Pulmonary arterial 5-hydroxytryptamine (serotonin) (5-HT) transporter (SERT)-, 5-HT receptor expression, and 5-HTinduced vasoconstriction can be increased in pulmonary hypertension. These variables were studied in normoxic and hypoxic Fawn-Hooded (FH) and Sprague-Dawley (SD) rats. Furthermore, we compared the functional effects of SERT inhibitors and 5-HT receptor antagonists against 5-HTinduced vasoconstriction of pulmonary arteries. SERT and 5-HT 1B expression was greater in FH rat lungs than in SD rats, as was 5-HT-mediated vasoconstriction. However, these inhibitors potentiated responses to 5-HT in FH vessels. After exposure of rats to 2 weeks of hypoxia, there was increased 5-HTmediated vasoconstriction and a profound decrease in SERT expression in both the FH and SD rat lung. Accordingly, citalopram had no effect on 5-HT-induced constriction in SD rat vessels and markedly less effect in FH rat vessels. Ketanserin, SB224289, and LY393558 inhibited responses to 5-HT in all hypoxic rat vessels. LY393558 was the most potent antagonist, and there was synergy between the effects of fluoxetine and SB224289 when given simultaneously. The results suggest that, in FH rats, SERT inhibitors may increase pulmonary vasoconstriction, but this can be inhibited by simultaneous 5-HT 1B receptor antagonism. There is synergy between the inhibitory effects of 5-HT 1B receptor antagonists and SERT inhibitors on 5-HT-induced pulmonary vasoconstriction.Pulmonary arterial hypertension (PAH) is characterized by sustained elevation in pulmonary artery pressure. Familial PAH can be related to heterozygous germline mutations in the gene encoding the bone morphogenetic protein type II receptor and/or polymorphisms in the gene encoding the 5-hydroxytryptamine (serotonin) (5-HT) transporter (SERT) (Lane et al., 2000;Eddahibi et al., 2001). Idiopathic PAH has no demonstrable cause and PAH can also occur secondary to many cardiorespiratory disorders. Regardless of the type of PAH, the elevated pulmonary vascular resistance is associated with remodeling of muscular pulmonary arteries and arterioles that exhibit smooth muscle proliferation, medial hypertrophy, and fibrosis (Fishman, 1998).SERT mRNA is elevated in platelets from patients with PAH (Eddahibi et al., 2001). A polymorphism with long and short forms (Lesch et al., 1996) affects SERT function with the long allele inducing an increased rate of SERT gene transcription. The SERT polymorphism can also predict the severity of PAH in patients with chronic obstructive pulmonary disease (Eddahibi et al., 2003). Hence, it has been hypothesized that inhibitors of SERT may be useful in the treatment of PAH. One mechanism of action of SERT inhibitors treating clinical depression, however, is to cause an extracellular accumulation of 5-HT and increased 5-HT receptor activation (Slattery et al., 2004). The possibility that Article, publication date, and citation information can be found at
Background-Increased serotonin (5-hydroxytryptamine, 5-HT) transporter activity has been observed in human familial pulmonary hypertension. Methods and Results-We investigated pulmonary hemodynamics and the development of hypoxia-induced pulmonary hypertension and pulmonary vascular remodeling in mice overexpressing the gene for the 5-HT transporter (5-HTTϩ mice). Right ventricular pressure was elevated 3-fold in normoxic 5-HTTϩ mice compared with their wild-type controls. Hypoxia-induced increases in right ventricular hypertrophy and pulmonary vascular remodeling were also potentiated in the 5-HTTϩ mice. 5-HTT-like immunoreactivity, protein, and binding sites were markedly increased in the lungs from the 5-HTTϩ mice. Hypoxia, however, decreased 5-HT transporter immunoreactivity, mRNA transcription, protein, and binding sites in both wild-type and 5-HTTϩ mice. Conclusions-Increased
The antagonist ligand BODIPY-FL-prazosin (QAPB) fluoresces when bound to bovine ␣ 1a -adrenoceptors (ARs). Data indicate that the receptor-ligand complex is spontaneously internalized by -arrestin-dependent endocytosis. Internalization of the ligand did not occur in -arrestin-deficient cells; was blocked or reversed by another ␣ 1 ligand, phentolamine, indicating it to reflect binding to the orthosteric recognition site; and was prevented by blocking clathrin-mediated endocytosis. The ligand showed rapid, diffuse, low-intensity, surface binding, superseded by punctate intracellular binding that developed to equilibrium in 50 to 60 min and was reversible on ligand removal, indicating a dynamic equilibrium. In cells expressing a human ␣ 1a -AR-enhanced green fluorescent protein (EGFP) 2 fusion protein, BODIPY-R-558/568-prazosin (RQAPB) colocalized with the fusion, indicating that the ligand gained access to all compartments containing the receptor, and, conversely, that the receptor has affinity for the ligand at all of these sites. The distribution of QAPB binding sites was similar for receptors with or without EGFP2, validating the fusion protein as an indicator of receptor location. The ligand partially colocalized with -arrestin in recycling and late endosomes, indicating receptor transit without destruction. Organelles containing receptors showed considerable movement consistent with a transportation function. This was absent in -arrestin-deficient cells, indicating that both constitutive receptor internalization and subsequent intracellular transportation are -arrestin-dependent. Calculations of relative receptor number indicate that at steady state, less than 30% of receptors reside on the cell surface and that recycling is rapid. We conclude that ␣ 1a -ARs recycle rapidly by an agonist-independent, constitutive, -arrestin-dependent process and that this can transport "␣-blockers" into cells carrying these receptors.
The basis of the selectivity of fluorochromes routinely used to visualize the endoplasmic reticulum (ER) in live cells remains obscure. To clarify this, interactions of living cells with fluorochromes of varied physicochemical properties were analyzed experimentally and numerically using a quantitative structure activity relationship analysis (QSAR). Routine selective ER probes were found to be amphipathic, lipophilic cations with moderate-sized conjugated systems. The moderately lipophilic character permits probe uptake by passive diffusion without nonspecific accumulation in biomembranes. The moderately amphipathic character favors uptake into the ER, perhaps owing to its high concentration of zwitterionic lipid head-groups. The QSAR model rationalizes the impractical character of some ER probes mentioned in the literature, and could permit design of novel ER probes with different emission colors. The possibility of using the QSAR model as a tool to predict the accumulation of xenobiotics in the ER of living cells is illustrated by the localization of certain antipsychotic drugs in cultured cells.
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