Classically, nitric oxide (NO) formed by endothelial NO synthase (eNOS) freely diffuses from its generation site to smooth muscle cells where it activates soluble guanylyl cyclase (sGC), producing cGMP. Subsequently, cGMP activates both cGMPand cAMP-dependent protein kinases [cGMP-dependent protein kinase (PKG) and cAMP-dependent protein kinase (PKA), respectively], leading to smooth muscle relaxation. In endothelial cells, eNOS has been localized to caveolae, small invaginations of the plasma membrane rich in cholesterol. Membrane cholesterol depletion impairs acetylcholine (ACh)-induced relaxation due to alteration in caveolar structure. Given the nature of NO to be more soluble in a hydrophobic environment than in water, and assuming that colocalization of components in a signal transduction cascade seems to be a critical determinant of signaling efficiency by eNOS activation, we hypothesize that sGC, PKA, and PKG activation may occur at the plasma membrane caveolae. In endothelium-intact rat aortic rings, the relaxation induced by ACh, by the sGC activator 3-(5Ј-hydroxymethyl-2Јfuryl)-1-benzyl indazole (YC-1), and by 8-bromocGMP was impaired in the presence of methyl-␤-cyclodextrin, a drug that disassembles caveolae by sequestering cholesterol from the membrane. sGC, PKG, and PKA were colocalized with caveolin-1 in aortic endothelium, and this colocalization was abolished by methyl-␤-cyclodextrin. Methyl-␤-cyclodextrin efficiently disassembled caveolae in endothelium. In summary, our results provide evidence of compartmentalization of sGC, PKG, and PKA in endothelial caveolae contributing to NO signaling cascade, giving new insights by which the endothelium mediates vascular smooth muscle relaxation.

Uridine adenosine tetraphosphate (Up(4)A) has been recently reported as an endothelium-derived vasoconstrictor and plasma levels of this dinucleotide are increased in juvenile hypertensive subjects. This study aimed to evaluate the vascular actions of Up(4)A, typify the putative purinergic receptors that might mediate these effects and characterize the intracellular signaling pathways that may govern Up(4)A responses. Up(4)A induced a modest endothelium-dependent relaxation of rat aortic rings contracted with phenylephrine. From baseline, Up(4)A induced concentration-dependent contractions that were significantly potentiated by endothelium removal or nitric oxide synthase inhibition. The contractile response induced by Up(4)A was not tachyphylactic and was significantly reduced in the presence of P1 or P2X receptor antagonists, L-type Ca(2+) channel blocker and Rho-kinase inhibitor. Up(4)A-induced contraction apparently involves superoxide anion formation since it was significantly reduced by treatment with apocynin or tempol. This study presents the unique findings that the endogenous compound Up(4)A is able to induce relaxation in addition to contraction of rat aorta. Up(4)A-induced contraction is modulated by nitric oxide production, mediated by P1 and P2X receptor activation, and involves L-type Ca(2+) channels, Rho-kinase pathway and superoxide formation.

We hypothesized that the 5-hydroxytryptamine (5-HT; serotonin) system is present and functional in veins. In vena cava (VC), the presence of the 5-HT synthesis rate-limiting enzyme tryptophan hydroxylase-1 mRNA and accumulation of the 5-HT synthesis intermediate 5-hydroxytryptophan after incubation with tryptophan supported the ability of veins to synthesize 5-HT. The presence of 5-HT and its metabolite 5-hydroxyindole acetic acid was measured by high-performance liquid chromatography in VC and jugular vein (JV), and it was compared with similarly sized arteries aorta (RA) and carotid (CA), respectively. In rats treated with the monoamine oxidase-A (MAO-A) inhibitor pargyline to prevent 5-HT metabolism, basal 5-HT levels were higher in veins than in arteries. 5-HT uptake was observed after exposure to exogenous 5-HT in all vessels. The presence of MAO-A and the 5-HT transporter (SERT) in VC was observed by immunohistochemistry and Western analysis. However, 5-HT uptake was not inhibited by the SERT inhibitors fluoxetine and/or fluvoxamine in VC and JV, as opposed to the inhibition in RA and CA. Moreover, studies performed in VC from mutant rats lacking SERT showed no differences in 5-HT uptake compared with VC from wild type. These data suggest the SERT is not functional under physiological conditions in veins. The differences in 5-HT handling between veins and arteries may represent alternative avenues for targeting the 5-HT system in the peripheral circulation for controlling vascular tone.5-Hydroxytryptamine (5-HT; serotonin) was first described as a substance that causes contraction of smooth muscle (Rapport et al., 1948;Erspamer and Asero, 1952). The function of 5-HT as a neurotransmitter is well established, as drugs that affect 5-HT concentration [e.g., Prozac (fluoxetine hydrochloride)] are widely used to treat conditions such as depression, anxiety, and obesity. However, its role in the cardiovascular system is far from being elucidated. For the last decade, accumulating evidence supports the involvement of 5-HT in the control of pulmonary circulation under normal and hypertensive conditions. However, a role for 5-HT in systemic vasculature is a matter of debate (for review, see Watts, 2005).In the periphery, platelets represent a large 5-HT storage site, and they may function as a buffer, keeping the free circulating 5-HT in low levels (Nilsson et al., 1985;Vanhoutte, 1991;Brenner et al., 2007). Indeed, platelet 5-HT uptake is decreased with age and in hypertension accompanied by an increase in free 5-HT circulating levels (Amstein et al., 1991;Brenner et al., 2007).5-HT is abundantly synthesized in the enterochromaffin cells of the intestine, representing more than 95% of total body 5-HT. 5-HT is also synthesized in the raphe nuclei of the brain, pineal gland, and in endothelial cells lining the lung. Potential sites of 5-HT synthesis in the systemic vasculature have not yet been identified. 5-HT is synthesized from the essential amino acid tryptophan in a two-step pathway. The hydroxylation o...

1. Our goal was to investigate the body distribution of serotonin (5-hydroxytryptamine; 5-HT) in rats infused with 5-HT (25 microg/kg per min) for 7 days and the contribution of the 5-HT transporter (SERT) for 5-HT uptake into the tissues. 2. Mini-osmotic pumps containing 5-HT or vehicle were implanted in rats knocked out for SERT (SERT-KO) or in wild-type (WT) rats. On the 8th day, tissues were harvested for measurements of 5-HT by high-performance liquid chromatography (HPLC). The 5-HT metabolite 5-hydroxyindole acetic acid (5-HIAA) was also measured by HPLC, because an increase in 5-HIAA in tissues from rats receiving 5-HT reflects 5-HT uptake followed by metabolism. 3. In WT rats infused with 5-HT, an increase in 5-HT or 5-HIAA was observed in the heart, pancreas, thyroid, adrenal gland, kidney, seminal vesicle, bladder, prostate, liver, oesophagus, stomach, femur, trachea, lung and spleen compared with vehicle-infused rats. An increase in 5-HT and 5-HIAA was not observed in aorta, vena cava and jejunum. In tissues from SERT-KO rats infused with 5-HT, the content of 5-HT or 5-HIAA was decreased in most of the tissues studied compared with 5-HT-infused WT rats. Although 5-HT uptake in the kidney, seminal vesicle, prostate, jejunum and trachea is SERT dependent, it is SERT independent in the pancreas. The remaining tissues display SERT-dependent and -independent mechanisms for 5-HT uptake. 4. Altogether, tissues from different systems, such as the cardiovascular, endocrine, genitourinary and gastrointestinal, accumulate 5-HT mainly via SERT and, thus, these systems are potential targets for drugs that interfere with 5-HT homeostasis.

This study tested the hypothesis that nitric oxide (NO) synthase inhibition in mice would result in hypertension characterized by increased agonist-induced vasoconstrictor responsiveness and attenuated endothelium-dependent vasodilation. Administration of N-nitro-L-arginine (L-NNA), an NO synthase inhibitor (1 g/L, 4 weeks), via drinking water to mice resulted in significant elevations in blood pressure. Phenylephrine-induced contraction was significantly increased in aortic rings from L-NNA-treated mice compared with rings from control mice. Aortic rings from control mice showed a concentration-dependent relaxation to acetylcholine whereas those obtained from L-NNA-treated mice showed a biphasic response, contracting at lower concentrations while relaxing at higher concentrations. Aortic rings from L-NNA-treated mice had decreased relaxation to acetylcholine and increased sensitivity to sodium nitroprusside compared with control rings. The relaxation induced by an NO-independent soluble guanylyl cyclase activator was not different between groups. In aortic rings from control and L-NNA-treated mice pre-contracted with phenylephrine, the administration of L-NNA to the organ bath caused additional and sustained contraction. When compared with the contraction induced by phenylephrine, L-NNA-induced contraction in aorta from control mice was significantly higher than that in aorta from L-NNA-treated mice. We conclude that mice treated with L-NNA develop hypertension and that a reduction in NO availability is responsible for the changes observed in vascular reactivity.