Michitoshi Sunako

Angiotensin II induces expression of the c-fos gene through protein kinase C activation and calcium ion mobilization in cultured vascular smooth muscle cells☆

Incubation of the serum-deprived cultures of rat vascular smooth muscle cells with angiotensin II, a potent vasoconstrictor, caused a rapid and transient increase in the c-fos mRNA level. The doses of this agonist necessary for the increase in the c-fos mRNA level coincided with those for the phospholipase C-mediated hydrolysis of phosphoinositides. Moreover, protein kinase C-activating 12-O-tetradecanoylphorbol-13-acetate and Ca2+-ionophore A23187 increased the c-fos mRNA level in an additive manner. These results suggest that angiotensin II induces expression of the c-fos gene through the activation of protein kinase C and Ca2+ mobilization in cultured vascular smooth muscle cells.

Stimulation of phospholipase C-mediated hydrolysis of phosphoinositides by endothelin in cultured rabbit aortic smooth muscle cells

Incubation of the [3H] inositol-labeled cultured rabbit vascular smooth muscle cells (VSMCs) with either endothelin or angiotensin II caused a rapid formation of inositol mono-, bis- and trisphosphates (IP1, IP2 and IP3, respectively). Time courses of the endothelin- and angiotensin II-induced formation of these inositol phosphates were similar. The maximal levels of IP1, IP2 and IP3 formation induced by endothelin were about 50%, 25% and 40%, respectively, of those induced by angiotensin II. The doses of endothelin necessary for the half maximal and maximal extents of the formation of IP1 were about 1 nM and 100 nM, respectively. Protein kinase C-activating 12-Q-tetradecanoylphorbol-13-acetate (TPA) inhibited the endothelin-induced formation of IP1 with the half maximal extent of inhibition seen at 3 nM. The inhibitory action of TPA was mimicked by another protein kinase C-activating phorbol ester, phorbol-12,13-dibutyrate, but not by 4 alpha-phorbol-12,13-didecanoate, known to be inactive for this enzyme. These results indicate that endothelin causes the phospholipase C-mediated hydrolysis of phosphoinositides, though to a lesser extent than angiotensin II, in cultured VSMCs and suggest that protein kinase C modulates the signaling mechanism of endothelin to the phospholipase C.

Stimulation of platelet-derived growth factor-induced DNA synthesis by angiotensin II in rabbit vascular smooth muscle cells

In cultured rabbit vascular smooth muscle cells (VSMCs), angiotensin II by itself had little mitogenic effect even in the presence of cell-free plasma-derived serum (PDS), but markedly stimulated the platelet-derived growth factor (PDGF)-induced DNA synthesis in the presence of PDS. The maximal extent of DNA synthesis induced by PDGF plus angiotensin II was about twice that induced by PDGF alone. The stimulatory effect of angiotensin II was dose-dependent with the maximal response seen at 1 microM and was inhibited by the specific angiotensin II receptor antagonist, [Sar1, Ile8]angiotensin II. In VSMCs, both PDGF and angiotensin II induced expression of the c-fos gene in dose-dependent manners. In contrast to the synergistic effect of angiotensin II and PDGF on DNA synthesis, they induced expression of the c-fos gene in an additive manner. These results suggest that angiotensin II may act as a growth regulator for VSMCs in addition to acting as a vasoconstrictor.

Mass analysis of 1,2-diacylglycerol in cultured rabbit vascular smooth muscle cells. Comparison of stimulation by angiotensin II and endothelin.

We compared the effects of angiotensin II and endothelin on mass levels of 1,2-diacylglycerol, an endogenous activator of protein kinase C, in cultured rabbit vascular smooth muscle cells with the effects of these vasoconstrictors on contractile responses of rabbit aortic strips. At a high concentration (1 /*M), both angiotensin II and endothelin induced a biphasic formation of 1,2-diacylglycerol with an early transient phase and a late sustained phase. At this high concentration, angiotensin II caused a transient contraction followed by a gradual relaxation, whereas endothelin caused a slowly developing and sustained contraction. At a low concentration (EC50 for the early phase of 1,2-diacylglycerol formation), angiotensin II also induced a biphasic formation of 1,2-diacylglycerol and caused a transient contraction, but endothelin induced a monophasic formation of 1,2-diacylglycerol with only an early peak. Despite a rapid decrease of 1,2-diacylglycerol, endothelin at this low concentration still caused a sustained contraction. At both the high and low concentrations, the 1,2-diacylglycerol level was sustained higher for angiotensin II, whereas the tension during the late tonic phase of contraction was greater for endothelin. These results suggest that the unique persistent nature of endothelininduced contraction is not attributed simply to the stimulatory effect of this peptide on the 1,2-diacylglycerol/protein kinase C pathway.

Endothelin-induced biphasic formation of 1,2-diacylglycerol in cultured rabbit vascular smooth muscle cells ― mass analysis with a radioenzymatic assay

Mass analysis of 1,2-diacylglycerol (DG) with a radioenzymatic assay revealed that endothelin induced a biphasic formation of DG with an early transient phase peaking at 30 sec and a late sustained phase peaking at 5 min in cultured rabbit vascular smooth muscle cells (VSMCs). The amounts of DG after the 30-sec and 5-min incubation with endothelin were 0.74 +/- 0.08 (mean +/- SE) nmol and 0.87 +/- 0.10 nmol/100 nmol of lipid phosphorus, representing 2.6- and 3.1-fold increases of the resting level, respectively. The EC50 values of endothelin for the early and late phases of DG formation were about 1 nM and 40 nM, respectively. In the [3H] inositol-labeled VSMCs, endothelin induced a rapid transient formation of inositol tris- and bisphosphates which peaked at 30 sec and a sustained formation of inositol monophosphate which peaked at 5 min. The EC50 values for the formation of these inositol phosphates were the same and about 1 nM. These results suggest that the early transient phase of DG is derived from the hydrolysis of polyphosphoinositides, while a large part of the late sustained phase of DG is from the reaction(s) other than the hydrolysis of phosphoinositides but its sources remain to be clarified.

Identification of a major GTP-binding protein in bovine aortic smooth muscle membranes as smg p21, a GTP-binding protein having the same effector domain as ras p21s.

At least two GTP-binding proteins (G proteins) with Mr values of about 20,000 were extracted from bovine aortic smooth muscle membranes by sodium cholate. The most abundant G protein (22K G) was purified to near homogeneity by successive column chromatographies of Ultrogel AcA-44, phenyl-Sepharose CL-4B, hydroxyapatite and Mono Q HR5/5. 22K G showed kinetic and physical properties very similar to those of smg p21, a G protein recently isolated from bovine brain and human platelet membranes, having the same effector domain as ras p21s. Moreover, 22K G was recognized specifically by the anti-smg p21 antibody. These results indicate that the major G protein in bovine aortic smooth muscle membranes is smg p21.

Increased 201Tl uptake by the chest wall following cardioversion.

A patient with acute inferior infarct is described who showed increased 201Tl uptake in the anterior chest wall following cardioversion which mimicked increased lung uptake of the tracer. The site of accumulation of 201Tl corresponded to that of 99mTc-pyrophosphate (PYP) and the right sided catheterization revealed almost normal hemodynamic data.