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 (pEC(50) 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 (3x10(-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, 3x10(-6)M) to 28+/-2% and 20+/-2% respectively and from 94+/-2% (NA, 3x10(-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 Ca2+-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 Ca2+-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 Ca2+-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 (3x10(-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.