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.