Jian-Ming Pei

k-opioid receptor agonist inhibits the cholera toxin sensitive G-protein in the heart

Summary: To explore the signaling mechanisms of the negative modulation of &bgr;‐adrenoceptors by &kgr;‐opioid receptors (&kgr;‐OR) in the heart, the possibility of the interaction at the level of G protein and receptor was determined. Cholera toxin, an activator of the stimulatory G protein (Gs), elevated electrically induced intracellular Ca2+ ([Ca2+]i) transients and induced ribosylation of the &agr;‐subunit of Gs (Gs&agr;) in rat ventricular myocytes. The effects were significantly attenuated by U50,488H, a specific agonist of &kgr;‐OR, and were abolished by nor‐binaltorphimine, a selective &kgr;‐OR antagonist. The content of Gs&agr;, however, was not affected by U50,488H. Receptor binding experiments showed that neither Bmax nor Kd of the binding of [3H]CGP‐12177, a &bgr;‐adrenoceptor antagonist, was affected by U50,488H. The current study provides the first evidence that &kgr;‐OR stimulation inhibits the ribosylation of the &agr;‐subunit of the Gs protein, thus inhibiting the action of cholera toxin on the protein.

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

The role of protein kinase C‐epsilon (PKC‐ε) in the development of κ‐opioid receptor (κ‐OR) tolerance to the effects of trans‐(±)‐3,4‐dichloro‐N‐methyl‐N‐(2‐[1‐pyrrolidinyl]cyclohexyl) (U50,488H), the selective agonist of κ‐OR, was determined in rat ventricular myocytes. Incubation of ventricular myocytes with 1u2003μM U50,488H for 24u2003h significantly attenuated the inhibitory effects of 30u2003μM U50,488H on the electrically‐induced [Ca2+]i transient and forskolin‐stimulated cyclic AMP accumulation, indicating the development of tolerance to the κ‐OR agonist. Chronic treatment of ventricular myocytes with U50,488H also induced translocation of PKC‐ε to the particulate fraction. On the other hand, administration of 30u2003μM U50,488H for 10u2003min induced translocation of PKC‐α to the particulate fraction in naïve ventricular myocytes, but not in cells pretreated with 1u2003μM U50,488H for 24u2003h. In ventricular myocytes incubated for 24u2003h with 1u2003μM U50,488H together with 1u2003μM chelerythrine or 1u2003μM GF109203X, PKC inhibitors, or 0.1u2003μM εV1‐2 peptide, a selective inhibitor of PKC‐ε, 30u2003μM U50,488H still produced the inhibitory effect on the electrically‐induced [Ca2+]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 εV1‐2 peptide alone did not show an altered [Ca2+]i response to U50,488H. These results indicate that activation of PKC‐ε is a critical step in the development of tolerance in the κ‐OR.

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

To study the effects of κ-opioid receptor stimulation on intracellular Ca2+ concentration ([Ca2+]i) homeostasis during extracellular acidosis, we determined the effects of κ-opioid receptor stimulation on [Ca2+]iresponses during extracellular acidosis in isolated single rat ventricular myocytes, by a spectrofluorometric method. U-50488H (10-30 μM), a selective κ-opioid receptor agonist, dose dependently decreased the electrically induced [Ca2+]itransient, which results from the influx of Ca2+ and the subsequent mobilization of Ca2+ from the sarcoplasmic reticulum (SR). U-50488H (30 μM) also increased the resting [Ca2+]iand inhibited the [Ca2+]itransient induced by caffeine, which mobilizes Ca2+ from the SR, indicating that the effects of the κ-opioid receptor agonist involved mobilization of Ca2+ from its intracellular pool into the cytoplasm. The Ca2+responses to 30 μM U-50488H were abolished by 5 μM nor-binaltorphimine, a selective κ-opioid receptor antagonist, indicating that the event was mediated by the κ-opioid receptor. The effects of the agonist on [Ca2+]iand the electrically induced [Ca2+]itransient 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 μM 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 κ-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+]iand pHi increased. Interestingly, the effects of U-50488H on [Ca2+]iand the electrically induced [Ca2+]itransient 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+]iincreased; 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+]iand electrically induced [Ca2+]itransients to 30 μM U-50488H were also significantly attenuated. Results from the present study demonstrated for the first time that extracellular acidosis antagonizes the effects of κ-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 κ-opioid receptor stimulation.

κ-Opioid receptor stimulation increases the expression of NA+-H+ exchange gene in the heart

Kappa-opioid receptor (OR) stimulation increases intracellular pH (pHi) via activating the Na+-H+ exchange (NHE). In the present study, we determined the expression of the gene of NHE1, the predominant NHE isoform in the heart, and intracellular pH (pHi) upon kappa-OR stimulation in the rat heart. We found that 1 microM U50,488H (trans-3,4-dichloro-N-methyl-N-(2-(1 pyrrolidinyl)cyclohexyl)benzeneacetamide), a selective kappa-OR agonist, increased the expression of the NHE1 gene. We also found that U50,488H dose-dependently increased pHi in the heart. The effects were abolished by 1 microM nor-binaltorphimine (nor-BNI), a selective kappa-OR antagonist, indicating that the events were kappa-OR mediated. The effects on both NHE1 gene expression and pHi were also abolished by 5 microM chelerythrine and 5 microM BSM (bisyndolylmaleimide), protein kinase C (PKC) inhibitors, indicating that PKC mediated the actions. In addition, the effect of U50,488H on pHi was blocked by 10 microM EIPA (ethylisopropyl amiloride), a NHE1 inhibitor, indicating that NHE1 also mediated the action of U50,488H. The present study provides evidence for the first time that kappa-OR stimulation increased the NHE1 gene expression in the heart via a PKC dependent pathway. Kappa-OR stimulation also increases pHi via PKC and NHE in the heart.