JoEllen Welsh

1,25(OH)2D3 increases membrane associated protein kinase C in MDBK cells

To determine whether 1,25-dihydroxycholecalciferol [1,25(OH)2D3] affects protein kinase C (PKC) activity in kidney, as has been demonstrated in HL-60 cells we measured 1,25(OH)2D3 binding, PKC activity and PKC immunoreactivity in Madin Darby bovine kidney (MDBK) cells, a normal renal epithelial cell line derived from bovine kidney. Our data demonstrate that MDBK cells exhibit specific high affinity binding for 1,25(OH)2D3, indicating the presence of the vitamin D receptor (VDR). Treatment of MDBK cells with 1,25(OH)2D3 for 24 h increased membrane PKC activity and immunoreactivity. The effect of 1,25(OH)2D3 was dose-dependent, with a peak effect observed at 10(-7)M 1,25(OH)2D3. The 1,25(OH)2D3 induced increase in membrane PKC was paralleled by a comparable decrease in cytosolic PKC activity and amount. Although time course studies were consistent with a VDR mediated effect of 1,25(OH)2D3 on PKC protein synthesis, total PKC activity was not increased by 1,25(OH)2D3, suggesting an effect on PKC translocation or localization. These results suggest that 1,25(OH)2D3 modulates PKC mediated events in kidney, a classic target for this steroid hormone.

Modulation and phosphorylation of calbindin-D28K correlates with protein kinase C activation

Calbindin-D28K is a vitamin D3 dependent calcium-binding protein expressed in renal distal tubules. We previously reported that 24 h treatment with 1 alpha, 25-dihydroxyvitamin D3 (1,25-D3), the active form of vitamin D3, induces calbindin-D28K and activates protein kinase C (PKC) in MDBK (Madin-Darby bovine kidney) cells. In contrast, 24 h treatment with the phorbol ester 12-O-tetradecanoylphorbol-3-acetate (TPA) downregulates calbindin-D28K and PKC activity. In the present studies, we demonstrate that TPA rapidly enhances calbindin-D28K expression in MDBK cells in the absence of 1,25-D3. The enhancement of calbindin-D28K expression is preceded by activation and translocation of PKC alpha. Further, we show that PKC directly phosphorylates calbindin-D28K in a calcium- and phospholipid-dependent manner in vitro. In MDBK cells, the calbindin-D28K antibody immunoprecipitates a 28 kDa protein for which phosphorylation is enhanced after treatment for 1 h with TPA or 24 h with 1,25-D3. Consistent with amino acid sequence analysis of calbindin-D28K indicating two threonine residues that fit the consensus for PKC phosphorylation, TPA-treated MDBK cells exhibit enhanced expression of a phosphothreonine-containing protein that co-migrates with calbindin-D28K. These studies offer the first report that calbindin-D28K is a phosphoprotein and implicate the PKC signal transduction pathway in its regulation.

TPA decreases 1,25(OH)2D3 binding and calbindin D-28K in renal (MDBK) cells

The effect of the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) on vitamin D receptors (VDRs) was studied in MDBK cells, a normal bovine renal epithelial cell line. 24 h treatment of MDBK cells with TPA resulted in down-regulation of VDR number, with no change in the binding affinity for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) or approximate molecular weight determined by fast protein liquid chromatography (FPLC). TPA treatment also reduced the level of calbindin D-28K, a vitamin D-dependent renal protein. 4 alpha-Phorbol 12,13-didecanoate (4 alpha-PDD), an inactive phorbol ester, did not affect either 1,25(OH)2D3 binding or calbindin D-28K levels. TPA elicited a significant decrease in membrane-associated protein kinase C (PKC) activity which coincided with the reduction in VDR number and calbindin D-28K. These data support a link between TPA, PKC activity and vitamin D actions in kidney.

The regulation of adenylate cyclase in liver membranes of lean and obese mice

The modulation of adenylate cyclase by guanosine triphosphate (GTP) and hormones was examined in liver membranes of lean and ob/ob mice, to determine whether a defective regulation of cyclase similar to that found in adipocyte membranes was present. In conjunction with GTP, glucagon was a powerful stimulant of cyclase in both types of membranes. In contrast, GTP alone or in conjunction with isoproterenol and norepinephrine stimulated significantly less in the membranes of the lean than in those of the obese mouse. In addition, low concentrations of norepinephrine elicited an inhibitory response in membranes of the lean mouse, but not in those of the obese. This inhibitory effect of norepinephrine was abolished by the alpha 2-antagonist yohimbine and by treatment with pertussis toxin, but not by propranolol or treatment with cholera toxin. These data show that it is possible to demonstrate inhibitory effects of guanine nucleotides on cyclase in the membranes from lean but not those from obese mice and suggest a defect in inhibitory regulation in the tissue of the obese.

Vitamin D metabolism in magnesium deficient chicks

Abstract To determine if alterations in vitamin D metabolism are associated with the development of hypocalcemia in magnesium (Mg) deficiency, we measured plasma Mg, calcium (Ca), phosphorus (P) and vitamin D metabolites in growing chicks pair-fed diets adequate (1000ppm) or low (130ppm) in Mg for up to 15 days. A third group received a low Ca (0.2%) diet adequate in Mg and served as a positive control group in which the classical response to hypocalcemia was demonstrated. A progressive decline in plasma Mg in the Mg deficient chicks was associated with progressive and severe hypocalcemia. There was no rise in plasma 1,25-dihydroxycholecalcifetol (DHCC) in response to hypocalcemia in the Mg deficient chicks. In contrast, chicks fed low dietary Ca with adequate Mg exhibited a 3-fold elevation in circulating 1,25-DHCC and maintained plasma calcium slightly below that of control animals. The failure of the Mg deficient chicks to increase 1,25-DHCC production in response to hypocalcemia was not associated with inadequate circulating 25-hydroxycholecalciferol or increased plasma 24,25-DHCC. 1α-hydroxylase activity, measured in isolated renal tubules, was not elevated in the Mg deficient chicks, compared to controls. A significant correlation between plasma 1,25-DHCC and 1α-hydroxylase activity suggested that metabolic turnover of 1,25-DHCC was not significantly altered by Mg deficiency. The data suggest that the progression of hypocalcemia during Mg deficiency may be related to insufficient production of 1,25-DHCC in response to the prevailing hypocalcemia.