The role of diacylglycerol and activation of protein kinase C in alpha 1A-adrenoceptor-mediated contraction to noradrenaline of rat isolated epididymal vas deferens.
1. The mechanism of contraction to noradrenaline (pEC50 5.6 +/- 0.1) in the rat epididymal vas deferens (mediated via alpha 1A-adrenoceptors) has been studied in functional experiments. 2. Contractions to noradrenaline at 10(-6) M were potentiated by the diacylglycerol (DAG) kinase inhibitor R 59022 (3 x 10(-7) M) from 49 +/- 4% to 63 +/- 3% maximum response and the time taken from initiation of contraction to the maximum response was reduced from 16 +/- 2 s to 9 +/- 1 s. The same contractions were not significantly potentiated by the DAG lipase inhibitor, U-57,908, 10(-5) M (51 +/- 2% control and 53 +/- 4% in the presence of U-57,908) nor was the time taken from initiation of contraction to the maximum response significantly altered (17 +/- 1 s control and 16 +/- 1 s in the presence of U-57,908). 3. Concentration-dependent contractions to noradrenaline (NA) were reduced by staurosporine (10(-7) M) and the selective protein kinase C inhibitor, calphostin C (10(-6) M) from 68 +/- 2% (NA, 3 x 10(-6) M) to 28 +/- 2% and 20 +/- 2% respectively and from 94 +/- 2% (NA, 3 x 10(-5) M) to 50 +/- 2% and 44 +/- 2% respectively. Contractions to K+ (40 +/- 2% maximum response to NA) were also significantly reduced by staurosporine (10(-7) M) (35 +/- 2%) but not by calphostin C (43 +/- 3%). 4. The phorbol ester, phorbol-12,13-dibutyrate (PDBu), produced a phasic, concentration-dependent contraction (10(-7) M - 10(-4) M) which was 41 +/- 2% of the maximum response to NA at 10(-4) M PDBu. The contraction to PDBu (10(-5) M) was reduced by calphostin C (10(-6) M) from 33 +/- 5% to 4 +/- 1% maximum response to NA. 5. Non-cumulative contractions to NA (10(-8) M - 10(-4) M) were abolished in Ca(2+)-free Krebs solution containing EGTA (1 mM) and were reduced in the presence of nifedipine (10(-6)M) in normal Krebs solution by 91 +/- 2% at 10(-4)M NA. The contraction to PDBu (10(-5)M, 33 +/- 5% maximum response to NA) was also abolished in Ca(2+)-free Krebs solution containing EGTA (1 mM) or by the presence of nifedipine (10(-6)M) in normal Krebs solution. 6. When NA (10(-4)M) was added to vasa deferentia in Ca(2+)-free Krebs solution containing EGTA (1 mM), following its wash out (and with EGTA later removed from the Krebs solution), readdition of Ca2+ (2.5 mM) to the Krebs solution produced no response. Cyclopiazonic acid (10(-5)M), which can deplete Ca2+ from intracellular stores, also produced no contraction. Therefore influx of extracellular Ca2+ is not a consequence of depletion of intracellular Ca2+ stores (capacitative Ca2+ influx). 7. Pre-incubation of tissues for 30 min with either cyclopiazonic acid (10(-5)M) or ryanodine (10(-4)M), which can both deplete intracellular Ca2+ stores, did not reduce the contractions to NA (3 x 10(-6)M). Pre-incubation of vasa deferentia with cyclopiazonic acid (1 or 3 min, when any rise in [Ca2+]i produced by cyclopiazonic acid might still exist) did not potentiate the contraction to PDBu (10(-5)M). Thus mobilization of intracellular Ca2+ may not be required for the activation of protein kinase C involved in these contractions. 8. In conclusion, the contraction of the rat epididymal vas deferens to NA mediated by alpha 1A-adrenoceptors appears to depend upon activation of protein kinase C by diacylglycerol, resulting in the influx of extracellular Ca2+ through voltage-gated Ca2+ channels. There was no evidence for a role of inositol trisphosphate in the contraction to noradrenaline in this tissue.
Burt
R P
RP
Department of Pharmacology, University College London.
Chapple
C R
CR
Marshall
I
I
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002273:Carcinogens / Q000494:pharmacology; D003512:Cyclohexanones / Q000494:pharmacology; D004075:Diglycerides / Q000378:metabolism*; D008071:Lipoprotein Lipase / Q000037:antagonists & inhibitors; D008297:Male; D009281:Naphthalenes / Q000494:pharmacology; D009638:Norepinephrine / Q000494:pharmacology*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010972:Platelet Activating Factor / Q000037:antagonists & inhibitors; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011744:Pyrimidinones / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D018340:Receptors, Adrenergic, alpha-1 / Q000187:drug effects / Q000378:metabolism*; D019311:Staurosporine / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D014649:Vas Deferens / Q000187:drug effects / Q000378:metabolism*
C011439:1,2-diacylglycerol; D002273:Carcinogens; D003512:Cyclohexanones; D004075:Diglycerides; D009281:Naphthalenes; D010972:Platelet Activating Factor; D011744:Pyrimidinones; D018340:Receptors, Adrenergic, alpha-1; D013844:Thiazoles; D015240:Phorbol 12,13-Dibutyrate; C036535:1,6-bis(cyclohexyloximinocarbonyl)hexane; C047505:R 59022; D011493:Protein Kinase C; D008071:Lipoprotein Lipase; D019311:Staurosporine; C058819:calphostin C; D002118:Calcium; D009638:Norepinephrine