1 The mechanism of contraction to phenylephrine in the rat spleen (mediated via alpha(1B)-adrenoceptors) has been studied in functional experiments. 2 The concentration-dependent contraction of the rat spleen to cumulative additions of phenylephrine (pD(2) 4.8 +/- 0.1) was not significantly reduced by the selective protein kinase C (PKC) inhibitor, calphostin C (10(-6) M) or potentiated by the DAG kinase inhibitor, R59022 (10(-6) M). 3 Contraction of the rat spleen in normal Krebs solution containing Ca2+ (2.5 mM) to a single concentration of phenylephrine (3 x 10(-4) M) produced a maximal response consisting of an initial phasic component and a more slowly developing tonic component. However in Ca2+-free Krebs solution (containing EGTA), phenylephrine (3 x 10(-4) M) produced only a phasic contraction which was reduced to 46 +/- 3% maximum response to phenylephrine in normal Krebs solution. 4 In some tissues after the contraction to phenylephrine (3 x 10(-4) M) in Ca2+-free Krebs solution (containing EGTA), the phenylephrine was washed out and the tissue was allowed to recover. After 2 h, upon addition of Ca2+ (2.5 mM) to the Krebs solution (EGTA now removed) a tonic contraction developed in the tissue (97 +/- 4% maximum response to phenylephrine). 5 Cyclopiazonic acid produced a tonic contraction of the rat spleen with a maximum effect at 10(-5) M (202 +/- 8% maximum response compared with that to phenylephrine). The contraction to CPA (10(-5) M) was reduced in Ca2+-free Krebs solution containing EGTA (30 +/- 4% of the maximum response to phenylephrine). One hour after the end of the contraction in Ca2+-free Krebs solution (EGTA now removed), upon addition of Ca2+(2.5 mM) to the Krebs solution a tonic contraction developed in the tissue (263 +/- 12% maximum response to phenylephrine). 6 In Ca2+-free Krebs solution, after the spleen had been incubated with cyclopiazonic acid for 30 min, the subsequent contraction to phenylephrine (3 x 10(-4) M) was reduced from 46 +/- 3% to 9 +/- 2% maximum response to phenylephrine. 7 Cumulative contractions to phenylephrine and the contraction to cyclopiazonic acid (10(-5) M) in the spleen were not significantly affected by nifedipine (10(-6) M). The non-selective Ca2+ channel blocker, SK&F 96365 (3 x 10(-5) M) reduced the maximum response for the cumulative additions of phenylephrine to 35 +/- 1% and the contraction to CPA (10(-5) M) from 202 +/- 8% to 108 +/- 8% maximum response to phenylephrine. 8 The tyrosine kinase inhibitors genistein (3 x 10(-5) M) and tyrphostin 23 (10(-4) M), reduced the maximum response to phenylephrine in the spleen to 51 +/- 4% and 44 +/- 5% respectively and the maximum contraction to cyclopiazonic acid (3 x 10(-6) M) in the spleen from 132 +/- 6% to 82 +/- 5% and 80 +/- 7% maximum response to phenylephrine respectively without affecting contractions to K+ 9 In conclusion, these results are consistent with the contraction of the rat spleen to phenylephrine consisting of an initial phasic contraction due to release of intracellular Ca2+ and a larger tonic contraction due to capacitative Ca2+ influx through non-voltage-gated Ca2+ channels and which may involve a tyrosine kinase. This suggests that inositol triphosphate but not diacylglycerol is involved in the contraction.
