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S. Ishikawa and T. Saito

ABSTRACT

The effect of extracellular calcium (Ca2+) on the cellular action of arginine vasopressin (AVP) was examined using an Na+, K+-ATPase inhibitor in rat renal papillary collecting tubule cells in culture. The pretreatment of cells with ouabain enhanced basal and AVP-induced cAMP production in a dose-dependent manner. The augmentation by ouabain of cellular cAMP production in response to AVP was totally abolished by co-treatment with cobalt, lanthanum, verapamil or Ca2+-free medium containing 1 mmol EGTA/l, each blocking cellular Ca2+ uptake by different mechanisms. Two other findings indicated that ouabain directly stimulated cellular Ca2+ mobilization; namely, that ouabain significantly increased 45Ca2+ influx and cellular free Ca2+ concentration ([Ca2+]i) determined by Fura-2 fluorescence. The ouabain-induced increase in [Ca2+]i was completely blocked by either cobalt or Ca2+-free medium containing 1 mmol EGTA/l. AVP at 0·1 μmol/l increased [Ca2+]i to 177·1 ±26·2 nmol/l from 92·2 ± 8·0 nmol/l (P<0·01) in renal papillary collecting tubule cells, and ouabain significantly enhanced the AVP-induced increase in [Ca2+]i. The increase of cellular free Ca2+ induced by ouabain probably binds to calmodulin to form an active complex of Ca2+-calmodulin in the cell, since two chemically dissimilar antagonists of calmodulin attenuated the enhancement by ouabain of cAMP production in response to AVP. These results therefore indicate that ouabain increases cellular Ca2+ uptake and enhances AVP-induced cellular free Ca2+ mobilization and its own second messenger cAMP production in renal papillary collecting tubule cells, and that extracellular Ca2+ is an important source for ouabain-mobilized cellular Ca2+.

Journal of Endocrinology (1989) 121, 467–477

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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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Y Tsuboi, S Ishikawa, G Fujisawa, K Okada and T Saito

Abstract

The present study was undertaken to determine whether a non-peptide arginine vasopressin (AVP) antagonist (5-dimethylamino - 1- [4-(2-methylbenzoylamino)benzoyl]-2,3,4,5-tetrahydro-1H-benzazepine; OPC-31260) antagonizes the antidiuretic action of endogenous and exogenous AVP in conscious rats. OPC-31260, given orally at a dose of 5 mg/kg or higher, increased urinary volume (UV) and reduced urinary osmolality (Uosm) in a dose-dependent manner, in rats acutely denied access to water. Minimal Uosm was obtained 1–2 h after oral administration of OPC-31260. OPC-31260 caused sustained water diuresis for more than 12 h when water was available ad libitum since OPC-31260 (30 mg/kg) reduced Uosm to less than 230 mOsmol/kg H2O, significantly less than the control value of 600 mOsmol/kg H2O. Water deprivation for 24 h increased plasma AVP levels to 7·2 pmol/l and increased Uosm to 2160 mOsmol/kg H2O. In such water-deprived rats, oral administration of OPC-31260 at 100 mg/kg was diuretic; it markedly increased free water clearance and decreased Uosm to 202 mOsmol/kg H2O.

In homozygous Brattleboro rats (with inherited AVP deficiency), given free access to water, subcutaneous infusion of the V2 agonist 1-deamino-8-D-AVP (dDAVP) at a rate of 1 ng/h markedly decreased UV to 12.6 from 148·7 ml/day and increased Uosm to 1762 from 231 mOsmol/kg H2O. OPC-31260 (30 mg/kg) promptly increased UV and reduced Uosm to levels similar to those before the administration of dDAVP; repeated OPC-31260 treatment had sustained effects. These results indicate that OPC-31260 is an orally effective non-peptide AVP antagonist to the antidiuretic action of AVP in the conscious rat.

Journal of Endocrinology (1994) 143, 227–234

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T Yoshida, K Yamanaka, S Atsumi, H Tsumura, R Sasaki, K Tomita, E Ishikawa, H Ozawa, K Watanabe and T Totsuka

Abstract

This paper describes a novel mutant mouse that has been spontaneously derived from the Snell's dwarf (DW/J) mouse. It was named the 'growth-retarded mouse' because of a characteristic growth pause followed by the delayed onset of pubertal growth. The onset of the increase in pituitary GH content that normally occurs concomitant with pubertal growth was also delayed in the growth-retarded mice. The serum concentration of thyroxine was very low in these mice from the neonatal period through adulthood, and a supplement of tri-iodothyronine was effective in shortening the growth pause and commencing the suppressed pubertal growth. Histological and immunohistochemical studies revealed that the anterior pituitary gland of the growth-retarded mouse contains clustered unusual chromophobic cells which are not reactive to various antisera against anterior pituitary hormones and the gland becomes enlarged with age. Breeding data indicated that these characteristics of the mice show an autosomal recessive inheritance and the gene responsible was designated as 'grm'. Partial linkage analysis utilizing microsatellite polymorphism demonstrated that the grm gene does not identify with the lit or hyt genes. Based on comparison of the hormonal status and growth pattern between growth-retarded, dwarf and normal mice, we have suggested the existence of a mutual interaction, possibly positive feedback regulation, between the pituitary and thyroid glands, that develops or matures the hormonal network which is responsible for rapid somatic growth and metabolic changes at puberty in mice.

Journal of Endocrinology (1994) 142, 435–446