ABSTRACT
Guanyl nucleotide regulation of parathyroid hormone (PTH)-activated adenylate cyclase was studied in membrane preparations of cultured opossum kidney cells. Guanosine triphosphate (GTP) (100 microM) decreased PTH-stimulated activity by 70%. Pertussis toxin enhanced PTH stimulation in intact cells and membranes, completely blocked the inhibitory effect of GTP, and catalyzed the [32P]ADP-ribosylation of a 38,000-dalton protein migrating in the position of the alpha-subunit of the inhibitory GTP-regulatory protein Ni. Cholera toxin was used to identify the alpha-subunit of the stimulatory GTP-binding protein Ns, a 42,000-dalton protein. We tested the idea that Ni may be involved in mediating the reduced response of opossum kidney cells to PTH after pretreatment with the hormone (desensitization). GTP inhibited PTH-stimulated activity to approximately the same degree in membranes from PTH-pretreated cells and control cells whether or not the cells had also received pertussis toxin. We conclude that GTP inhibits PTH action in opossum kidney cells through Ni but that PTH-induced desensitization is not mediated by Ni.
Subject(s)
Adenylyl Cyclases/metabolism , Guanosine Triphosphate/physiology , Kidney/enzymology , Parathyroid Hormone/physiology , Adenylate Cyclase Toxin , Adenylyl Cyclase Inhibitors , Cell Line , Cholera Toxin , Osmolar Concentration , Parathyroid Hormone/antagonists & inhibitors , Parathyroid Hormone/pharmacology , Pertussis Toxin , Virulence Factors, BordetellaSubject(s)
Parathyroid Hormone/chemical synthesis , Adenylyl Cyclases/metabolism , Amino Acid Sequence , Animals , Biological Assay , Chromatography, Gel , Dogs , Electrophoresis, Polyacrylamide Gel , Humans , Indicators and Reagents , Isoelectric Focusing , Kidney/enzymology , Parathyroid Hormone/isolation & purification , Parathyroid Hormone/pharmacologyABSTRACT
Antisera directed toward the carboxyl-terminal region of human parathyrin (parathyroid hormone), for use in daignostically applicable radioimmunoassays of the hormone in serum, are scarce, largely because of the lack of suitable immunogens of human origin. We produced four antisera in goats and guinea pigs by immunization with recently discovered carboxyl-terminal fragments of human parathyrin extracted from parathyroid tumors. Here, we report results of radioimmunoassays of nearly 200 normal and pathological sera with one of these antisera; we observed almost complete differentiation between concentrations of parathyrin in serum of healthy normal subjects and patients with primary, secondary (due to chronic renal failure), or "ectopic" hyperparathyroidism (due to nonparathyroid cancer). The availability of a new immunogen should now make possible the deliberate production of large quantities of diagnostically applicable parathyrin antisera directed toward the carboxyl-terminal region of human parathyrin. This should, in turn, lead to more widespread availability of this useful radioimmunoassay.
