Hidenori Yazawa

Characterization of Muscarinic Cholinoceptor in Primary Culture of Smooth Muscle Cells from Human Prostate

We obtained a primary culture of prostatic cells by an explant method from patients with benign prostatic hypertrophy (BPH). Ultrastructural morphology and growth characteristics of these cells conformed to those reported for smooth muscle cells isolated from vascular and visceral tissue sources. The cells retained their original character including the presence of androgen receptor, acid phosphatase and normal chromosomal number. [3H]-methyl-quinuclidinyl benzilate (QNB) saturation experiments showed the existence of a homogeneous population of binding sites with a high affinity and low capacity (KD = 0.17 +/- 0.05 nM., Bmax = 15,000 sites per cell). Inhibition of [3H]-methyl-QNB binding by nonlabelled compounds showed these [3H]-methyl-QNB binding sites to be M2 muscarinic cholinoceptors. cAMP formation induced by forskolin and isoproterenol was inhibited by carbamoyl choline and oxotremorine. These results suggest that prostatic smooth muscle cells contain M2 muscarinic cholinoceptors and that these cholinoceptors couple adenylate cyclase inhibition.

Mitogenic activity of endothelin on human cultured prostatic smooth muscle cells.

The effects of endothelins on human prostatic smooth-muscle cell growth were examined. Endothelin-1 and endothelin-3 induced a concentration-dependent increase in DNA synthesis and also promoted cell growth. Use of subtype selective antagonists BQ-123 ((cyclo(D-Trp-D-Asp(ONa)-Pro-D-Val-Leu); endothelin ET(A) receptor selective) and BQ-788 ((N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methyl Leu-D-Trp-(COOMe)-D-Nle-ONa); endothelin ET(B) receptor selective), indicated that mitogenic effects of endothelin were mediated through activation of both endothelin ET(A) and ET(B) receptors. The mitogenic effects of endothelin-1 and endothelin-3 were significantly inhibited by pretreatment of the cells with pertussis toxin. However, mitogenesis due to basic fibroblast growth factor was not affected. In conclusion, endothelin has mitogenic effects on human prostatic smooth muscle cells through activation of both endothelin ET(A) and ET(B) receptors via different signalling pathways from basic fibroblast growth factor. This may contribute to smooth muscle hyperplasia associated with benign prostatic hyperplasia.

Oxytocin receptors expressed and coupled to Ca2+ signalling in a human vascular smooth muscle cell line.

1 In a human vascular smooth muscle cell line (HVSMC), binding experiments with [3H]‐arginine8‐vasopressin (AVP) have shown the existence of a homogeneous population of binding sites with affinity (Kd value) of 0.65 nM and a maximum number of binding sites (Bmax) of 122 fmol mg−1 protein. 2 Nonlabelled compounds compete for [3H]‐AVP binding in the HVSMC membrane with an order of potency of oxytocin > lyspressin ≥ AVP > Thr4, Gly7‐oxytocin > (β‐mercapto‐β‐β‐cyclopentamethylenepropionyl‐O‐Me Tyr2, Arg8) vasopressin > desmopressin > OPC21268 > OPC31260. This order was markedly different from that observed in rat vascular smooth muscle cells (A10), a well‐established V1A receptor system. 3 In HVSMC both oxytocin and AVP increased inositol 1,4,5‐trisphosphate (IP3) production and [Ca2+]i response, but the efficacy of the responses was greater for oxytocin than AVP. 4 Reverse transcription‐polymerase chain reaction (RT‐PCR) assay detected only oxytocin receptor but not V1A or V2 receptors in HVSMC, whereas only V1A receptors were found in A10 cells. 5 In conclusion, in HVSMC only oxytocin receptors are expressed among the vasopressin receptor family, and they coupled to phosphatidyl inositol (PI) turnover/Ca2+ signalling. This unexpected observation should provide new insight into the functional role of the oxytocin receptor in a human vascular smooth muscle cell line.

Endothelin receptors and their cellular signal transduction mechanism in human cultured prostatic smooth muscle cells

Endothelin (ET) receptors, and their cellular signal transduction mechanism, were characterized in a primary culture of human prostatic smooth muscle cells (HP cell). [125I]‐ET‐1 and [125I]‐ET‐3 binding studies revealed that both ETA and ETB receptors were present in the HP cells, and the ratio of ETA to ETB receptors was 1.4:1. Analysis of ET receptor mRNA by reverse transcription‐polymerase chain reaction also demonstrated that HP cells express both ETA and ETB receptors. ET‐1 and ET‐3 increased intracellular free Ca2+ concentration ([Ca2+]i) in the HP cells in a concentration‐dependent manner. Use of subtype selective antagonists BQ‐123 and BQ‐788, indicated that both ETA and ETB receptors were coupled to an increase in [Ca2+]i. Pretreatment of the cells with pertussis toxin resulted in a significant but partial attenuation of the [Ca2+]i increase mediated through the ETA and ETB receptors. However, sensitivity to pertussis toxin (PTX) was significantly different between them. In conclusion, HP cells possess ETA and ETB receptors. Further, these two endothelin receptor subtypes evoke an increase in [Ca2+]i possibly via the action of different GTP‐binding proteins.