Background-Angiotensin II-induced hypertension is associated with NAD(P)H oxidase-dependent superoxide production in the vessel wall. Vascular superoxide level is also increased in deoxycorticosterone acetate (DOCA)-salt hypertension, which is associated with a markedly depressed plasma renin activity because of sodium retention. However, the mechanisms underlying superoxide production in low-renin hypertension are undefined. Methods and Results-This study investigated (1) whether and how endothelin-1 (ET-1), which is increased in DOCA-salt hypertensive rats, contributes to arterial superoxide generation and (2) the effect of gene transfer of manganese superoxide dismutase and endothelial nitric oxide synthase. Both superoxide and ET-1 levels were significantly elevated in carotid arteries of DOCA-salt rats compared with that of the sham-operated controls. ET-1 concentration-dependently stimulated superoxide production in vitro in carotid arteries of normotensive rats. The increase in arterial superoxide in both ET-1-treated normotensive and DOCA-salt rats was reversed by a selective ET A receptor antagonist, ABT-627, the flavoprotein inhibitor diphenyleneiodonium, and the NADPH oxidase inhibitor apocynin but not by the nitric oxide synthase inhibitor N -L-arginine methyl ester or the xanthine oxidase inhibitor allopurinol. Furthermore, in vivo blockade of ET A receptors significantly reduced arterial superoxide levels, with a concomitant decrease of systolic blood pressure in DOCA-salt rats. Ex vivo gene transfer of manganese superoxide dismutase or endothelial nitric oxide synthase also suppressed superoxide levels in carotid arteries of DOCA-salt rats. Conclusions-These findings suggest that ET-1 augments vascular superoxide production at least in part via an ET A /NADPH oxidase pathway in low-renin mineralocorticoid hypertension.

Abstract-Arteries from deoxycorticosterone acetate (DOCA)-salt and N -nitro-L-arginine (L-NNA) hypertensive but not normotensive rats develop spontaneous tone. LY294002 and wortmannin, phosphoinositide 3-kinase (PI3-kinase) inhibitors, eliminate spontaneous tone. We hypothesized that PI3-kinase protein and/or activity was increased in hypertension and contributed to the observed enhanced contractility. PI3-kinase activity assays revealed 2-fold higher activity in thoracic aorta from DOCA-salt [systolic blood pressure (SBP)ϭ184Ϯ5 mm Hg] compared with sham rats (SBPϭ111Ϯ2 mm Hg). Western analyses of aortic homogenates revealed the presence of p85␣, p110␣, p110␤, and p110␦ but not p110␥ PI3-kinase subunits; p110␦ protein was elevated in aorta of hypertensive rats as compared with sham. Aortic homogenates from L-NNA rats also had elevated p110␤ protein density, but neither L-NNA nor DOCA-salt had differences in p85␣ and p110␣. Total Akt density was unaltered, but pAkt was significantly lower in homogenates from DOCA-salt rats. LY294002 (20 mol/L) and nifedipine (50 nmol/L) abolished Ca 2ϩ -induced spontaneous tone in aorta from DOCA-salt rats. However, LY294002 did not alter BayK8644-induced contraction, indicating that LY294002 does not inhibit L-type Ca 2ϩ channels directly. PTEN (phosphatase and tensin homolog) and pPTEN were expressed but not different in aorta from DOCA-salt and sham rats. LY294002 corrected the enhanced contraction to KCl and norepinephrine in aorta from DOCA-salt rats. These data support an increase in PI3-kinase activity and p110␦ density in aorta from L-NNA and DOCA-salt rats. Importantly, this increase contributes to the enhanced contractility observed in two models of hypertension. Key Words: phosphoinositide 3-kinase Ⅲ artery Ⅲ deoxycorticosterone acetate-salt hypertension Ⅲ phosphatase and tensin homolog Ⅲ N -nitro-L-arginine hypertension P hosphoinositide 3-kinase (PI3-kinase) is a multifaceted enzyme, possessing both lipid and protein kinase activity. It participates in multiple signaling pathways, receiving input from a variety of receptors ultimately leading to changes in gene expression, metabolism, cell division, apoptosis, cellular trafficking, and contractility.

It is hypothesized that selective muscarinic M1 subtype activation could be a strategy to provide cognitive benefits to schizophrenia and Alzheimer's disease patients while minimizing the cholinergic side effects observed with nonselective muscarinic orthosteric agonists. Selective activation of M1 with a positive allosteric modulator (PAM) has emerged as a new approach to achieve selective M1 activation. This manuscript describes the development of a series of M1-selective pyridone and pyridine amides and their key pharmacophores. Compound 38 (PF-06767832) is a high quality M1 selective PAM that has well-aligned physicochemical properties, good brain penetration and pharmacokinetic properties. Extensive safety profiling suggested that despite being devoid of mAChR M2/M3 subtype activity, compound 38 still carries gastrointestinal and cardiovascular side effects. These data provide strong evidence that M1 activation contributes to the cholinergic liabilities that were previously attributed to activation of the M2 and M3 receptors.

We tested the hypothesis that the 5-HT transporter (5-HTT) is present and functional in peripheral arterial smooth muscle. In aorta and mesenteric resistance arteries, real time RT-PCR and western analyses indicated the presence of 5-HTT mRNA and a 74 kDa 5-HTT protein. Immunohistochemistry localized the transporter to smooth muscle and endothelial cells. 5-HT and the metabolite 5-hydroxyindole acetic acid (5-HIAA) were detected in aorta, carotid, and superior mesenteric arteries using HPLC; the MAOA inhibitor pargyline significantly increased (over 400%) arterial 5-HT concentration. 5-HT was taken up by arteries in a time-dependent manner and uptake was independent of the endothelium, sympathetic nerves, and norepinephrine transporter. 5-HT-induced contraction of normal aorta was potentiated by the 5-HTT inhibitor fluvoxamine. A change in arterial 5-HTT function occurs in deoxycorticosterone (DOCA)-salt hypertension as the potency and threshold of 5-HT in contracting aorta from the DOCA-salt rat was increased by fluoxetine and fluvoxamine (1 micromol/L; DOCA fluvoxamine -log EC50 [mol/L] = 6.85 +/- 0.08, DOCA-control = 6.44 +/- 0.08); expression of transporter was significantly increased in aorta of DOCA salt rats (145% Sham). These studies show for the first time the presence of the 5-HTT in peripheral arterial smooth muscle and raise the question as to the function of the 5-HTT in regulating peripheral effects of 5-HT.