Contraction of urethral smooth muscle following ₁-adrenoceptor stimulation is mediated via activation of phospholipase C which produces the second messengers IP₃ and DAG. These cause smooth muscle contraction by eliciting intracellular calcium release (IP₃ pathway) and extracellular calcium influx via the DAG pathway. Agonist-specific coupling of G-protein coupled receptors to different second messenger pathways has been demonstrated for -adrenoceptors (Rudling et al., 2000; Richardson et al., 2003). The aim of this study was to investigate whether using different ₁-adrenoceptors agonists, the endogenous agonist noradrenaline and the synthetic ₁-adrenoceptor agonist phenyleprine activate differing second messenger pathways.

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 noradrenaline and 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 mean maximum responses for noradrenaline or phenylephrine. Paired Student’s t-test was used for statistical comparisons.

Noradrenaline produced concentration-dependant contractions of the urethra with a mean pEC₅₀ of 5.51±0.11 and a mean maximum response of 0.92±0.06g (n=25). Inhibiting the DAG pathway with calphostin C (1µM) a protein kinase C inhibitor, significantly reduced maximum responses to noradrenaline by 41.3±6.4% (n=9, p=0.006). The L-type calcium channel inhibitor nifedipine (1µM) also significantly reduced contractile responses to noradrenaline by 51.6±6.4% (p=0.003) and significantly reduced tissue sensitivity pEC₅₀ 4.79±0.34, (p=0.02, n = 6). Inhibiting the IP₃ pathway with the IP₃ receptor antagonist 2-aminoethoxydiphenylborane, (2-APB, 30µM), and the ryanodine receptor with ryanodine (30µM) had no effect upon tissue sensitivity or maximum responses to noradrenaline (n=9 and n=10 respectively).

Phenylephrine was also a full agonist on this tissue with mean pEC₅₀  of 5.47 ± 0.08 and a mean maximum response of 1.00±0.19g (n=10). In contrast to that observed with noradrenaline, 2-APB (30µM) reduced urethral sensitivity to phenylephrine (pEC₅₀ 5.07 ± 0.17, p=0.04) and depressed maximum responses by 51 ± 9.7% (p=0.007, n=5). Ryanodine (30µM) 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). Calphostin C (1µM) reduced the potency of phenylephrine (pEC₅₀ of 5.09±0.04, n=7, p=0.05) but failed to significantly reduce maximum responses. Nifedipine (1µM) had no effect on contractile responses to phenylephrine (n=5).

These data suggest that different ₁–adrenoceptor agonists activate distinct second messenger pathways in this tissue, with phenylephrine evoking smooth muscle contraction via activation of the IP₃ pathway and subsequent release of calcium from intracellular stores, and noradrenaline stimulating the DAG pathway resulting in the influx of extracellular calcium.

Richardson et al., (2003) Naunyn-Schmiedebergs Arch. Pharmacol. 367, 333-341.
Rudling et al., (2000) Br.J.Pharmacol. 131, 933-941.