Hypoxia inhibits adenylyl cyclase catalytic activity in a porcine model of persistent pulmonary hypertension of the newborn.

Persistent pulmonary hypertension of the newborn (PPHN) features hypoxemia, pulmonary vasoconstriction, and impaired cardiac inotropy. We previously reported low basal and stimulated cAMP in hypoxic pulmonary artery smooth muscle cells (PASMCs). We now examine pulmonary arterial adenylyl cyclase (AC) activity and regulation in hypoxic PPHN. PPHN was induced in newborn swine by normobaric hypoxia (fraction of inspired oxygen 0.10) for 72 h and compared with age-matched normoxic controls. We studied relaxation of pulmonary arterial (PA) rings to AC activator forskolin and cGMP activator sodium nitroprusside (SNP) by isometric myography, ATP content, phosphodiesterase activity, AC content, isoform expression, and catalytic activity in presence or absence of Gαs-coupled receptor agonists, forskolin, or transnitrosylating agents in human and neonatal porcine PASMCs and HEK293T stably expressing AC isoform 6, after 72 h hypoxia (10% O2) or normoxia (21% O2). Relaxation to forskolin and SNP were equally impaired in PPHN PA. AC-specific activity decreased in hypoxia. PASMC from PPHN swine had reduced AC activity despite exposure to normoxia in culture; transient hypoxia in vitro further decreased AC activity. Prostacyclin receptor ligand affinity decreased, but its association with Gαs increased in hypoxia. Total AC content was unchanged by hypoxia, but AC6 increased in hypoxic cells and PPHN pulmonary arteries. Impairment of AC6 activity in hypoxia was associated with nitrosylation. PPHN PA relaxation is impaired because of loss of AC activity. Hypoxic AC is inhibited because of S-nitrosylation; inhibition persists after removal from hypoxia. Downregulation of AC-mediated relaxation in hypoxic PA has implications for utility of Gαs-coupled receptor agonists in PPHN treatment.

Thromboxane receptor hyper-responsiveness in hypoxic pulmonary hypertension requires serine 324.

BACKGROUND AND PURPOSE: Dysregulation of the thromboxane A2 (TP) receptor, resulting in agonist hypersensitivity and hyper-responsiveness, contributes to exaggerated vasoconstriction in the hypoxic pulmonary artery in neonatal persistent pulmonary hypertension. We previously reported that hypoxia inhibits TP receptor phosphorylation, causing desensitization. Hence, we examined the role of PKA-accessible serine residues in determining TP receptor affinity, using site-directed mutational analysis. EXPERIMENTAL APPROACH: Vasoconstriction to a thromboxane mimetic and phosphorylation of TP receptor serine was examined in pulmonary arteries from neonatal swine with persistent pulmonary hypertension and controls. Effects of hypoxia were determined in porcine and human TP receptors. Human TPα serines at positions 324, 329 and 331 (C-terminal tail) were mutated to alanine and transiently expressed in HEK293T cells. Saturation binding and displacement kinetics of a TP antagonist and agonist were determined in porcine TP, wild-type human TPα and all TP mutants. Agonist-elicited calcium mobilization was determined for each TP mutant, in the presence of a PKA activator or inhibitor, and in hypoxic and normoxic conditions. KEY RESULTS: The Ser324A mutant was insensitive to PKA activation and hypoxia, had a high affinity for agonist and increased agonist-induced calcium mobilization. Ser329A was no different from wild-type TP receptors. Ser331A was insensitive to hypoxia and PKA with a decreased agonist-mediated response. CONCLUSIONS AND IMPLICATIONS: In hypoxic pulmonary hypertension, loss of site-specific phosphorylation of the TP receptor causes agonist hyper-responsiveness. Ser324 is the primary residue phosphorylated by PKA, which regulates TP receptor-agonist interactions. Ser331 mutation confers loss of TP receptor-agonist interaction, regardless of PKA activity.

Milrinone attenuates thromboxane receptor-mediated hyperresponsiveness in hypoxic pulmonary arterial myocytes.

BACKGROUND AND PURPOSE: Neonatal pulmonary hypertension (PPHN) is characterized by pulmonary vasoconstriction, due in part to dysregulation of the thromboxane prostanoid (TP) receptor. Hypoxia induces TP receptor-mediated hyperresponsiveness, whereas serine phosphorylation mediates desensitization of TP receptors. We hypothesized that prostacyclin (IP) receptor activity induces TP receptor phosphorylation and decreases ligand affinity; that TP receptor sensitization in hypoxic myocytes is due to IP receptor inactivation; and that this would be reversible by the cAMP-specific phosphodiesterase inhibitor milrinone. EXPERIMENTAL APPROACH: We examined functional regulation of TP receptors by serine phosphorylation and effects of IP receptor stimulation and protein kinase A (PKA) activity on TP receptor sensitivity in myocytes from neonatal porcine resistance pulmonary arteries after 72 h hypoxia in vitro. Ca(2+) response curves to U46619 (TP receptor agonist) were determined in hypoxic and normoxic myocytes incubated with or without iloprost (IP receptor agonist), forskolin (adenylyl cyclase activator), H8 (PKA inhibitor) or milrinone. TP and IP receptor saturation binding kinetics were measured in presence of iloprost or 8-bromo-cAMP. KEY RESULTS: Ligand affinity for TP receptors was normalized in vitro by IP receptor signalling intermediates. However, IP receptor affinity was compromised in hypoxic myocytes, decreasing cAMP production. Milrinone normalized TP receptor sensitivity in hypoxic myocytes by restoring PKA-mediated regulatory TP receptor phosphorylation. CONCLUSIONS AND IMPLICATIONS: TP receptor sensitivity and EC(50) for TP receptor agonists was regulated by PKA, as TP receptor serine phosphorylation by PKA down-regulated Ca(2+) mobilization. Hypoxia decreased IP receptor activity and cAMP generation, inducing TP receptor hyperresponsiveness, which was reversed by milrinone.

Dopaminergic regulation of glucose-induced insulin secretion through dopamine D2 receptors in the pancreatic islets in vitro.

The stimulatory effect of dopamine through dopamine D2 receptor on glucose-induced insulin secretion was studied in the pancreatic islets in vitro. Dopamine significantly stimulated insulin secretion at a concentration of 10-8 M in the presence of high glucose (20 mM). The higher concentrations of dopamine (10(-7)-10(-4)) inhibited glucose-induced insulin secretion in the presence of both 4 mM and 20 mM glucose. Stimulatory and inhibitory effect of dopamine on glucose-induced insulin secretion was reverted by the addition of dopamine D2 receptor antagonists such as butaclamol and sulpiride. Norepinephrine (NE) at 10(-4) M concentration inhibited the dopamine uptake as well as its stimulatory effect at 10(-8) M concentration on glucose induced insulin secretion. Our results suggest that dopamine exerts a differential effect on glucose-induced insulin secretion through dopamine D2 receptor and it is essential for the regulation of glucose-induced insulin secretion by pancreatic islets.

Potent suppressive effect of green tea polyphenols on tobacco-induced mutagenicity.

Antimutagenic activity of green tea (Camellia sinensis) was studied using Salmonella typhimurium strains (TA 102) (Ames test). Aqueous tobacco extract was found to be mutagenic to S. typhimurium TA 102 at concentration of 50 mg/plate. Green tea polyphenols was found to inhibit the mutagenicity of tobacco in a concentration-dependent manner. Concentrations needed for 50% inhibition of mutagen-induced revertant formation was found to be 5 mg/plate. Green tea polyphenols was also found to inhibit the urinary mutagenicity in rats induced by tobacco extract. Moreover green tea polyphenols were found to inhibit in vitro nitrosation reaction produced by reaction sodium nitrite and methyl urea and further inhibition of mutagenicity indicating that green tea has dual action to bring out a reduction in the mutagenic and carcinogenic potential of tobacco.