h-89 and 1-3-dipropyl-8-cyclopentylxanthine

Adenosine A(1) receptor (A(1)R)-induced translocation of PKCε to transverse (t) tubular membranes in isolated rat cardiomyocytes is associated with a reduction in β(1)-adrenergic-stimulated contractile function. The PKCε-mediated activation of protein kinase D (PKD) by endothelin-1 is inhibited by β(1)-adrenergic stimulated protein kinase A (PKA) suggesting a similar mechanism of A(1)R signal transduction modulation by adrenergic agonists may exist in the heart. We have investigated the influence of β(1)-adrenergic stimulation on PKCε translocation elicited by A(1)R. Immunofluorescence imaging and Western blotting with PKCε and β-COP antibodies were used to quantify the co-localization of PKCε and t-tubular structures in isolated rat cardiomyocytes. The A(1)R agonist CCPA increased the co-localization of PKCε and t-tubules as detected by imaging. The β(1)-adrenergic receptor agonist isoproterenol (ISO) inhibited this effect of CCPA. Forskolin, a potent activator of PKA, mimicked, and H89, a pharmacological PKA inhibitor, and PKI, a membrane-permeable PKA peptide PKA inhibitor, attenuated the negative effect of ISO on the A(1)R-mediated PKCε translocation. Western blotting with isolated intact hearts revealed an increase in PKCε/β-COP co-localization induced by A(1)R. This increase was attenuated by the A(1)R antagonist DPCPX and ISO. The ISO-induced attenuation was reversed by H89. It is concluded that adrenergic stimulation inhibits A(1)R-induced PKCε translocation to the PKCε anchor site RACK2 constituent of a coatomer containing β-COP and associated with the t-tubular structures of the heart. In that this translocation has been previously associated with the antiadrenergic property of A(1)R, it is apparent that the interactive effects of adenosine and β(1)-adrenergic agonists on function are complex in the heart.

Topics: Adenosine; Adenosine A1 Receptor Antagonists; Adrenergic beta-1 Receptor Agonists; Animals; Colforsin; Cyclic AMP-Dependent Protein Kinases; Intracellular Signaling Peptides and Proteins; Isoproterenol; Isoquinolines; Membrane Proteins; Myocardium; Myocytes, Cardiac; Organ Culture Techniques; Protein Kinase C-epsilon; Protein Kinase Inhibitors; Protein Transport; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A1; Receptors, Adrenergic, beta-1; Signal Transduction; Sulfonamides; Xanthines

Delta-opioid receptor (DOPr) activation fails to produce cellular physiological responses in many brain regions, including the periaqueductal gray (PAG), despite neural expression of high densities of the receptor. Previous histochemical studies have demonstrated that a variety of stimuli, including chronic morphine treatment, induce the translocation of DOPr from intracellular pools to the surface membrane of CNS neurons. PAG neurons in slices taken from untreated mice exhibited mu-opioid receptor (MOPr) but not DOPr-mediated presynaptic inhibition of GABAergic synaptic currents. In contrast, after 5-6 d of chronic morphine treatment, DOPr stimulation inhibited synaptic GABA release onto most neurons. Shorter exposure to morphine in vitro (upto 4 h) or in vivo (18 h) did not induce functional DOPr responses. DOPr-mediated presynaptic inhibition could not be induced in slices from untreated animals by increasing synaptic activity in vitro using high extracellular potassium concentrations or activation of protein kinase A. Induction of functional DOPr signaling by chronic morphine required MOPr expression, because no DOPr receptor responses were observed in MOPr knock-out mice. DOPr agonists also had no effect on miniature IPSCs in beta-arrestin-2 knock-out mice after chronic morphine. These results suggest that induction of DOPr-mediated actions in PAG by chronic morphine requires prolonged MOPr stimulation and expression of beta-arrestin-2.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Arrestins; beta-Arrestin 2; beta-Arrestins; Dose-Response Relationship, Drug; Drug Administration Schedule; Drug Interactions; Electric Stimulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Excitatory Amino Acid Antagonists; G Protein-Coupled Inwardly-Rectifying Potassium Channels; gamma-Aminobutyric Acid; Glycine Agents; In Vitro Techniques; Isoquinolines; Male; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Knockout; Morphine; Narcotic Antagonists; Narcotics; Neurons; Oligopeptides; Patch-Clamp Techniques; Periaqueductal Gray; Protein Kinase Inhibitors; Receptors, Opioid, delta; Receptors, Opioid, mu; Strychnine; Sulfonamides; Synaptic Transmission; Time Factors; Xanthines

With the nystatin-perforated whole-cell patch-clamp recording technique, the modulatory effects of adenosine on GABA-activated whole-cell currents were investigated in neurons acutely dissociated from the superficial laminae (laminae I and II) of the rat spinal dorsal horn. The results showed that: (1) GABA acted on GABA(A) receptor and elicited inward Cl(-) currents (I(GABA)) at a holding potential (V(H)) of -40 mV; (2) adenosine suppressed GABA-induced Cl(-) current with affecting neither the reversal potential of I(GABA) nor the apparent affinity of GABA to its receptor; (3) N6-cyclo-hexyladenosine, a selective A(1) adenosine receptor agonist, mimicked the suppressing effect of adenosine on I(GABA), whereas 8-cyclopentyl-1,3-dipropylxanthine, a selective A(1) adenosine receptor antagonist, blocked the suppressing effect of adenosine; (4) chelerythrine, an inhibitor of protein kinase C, reduced the suppressing effect of adenosine on I(GABA); (5) pretreatment with 1,2-bis-(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxy-methyl) ester, a Ca(2+) chelator, did not affect adenosine-induced suppression of I(GABA). The results indicate that: (1) the suppression of adenosine on I(GABA) is mediated by adenosine A(1) receptor and through a Ca(2+)-independent protein kinase C transduction pathway; (2) the interactions between adenosine and GABA might be involved in the modulation of nociceptive information transmission at spinal cord level.

Topics: Adenosine; Alkaloids; Analgesics; Animals; Animals, Newborn; Benzophenanthridines; Bicuculline; Chelating Agents; Diglycerides; Dose-Response Relationship, Drug; Drug Interactions; Egtazic Acid; Electric Conductivity; Enzyme Inhibitors; GABA Agonists; GABA Antagonists; gamma-Aminobutyric Acid; Isoquinolines; Lithium; Membrane Potentials; Muscimol; Neural Inhibition; Patch-Clamp Techniques; Phenanthridines; Posterior Horn Cells; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Sprague-Dawley; Spinal Cord; Sulfonamides; Xanthines

Chronic treatment with opioids induces adaptations in neurons leading to tolerance and dependence. Studies have implicated the midbrain periaqueductal gray (PAG) in the expression of many signs of withdrawal. Patch-clamp recording techniques were used to examine whether augmentation of adenylyl cyclase signalling produces hyperexcitation in GABAergic nerve terminals within the mouse PAG. Both the rate of mIPSCs and the amplitude of evoked IPSCs during naloxone-precipitated withdrawal was profoundly enhanced in chronically morphine treated mice, compared to vehicle treated controls, in the presence but not the absence an adenosine A(1) receptor antagonist DPCPX. Enhanced GABAergic transmission in the presence of DPCPX was abolished by blocking protein kinase A. Inhibitors of cAMP transport, phosphodiesterase and nucleotide transport mimicked the effect of DPCPX. Coupling efficacy of micro-receptors to presynaptic inhibition of GABA release was increased in dependent mice in the presence of DPCPX. The increased coupling efficacy was abolished by blocking protein kinase A, which unmasked an underlying micro-receptor tolerance. These findings indicate that enhanced adenylyl cyclase signalling following chronic morphine treatment produces (1) GABAergic terminal hyperexcitability during withdrawal that is retarded by a concomitant increase in endogenous adenosine, and (2) enhanced micro-receptor coupling to presynaptic inhibition that overcomes an underlying tolerance.

Topics: Action Potentials; Adenosine; Affinity Labels; Animals; Colforsin; Cyclic AMP; Dipyridamole; Dose-Response Relationship, Drug; Drug Interactions; Enkephalins; Enzyme Inhibitors; gamma-Aminobutyric Acid; In Vitro Techniques; Isoquinolines; Male; Mesencephalon; Mice; Mice, Inbred C57BL; Morphine; Morphine Dependence; Naloxone; Narcotic Antagonists; Narcotics; Neural Inhibition; Neurons; Patch-Clamp Techniques; Periaqueductal Gray; Probenecid; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Substance Withdrawal Syndrome; Sulfonamides; Synaptic Transmission; Thioinosine; Time Factors; Uricosuric Agents; Vasodilator Agents; Xanthines