The role of diacylglycerol and activation of protein kinase C in α1A-adrenoceptor-mediated contraction to noradrenaline of rat isolated epididymal vas deferens

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Abstract
1 The mechanism of contraction to noradrenaline (pEC50 5.6±0.1) in the rat epididymal vas deferens (mediated via α1A-adrenoceptors) has been studied in functional experiments. 2 Contractions to noradrenaline at 10−6m were potentiated by the diacylglycerol (DAG) kinase inhibitor R 59022 (3 × 10−7m) 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, 10−5m (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 (10−7m) and the selective protein kinase C inhibitor, calphostin C (10−6m) from 68±2% (NA, 3 × 10−6m) to 28±2% and 20±2% respectively and from 94±2% (NA, 3 × 10−5m) to 50±2% and 44±2% respectively. Contractions to K+ (40±2% maximum response to NA) were also significantly reduced by staurosporine (10−7m) (35±2%) but not by calphostin C (43±43%). 4 The phorbol ester, phorbol-12, 13-dibutyrate (PDBu), produced a phasic, concentration-dependent contraction (10−7M-10−4m) which was 41±2% of the maximum response to NA at 10−4m PDBu. The contraction to PDBu (10−5m) was reduced by calphostin C (10−6m) from 33±5% to 4±1% maximum response to NA. 5 Non-cumulative contractions to NA (10−8M-10−4m) were abolished in Ca2+-free Krebs solution containing EGTA (1 mM) and were reduced in the presence of nifedipine (10−6m) in normal Krebs solution by 91±2% at 10−4m NA. The contraction to PDBu (10−5m, 33±5% maximum response to NA) was also abolished in Ca2+-free Krebs solution containing EGTA (1 mM) or by the presence of nifedipine (10−6m) in normal Krebs solution. 6 When NA (10−4m) 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 (10−5m), 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 (10−5m) or ryanodine (10−4m), which can both deplete intracellular Ca2+ stores, did not reduce the contractions to NA (3 × 10−6m). Preincubation 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 (10−5m). 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 α1A-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.