Anti-allergic action of anti-malarial drug artesunate in experimental mast cell-mediated anaphylactic models.

BACKGROUND: Allergy is an acquired hypersensitivity reaction of the immune system mediated by cross-linking of allergen-specific IgE-bound high-affinity IgE receptors, leading to immediate mast cell degranulation. Artesunate is a semi-synthetic derivative of artemisinin, an active component of the medicinal plant Artemisia annua. Artesunate is a clinically effective anti-malarial drug and has recently been shown to attenuate allergic asthma in mouse models. This study investigated potential anti-allergic effects of artesunate in animal models of IgE-dependent anaphylaxis. METHODS: Anti-allergic actions of artesunate were evaluated in passive cutaneous anaphylaxis and passive systemic anaphylaxis mouse models, and in ovalbumin-induced contraction of bronchial rings isolated from sensitized guinea pigs. Direct mast cell-stabilizing effect of artesunate was examined in RBL-2H3 mast cell line and in mature human cultured mast cells. Anti-allergic signaling mechanisms of action of artesunate in mast cells were also investigated. RESULTS: Artesunate prevented IgE-mediated cutaneous vascular hyperpermeability, hypothermia, elevation in plasma histamine level, and tracheal tissue mast cell degranulation in mice in a dose-dependent manner. In addition, artesunate suppressed ovalbumin-mediated guinea pig bronchial smooth muscle contraction. Furthermore, artesunate concentration-dependently blocked IgE-mediated degranulation of RBL-2H3 mast cells and human culture mast cells. Artesunate was found to inhibit IgE-induced Syk and PLCγ1 phosphorylation, production of IP(3) , and rise in cytosolic Ca(+2) level in mast cells. CONCLUSIONS: We report here for the first time that artesunate possesses anti-allergic activity by blocking IgE-induced mast cell degranulation, providing a foundation for developing artesunate for the treatment of allergic asthma and other mast cell-mediated allergic disorders.

Hydrogen sulphide in the hypothalamus causes an ATP-sensitive K+ channel-dependent decrease in blood pressure in freely moving rats.

Hydrogen sulfide (H2S) is a naturally occurring gas that may act as an endogenous signaling molecule. In the brain, H2S is mainly produced by cystathionine beta-synthase (CBS) and its cellular effects have been attributed to interactions with N-methyl-D-aspartate (NMDA) receptors and cyclic adenosine 3',5'-monophosphate (cAMP). In contrast, direct vasodilator actions of H2S are most probably mediated by opening smooth muscle ATP-sensitive K+ (K(ATP)) channels. In the hypothalamus, K(ATP) channel-dependent mechanisms are involved in CNS-mediated regulation of blood pressure. In this report, we investigated the hypothesis that H2S may act via K(ATP) channels in the hypothalamus to regulate blood pressure. Mean arterial blood pressure (MAP) and heart rate were monitored in freely moving rats via a pressure transducer placed in the femoral artery. Drugs were infused via a cannula placed in the posterior hypothalamus. Infusion of 200 microM sodium hydrogen sulfide (NaHS), an H2S donor, into the hypothalamus of freely moving rats reduced MAP and heart rate. Infusion of 300 nM to 3 microM gliclazide dose-dependently blocked the effect of 200 microM NaHS. Infusion of the CBS activator, s-adenosyl-L-methionine (0.1 mM and 1 mM), likewise decreased MAP. Infusion of the CBS inhibitors aminooxyacetic acid (10 mM) and hydroxylamine (20 mM) increased MAP but did not block the effects of infusion of 200 microM NaHS. These data indicate that actions of H2S in the hypothalamus decrease blood pressure and heart rate in freely moving rats. This effect appears to be mediated by a K(ATP) channel-dependent mechanism and mimicked by endogenous H2S.

Hydrogen sulfide: neurochemistry and neurobiology.

Current evidence suggests that hydrogen sulfide (H2S) plays an important role in brain functions, probably acting as a neuromodulator as well as an intracellular messenger. In the mammalian CNS, H2S is formed from the amino acid cysteine by the action of cystathionine beta-synthase (CBS) with serine (Ser) as the by-product. As CBS is a calcium and calmodulin dependent enzyme, the biosynthesis of H2S should be acutely controlled by the intracellular concentration of calcium. In addition, it is also regulated by S-adenosylmethionine which acts as an allosteric activator of CBS. H2S, as a sulfhydryl compound, has similar reducing properties as glutathione. In neurons, H2S stimulates the production of cAMP probably by direct activation of adenylyl cyclase and thus activate cAMP-dependent processes. In astrocytes, H2S increases intracellular calcium to an extent capable of inducing and propagating a "calcium wave", which is a form of calcium signaling among these cells. Possible physiological functions of H2S include potentiating long-term potentials through activation of the NMDA receptors, regulating the redox status, maintaining the excitatory/inhibitory balance in neurotransmission, and inhibiting oxidative damage through scavenging free radicals and reactive species. H2S is also involved in CNS pathologies such as stroke and Alzheimer's disease. In stroke, H2S appears to act as a mediator of ischemic injuries and thus inhibition of its production has been suggested to be a potential treatment approach in stroke therapy.

Effects of kappa-opioid receptor stimulation in the heart and the involvement of protein kinase C.

1. The role of protein kinase C (PKC) in mediating the action of kappa-receptor stimulation on intracellular Ca2+ and cyclic AMP production was determined by studying the effects of trans-(+/-)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl] cyclohexyl) benzeneacetamide methanesulphonate (U50,488H), a selective kappa-receptor agonist, and phorbol 12-myristate 13-acetate (PMA), a PKC agonist, on the electrically-induced [Ca2+]i transient and forskolin-stimulated cyclic AMP accumulation in the presence and absence of a PKC antagonist, staurosporine or chelerythrine, in the single rat ventricular myocyte. 2. U50,488H at 2.5-40 microM decreased both the electrically-induced [Ca2+]i transient and forskolin-stimulated cyclic AMP accumulation dose-dependently, effects which PMA mimicked. The effects of the kappa-agonist, that were blocked by a selective kappa-antagonist, nor-binaltorphimine, were significantly antagonized by the PKC antagonists, staurosporine and/or chelerythrine. The results indicate that PKC mediates the actions of kappa-receptor stimulation. 3. To determine whether the action of PKC was at the sarcoplasmic reticulum (SR) or not, the [Ca2+]i transient induced by caffeine, that depletes the SR of Ca2+, was used as an indicator of Ca2+ content in the SR. The caffeine-induced [Ca2+]i transient was significantly reduced by U50,488H at 20 microM. This effect of U50,488H on caffeine-induced [Ca2+]i transient was significantly attenuated by 1 microM chelerythrine, indicating that the action of PKC involves mobilization of Ca2+ from the SR. When the increase in IP3 production in response to K-receptor stimulation with U50,488H in the ventricular myocyte was determined, the effect of U50,488H was the same in the presence and absence of staurosporine, suggesting that the effect of PKC activation subsequent to kappa-receptor stimulation does not involve IP3. The observations suggest that PKC may act directly at the SR. 4. In conclusion, the present study has provided evidence for the first time that PKC may be involved in the action of kappa-receptor stimulation on Ca2+ in the SR and cyclic AMP production, both of which play an essential role in Ca2+ homeostasis in the heart.

[Immobilization stress induced changes of ventricular electric stability in damaged heart depends on the extent of free radical damage].

Following persistent immobilization stress, differences in changes of ventricular electric stability (VES) between normal (Nor) rats and rats with myocardial damage induced by doxorubicin (Dox) were studied and compared. The latencies of arrhythmias due to ivgtt aconitine (at a rate of 0.8 microgram/min) were significantly shorter in Dox rats than in Nor rats, i.e., it was easier for Dox rats to develop arrhythmias. Persistent immobilization stress caused an initial decrease followed by a subsequent increase of VES in Nor rats. However, Dox rats following a 8 h' stress show no significant changes in latency and duration of arrhythmias, in contrast with the shortening of the two indexes in rats only immobilized for 2 h, indicating that the increased VES induced by prolonged stress in Nor rats was eliminated by Dox. Both heart rate and cAMP content in myocardial tissue of Dox rats show no obvious changes in 2 h' stress, but significantly decreased in 8 h' immobilization rats. Thus, sympathetic nervous system probably doesn't seem to account for the changes of VES in Dox rats. It was found also that 2 h' stress induced a decrease in the activity of SOD and an increase of MDA content in the myocardial tissue of Dox rats, but 8 h' stress did not do any good to the rats. These data suggest that changes of VES caused by prolonged immobilization in Dox rats have a close relationship with the extent of free radicals in myocardial tissue.

H2S releasing aspirin protects amyloid beta induced cell toxicity in BV-2 microglial cells.

ß-Amyloid (Aß) plaques are characteristic hallmarks of Alzheimer's disease. In the present study, we examined the neuroprotective effects of S-aspirin, a hydrogen sulfide (H(2)S)-releasing aspirin, on Aß-induced cell toxicity. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay showed that S-aspirin, but not aspirin, significantly increased cell viability in BV-2 microglial cells, indicating that S-aspirin may protect cells against injury via releasing H(2)S. S-aspirin at 2.5-10 µM significantly increased cell viability and decreased lactate dehydrogenase release in Aß-treated BV-2 microglial cells. Western blotting analysis showed that S-aspirin suppressed the protein expression levels of cyclooxygenase-2 and growth arrest DNA damage (GADD). These data suggest that S-aspirin may protect microglial cells by inhibition of Aß-induced inflammation and cell cycle re-entry. To study whether S-aspirin can protect mitochondria function, mitochondria membrane potential was measured with molecular probe JC-1. It was found that S-aspirin protected mitochondria from Aß-induced loss of mitochondrial member potential. (ΔΨm). In addition, S-aspirin also prevented Aß-induced activation of p38-mitogen activated protein kinase (MAPK). In conclusion, our results suggest that S-aspirin may protect microglial injury via inhibition of inflammation, prevention of mitochondria function, and stimulation of cell growth via stimulating p38-MAPK pathway. Our study may suggest that S-aspirin may have potential therapeutic value for the treatment of Alzheimer's disease.

Hydrogen sulfide protects neurons against hypoxic injury via stimulation of ATP-sensitive potassium channel/protein kinase C/extracellular signal-regulated kinase/heat shock protein 90 pathway.

Cerebral hypoxia is one of the main causes of cerebral injury. This study was conducted to investigate the potential protective effect of H(2)S in in vitro hypoxic models by subjecting SH-SY5Y cells to either oxygen-glucose deprivation or Na(2)S(2)O(4) (an oxygen scavenger) treatment. We found that treatment with NaHS (an H(2)S donor, 10-100 microM) 15 min prior to hypoxia increased cell viability in a concentration-dependent manner. Time-course study showed that NaHS was able to exert its protective effect even when added 8 h before or less than 4 h after hypoxia induction. Interestingly, endogenous H(2)S level was markedly reduced by hypoxia induction. Over-expression of cystathionine-beta-synthase prevented hypoxia induced cell apoptosis. Blockade of ATP-sensitive K(+) (K(ATP)) channels with glibenclamide and HMR-1098, protein kinase C (PKC) with its three specific inhibitors (chelerythrine, bisindolylmaleide I and calphostin C), extracellular signal-regulated kinase 1/2 (ERK1/2) with PD98059 and heat shock protein 90 (Hsp90) with geldanamycin and radicicol significantly attenuated the protective effects of NaHS. Western blots showed that NaHS significantly stimulated ERK1/2 activation and Hsp90 expression. In conclusion, H(2)S exerts a protective effect against cerebral hypoxia induced neuronal cell death via K(ATP)/PKC/ERK1/2/Hsp90 pathway. Our findings emphasize the important neuroprotective role of H(2)S in the brain during cerebral hypoxia.

Enhanced responses to 17beta-estradiol in rat hearts treated with isoproterenol: involvement of a cyclic AMP-dependent pathway.

We determined the effects of 17beta-estradiol, the most effective estrogen, acutely administered, on the heart/ventricular myocyte with or without treatment with isoproterenol (Iso). At 0.1 to 1 nM, 17beta-estradiol, which itself had no effect, reduced the heart rate and developed pressures in the isolated perfused heart treated with 10(-7) M Iso. One nanomolar 17beta-estradiol also inhibited the cyclic AMP (cAMP) production in Iso-treated ventricular myocytes. At 10 nM to 1 microM, 17beta-estradiol itself reduced the heart rate and incidence of ischemia/reperfusion-induced arrhythmias, with the exception of diastolic pressure. The effects of 17beta-estradiol on heart rate, systolic and mean pressures, and arrhythmias were significantly enhanced in the heart/ventricular myocyte treated with Iso. Tamoxifen, an estrogen receptor antagonist, did not antagonize the effect of 17beta-estradiol on the Ca(2+) current in ventricular myocytes treated with Iso, nor did it alter the effect of the hormone on the cAMP production augmented by Iso and forskolin. The effects of 17beta-estradiol on Ca(2+) current in the presence or absence of tamoxifen and/or Iso were similar in male rats, which do not possess the estrogen receptor, and female rats, which have the estrogen receptor. In conclusion, we have shown for the first time that estrogen at physiological concentrations modulates negatively the stimulatory actions of Iso on the heart rate and cardiac contractility. The effects may result from activation of an unknown membrane receptor and the adenylate cyclase/cAMP pathway, which enhances Ca(2+) influx across the L-type Ca(2+) channel.

Acidosis antagonizes intracellular calcium response to kappa-opioid receptor stimulation in the rat heart.

To study the effects of kappa-opioid receptor stimulation on intracellular Ca2+ concentration ([Ca2+]i) homeostasis during extracellular acidosis, we determined the effects of kappa-opioid receptor stimulation on [Ca2+]i responses during extracellular acidosis in isolated single rat ventricular myocytes, by a spectrofluorometric method. U-50488H (10-30 microM), a selective kappa-opioid receptor agonist, dose dependently decreased the electrically induced [Ca2+]i transient, which results from the influx of Ca2+ and the subsequent mobilization of Ca2+ from the sarcoplasmic reticulum (SR). U-50488H (30 microM) also increased the resting [Ca2+]i and inhibited the [Ca2+]i transient induced by caffeine, which mobilizes Ca2+ from the SR, indicating that the effects of the kappa-opioid receptor agonist involved mobilization of Ca2+ from its intracellular pool into the cytoplasm. The Ca2+ responses to 30 microM U-50488H were abolished by 5 microM nor-binaltorphimine, a selective kappa-opioid receptor antagonist, indicating that the event was mediated by the kappa-opioid receptor. The effects of the agonist on [Ca2+]i and the electrically induced [Ca2+]i transient were significantly attenuated when the extracellular pH (pHe) was lowered to 6.8, which itself reduced intracellular pH (pHi) and increased [Ca2+]i. The inhibitory effects of U-50488H were restored during extracellular acidosis in the presence of 10 microM ethylisopropyl amiloride, a potent Na+/H+ exchange blocker, or 0.2 mM Ni2+, a putative Na+/Ca2+ exchange blocker. The observations indicate that acidosis may antagonize the effects of kappa-opioid receptor stimulation via Na+/H+ and Na+/Ca2+ exchanges. When glucose at 50 mM, known to activate the Na+/H+ exchange, was added, both the resting [Ca2+]i and pHi increased. Interestingly, the effects of U-50488H on [Ca2+]i and the electrically induced [Ca2+]i transient during superfusion with glucose were significantly attenuated; this mimicked the responses during extracellular acidosis. When a high-Ca2+ (3 mM) solution was superfused, the resting [Ca2+]i increased; the increase was abolished by 0.2 mM Ni2+, but the pHi remained unchanged. Like the responses to superfusion with high-concentration glucose and extracellular acidosis, the responses of the [Ca2+]i and electrically induced [Ca2+]i transients to 30 microM U-50488H were also significantly attenuated. Results from the present study demonstrated for the first time that extracellular acidosis antagonizes the effects of kappa-opioid receptor stimulation on the mobilization of Ca2+ from SR. Activation of both Na+/H+ and Na+/Ca2+ exchanges, leading to an elevation of [Ca2+]i, may be responsible for the antagonistic action of extracellular acidosis against kappa-opioid receptor stimulation.

The role of phosphodiesterase in mediating the effect of protein kinase C on cyclic AMP accumulation upon kappa-opioid receptor stimulation in the rat heart.

This study determined whether phosphodiesterase (PDE) was activated by protein kinase C (PKC) upon kappa-receptor stimulation, and if so, to identify the isozyme. We first studied the effects of trans-(+/-)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl] cyclohexyl) benzeneacetamide methanesulphonate (U50,488H), a selective kappa-opioid receptor (OR) agonist, and phorbol-12-myristate-13-acetate (PMA), a PKC activator, on cAMP accumulation and PDE activity in rat ventricular myocytes when PKC and PDE were inhibited by respective inhibitors. Like PMA, U50,488H decreased the forskolin-stimulated cAMP accumulation and dose-dependently stimulated the PDE activity, which were antagonized by 10(-6) M chelerythrine and bisindolylmaleimide I, selective PKC antagonists. In addition, 3-isobutyl-1-methylxanthine, a PDE inhibitor, dose-dependently attenuated the inhibition on forskolin-stimulated cAMP accumulation and abolished the stimulation on PDE activity by U50,488H and PMA. The observations suggest that PKC may enhance cAMP degradation through activating PDE upon kappa-OR stimulation. To identify the isozyme(s) mediating the effect of PKC upon kappa-OR stimulation, selective inhibitors were used. We found that 10(-5) M Ro-20-1724, a selective cAMP-specific PDE (PDE-IV) inhibitor, abolished the inhibitory effects of U50,488H and PMA, whereas 8-methoxymethyl-3-isobutyl-1-methylxanthine, erythro-9-(2-hydroxy-3-nonyl) adenine, cilostamide, and zaprinast, selective inhibitors of Ca(2+)/calmodulin-dependent PDE (PDE-I), cGMP-stimulated PDE (PDE-II), cGMP-inhibited PDE (PDE-III), and cGMP-specific PDE (PDE-V), respectively, had no effect. Moreover, rolipram, another selective PDE-IV inhibitor, also dose-dependently attenuated the inhibition on forskolin-stimulated cAMP accumulation and stimulation on PDE activity by U50,488H and PMA. In conclusion, this study has provided evidence for the first time that PKC and PDE-IV mediate the action of kappa-OR.

Phospholipase C inhibitors attenuate arrhythmias induced by kappa-receptor stimulation in the isolated rat heart.

To determine whether the phospholipase C (PLC)/inositol 1,4,5 trisphosphate (IP3)/Ca2+ pathway mediates cardiac arrhythmias induced by kappa-opioid receptor stimulation, the effects of U50,488H, a selective kappa-opioid receptor agonist, on cardiac rhythm in a isolated perfused rat heart, intracellular calcium ([Ca2+]i) in a single ventricular myocyte and IP3 production in myocytes were studied in the presence and absence of PLC inhibitors. U50,488H, the effects of which had been shown to be abolished by a selective kappa-receptor antagonist, nor-binaltorphimine, induced arrhythmias dose-dependently and increased both [Ca2+]i and IP3-production in the heart. More importantly, the effects of U50,488H were blocked by PLC inhibitors, neomycin and streptomycin. To further confirm the selectivity of action of the PLC inhibitor, the effects of another PLC inhibitor U73122 and its inactive structural analog, U73343, on cardiac rhythm in the isolated perfused rat heart were compared. The former did, while the latter did not, block the arrhythmogenic effect of U50,488H. We also determined whether the effects of kappa-receptor stimulation involves a pertussis toxin (PTX)-sensitive G-protein. We found that pretreatment with PTX at 4 microg/l for 10 min, a treatment shown to affect PTX sensitive G-protein-mediated functions, attenuated significantly the U50,488H-induced arrhythmias. The present study provides evidence that kappa-receptor stimulation-induced cardiac arrhythmias involves, at least partly, the PLC/IP3/Ca2+ pathway as well as a PTX sensitive G-protein.

Effects of U50488 and bremazocine on [Ca2+]i and cAMP in naive and tolerant rat ventricular myocytes: evidence of kappa opioid receptor multiplicity in the heart.

To explore the existence of multiplicity of kappa receptor in the heart, two series of experiments were performed. In the first we studied the antagonistic actions of nor-BNI, a selective kappa 1 antagonist, and quadazocine, a preferential kappa 2 antagonist, against the effects of U50488, a selective kappa 1 agonist, and bremazocine, a universal agonist preferentially binding to kappa 2 receptor, on the electrically stimulated [Ca2+]i transient and forskolin-stimulated cAMP accumulation in the rat ventricular myocyte. In the second series of experiments, we determined and compared the effects of above two kappa receptor agonists in the ventricular myocytes made insensitive to kappa 1 and kappa 2 agonists by prior exposure to the respective agonists. At the concentration range of 3 x 10(-6)-3 x 10(-5) M, both U50488 and bremazocine dose-dependently inhibited the [Ca2+]i transient induced by electrical stimulation. The inhibitory effects of U50488 and bremazocine were antagonized by nor-BNI and quadazocine. The antagonistic actions of nor-BNI were significantly greater against the effects of U50488, but smaller against the effects of bremazocine than those of quadazocine. At 1 x 10(-6)-5 x 10(-5) M, both U50488 and bremazocine dose-dependently and significantly inhibited the forskolin-induced cAMP accumulation. The inhibitory effect of 30 microM U50488 on cAMP accumulation was significantly attenuated by 5 microM nor-BNI, but not by quadazocine at the same concentration; whereas the effect of 30 microM bremazocine was significantly blocked by 5 microM quadazocine, but not by nor-BNI at the same concentration. The inhibitory effect of 30 microM U50488 on electrically stimulated [Ca2+]i was abolished by preincubation of myocytes with 10(-6) M U50488 for 24 h, but not with 10(-6) M bremazocine for h; whereas the inhibitory effect of 30 microM bremazocine on electrically stimulated [Ca2+]i transient was significantly attenuated after incubation of the myocyte with 10(-6) M bremazocine for 24 h, but not with 10(-6) M U50488 for 24 h. The observations indicate the existence of kappa receptor subtypes in the rat heart.

kappa -opioid receptor stimulation induces arrhythmia in the isolated rat heart via the protein kinase C/Na(+)-H(+)exchange pathway.

The present study attempted to determine whether the protein kinase C (PKC)/Na(+)-H(+)exchange (NHE) pathway would mediate the arrhythmogenic action of kappa -opioid receptor (OR) stimulation. We first determined the effects of U50,488H, a selective kappa -OR agonist, on PKC activity and cardiac rhythm in the isolated perfused rat heart, and intracellular pH (pH(i)), and Ca(2+)([Ca(2+)](i)) and Na(+)([Na(+)](i)) concentrations in the isolated ventricular myocyte. At 5-40 microm U50,488H concentration dependently increased the particulate PKC activity and pH(i), and induced arrhythmia. 40 microm U50,488H also increased [Na(+)](i)and [Ca(2+)](i). The arrhythmogenic effects of 40 microm U50,488H were abolished by nor-binaltorphimine, a selective kappa -OR antagonist. Blockade of PKC and NHE with respective blockers, 1 microm bisindolylmaleimide I or 0.5 microm calphostin C, and 1 microm 5-[N -methyl- N -isobutyl]amiloride or 1 microm 5-([N -ethyl- N -isopropopyl]amiloride, abolished and significantly attenuated, respectively, the effects of kappa -OR stimulation on pH(i), [Na(+)](i)and [Ca(2+)](i), and arrhythmia. To determine the role of pH(i), we observed U50,488H-induced arrhythmia at pH(i)6.8. At this pH(i), the pH(i)increased gradually both in the presence and absence of 40 microm U50,488H to a similar extent. While the increase in response to U50,488H was significantly less at pH(i)6.8 (from 0.09 to 0.10) than that at pH(i)7.1 (from 0.01 to 0.18), the arrhythmia induced by the agonist was the same at both high and low pHs. On the other hand, 5 microm monensin, a sodium ionophore, increased [Na(+)](i)and [Ca(2+)](i), and induced arrhythmia to similar extents as U50,488H. PKC and NHE inhibitors, that significantly attenuated the effects induced by U50,488H, had no effect on those induced by monensin. In conclusion, kappa -OR stimulation induces arrhythmia via PKC/NHE. [Na(+)](i)and [Ca(2+)](i), but not pH(i), may be directly responsible for arrhythmia induced by kappa -OR stimulation.

Impaired [Ca(2+)](i) and pH(i) responses to kappa-opioid receptor stimulation in the heart of chronically hypoxic rats.

kappa-Opioid receptor (kappa-OR) stimulation with U50,488H, a selective kappa-OR agonist, or activation of protein kinase C (PKC) with 4-phorbol 12-myristate 13-acetate (PMA), an activator of PKC, decreased the electrically induced intracellular Ca(2+) ([Ca(2+)](i)) transient and increased the intracellular pH (pH(i)) in single ventricular myocytes of rats subjected to 10% oxygen for 4 wk. The effects of U50,488H were abolished by nor-binaltorphimine, a selective kappa-OR antagonist, and calphostin C, a specific inhibitor of PKC, while the effects of PMA were abolished by calphostin C and ethylisopropylamiloride (EIPA), a potent Na(+)/H(+) exchange blocker. In both right hypertrophied and left nonhypertrophied ventricles of chronically hypoxic rats, the effects of U50,488H or PMA on [Ca(2+)](i) transient and pH(i) were significantly attenuated and completely abolished, respectively. Results are first evidence that the [Ca(2+)](i) and pH(i) responses to kappa-OR stimulation are attenuated in the chronically hypoxic rat heart, which may be due to reduced responses to PKC activation. Responses to all treatments were the same for right and left ventricles, indicating that the functional impairment is independent of hypertrophy. kappa-OR mRNA expression was the same in right and left ventricles of both normoxic and hypoxic rats, indicating no regional specificity.