CYTOPLASMIC AND NUCLEAR SIGNALING PATHWAYS OF V-1-VASCULAR VASOPRESSIN RECEPTORS

We studied the cytoplasmic and nuclear signaling pathways of V1-vascul ar AVP receptors of human platelets, primary cultures of renal glomeru lar mesangial cells, and established cultures of the A7r5 aortic smoot h muscle cell line. The immediate transmembrane signals are triggered by the formation of ligand-receptor complexes as illustrated by bindin g experiments with [HS-3]AVP (K(d) = 2.50 nM), d(CH2)5Tyr(Me)AVP (K(d) = 0.62 nM), the linear V1 antagonist cetyl-D-Tyr(Et)-Phe-Val-Asn-Lys- Pro-[I-125]Tyr-NH2 (K(d) = 1.42 nM) or by fluorescence experiments wit h linear antagonists like phenylacetyl-D-Tyr(Et)-Phe-Gln-Asn-Lys-Pro-A rg-NH2 coupled to biotin and made fluorescent by labeling with tetrame thylrhodamine-avidin. We used several approaches (radioreceptor bindin g, radioactive labeling, autoradiographic, enzymatic, photoaffinity la beling, and immunoblotting procedures) to identify the guanine nucleot ide regulatory protein coupled to V1-vascular vasopressin receptors. A VP-stimulated GTPase activity of human platelet membranes was blocked by pretreatment with antibodies specific for the C-terminal of the new ly described G(qalpha) protein. In the presence Of MgCl2, AVP increase d labeling by the photoreactive GTP analog [alpha-P-32]azidoanilido GT P of a platelet membrane protein of apparent molecular mass of 42 kDa. AVP effect was reversed by the specific V1-vascular antagonist d(CH2) 5Tyr(Me)AVP and labeling was completely abolished by GTP(gammaS). Immu noblotting of platelet proteins with various antibodies specific for t he C-terminal of the alpha subunits of the three families of G protein s revealed that the 42 kDa protein labeled with [alpha-P-32]azidoanili do GTP was specifically labeled only by antibodies specific for the al pha subunit of G. thus suggesting that V1-vascular AVP receptors are c oupled in a divalent cation-dependent manner to a G protein belonging to the G(q/11) family. V1-vascular AVP receptors activate not only pho spholipases C and D but also phospholipase A2. In fura-2 loaded A7r5 c ells, the phospholipase A2 inhibitor aristolochic acid reduced AVP-ind uced [Ca2+]i transients. The role of arachidonic acid (AA) metabolites in AVP-induced calcium mobilization was precised as follows. AA poten tiated AVP-induced influx of extracellular Ca2+ and mobilization of in tracellular Ca2+. The cyclooxygenase inhibitor indomethacin reduced AA - and AVP-induced influx of extracellular Ca2+ but not intracellular C a2+ mobilization. AVP-induced [Ca2+]i transients were not altered by l ipoxygenase inhibitors but were reduced in a dose-dependent fashion by ketoconazole, an inhibitor of cytochrome P-450 epoxygenases. The inhi bitory action of ketoconazole was noted both in the presence and absen ce of extracellular Ca2+. Among different epoxygenase metabolites test ed, 5,6-epoxyeicosatricnoic acid (5,6-EET) potentiated AVP-induced [Ca 2+]i transients. Reverse-phase HPLC analysis of lipid extracts from A7 r5 cells prelabeled with [C-14]AA isolated a radioactive peak eluting with epoxygenase products. This peak was increased after AVP stimulati on and blocked by ketoconazole. Gas chromatography-mass spectrometry a nalysis of cell extracts isolated a compound with epoxyeicosatrienoic characteristics. The secondary nuclear signaling pathways of V1-vascul ar AVP receptors modulate gene expression. To investigate whether vaso pressin (AVP) modulates immediate-early gene expression associated wit h growth in vascular smooth muscle cells, the expression of the proto- oncogenes c-fos and c-jun was studied in cultured A7r5 aortic smooth m uscle cells made quiescent by being grown in serum-free media for 48 h s. AVP stimulation (10(-12) to 10(-6) M) of A7r5 cells raised in a dos e-dependent fashion mRNA levels of c-fos and c-jun without altering th e expression of the constitutive gene GAPDH. AVP induction of c-fos an d c-jun mRNA expression was rapid and transient as it was over within 3 hs. AVP effect was specific and blocked by the V1-vascular antagonis t d(CH2)5Tyr(Me)AVP. The mRNA stimulating action of AVP required the p resence of Ca2+ and involved activation of phospholipases A2 and C as well as protein kinase C. All these cytoplasmic and nuclear signals ex plain the major cellular effects of activation of V1-vascular AVP rece ptors, i.e., cell contraction and growth.