Prevention of α2-adrenergic inhibition on ADH action by pertussis toxin in rabbit CCT

C. P. Ribeiro, F. Ribeiro-Neto, J. B. Field, Wadi N. Suki

Research output: Contribution to journalArticle

12 Scopus citations

Abstract

The present studies were performed to investigate the mechanism whereby α2-adrenergic receptor occupancy inhibits the hydrosmotic action of antidiuretic hormone (ADH) in isolated cortical collecting tubules (CCT). The ADH-ribosyltransferase activity of pertussis toxin (PT) was used to promote covalent modification in CCT N(i), the inhibitory regulatory protein of adenylate cyclase, which presumably mediates the α2-adrenergic inhibition of water flow. Tubules preincubated with PT were studied after the addition of ADH and then after the superimposition of clonidine. In these studies, the inhibition of J(v) (water absorption, nl·mm-1·min-1) and P(f) (water permeability coefficient, cm/s), by the addition of 10-4 M clonidine to the bath, was attenuated by PT in a concentration-dependent manner. Reversal of the inhibitory action of clonidine was accomplished with a concentration of 1.0 μg/ml PT. To further elucidate the molecular basis of N(i)-mediated transduction of the α2-adrenergic signal, ADP-ribosylation studies were undertaken in membrane preparations of dissected CCT segments. PT ADP ribosylated a 40,000 M(r) peptide which was proportional to the amount of membrane protein added. Furthermore, pretreatment of CCT during dissection with 0.5 μg/ml PT dramatically decreased the susceptibility of the subunit of N(i) (α1) to be subsequently ADP ribosylated by PT, when compared with CCT preparations not previously treated with PT. Cholera toxin ADP ribosylated a 42,000 M(r) peptide from CCT membranes and PT pretreatment did not interfere with the reaction. We conclude that CCT segments have both the pertussis and cholera toxin substrates and the effect of clonidine to attenuate ADH action is mediated through N(i).

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Volume253
Issue number1
StatePublished - Jan 1 1987

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

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