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S. Ishikawa, T. Saito and T. Kuzuya

ABSTRACT

The effect of calmodulin on the stimulation of cyclic AMP production by arginine vasopressin (AVP), prostaglandin E2 (PGE2) and forskolin was examined in cultured renal papillary collecting tubule cells of the rat. In the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine submaximal concentrations of AVP (1 nmol/l), PGE2 (20 nmol/l) and forskolin (240 nmol/l) significantly increased cellular cyclic AMP accumulation by 2·3-, 6·0- and 8·4-fold respectively. Two chemically dissimilar inhibitors of calmodulin, namely trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7), attenuated the AVP-, PGE2- and forskolin-stimulated cellular production of cyclic AMP in a dose-related manner. Cellular production of cyclic AMP was inhibited by 50% (ID50) by doses ranging from 16 to 28 μmol trifluoperazine/1 and 35 to 44 μmol W-7/1. Basal accumulation of cellular cyclic AMP was also decreased by treatment with either trifluoperazine or W-7, but the effective dose was higher than that which inhibited cellular cyclic AMP production stimulated by AVP, PGE2 and forskolin. Since forskolin directly activates adenylate cyclase at a site of the catalytic unit and the cellular action of AVP to activate adenylate cyclase is mediated through receptor-guanine nucleotide regulatory-catalytic units, the present study indicates calmodulin regulation of basal, AVP-, PGE2-and forskolin-activated adenylate cyclase in the papillary collecting tubule cells. The inhibition of AVP- or PGE2-induced cellular cyclic AMP production by treatment with either Ca2+-free medium or verapamil, a blocker of cellular Ca2+ uptake, was demonstrated and suggests that an increase in cytosol Ca2+, which interacts with calmodulin to form an active complex is, at least in part, due to the increased cellular influx of Ca2+ from the extracellular space.

J. Endocr. (1985) 107, 15–22

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S. Ishikawa, T. Saito and T. Kuzuya

ABSTRACT

The effect of potassium (K)-free medium on the stimulation of cyclic AMP (cAMP) production by arginine vasopressin (AVP) and forskolin was examined in rat renal papillary collecting tubule cells in culture. All experiments were performed in the presence of 3-isobutyl-l-methylxanthine (0·5 mmol/l). Cellular cAMP levels in response to 1 nmol and 0·1 μmol AVP/1 were 430·9 ± 42·1 (s.e.m.) and 501·8± 43·6 fmol/μg protein per 10 min respectively; these levels were significantly (P <0·01) higher than those in the vehicle-treated group (126·6 ± 23·3 fmol/μg protein per 10 min). The cellular cAMP response to 1 nmol AVP/1 was significantly attenuated after 24 and 72 h of exposure of cells to K-free medium, cellular concentrations of cAMP being 280·2 ± 37·1 and 233·0 ± 9·6 fmol/μg protein per 10 min respectively. The response of cAMP to AVP remained unchanged when the cells were preincubated with K-free medium for 1 h. Similarly, forskolin (20 nmol/l)-stimulated cellular cAMP production was also significantly impaired after 24 or 72 h of exposure of cells to K-free medium. When the cells preincubated in K-free medium were again exposed for 1 h to K-replete medium containing 5 mmol KC1/1, cellular cAMP production in response to AVP or forskolin recovered totally. Cellular protein and ATP content and cellular viability were not altered by exposure of cells to K-free medium for 24 h, and thus the impaired cAMP response to AVP or forskolin in the K-depleted cells was independent of altered cellular viability and source of ATP. The present results indicate that the K ion is an important factor for AVP– and forskolin-activated adenylate cyclase at the catalytic unit in the renal papillary collecting tubule cells.

J. Endocr. (1987) 113, 199–204

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T Usui, Y Ikeda, T Tagami, K Matsuda, K Moriyama, K Yamada, H Kuzuya, S Kohno and A Shimatsu

Some plant compounds or herb mixtures are popular alternatives to conventional therapies and contain organic compounds that bind to some nuclear receptors, such as the estrogen receptor (ER), to exert various biological effects. We studied the effect of various herbal extracts on ERalpha and ERbeta isoforms. One herbal extract, Rhei rhizoma (rhubarb), acts as an agonist to both ERalpha and ERbeta. The phytochemical lindleyin, a major component of rhubarb, might contribute to this estrogenic activity through ERalpha and ERbeta. 4-Hydroxytamoxifen, an ER antagonist, completely reversed the estrogenic activity of lindleyin. Lindleyin binds to ERalpha in vitro, as demonstrated using a fluorescent polarization assay. The in vivo effect of rhubarb extract was studied using a vitellogenin assay system in the freshwater fish, Japanese medaka (Oryzias latipes). There were marked increases in serum vitellogenin levels in male medaka exposed to rhubarb extract. We conclude that lindleyin, a component of some herbal medicines, is a novel phytoestrogen and might trigger many of the biological responses evoked by the physiological estrogens.