ABSTRACT
Incubation of the toad bladder epithelia with 100 nM 12-O-tetradecanoylphorbol 13-acetate (TPA) for 1, 3 and 5 min decreased cytosolic protein kinase C (PKC) activity to 85, 80 and 75% of control, while membrane-associated PKC activity increased to 127, 140 and 126% of control, respectively. Long-term treatment of epithelial cells with TPA caused downregulation of PKC with a loss of 80% of the enzymic activity. Incubation with vasopressin (AVP) for 2 min decreased cytosolic PKC activity by 20%, whereas the activity in the membrane fraction increased by 33%. PKC translocation did not occur when epithelia were stimulated with [deamino1-D-arginine8]-vasopressin which binds more specifically to the V2 receptor. Staurosporine inhibited PKC activity as well as the effect of AVP on translocation. Phorbol esters decreased the response to AVP on water transport, whereas staurosporine greatly increased the hormonal response. We conclude that TPA induces an intracellular translocation and downregulation of PKC. The translocation of PKC by AVP and the inhibition of AVP-stimulated water flow by TPA suggest a significant negative feedback loop involving PKC to modulate the action of AVP.
Subject(s)
Arginine Vasopressin/pharmacology , Protein Kinase C/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Urinary Bladder/enzymology , Animals , Anura , Biological Transport/drug effects , Cell Membrane/enzymology , Cytosol/enzymology , Deamino Arginine Vasopressin/pharmacology , Enzyme Inhibitors/pharmacology , Epithelium/enzymology , Female , Protein Kinase C/antagonists & inhibitors , Renal Agents/pharmacology , Staurosporine/pharmacology , Urinary Bladder/ultrastructureABSTRACT
22 patients with chronic renal failure, maintained on chronic maintenance dialysis with a dialysate calcium of 1.5 mmol/l, were randomly selected for measurement of pre- and postdialysis plasma calcium fractions. Total, ionized and protein-bound calcium fractions in plasma showed significant increases following dialysis, but with correction for hemoconcentration due to ultrafiltration from dialysis, only protein-bound calcium showed a significant increase. Change of in vitro pH from 7.35 to 7.44 did not influence calcium binding. Similarly, in vitro addition of calcium to pre- and postdialysis blood samples did not result in significant differences for protein-bound calcium fractions. Comparison of dissociation constants for protein-bound calcium between pre- and postdialysis samples showed a significant change. Our results indicate that dialysis alters the affinity of serum protein by some unexplained mechanism and may contribute to the genesis of dialysis-bone disease.
