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Muscarinic receptors of human prostatic stromal cells in vitro
1992
Muscarhfic receptors of human prostatic stroma| cells in vitro Patei, M.K., Schachter, M. a n d Sever, P.S. Department of Clinical Pharmacology, St. MmT. "s Hospital Medical Sch~i, Queen Elizabeth The Queen 34other Wing, London W2 INY, U.K.
Benign prostatic hyperplasia is a commonly occurring urological disease in man, which is considered to develop primarily from the prolileration of smooth muscle cells in the prostate gland. The hypertrophy of the smooth muscle cells represents the main cause for the obstruction of the prostatic bladder outlet. The obstruction comprises a static component, due to the enlarged prostate, in addition to a dynamic constrictor component. Classical pharmacological and radioligand studies have shown the presence of significant numbers of al-adrenergic receptors located on the smooth muscle cells and muscarinic receptors localised exclusively on the prostatic epithelium (Hedlund et al., 1985). Recent observations on the pharmacology of human prostatic stromal cells have shown that these cultured cells expr~'ss a wide range of functional receptors for many agonists, including bradykinin, CGRP, endothelin, histamine, neuropeptide Y and thrombin (Patel et al., submitted). We now report on the changes in intracellular calcium, [Ca 2+ ]i, of these cultured human prosta~c stromal cells in response to muscarinic agents. f¢"..~2 + ! t~ Ji "-,,-- meas~.A i,'~ suspensions of human prostatic stromal cells which were loaded ,:Ath Fura 2. !~.uorescenee measurements were performed in a PT! dual wavelength spectrofluorimeter a n d [Ca2+]i was calculated according to the standard method of Grynkiewicz et al., 1985. Intermediate sized increases in [Ca2+]i have been observed in response to carbachol (interquartile range [IQR] 137-242nM, median 219nM. n = 8). Agop_ist studies conducted with McN-A-343, (4-hydroxy-2-butynyl)trimethylammonium chloride m-chlorocarbanilate, and pilocarpine which are selective M 1 and M 2 agonists respectively, have shown only small increases with pilocarpine (IQR 78-125nM, median 108nM, n - - 8 ) or negligible increases with McN-A-343 (IQR 34-407nM, median 45nM, n = 8). Studies with the selective antagonists pirenzepine (MI), methoch-am/n¢ (hi 2) and 4-DAMP,4-diphenylacetoxy-N-methylpiperidine meth/odide, (M3) b3dicate that the prostatic stromal cells possess a non-M~ receptor type. It has not been possible to distinguish whether the muscarin/c response of the human prostatic stromal cells is mediated by either M 2 :~r M 3 receptors since there is a similar inhibition of the rise in [Ca2+]i when these cells are preincubated with either methoctramine or 4-DAMP. It is particularly interesting that muscarinic receptors are expressed in human prostatic stromal cells in vitro, although they are not expressed in these cells in vivo. It seems that the prostatic stromal cells acquire the ability to express muscarinic receptors as a result of growth under culture conditions. The significance of the expression of non-Ma receptors in this cell type ren, ains to be elucidated. Further experiments, using other muscarinic agonists and antagonists, are being conducted in order to further characterise the muscarinic receptors expressed on these cells. It should be noted that calculation of inhibition constants using standard pharm.aco!ogical techniques is hampered by the interexper:~ental variation that has been observed in the intracellular calcium experiments. ,~,~.e
References Grynkiewicz, G., M. Poenie aed R.Y. Tsien, 1985, J. Biol. Chem. 260, 3440. Hedlund, H., K-E. Andersson and B. Larsson, 1985, J. Urol. 134, 1291. Patel, M.IC, M. Sehachter and P.S. Sever, (submitted for pubfication).
Muscarhfic receptors of human prostatic stroma| cells in vitro Patei, M.K., Schachter, M. a n d Sever, P.S. Department of Clinical Pharmacology, St. MmT. "s Hospital Medical Sch~i, Queen Elizabeth The Queen 34other Wing, London W2 INY, U.K.
Benign prostatic hyperplasia is a commonly occurring urological disease in man, which is considered to develop primarily from the prolileration of smooth muscle cells in the prostate gland. The hypertrophy of the smooth muscle cells represents the main cause for the obstruction of the prostatic bladder outlet. The obstruction comprises a static component, due to the enlarged prostate, in addition to a dynamic constrictor component. Classical pharmacological and radioligand studies have shown the presence of significant numbers of al-adrenergic receptors located on the smooth muscle cells and muscarinic receptors localised exclusively on the prostatic epithelium (Hedlund et al., 1985). Recent observations on the pharmacology of human prostatic stromal cells have shown that these cultured cells expr~'ss a wide range of functional receptors for many agonists, including bradykinin, CGRP, endothelin, histamine, neuropeptide Y and thrombin (Patel et al., submitted). We now report on the changes in intracellular calcium, [Ca 2+ ]i, of these cultured human prosta~c stromal cells in response to muscarinic agents. f¢"..~2 + ! t~ Ji "-,,-- meas~.A i,'~ suspensions of human prostatic stromal cells which were loaded ,:Ath Fura 2. !~.uorescenee measurements were performed in a PT! dual wavelength spectrofluorimeter a n d [Ca2+]i was calculated according to the standard method of Grynkiewicz et al., 1985. Intermediate sized increases in [Ca2+]i have been observed in response to carbachol (interquartile range [IQR] 137-242nM, median 219nM. n = 8). Agop_ist studies conducted with McN-A-343, (4-hydroxy-2-butynyl)trimethylammonium chloride m-chlorocarbanilate, and pilocarpine which are selective M 1 and M 2 agonists respectively, have shown only small increases with pilocarpine (IQR 78-125nM, median 108nM, n - - 8 ) or negligible increases with McN-A-343 (IQR 34-407nM, median 45nM, n = 8). Studies with the selective antagonists pirenzepine (MI), methoch-am/n¢ (hi 2) and 4-DAMP,4-diphenylacetoxy-N-methylpiperidine meth/odide, (M3) b3dicate that the prostatic stromal cells possess a non-M~ receptor type. It has not been possible to distinguish whether the muscarin/c response of the human prostatic stromal cells is mediated by either M 2 :~r M 3 receptors since there is a similar inhibition of the rise in [Ca2+]i when these cells are preincubated with either methoctramine or 4-DAMP. It is particularly interesting that muscarinic receptors are expressed in human prostatic stromal cells in vitro, although they are not expressed in these cells in vivo. It seems that the prostatic stromal cells acquire the ability to express muscarinic receptors as a result of growth under culture conditions. The significance of the expression of non-Ma receptors in this cell type ren, ains to be elucidated. Further experiments, using other muscarinic agonists and antagonists, are being conducted in order to further characterise the muscarinic receptors expressed on these cells. It should be noted that calculation of inhibition constants using standard pharm.aco!ogical techniques is hampered by the interexper:~ental variation that has been observed in the intracellular calcium experiments. ,~,~.e
References Grynkiewicz, G., M. Poenie aed R.Y. Tsien, 1985, J. Biol. Chem. 260, 3440. Hedlund, H., K-E. Andersson and B. Larsson, 1985, J. Urol. 134, 1291. Patel, M.IC, M. Sehachter and P.S. Sever, (submitted for pubfication).