Primary cultures of smooth muscle cells (SMCs) were derived from the human prostate obtained from four patients with symptomatic benign prostatic hyperplasia (BPH) undergoing open prostatectomy, using collagenase digestion and preferential adhesion techniques. SMCs attached to the plastic substrate by 6 to 10 h, proliferated by 2 to 3 days, and reached confluency by 10 to 14 days with a “hill-and-vallery” pattern. Immunocytochemical staining of cultured SMCs using anti human muscle-specific actin monoclonal antibody (HHF35), confirmed that more than 90% primary cultured cells were positive for HHF-35. Transmission electron microscopy of SMCs revealed dense myofibrils (6-8 nm width) showing focal density, and was compatible to smooth muscle cells. This primary culture will be utilized to provide a useful model to investigate the character of smooth muscle cells and their role in the development of BPH.

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Okadaic acid (OA) induced marked shape changes of human platelets but neither granule secretion nor increase in [Ca²⁺] i occurred. Morphological changes induced by OA were unique and different from those observed with thrombin mainly in two respects. One is that the pseudopods formed by OA treatment were longer and straighter than those by thrombin. The other is that no granule centralization was observed with OA-treatment, although central condensation of cytofilaments was observed as was with thrombin. The immunocytochemical analysis employing anti-myosin antibody revealed that myosin was distributed in the elongated pseudopods. Since OA induced phosphorylation of the 20-kDa myosin light chain (MLC20) corresponding to the change in shape of platalets but did not induce phosphorylation of the 47-kDa protein (pleckstrin) (Higashihara M. et al. FEBS Lett. 307: 206-210, 1992), these results suggest that phosphorylation of MLC20 plays an important role in OA-induced unique morphological changes of platelets.

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The contractile effects of 5-hydroxytryptamine (5-HT) and influences of several kinds of divalent cations were investigated on longitudinal muscle strips of the guinea-pig isolated distal colon. 5-HT (10nM-10μM) produced phasic contractions which were partially inhibited by atropine (1μM) and markedly inhibited by tetrodotoxin (1μM), indicating that 5-HT acts mainly on the myenteric plexus and releases transmitters to cause contraction of the longitudinal muscle.
The contractile response to 5-HT (3μM) was almost completely inhibited by spantide (10μM), a substance P antagonist, in the presence of atropine (1μM), while spantide alone did not block 5-HT-induced contraction.
Of several divalent cations including Cd²⁺, Co²⁺, Mg²⁺, Mn²⁺, Ni²⁺, Sr²⁺ and Zn²⁺, Cd²⁺ ions (10μ-100μM), which block L-and N-type Ca²⁺channels, were most effective inhibitor of the 5-HT-induced contractions. While Sr²⁺ and Co²⁺ at a concentration of 100μM did not have a significant effect. The order effectiveness of inhibition was Cd²⁺ >>Mn²⁺>Mg²⁺=Ni²⁺=Zn²⁺.
Bay K 8644 (1μM), a L-type Ca²⁺channel activator, did not influence the contractile response of the longitudinal muscle strip to 5-HT (3μM).
The present results suggest that 5-HT may mainly act on N-type Ca²⁺channels in the myenteric neurones and cause the release of at least acetylcholine and substance P to induce contractions of the longitudinal muscle in the guinea-pig distal colon.

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The true role of the extrinsic cholinergic nerve fibers proliferating abundantly in the aganglionic colon of Hirschsprung's disease has not yet been established.
Electrophysiological and pharmacological studeis using superfusion method were carried out on rectal muscle strips of piebald lethal mice, one of murine models of Hirschsprung's disease and their siblings as a control. The muscle strips were preincubated with [³H] choline in Krebs solution. The contractions and the release of [³H] acetylcholine evoked by electrical transmural stimulation of the muscle strips were measured. In addition, the contractility of muscle strips treated with acetylcholinesterase blocking agents were recorded.
In the aganglionic colon, as in the normal colon, the release of [³H] acetylcholine evoked by electrical transmural stimulation was recognized. However, no contractions were evoked in the aganglionic colon.
The uptake of [³H] choline in the aganglionic colon was larger than in the normal colon presumably due to the proliferated extrinsic cholinergic nerve fibers. On the other hand, the release of [³H] acetylcholine evoked by electrical transmural stimulation was smaller in the aganglionic colon.
In the aganglionic colon, no contractions were evoked by acetylcholinesterase blocking agents, indicating that there is no spontaneous release of acetylcholine. In the presence of low concentration of acetylcholinesterase blocking agents, the form of contractions evoked by electrical transmural stimulation in the aganglionic colon was different from those in the normal colon.
These results have indicated that in the murine aganglionic colon, the proliferated extrinsic cholinergic nerve fibers may not directly regulate the smooth muscles through the functional neuro-muscular junction.
In conclusion, the proliferated extrinsic cholinergic nerve fibers is not related to the narrowing of the aganglionic colon.

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