Masakuni Suematsu

Inhibition of Endothelial Nitric Oxide Synthase Activity by Protein Kinase C

Nitric oxide (NO) is an important molecular messenger accounting for endothelium-derived relaxing factor. Recently, NO synthase (NOS) from cultured endothelial cells has been purified and molecularly cloned. To evaluate the effect of phosphorylation by protein kinase C (PKC) and cyclic AMP-dependent protein kinase (PKA) on endothelial constitutive NOS catalytic activity, we incubated purified endothelial NOS with PKC or PKA. Endothelial NOS was stoichiometrically phosphorylated by PKC and PKA. In intact bovine aortic endothelial cells (BAECs), NOS was phosphorylated by stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA). NOS activity measured by the conversion of [3H]arginine to [3H]citrulline in homogenates of BAECs treated with TPA or phorbol 12,13-dibutyrate was reduced by 30%, whereas dibutylyl cyclic AMP did not affect NOS activity. Moreover, we measured NO release from cultured BAECs by a chemiluminescence method to examine the effect of PKC and PKA on endothelial NOS activity. In cultured BAECs, ATP gamma S and A23187 induced NO release in time- and dose-dependent manners. Phorbol esters such as TPA and phorbol 12,13-dibutyrate dose dependently inhibited NO release stimulated by A23187 as well as ATP gamma S. Reduction of NO release by TPA was almost completely prevented by pretreatment with staurosporine, an inhibitor of PKC. NO release by A23187 was increased in PKC-downregulated BAECs. In contrast, dibutylyl cyclic AMP or 8-bromo cyclic GMP had no effect on NO release from BAECs induced by A23187 or ATP gamma S. These results indicate that phosphorylation of NOS by PKC is associated with a reduction of its catalytic activity in vascular endothelial cells.

High density lipoprotein reverses inhibitory effect of oxidized low density lipoprotein on endothelium-dependent arterial relaxation.

We have recently reported that oxidized low density lipoprotein (ox-LDL) inhibits endothelium-dependent arterial relaxation through its increased lysophosphatidylcholine (LPC). In this study we examined whether high density lipoprotein (HDL) has any effect on the inhibition of endothelium-dependent relaxation by ox-LDL in isolated strips of rabbit thoracic aorta. Both low density lipoprotein (LDL) and HDL were isolated from normal human plasma, and LDL was oxidized by exposure to copper. Preincubation of arterial strips with ox-LDL (0.1-0.5 mg protein/ml) inhibited endothelium-dependent relaxation to acetylcholine (ACh) in a concentration-dependent manner. HDL (1 mg protein/ml) by itself had no effect on the relaxation to ACh. In the presence of HDL, the inhibition by ox-LDL was markedly reduced. Although synthetic L-alpha-palmitoyl LPC (5 micrograms/ml) completely abolished a relaxation to ACh, the preincubation of arterial strips with HDL completely prevented the LPC-induced inhibition. Moreover, a relaxation to ACh was almost completely recovered when the strips were washed with buffer containing HDL even after LPC-induced inhibition had occurred. HDL markedly reduced the incorporation of [1-14C]palmitate-labeled LPC ([14C]LPC) into cultured bovine aortic endothelial cells and promoted the release of cell-incorporated [14C]LPC into the medium, resulting in a reduction of the remaining [14C]LPC in the cells. Agarose electrophoresis after incubation of a mixture of ox-LDL labeled with [14C]LPC and unlabeled HDL demonstrated a transfer of [14C]LPC from ox-LDL to HDL. These results indicate that HDL reverses the ox-LDL-induced impairment of endothelium-dependent relaxation by removing LPC from ox-LDL and preventing LPC from acting on the endothelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Myristoylation of endothelial cell nitric oxide synthase is important for extracellular release of nitric oxide

Endothelial cell nitric oxide synthase (NOS) is known to have a N-myristoylation consensus sequence. Such a consensus sequence is not evident in the macrophage, smooth muscle and neuronal NOS. A functional role for this N-terminal myristoylation is not clear yet. In the present study, we examined the effect of N-terminal myristoylation on the NOS activity determined by the conversion of L-[3H]arginine to L-[3H]citrulline and extracellular NO release determined by nitrite production in the conditioned medium from the COS-7 cells transfected with wild type bovine aortic endothelial cell (BAEC) NOS cDNA or nonmyristoylated BAEC-NOS mutant cDNA. NOS activity of wild type BAEC-NOS in COS-7 cells was localized in the particulate fraction and that of mutant NOS was in the cytosolic fraction. In contrast, nitrite production from COS-7 cells transfected with wild type BAEC-NOS cDNA was greater than that of mutant cDNA in a time dependent and a concentration dependent manner. These results suggest that membrane localization of NOS with myristoylation facilitates extracellular transport of NO and leads to enhanced NO signaling on the vascular smooth muscle cells and the intravascular blood cells including neutrophils, macrophages and platelets.

Augmented receptor-mediated Ca2+ mobilization causes supersensitivity of contractile response to serotonin in atherosclerotic arteries.

We have previously reported that atherosclerotic arteries obtained from Watanabe heritable hyperlipidemic (WHHL) rabbits exhibit a marked increase of contractile response to serotonin (5-hydroxytryptamine [5-HT]) and ergonovine and that these augmented contractile responses to specific agonists may play an important role in the pathogenesis of vasospasm. In the present study, we investigated whether supersensitivity to 5-HT in atherlosclerotic arteries was due to an increase in 5-HT receptor-mediated Ca2+ mobilization or to an increase in Ca2+ sensitivity of the contractile elements. We measured simultaneously both isometric tension and [Ca2+]i in fura 2-loaded aortic smooth muscle strips from control and WHHL rabbits. Muscle tension in the high K+ (72.7 mmol/L)-stimulated states and [Ca2+]i in both resting and high K(+)-stimulated states did not differ between control and WHHL rabbits. In atherosclerotic aortas from WHHL rabbits, the dose-response curves of both tension and [Ca2+]i for 5-HT were shifted to the left at lower threshold concentrations and one-half maximally effective dose. The maximum response of contraction produced by 5-HT in WHHL rabbits was augmented compared with that in control rabbits (123 +/- 17% versus 33 +/- 7% of the 72.7 mmol/L K(+)-induced contraction, P < .001). The maximum response of [Ca2+]i produced by 5-HT was also augmented in WHHL rabbits compared with control rabbits (29 +/- 4% versus 10 +/- 0.9% of the 72.7 mmol/L K(+)-induced [Ca2+]i, P < .001). In contrast, the responses of contraction and [Ca2+]i to phenylephrine were similar between control and WHHL rabbits.(ABSTRACT TRUNCATED AT 250 WORDS)

Transforming growth factor-beta 1 potentiated alpha 1-adrenergic and stretch-induced c-fos mRNA expression in rat myocardial cells.

Since transforming growth factor-beta 1 (TGF-beta 1) has been recently shown to be expressed in the heart by mechanical stretch and ischemic injury, we examined the modulation of c-fos mRNA expression and amino acid uptake by TGF-beta 1 in rat myocardial cells. Pretreatment with TGF-beta 1 potentiated norepinephrine (NE)-induced and stretch-induced (+10% and +20% elongation, for 30 minutes) c-fos mRNA expression by 2.2-fold, whereas TGF-beta 1 alone did not induce c-fos mRNA expression in Northern blot analysis. NE-induced [14C]phenylalanine uptake was also potentiated with TGF-beta 1 pretreatment. The effect of TGF-beta 1 on the NE action was not blocked by propranolol but by prazosin. The protein kinase C activators (12-O-tetradecanoylphorbol 13-acetate [TPA], phorbol 12,13-dibutyrate, and 1-oleyl-2-acetyl-rac-glycerol) induced c-fos mRNA expression, which was also potentiated by TGF-beta 1. Cycloheximide (protein synthesis inhibitor) could not suppress the TGF-beta 1 actions. In nonmuscle cells, TGF-beta 1 modified neither adrenergic nor TPA-induced c-fos mRNA expression. These data suggested that TGF-beta 1 potentiated the c-fos mRNA expression and amino acid incorporation by modification of the alpha 1-adrenergic and stretch-activated protein kinase C pathway. This mechanism did not require protein synthesis.

Activation and Inhibition of Nitric Oxide Synthase from Cultured Bovine Aortic Endothelial Cells by Phospholipids and Arachidonic Acid

Nitric oxide (NO) acts as a novel regulator of cell functions and a signal molecule in the control of vascular tone. NO is generated from the terminal guanidino nitrogen of L-arginine through the action of NO synthase (NOS).’ The particulate NOS has been purified and molecularly cloned from bovine aortic endothelial cells (BAECS).~ Lipids play an important role in modulating functions of the arterial wall, such as endothelium-dependent rela~ation.”~ In the present study, we investigated effects of lipids on constitutive NOS activity purified from BAECs.

Comparison of medium-dose losartan/hydrochlorothiazide and maximal-dose angiotensin II receptor blockers in the treatment of Japanese patients with uncontrolled hypertension: the Kobe-CONNECT Study.

The objective of this study is to examine the effects of thiazide diuretics, plus medium-dose losartan versus maximal-dose angiotensin II receptor blockers (ARBs) on blood pressure (BP) in Japanese patients with uncontrolled hypertension despite the use of medium-dose ARBs. Hypertensive patients in whom BP was inadequately controlled by treatment with medium-dose ARBs alone or with calcium-channel blockers were enrolled. Patients were randomly assigned to a fixed-dose combination of 50 mg per day losartan and 12.5 mg per day hydrochlorothiazide (HCTZ; n=98), or to a maximal dose of current ARBs (n=95). The reduction in office BP from baseline was significantly larger in the losartan/HCTZ group than in the maximal-dose ARB group (systolic BP −22.7±13.7 vs. −11.7±13.0 mm Hg, diastolic BP −9.6±10.9 vs. −4.5±11.0 mm Hg; P<0.01, respectively). The proportion of patients in whom the therapeutic target BP was achieved was greater in the losartan/HCTZ group than in the maximal-dose ARB group (59.2 vs. 26.3%; P<0.001). Both early-morning and evening BP were controlled more effectively over 1 year of treatment in the losartan/HCTZ group than in the maximal-dose ARB group (the mean BP difference between the groups, early-morning: 5.6 mm Hg (P=0.001), evening: 3.8 mm Hg (P=0.049)). Adverse changes in serum potassium and uric acid were observed in the losartan/HCTZ group; however, both changes were very slight, and the values were still within the normal range. The concomitant usage of losartan and HCTZ had no influence on glucose metabolism and lipid profiles. Declines in plasma N-terminal pro-brain natriuretic peptide levels and urinary albumin excretion were observed in the losartan/HCTZ group, but not in the maximal-dose ARB group. Switching from medium-dose ARBs to losartan plus HCTZ reduced both office and home BP efficiently in patients with uncontrolled hypertension.