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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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P Hawkins, C Steyn, HH McGarrigle, T Saito, T Ozaki, LL Stratford, DE Noakes and MA Hanson

The fetal hypothalamic-pituitary-adrenal (HPA) axis has numerous key roles in development. Epidemiological data have linked adverse prenatal nutrition with altered organ development and increased incidence of disease in adult life. We studied HPA axis development in resting and stimulated states in late gestation fetal sheep, following 15% reduction in maternal nutritional intake over the first 70 days of gestation (dGA). Fetuses from control (C) and nutrient-restricted (R) ewes were chronically catheterised and response profiles for ACTH and cortisol were determined at 113-116 and 125-127 dGA after administration of corticotrophin releasing hormone (CRH) and arginine vasopressin (AVP). At 126-128 dGA cortisol profiles were also determined following ACTH administration. Basal ACTH and cortisol concentrations were not different between C and R fetuses. In R fetuses, ACTH response to CRH+AVP was significantly smaller at 113-116 dGA (P<0.01), and cortisol responses were smaller at both 113-116 dGA (P<0.01) and 125-127 dGA (P<0.0001). Cortisol response to ACTH was also smaller in R fetuses (P<0.001). We conclude that, in late gestation fetal sheep, pituitary and adrenal responsiveness is reduced following modest maternal nutrient restriction in early gestation.

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T Higuchi, C-X Liu, H Saito, H Negoro and S Matsukawa

Abstract

Oxytocin (OT) is a neurohypophysial hormone with potent stimulating activity of the pregnant uterus, but its physiological role in parturition is still unclear. Recently, OT was found to be synthesized in the pregnant uterus, indicating that OT originating from the uterus, not from the posterior pituitary gland, may trigger the onset of labour. In order to define the factors responsible for the induction of uterine OT, the effect of ovarian steroid hormones and conceptus on the induction of OT mRNA in the rat uterus was examined by Northern and dot blot hybridization analysis. OT mRNA in the uterus started to increase on day 14 of pregnancy and showed very high levels at the time of parturition. Uterine OT mRNA was not altered by any steroid treatment, oestradiol-17β (0·2 μg), progesterone (4 mg) or both in combination, for 6 days. The gravid horn of the uterus had 3·6-fold as much OT mRNA as the non-gravid horn on day 21 of pregnancy in hemipregnant rats with one ligated oviduct. The ovarian steroid hormones could not induce accumulation of OT mRNA in the uterus of ovariectomized rats, at least under the conditions used, but the presence of a conceptus may be critical for the very high levels of OT mRNA.

Journal of Endocrinology (1995) 146, 81–85

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H Otsubo, S Hyodo, H Hashimoto, M Kawasaki, H Suzuki, T Saito, T Ohbuchi, T Yokoyama, H Fujihara, T Matsumoto, Y Takei and Y Ueta

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J Ogino, K Sakurai, K Yoshiwara, Yoichi Suzuki, N Ishizuka, N Seki, Yoshifumi Suzuki, H Koseki, T Shirasawa, N Hashimoto, K Yagui and Y Saito

Several mutations of the tyrosine kinase domain of insulin receptor (IR) have been clinically reported to lead insulin resistance and insulin hypersecretion in humans. However, it has not been completely clarified how insulin resistance and pancreatic β-cell function affect each other under the expression of mutant IR. We investigated the response of pancreatic β-cells in mice carrying a mutation (P1195L) in the tyrosine kinase domain of IR β-subunit. Homozygous (Ir P1195L/P1195L) mice showed severe ketoacidosis and died within 2 days after birth, and heterozygous (Ir P1195L/wt) mice showed normal levels of plasma glucose, but high levels of plasma insulin in the fasted state and after glucose loading, and a reduced response of plasma glucose lowering effect to exogenously administered insulin compared with wild type (Ir wt/wt) mice. There were no differences in the insulin receptor substrate (IRS)-2 expression and its phosphorylation levels in the liver between Ir P1195L/wt and Ir wt/wt mice, both before and after insulin injection. This result may indicate that IRS-2 signaling is not changed in Ir P1195L/wt mice. The β-cell mass increased due to the increased numbers of β-cells in Ir P1195L/wt mice. More proliferative β-cells were observed in Ir P1195L/wt mice, but the number of apoptotic β-cells was almost the same as that in Ir wt/wt mice, even after streptozotocin treatment. These data suggest that, in Ir P1195L/wt mice, normal levels of plasma glucose were maintained due to high levels of plasma insulin resulting from increased numbers of β-cells, which in turn was due to increased β-cell proliferation rather than decreased β-cell apoptosis.