Second messenger pathways involved in 1 adrenoceptor mediated contraction in the pig urethra R.L.Scott1, C.R. Chapple2, R. Chess-Williams1. 1Department of Biomedical Science, University of Sheffield, Sheffield, S10 2TN and 2Department of Urology, Royal Hallamshire Hospital, Sheffield, S10 2JF, UK.	Print Abstract Search PubMed for: Scott RL Chappel CR Chess-Williams R

Contraction of urethral smooth muscle following ₁-adrenoceptor stimulation is mediated via activation of phospholipase C. This enzyme produces the second messengers IP₃ and DAG, which can cause contraction by eliciting intracellular calcium release (IP₃) and extracellular calcium influx (DAG) respectively. However, the contribution of each pathway to the development of contraction may vary between tissues (Marshall et al., 1999). Furthermore, in some tissues (e.g. prostate) the tyrosine kinase pathway may also be involved in mediating contraction (Hawthorn et al., 1999). The present study investigates the intracellular pathways mediating contraction of the pig urethra following ₁ adrenoceptor stimulation with phenylephrine.

Strips of urethral circular smooth muscle were set up under 1g tension in gassed Krebs-bicarbonate solution at 37°C. Concentration-response curves were obtained to phenylephrine in the absence and presence of drugs that interfere with the second messenger pathways or calcium movements. Results are expressed as the mean ± sem for pEC50 values and maximum responses for phenylephrine. Paired Student's t-test was used for statistical comparisons.

Phenylephrine produced concentration-dependant contractions of the urethra with a mean pEC50 of 5.47 ± 0.08 and a mean maximum response of 1.00 ± 0.19g (n = 10). Inhibiting the IP₃ pathway with the IP₃ receptor antagonist 2-Aminoethoxydiphenylborane (30µM) reduced sensitivity to phenylephrine pEC50 5.07 ± 0.17 (p = 0.04) and depressed maximum responses by 51 ± 9.65% (P = 0.007) (n = 5). Inhibiting the DAG pathway with calphostin C (1µM), a protein kinase C inhibitor, reduced the potency of phenylephrine (pEC50 of 5.09 ± 0.04, n = 7, P = 0.05), but failed to significantly reduce maximum responses. Preventing breakdown of DAG by inhibiting DAG kinase with R59022 (300nM) also failed to potentiate urethral responses to phenylephrine. Inhibiting tyrosine kinase with genistein (30µM) reduced maximum responses to phenylephrine by 30.47 ± 7.26% (p = 0.03) and reduced tissue sensitivity p EC50 5.32 ± 0.08 (p = 0.03, n = 7).

The L-type calcium channel inhibitor nifedipine (1µM) had no effect on contractile responses to phenylephrine (n = 5). Ryanodine (30µM), which inhibits intracellular calcium release, depressed maximum contractions to phenylephrine by 82.4 ± 4.2% (n = 6, P = 0.001) and reduced the pEC50 value to 4.77 ± 0.29 (P=0.005). Similarly, depletion of intracellular calcium stores with cyclopiazonic acid (100µM) significantly reduced maximum responses (41.5 ± 12.0%, n = 6, p=0.003).These data suggest that contraction of the pig urethra following ₁-adrenoceptor stimulation is mediated primarily via the IP₃ pathway and thus associated with release of calcium from intracellular stores, with DAG and the tyrosine kinase pathways making only a minor contribution to contraction.

Hawthorn et al., (1999) Br. J. Pharmacol. 126, 222P.
Marshall et al., (1999) Eur. Urol 36 (suppl 1) 42-47.