Diphenylamine-2-carboxylate analogues block Cl- conductances in A7r5 cells by affecting cellular Ca2+ homeostasis.
We have investigated the cellular signalling pathway by which vasopressin stimulates a Ca2(+)-dependent Cl- conductance and the effects of two known Cl- channel blockers in cultured rat A7r5 aortic smooth muscle cells using anion efflux and fluorescent Ca2+ imaging studies. Addition of vasopressin (100 nM) to A7r5 cells enhanced 125I (Cl- substitute) efflux from the cells through a V1 receptor-mediated pathway. Maximal increases in the rate of efflux were observed 1 min following addition of vasopressin (4-fold above basal levels). Activation of the V1 pathway was demonstrated by an increase in inositol trisphosphate (IP3) formation and lack of cAMP accumulation by the cells following the addition of vasopressin. Fluorescent ratio imaging with fura-2 revealed that addition of vasopressin to the cells results in an increase of [Ca2+]i which peaks within 20 s and does not return to resting levels during the 100 s observation period. The addition of a Ca2+ ionophore mimicked the vasopressin-induced efflux from the cells. 5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and a chloro-substituted compound (cpd 149) inhibited the vasopressin-stimulated 125I efflux from the cells. The concentrations of NPPB and cpd 149 required to inhibit 125I efflux from the cells were similar to those which also attenuated vasopressin-induced Ca2+ transients in the cells. NPPB and cpd 149 had no effects on the ionomycin stimulated efflux. The mechanism(s) by which cpd 149 exerts its effect on stimulated efflux was examined by measuring its action on vasopressin-induced changes in IP3. Compound 149 inhibited IP3 generation in response to vasopressin.(ABSTRACT TRUNCATED AT 250 WORDS)
Merck Frosst Centre for Therapeutic Research, Kirkland, Que., Canada.
D J
DJ
Pon
M
M
Flezar
D L
DL
Litster
S
S
Heisler
D000818:Animals; D001013:Aorta, Thoracic / Q000187:drug effects / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D018118:Chloride Channels; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D006706:Homeostasis / Q000187:drug effects*; D007295:Inositol Phosphates / Q000378:metabolism; D007455:Iodine / Q000378:metabolism; D007457:Iodine Radioisotopes; D008565:Membrane Proteins / Q000037:antagonists & inhibitors*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D009579:Nitrobenzoates / Q000494:pharmacology; D051381:Rats; D014667:Vasopressins / Q000494:pharmacology; D062367:ortho-Aminobenzoates / Q000494:pharmacology*
D018118:Chloride Channels; D007295:Inositol Phosphates; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D009579:Nitrobenzoates; D062367:ortho-Aminobenzoates; D014667:Vasopressins; D005576:Colforsin; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; C001209:fenamic acid; D007455:Iodine; D000242:Cyclic AMP; D002118:Calcium