Objective-Asymmetric dimethylarginine (ADMA) is widely believed to be an endogenous nitric oxide synthase (eNOS) inhibitor. However, in this study, we examined our hypothesis that the long-term vascular effects of ADMA are not mediated by inhibition of endothelial NO synthesis. Methods and Results-ADMA was infused in wild-type and eNOS-knockout (KO) mice by osmotic minipump for 4 weeks. In wild-type mice, long-term treatment with ADMA caused significant coronary microvascular lesions. Importantly, in eNOS-KO mice, treatment with ADMA also caused an extent of coronary microvascular lesions that was comparable to that in wild-type mice. These vascular effects of ADMA were not prevented by supplementation of L-arginine, and vascular NO production was not reduced by ADMA treatment. Treatment with ADMA caused upregulation of angiotensin-converting enzyme (ACE) and an increase in superoxide production that were comparable in both strains and that were abolished by simultaneous treatment with temocapril (ACE inhibitor) or olmesartan (AT 1 receptor antagonist), which simultaneously suppressed vascular lesion formation. Key Words: asymmetric dimethylarginine Ⅲ arteriosclerosis Ⅲ nitric oxide Ⅲ endothelial nitric oxide synthase Ⅲ mice E ndothelium-derived nitric oxide (NO), synthesized from L-arginine by endothelial NO synthase (eNOS), has several important antiatherogenic actions. 1-5 Indeed, reduction of endothelial NO synthesis (endothelial dysfunction) predisposes the blood vessel to arteriosclerosis, 1-5 and the eNOS-deficient (eNOS-KO) mice exhibit accelerated vascular lesion formation. 6,7 As pharmacological tools to inhibit endothelial NO synthesis, synthetic L-arginine analogues have been used in vitro and in vivo. Among them, N -nitro-L-arginine methyl ester (L-NAME) is the most frequently used agent. [1][2][3][4][5] Long-term treatment with L-NAME is known to cause arteriosclerotic coronary lesions, especially at microvascular levels, in experimental animals. 8,9 This model with L-NAME is regarded as a useful animal model for examining the protective roles of endothelium-derived NO in the pathogenesis of arteriosclerosis. 8,9 See cover However, it is controversial whether these vascular effects of L-NAME are caused primarily by the inhibition of endothelial NO synthesis for the following reasons: first, the importance of endothelium-derived NO decreases as the vessel size becomes smaller, 10 whereas L-NAME-induced vascular lesions are prominent at microvascular levels; 8 second, long-term treatment with L-NAME does not reduce eNOS activity; 11 third, multiple actions of L-NAME other than simple inhibition of NO synthesis have been reported. 12,13 The most appropriate way to address this issue is to use mice that are deficient in the eNOS gene and to examine whether long-term treatment with L-NAME causes coronary vascular lesions in those mice. We have recently shown that treatment with L-NAME causes a comparable extent of Conclusions-These

Abstract:Pituitary adenylate cyclase-activating polypeptide (PACAP) causes both Ca2~release and Ca2 influx in bovine adrenal chromaffin cells. To elucidate the mechanisms of PACAP-induced Ca2~release, we investigated expression of PACAP receptors and measured inositol trisphosphates (lP 3), cyclic AMP, and the intracellular Ca 2~concentration in bovine adrenal medullary cells maintained in primary culture. RT-PCR analysis revealed that bovine adrenal medullary cells express the PACAP receptor hop, which is known to couple with both 1P 3 and cyclic AMP pathways. The two naturally occurring forms of PACAP, PACAP38 and PACAP27, both increased cyclic AMP and 1P3, and PACAP38 was more potent than PACAP27 in both effects. Despite the effects of PACAP on 1P3 production, the Ca 2~release induced by PA-CAP38 or by PACAP27 was unaffected by cinnarizine, a blocker of IF' 3 channels. The potencies of the peptides to cause Ca 2~release in the presence of cinnarizine were similar. The Ca2~release induced by PACAP38 or by PACAP27 was strongly inhibited by ryanodineand caffeine. In the presence of ryanodine and caffeine, PACAP38 was more potent than PACAP27. PACAP-induced Ca2~release was unaffected by Rp-adenosine 3',5'-cyclic monophosphothioate, an inhibitor of protein kinase A. Ca2~release induced by bradykinin and angiotensin Il was also inhibited by ryanodine and caffeine, but unaffected by cinnarizine. Afthough P 3 production stimulated by PACAP38 or bradykinin was abolished by the phospholipase C inhibitor, U-73122, Ca 2release in response to the peptides was unaffected by U-73122. These results suggest that PACAP induces Ca2~release from ryanodine/caffeine stores through anovel intracellular mechanism independent of both 1P 3 and cyclic AMP and that the mechanism may be the common pathway through which peptides release Ca 2~in adrenal chromaffin cells. Key Words: Pituitary adenylate cyclase-activating polypeptide-Adrenal medulla-Ca2~release-Fura-2-lnositol trisphosphatesCyclic AMP.

Abstract. Flavonoids are biologically active polyphenolic compounds widely distributed in plants. Recent research has focused on high dietary intake of flavonoids because of their potential to reduce the risks of diseases such as cardiovascular diseases, diabetes, and cancers. We report here the effects of plant flavonoids on catecholamine signaling in cultured bovine adrenal medullary cells used as a model of central and peripheral sympathetic neurons. Daidzein (0.01 -1.0 mM), a soy isoflavone, stimulated 14 C-catecholamine synthesis through plasma membrane estrogen receptors. Nobiletin (1.0 -100 mM), a citrus polymethoxy flavone, enhanced 14 Ccatecholamine synthesis through the phosphorylation of Ser19 and Ser40 of tyrosine hydroxylase, which was associated with 45 Ca 2+ influx and catecholamine secretion. Treatment with genistein (0.01 -10 mM), another isoflavone, but not daidzein, enhanced [ 3 H]noradrenaline uptake by SK-N-SH cells, a human noradrenergic neuroblastoma cell line. Daidzein as well as nobiletin (≥ 1.0 mM) inhibited catecholamine synthesis and secretion induced by acetylcholine, a physiological secretagogue. The present review shows that plant flavonoids have various pharmacological potentials on the catecholamine system in adrenal medullary cells, and probably also in sympathetic neurons.

Obesity is often associated with cardiovascular and metabolic disorders such as hypertension and hyperglycemia. Leptin, a protein product of the obese gene, regulates satiety and energy expenditure through its receptors in the hypothalamus. Recent studies have shown that leptin has extrahypothalamic and peripheral actions. The presence of leptin receptors has been reported in the adrenal medulla. In the present study, we examined the effects of leptin on catecholamine synthesis in cultured bovine adrenal medullary cells. Leptin (3-30 nM) caused a significant increase in (14)C-catecholamine synthesis from [(14)C] tyrosine, but not from [(14)C] DOPA. Incubation of cells with leptin resulted in an activation and phosphorylation of tyrosine hydroxylase. Leptin caused a transient activation of mitogen-activated protein kinases (MAPKs). U0126, an inhibitor of MAPK kinase, abolished the effect of leptin on (14)C-catecholamine synthesis. High concentrations of leptin (10-100 nM) produced an increase in intracellular Ca(2+) concentration, which was blocked by Cd(2+), an inhibitor of voltage-dependent Ca(2+) channels. Concurrent treatment of cells with leptin (10 nM) and acetylcholine (0.3 mM) potently enhanced the stimulatory effect of acetylcholine on (14)C-catecholamine synthesis. Leptin, however, failed to enhance the stimulatory effect of acetylcholine on the phosphorylation and activity of tyrosine hydroxylase. Acetylcholine (0.3 mM) decreased the intracellular pH (pHi). Leptin (10 nM) affected neither the basal pHi nor the acetylcholine-induced fall in pHi. These findings suggest that leptin phosphorylates and activates tyrosine hydroxylase and subsequently stimulates catecholamine synthesis through MAPK and probably Ca(2+) pathways in the adrenal medulla.