effect of kappa-opioid receptor stimulation on beta(1)-adrenoceptor stimulation may also involve CaMKIIdelta.

delta-Opioid receptor stimulation enhances the growth of neonatal rat ventricular myocytes via the extracellular signal-regulated kinase pathway.

1. The aims of the present study were to determine whether delta-opioid receptor stimulation enhanced proliferation of and to investigate the role of the extracellular signal-regulated kinase (ERK) pathway in ventricular myocytes from neonatal rats. 2. At concentratins ranging from 10 nmol/L to 10 micromol/L, [D-Ala2,D-Leu5]enkephalin (DADLE) concentration-dependently promoted myocardial growth and DNA synthesis and altered the cytoskeleton. 3. At 1 micromol/L, DADLE also increased the expression and phosphorylation of ERK. 4. These effects of 1 micromol/L DADLE were abolished by 10 micromol/L naltrindole, a selective delta-opioid receptor antagonist, 10 nmol/L U0126, a selective ERK antagonist, 1 micromol/L staurosporine, an inhibitor of protein kinase (PK) C, and 100 micromol/L Rp-adenosine 3',5'-cyclic monophosphorothioate triethylammonium salt hydrate (Rp-cAMPS), an inhibitor of PKA. 5. In conclusion, delta-opioid receptor stimulation enhances the proliferation and development of the ventricular myocytes of neonatal rats. The ERK pathway and related signalling mechanisms, namely PKC and PKA, are involved.

kappa-opioid receptor stimulation inhibits cardiac hypertrophy induced by beta1-adrenoceptor stimulation in the rat.

To test the hypothesis that kappa-opioid receptor stimulation inhibits cardiac hypertrophy induced by beta1-adrenoceptor stimulation, we determined the effects of trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide methanesulfonate salt (U50,488H), a selective kappa-opioid receptor agonist, on cardiac hypertrophy induced by isoprenaline, a selective beta-adrenoceptor agonist, in neonatal ventricular myocytes upon blockade of beta2-adrenoceptor. Hypertrophy of cardiomyocytes was determined by increases in (i) total protein content; (ii) [3H]leucine incorporation; and iii) cell size. 10 micromol/l isoprenaline increased all three parameters. The effects were abolished by 2 micromol/l propranolol, a beta-adrenergic receptor antagonist, or 300 nmol/l CGP20712A, a beta1-adrenoceptor antagonist, but not by 100 nmol/l ICI118,551, a beta2-adrenoceptor antagonist. The effects were also abolished by Rp-cAMPs 100 micromol/l, a protein kinase A inhibitor and not by pertussis toxin 5 mg/l. The effects of isoprenaline in the presence or absence of ICI118,551 were also abolished by 1 micromol/l U50,488H. The inhibitory effects of U50,488H were abolished by 1 micromol/l nor-binaltorphimine, a selective kappa-opioid receptor antagonist. U50,488H also abolished the increases in the amplitude and frequency of the spontaneous intracellular Ca2+ transient induced by 10 micromol/l isoprenaline in the presence or absence of ICI118,551, an effect also abolished by nor-binaltorphimine. In conclusion the results show that kappa-opioid receptor stimulation abolished both the cardiac hypertrophy and enhanced amplitude and frequency of the spontaneous intracellular Ca2+ transient induced by beta1-adrenoceptor stimulation.

Role of reverse mode Na+/Ca2+ exchanger in the cardioprotection of metabolic inhibition preconditioning in rat ventricular myocytes.

This study determined the role of the reverse mode Na(+)/Ca(2+) exchanger (NCX) in cardioprotection of metabolic inhibition preconditioning in isolated ventricular myocyctes. Activity of the reverse mode NCX was assessed by changes of [Ca(2+)](i) upon withdrawal of extracellular Na(+). [Ca(2+)](i) was measured by spectrofluorometry, using Fura-2 as Ca(2+) indicator. The amplitude of contraction and exclusion of trypan blue by myocytes served as indices of contractile function and viability, respectively. Firstly, NCX activity significantly decreased during simulated reperfusion after severe metabolic inhibition (index ischaemia) in myocytes subjected to metabolic inhibition preconditioning. This inhibitory effect on NCX activity correlated with the enhancing effect of metabolic inhibition preconditioning on cell viability following ischaemic insult. Treatment myocytes with E4031, an activator of reverse mode NCX, during index ischaemia and reperfusion attenuated the enhancing effects of metabolic inhibition preconditioning on cell contraction and viability. Secondly, NCX activity was significantly higher at the end of metabolic inhibition preconditioning. More importantly, E4031 pretreatment mimicked the beneficial effects of metabolic inhibition preconditioning in myocytes and ischaemic preconditioning in the isolated perfused heart, respectively, and these effects were abolished by KB-R7943, an inhibitor of reverse mode NCX. The results indicate that increased reverse mode NCX activity during preconditioning triggered cardioprotection, and reduced reverse mode NCX activity during reperfusion after index ischaemia conferred cardioprotection.

Cardioprotection by the female sex hormone -- interaction with the beta1-adrenoceptor and its signaling pathways.

Estrogen is a steroid and the predominant female sex hormone in the body. Ovariectomised (OVX) adult female rats exhibit greater myocardial injury compared to the sham rats following ischemic insult in the presence of beta-adrenoceptor stimulation. Estrogen replacement restores the response of OVX female rats to ischemic/beta-adrenoceptor stimulation to that of normal female rats, providing evidence for a cardioprotective role of estrogen during ischemic insult. The protective effect is due to down-regulation of the beta(1)-adrenoceptor. There is also evidence that estrogen suppresses the expression and activity of protein kinase A (PKA), a second messenger of the G(s) protein/adenylyl cyclase/cAMP/PKA pathway which ultimately influences contractile function. There is also preliminary evidence that estrogen may suppress the activity of Ca(2+)/calmodulin kinase II deltac isoform (CaMKII-deltac), another downstream second messenger of the beta(1)-adrenoceptor pathway, which is involved in PKA-independent cell apoptosis. Acute administration of estrogen at physiological level could inhibit myocardial beta(1)-adrenoceptor and attenuate Ca(2+) influx independent of the estrogen receptor. In addition, brain studies also show estrogen inhibits the activities activated by the beta-adrenoceptor in brain regions responsible for the regulation of arterial blood pressure. Thus, it can be appreciated that the interaction between estrogen and the beta(1)-adrenoceptor and its signaling pathways is a complex one. Estrogen plays an important role not only in reproduction but also in other regulatory functions such as cardioprotection.

Testosterone is required for delayed cardioprotection and enhanced heat shock protein 70 expression induced by preconditioning.

Ischemic preconditioning fails to confer immediate cardioprotection in the absence of testosterone, indicating that the hormone is required for the process. Here we set out to determine whether testosterone is also necessary for delayed cardioprotection and, if so, how it acts. Male Sprague Dawley rats (7-8 wk) underwent sham operation or gonadectomy without (G) or with testosterone replacement (GT) for 8 wk. Isolated ventricular myocytes were preconditioned either by metabolic inhibition or with U50,488H, a kappa-opioid receptor agonist. In intact rats, U50,488H was administered systemically and 24 h later the hearts were removed. Ventricular myocytes were then subjected to metabolic inhibition and anoxia and isolated hearts to regional ischemia, followed by reperfusion to induce injury. Both types of preconditioning significantly increased the viability and decreased the lactate dehydrogenase release in ventricular myocytes from sham rats. They also activated heat shock transcription factor-1 and increased heat shock protein 70 expression. In contrast, all these effects were absent in myocytes from G rats and were restored by testosterone replacement. Parallel results were found in isolated hearts. In addition, preconditioning improved contractile functions impaired by ischemic insults in sham and rats gonadectomized with testosterone replacement but not G rats. The effects of testosterone replacement in ventricular myocytes were abolished by androgen receptor blockade. In conclusion, preconditioning requires testosterone to increase heat shock protein 70 synthesis, which mediates delayed cardioprotection in the male. These effects of testosterone are mediated by the androgen receptor.

The K(Ca) channel as a trigger for the cardioprotection induced by kappa-opioid receptor stimulation -- its relationship with protein kinase C.

We first determined whether the cardioprotection resulting from kappa opioid receptor (kappa-OR) stimulation was blocked by the K(Ca) channel inhibitor, paxilline (Pax), administered before or during ischaemic insults in vitro. In isolated rat hearts, 30 min of ischaemia and 120 min of reperfusion induced infarction and increased lactate dehydrogenase (LDH) release. In isolated ventricular myocytes subjected to 5 min of metabolic inhibition and anoxia followed by 10 min of reperfusion, the percentage of live cells and the amplitude of the electrically induced intracellular Ca(2+) ([Ca(2+)](i)) transient decreased, while diastolic [Ca(2+)](i) increased. Pretreatment with 10 microM U50,488H, a kappa-OR agonist, attenuated the undesirable effects of ischaemic insults in both preparations. The beneficial effects of kappa-OR stimulation, that were abolished by 5 microM nor-BNI, a kappa-OR antagonist, were also abolished by 1 microM Pax administered before ischaemic insults or 20 microM atractyloside, an opener of the mitochondrial permeability transition pore. Activation of protein kinase C (PKC) with 0.1 microM phorbol 12-myristate 13-acetate decreased the infarct size and LDH release in isolated rat hearts subjected to ischaemia/reperfusion, and these effects were abolished by blockade of PKC with its inhibitors, 10 microM GF109203X or 5 microM chelerythrine, and more importantly by 1 microM Pax. On the other hand, the cardioprotective effects of opening the K(Ca) channel with 10 microM NS1619 were not altered by either PKC inhibitor. In conclusion, the high-conductance K(Ca) channel triggers cardioprotection induced by kappa-OR stimulation that involves inhibition of MPTP opening. The K(Ca) channel is located downstream of PKC.

Increased PKA activity and its influence on isoprenaline-stimulated L-type Ca2+ channels in the heart from ovariectomized rats.

We previously showed that oestrogen confers cardioprotection by downregulating the cardiac beta1-adrenoceptor (beta1-AR). The present study examined the effect of oestrogen on the post beta1-AR signalling cascade, with particular emphasis on the activity of protein kinase A (PKA) and its influence on the L-type Ca2+ channel. Three groups of adult female Sprague-Dawley rats were used: sham-operated controls, bilaterally ovariectomized (Ovx) rats, and Ovx rats with oestrogen replacement (Ovx + E2), which restored the oestrogen concentration to normal. The electrically induced intracellular Ca2+ transient (E[Ca2+]i), 45Ca(2+)-uptake through cardiac L-type Ca2+ channels (Ca2+ channels), heart rate and force of contraction in response to beta-AR stimulation with 10 nM isoprenaline (Iso) in hearts from Ovx rats were significantly greater than those of control and Ovx + E2 rats. The basal and Iso-induced PKA activities were also higher in hearts from Ovx rats. KT5720, a selective PKA inhibitor, completely inhibited its potentiating effect on basal Ca2+ channel activity in the Ovx rat heart. On the other hand, expression of G proteins (G(alpha)s and G(alpha)i1-3)), basal and forskolin-stimulated cAMP accumulation, and responsiveness of PKA to cAMP, were not altered by Ovx. Interestingly, the PKA inhibitor at the same concentration significantly reduced the increases in PKA activity and Ca2+ channel activity upon beta-AR stimulation in all three groups of rats and the inhibitions were significantly greater in the Ovx rat than in the other two groups of rats. This study provides the first evidence that, in addition to downregulation of beta1-AR shown previously, suppression of PKA activity, which is partly responsible for the suppressed Ca2+ channel activity, also determines the E[Ca2+]i and cardiac contractility following beta-AR stimulation in the female rat.

Protein tyrosine kinase-dependent modulation of voltage-dependent potassium channels by genistein in rat cardiac ventricular myocytes.

Effects of the isoflavone protein tyrosine kinase (PTK) inhibitor genistein on voltage-dependent K(+) currents, i.e., transient outward K(+) current (I(to)), sustained K(+) current (I(ss)), and inward rectifier K(+) current (I(K1)) were studied in rat cardiac ventricular myocytes. It was found that I(to) was reversibly inhibited by genistein in a concentration-dependent manner (IC(50)=28.1 microM), while I(ss) was suppressed by genistein with IC(50) of 18.5 microM. In addition, I(K1) (at -50 mV) was significantly decreased by 36.3+/-4.4% with 25 microM genistein. The inhibition of I(to), I(ss), and I(K1) by genistein was significantly reversed by the application of the protein tyrosine phosphatase inhibitor sodium orthovanadate (1 mM). However, I(to), I(ss), and I(K1) were not affected by the non-isoflavone PTK inhibitor tyrphostin A23 (100 microM) and PP2 (1 microM). These results indicate that activation of I(to), I(ss), and I(K1) channels is modulated by genistein-sensitive PTKs in rat ventricular myocytes.

Roles of KATP channels in delayed cardioprotection and intracellular Ca(2+) in the rat heart as revealed by kappa-opioid receptor stimulation with U50488H.

The effect of preconditioning with U50488 H (UP), a selective kappa-opioid receptor (kappa-OR) agonist, on infarct size and intracellular Ca2+ ([Ca2+]i) in the heart subjected to ischaemic insults were studied and evaluated. U50488 H administered intravenously reduced the infarct size 18-48 h after administration in isolated hearts subjected to regional ischaemia/reperfusion (I/R). The effect was dose dependent. A peak effect was reached at 10 mg x kg-1 U50488 H and at 24 h after administration. The effect of 10 mg x kg-1 U50488 H at 24 h after administration was abolished by nor-binaltorphimine (nor-BNI), a selective kappa-OR antagonist, indicating the effect was kappa-OR mediated. The infarct reducing effect of U50488 H was attenuated when a selective blocker of mitochondrial (5-hydroxydecanoic acid, 5-HD) or sarcolemmal (HRM-1098) ATP-sensitive potassium channel (KATP) was coadministered with U50488 H 24 h before ischaemia or when 5-HD was administered just before ischaemia. U50488 H also attenuated the elevation in [Ca2+]i and reduction in electrically induced [Ca2+]i transient in cardiomyocytes subjected to ischaemic insults. The effects were reversed by blockade of KATP channel, which abolished the protective effect of preconditioning with U50488 H. The results indicated that mitochondrial KATP channel serves as both a trigger and a mediator, while sarcolemmal KATP channel as a trigger only, of delayed cardioprotection of kappa-OR stimulation. The effects of these channels may result from prevention/attenuation of [Ca2+]i overload induced by ischaemic insults.

Role of protein kinase C-epsilon in the development of kappa-opioid receptor tolerance to U50,488H in rat ventricular myocytes.

The role of protein kinase C-epsilon (PKC-epsilon) in the development of kappa-opioid receptor (kappa-OR) tolerance to the effects of trans-(+/-)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]cyclohexyl) (U50,488H), the selective agonist of kappa-OR, was determined in rat ventricular myocytes. Incubation of ventricular myocytes with 1 microM U50,488H for 24 h significantly attenuated the inhibitory effects of 30 microM U50,488H on the electrically-induced [Ca(2+)](i) transient and forskolin-stimulated cyclic AMP accumulation, indicating the development of tolerance to the kappa-OR agonist. Chronic treatment of ventricular myocytes with U50,488H also induced translocation of PKC-epsilon to the particulate fraction. On the other hand, administration of 30 microM U50,488H for 10 min induced translocation of PKC-alpha to the particulate fraction in naïve ventricular myocytes, but not in cells pretreated with 1 microM U50,488H for 24 h. In ventricular myocytes incubated for 24 h with 1 microM U50,488H together with 1 microM chelerythrine or 1 microM GF109203X, PKC inhibitors, or 0.1 microM epsilonV1-2 peptide, a selective inhibitor of PKC-epsilon, 30 microM U50,488H still produced the inhibitory effect on the electrically-induced [Ca(2+)](i) transient as it did in naïve ventricular myocytes. Chronic treatment of ventricular myocytes with U50,488H and chelerythrine also attenuated the development of tolerance to acute U50,488H on cyclic AMP accumulation. Cells exposed to chelerythrine, GF109203X, or epsilonV1-2 peptide alone did not show an altered [Ca(2+)](i) response to U50,488H. These results indicate that activation of PKC-epsilon is a critical step in the development of tolerance in the kappa-OR.

Aprikalim reduces the Na+-Ca2+ exchange outward current enhanced by hyperkalemia in rat ventricular myocytes.

BACKGROUND: [corrected] Aprikalim, an adenosine triphosphate (ATP) sensitive K+ (K(ATP)) channel opener, attenuates the elevation of intracellular Ca2+ concentration ([Ca2+]i) and improves the contractile functions after hyperkalemic and hypothermic cardioplegia. There is evidence that cardioplegia increases the Na+-Ca2+ exchange activity without affecting Ca2+ influx through L-type Ca2+ channels or Ca2+ content in the sarcoplasmic reticulum, the intracellular Ca2+ store. METHODS: We measured the Na+-Ca2+ exchange outward current with the patch-clamp technique in single rat ventricular myocytes exposed to hyperkalemia and hypothermia in the presence of aprikalim. The intracellular calcium concentration ([Ca2+]i) during cardioplegia, and the contractile function and [Ca2+]i transients induced by electrical stimulation or caffeine during rewarming and reperfusion in single ventricular myocytes were also determined. Contraction and [Ca2+]i were determined with video tracking and spectrofluorometry, respectively. RESULTS: Aprikalim, 100 micromol/L, the effect of which was blocked by glibamclamide, a K(ATP) inhibitor, significantly attenuated the hyperkalemia-elevated Na+-Ca2+ exchange current by 26% and 11% at 22 degrees C and 4 degrees C, respectively. Aprikalim also attenuated significantly the [Ca2+]i elevated during cardioplegia. Furthermore aprikalim significantly attenuated the reduction in amplitude and prolongation in duration of contraction of myocytes after cardioplegia. The effects of aprikalim mimicked those of nickle (Ni2+), a Na+-Ca2+ exchange blocker. The electrically or caffeine-induced [Ca2+]i transients were unaltered by cardioplegia or aprikalim. CONCLUSIONS: Aprikalim attenuates the Na+-Ca2+ exchange outward current elevated by hyperkalemia, which may attenuate the [Ca2+]i elevation during hyperkalemia and improve the contractile function after cardioplegia in the ventricular myocyte. The study provides further support that addition of a K(ATP) channel opener to the cardioplegic solution may produce beneficial effects in open heart surgery.

Kappa-opioid receptor stimulation inhibits growth of neonatal rat ventricular myocytes.

The effects of trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide methanesulfonate salt (U50,488H), a selective kappa-opioid receptor agonist, on growth in neonatal ventricular myocytes were determined. In 15% serum culture medium, U50,488H at 0.1-1 microM significantly reduced the protein content, [3H]leucine uptake and cell size of the myocytes. The effect of U50,488H on protein content was abolished in the presence of 1 microM nor-binaltorphimine (nor-BNI), a selective kappa-opioid receptor antagonist. In a 0.4% serum medium, U50,488H at 0.1-1 microM had no effect on myocyte growth. Interestingly, 1 microM U50,488H abolished the stimulatory effects of 1 microM norepinephrine on protein content, [3H]leucine uptake and cell size of the myocytes in the low serum medium. The effect of U50,488H was abolished by 1 microM nor-BNI. With the exception of cell size, the effects of norepinephrine were completely abolished by blockade of both alpha- and beta-adrenoceptors, but only partially blocked by blockade of either adrenoceptors. These results provide first evidence that kappa-opioid receptor stimulation inhibits growth of the neonatal ventricular myocyte as a result of direct action as well as by inhibiting sympathetic stimulation of the heart. The stimulatory effects of sympathetic activity on growth occurs via both alpha- and beta-adrenoceptors.

Effects of U50,488H on transient outward and ultra-rapid delayed rectifier K+ currents in young human atrial myocytes.

The effects of trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide methanesulfonate salt (U50,488H), a selective kappa-opioid receptor agonist, on transient outward K+ current (Ito1) and ultra-rapid delayed rectifier K+ current (IKur) in young human atrial myocytes were evaluated with a whole-cell patch-clamp technique. At +10 mV, U50,488H decreased Ito1 in a concentration-dependent manner (IC50=12.4+/-3.5 microM), while at +50 mV, U50,488H produced biphasic effects on Ito1-increasing and decreasing the current at 1-3 and 10-30 microM, respectively. U50,488H at 10 microM shifted the midpoint (V0.5) of Ito1 activation in a depolarizing direction by approximately 5 mV, accelerated the inactivation, and slowed the recovery from inactivation of Ito1. In addition, U50,488H inhibited IKur in a concentration-dependent manner (IC50=3.3+/-0.6 microM). The effects of U50,488H on the two types of K+ currents were not antagonized by either 5 microM nor-binaltorphimine or 300 nM naloxone. These results indicate that U50,488H affects both Ito1 and IKur in young human atrial myocytes in an opioid receptor-independent manner.