The Inhibitory Effect of Leptin on Angiotensin II-Induced Vasoconstriction in Vascular Smooth Muscle Cells Is Mediated via a Nitric Oxide-Dependent Mechanism

Abstract

Leptin inhibits the contractile response induced by angiotensin (Ang) II in vascular smooth muscle cells (VSMCs) of the aorta. We studied in vitro and ex vivo the role of nitric oxide (NO) in the effect of leptin on the Ang II-induced vasoconstriction of the aorta of 10-wk-old Wistar rats. NO and nitric oxide synthase (NOS) activity were assessed by the Griess and (3)H-arginine/citrulline conversion assays, respectively. Stimulation of inducible NOS (iNOS) as well as Janus kinases/signal transducers and activators of transcription (JAK/STAT) and phosphoinositide 3-kinase (PI3K)/Akt signaling pathways were determined by Western blot. The contractile responses to Ang II were evaluated in endothelium-denuded aortic rings using the organ bath system. Changes in intracellular Ca(2+) were measured in VSMCs using fura-2 fluorescence. Leptin significantly (P < or = 0.01) stimulated NO release and NOS activity in VSMCs. Leptin's effect on NO was abolished by the NOS inhibitor, N(G)-monomethyl l-arginine, or the iNOS selective inhibitor L-N(6)-(1-iminoethyl)-lysine. Accordingly, leptin increased iNOS protein expression, with a comparable time course with that of NO production and NOS activity. Leptin also significantly increased STAT3 (P < or = 0.01) and Akt (P < or = 0.001) phosphorylation. Moreover, either the JAK2 inhibitor, AG490, or the PI3K inhibitor, wortmannin, significantly (P < or = 0.05) abrogated the leptin-induced increase in iNOS protein. Finally, both N(G)-monomethyl L-arginine and L-N(6)-(1-iminoethyl)-lysine inhibitors completely blunted (P < or = 0.001) the leptin-mediated inhibition of the Ang II-induced VSMC activation and vasoconstriction. These findings suggest that the endothelium-independent depressor action of leptin is mediated by an increase of NO bioavailability in VSMCs. This process requires the up-regulation of iNOS through mechanisms involving JAK2/STAT3 and PI3K/Akt pathways.

Targeted disruption of the iNOS gene improves adipose tissue inflammation and fibrosis in leptin-deficient ob/ob mice: role of tenascin C

“…In a first set of experiments, differentiated 3T3-L1 adipocytes were serum-starved for 24 h and quiescent cells were stimulated with recombinant murine leptin (10 nmol L −1 ) (450-31; PeproTech EC, Inc., Rocky Hill, NJ, USA) or tenascin C (10 nmol L −1 ) (3358-TC-050; R&D Systems, Minneapolis, MN, USA) for 24 h. In a second set of experiments, leptin (10 nmol L −1 )-treated 3T3-L1 adipocytes were incubated in the presence of the PI3K inhibitor wortmannin (Tocris, Ellisville, MO, USA) (10 μmol L −1 ), L-NAME, a nonspecific NOS inhibitor (N5751, Sigma) or L-NIL, selective inhibitor of iNOS (I8021; Sigma) (all 10 µmol L −1 ) for 24 h. The concentrations of leptin, tenascin C, and pharmacological inhibitors to perform the experiments were chosen on the basis of previous studies carried out in our laboratory 29 . One sample per experiment was used to obtain control responses in the presence of the solvent.…”

“…These cytokines also regulate leptin expression, sustaining a chronic proinflammatory state 25 . Since many biological functions of leptin are induced via NO [26][27][28][29][30][31] , our aim was to evaluate the effects of the disruption of the iNOS gene in wild type and ob/ob mice on AT inflammation and fibrosis 26,32 . To further confirm the potential participation of NO, the effect of the pharmacological inhibition of iNOS with N ω -nitro-L-arginine methyl ester hydrochloride (L-NAME), a non-selective NOS inhibitor, and L-N 6 -(1-iminoethyl)-lysine (L-NIL), a selective iNOS inhibitor, as well as the effect of iNOS gene silencing on the leptin-induced production of proinflammatory factors was analyzed in murine 3T3-L1 adipocytes.…”

Acylated and desacyl ghrelin stimulate lipid accumulation in human visceral adipocytes

“…29 Thirty micrograms of total protein were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions, transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and blocked in TBS with 0.05% Tween 20 (TBS-T) containing 5% non-fat dry milk for 1 h at room temperature. Blots were then incubated overnight at 4 1C with primary antibodies against GHS-R (Alpha Diagnostic International), aquaporin-7 (AQP7) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), FAS (Cell Signaling, Danver, MA, USA), adipophilin (Acris, Hiddenhausen, Germany), perilipin (Acris) or b-actin (Sigma).…”

Combined amylin–leptin treatment lowers blood pressure and adiposity in lean and obese rats

“…Additionally, LEP can act peripherally at a number of sites including the vasculature 48 possibly contributing to observed depressor actions of peripheral LEP. 45 LEP stimulates nitric oxide production, a vasodilator from vascular endothelial cells and vascular smooth muscle, both in vitro 45,49 and in vivo.…”