VASOPRESSIN-INDUCED CALCIUM SIGNALING IN CULTURED CORTICAL-NEURONS

Earlier autoradiographic studies from our laboratory detected vasopres sin recognition sites in the mammalian cerebral cortex [R.E. Brinton, K.W. Gee, J.K. Wamsley, T.P. Davis, H.I. Yamamura, Regional distributi on of putative vasopressin receptors in rat brain and pituitary by qua ntitative autoradiography, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 72 48-7252; C. Chen, R.D. Brinton, T.J. Shors, R.F. Thompson, Vasopressin induction of long-lasting potentiation of synaptic transmission in th e dentate gyrus, Hippocampus, 3 (1993) 193-204]. More recently, we hav e detected mRNA for the V1a vasopressin receptors (V1aRs) in cultured cortical neurons [R.S. Yamazaki, Q. Chen, S.S. Schreiber, R.D. Brinton , V1a Vasopressin receptor mRNA expression in cultured neurons, astrog lia, and oligodendroglia of rat cerebral cortex, Mel. Brain Res., 45 ( 1996) 138-140]. To determine whether these recognition sites are funct ional receptors, we have pursued the signal transduction mechanism ass ociated with the V1a vasopressin receptor in enriched cultures of cort ical neurons. Results of these studies demonstrate that exposure of co rtical neurons to the selective V-1 vasopressin receptor agonist, [Phe (2),Orn(8)]-vasotocin, (V-1 agonist) induced a significant accumulatio n of [H-3]inositol-1-phosphate ([H-3]IP1). V-1 agonist-induced accumul ation of [H-3]IP1 was concentration dependent and exhibited a linear d ose response curve. Time course analysis of V-1 agonist-induced accumu lation of [3H]IP, revealed a significant increase by 20 min which then decreased gradually over the remaining 60 min observation period. V-1 agonist-induced accumulation of [H-3]IP1 was blocked by a selective V 1a vasopressin receptor antagonist, (Phenylac(1), D-Tyr(Me)(2), Arg(6, 8), Lys-NH29)-vasopressin. Results of calcium fluorometry studies indi cated that V-1 agonist exposure induced a marked and sustained rise in intracellular calcium which was abolished in the absence of extracell ular calcium. The loss of the rise in intracellular calcium was not du e to a failure to induce PIP, hydrolysis since activation of the phosp hatidylinositol pathway occurred in the absence of extracellular calci um. V-1 agonist activation of calcium influx was then investigated. V- 1 agonist-induced Ca-45(2+) uptake was concentration dependent with a biphasic time course. Preincubation with the L-type calcium channel bl ocker, nifedipine, blocked V-1 agonist-induced calcium influx suggesti ng V-1 agonist-induced L-type calcium channel activation in cortical n eurons. Furthermore, V-1 agonist-induced calcium influx was blocked by both bisindolyleimide I (PKC inhibitor) and U-73122 (PLC inhibitor) s uggesting a modulation of V-1 agonist-induced L-type calcium channel a ctivation by downstream components of the phosphatidylinositol signali ng pathway such as protein kinase C. These results indicate that in cu ltured cortical neurons, V1a vasopressin receptor activation leads to induction of the phosphatidylinositol signaling pathway, influx of ext racellular calcium via L-type calcium channel activation, and a rise i n intracellular calcium which is dependent on V1a receptor activated i nflux of extracellular calcium. These data are the first to demonstrat e an effector mechanism for the V-1 vasopressin receptor in the cerebr al cortex and provide a potential biochemical mechanism that may under lie vasopressin enhancement of memory function. (C) 1998 Elsevier Scie nce B.V. All rights reserved.