[ARGININE]VASOPRESSIN HYDROLYZES PHOSPHOINOSITIDES IN THE MEDULLARY THICK ASCENDING LIMB OF MOUSE NEPHRON

NaCl reabsorption across the thick ascending limb of Henle's loop (TAL) is stimulated by several hormones, in particular vasopressin acting through V2 receptors and cyclic AMP production. This study used suspensions of medullary TAL (mTAL) tubules from the mouse nephron to investigate the possibility that, besides activating adenylyl cyclase, vasopressin also stimulates phospholipase C via V-1 receptor occupancy. Two different methods, phosphoinositide labelling and inositol trisphosphate (InsP(3)) radioimmunoassay, were used to show that [arginine]vasopressin (AVP) rapidly stimulated the formation of InsP(3), which peaked at 200%-250% of control within the first minute of incubation with 10 nmol/l vasopressin at 37 degrees C, and declined to basal level after 5-10 min. Dose/response curves for InsP(3), established at 30 degrees C and 37 degrees C using radioimmunoassay, showed a half-maximal stimulation of InsP(3) production at about 1 nmol/l AVP and a maximal response at 10 nmol/l. Similar values were obtained for the response to AVP in terms of cAMP accumulation. InsP(3) content in the presence of higher concentrations of AVP (1 mu mol/l) was significantly lower (P < 0.001) than in the presence of 10 nmo/l AVP, giving a bell-shaped appearance to the dose/response curve at 37 degrees C but not at 30 degrees C. The V-2 receptor agonist, 1-deamino-[8-D-Arg]vasopressin (dAVP) did not stimulate the formation of InsP(3), and the v(1) receptor antagonist d(CH2)(5)[Tyr(Me)(2)]AVP inhibited AVP-induced InsP(3) formation, which therefore appeared to be mediated by V-1 receptor occupancy. Under the same conditions, AVP also induced the formation of diradylglycerol via V-1 receptor activation, with an analogous dose/response curve. These results show that AVP, in addition to its well-known action through V-2 receptors, also acts on the mouse mTAL through a V-1-mediated stimulation of phospholipase C. Cyclic AMP controls this transduction pathway: dAVP (10 nmol/l), dibutyryl-cAMP (1 mmol/l and 0.1 mmol/l) and forskolin (1 mu mol/l) decreased the InsP(3) formation induced by AVP. Dibutyryl-cAMP itself at 37 degrees C also reduced the diglyceride content.