Introductory ParagraphThe coupling of hemoglobin sensing of physiological oxygen gradients to stimulation of nitric oxide (NO) bioactivity is an established principle of hypoxic blood flow. One mechanism proposed to explain this O 2 sensing/NO bioactivity linkage postulates an essential role for the conserved hemoglobin β93Cys residue and, specifically, for S-nitrosation of β93Cys to form S-nitrosohemoglobin (SNO-Hb) 1 . The SNO-Hb hypothesis, which conceptually linked hemoglobin and NO biology, has been debated intensely in recent years 2,3 . This debate has precluded a consensus on physiological mechanisms and on assessment of the potential role of SNO-Hb in pathology. Here we describe novel mouse models that express exclusively either human wild type hemoglobin or human hemoglobin in which the β93cys residue is replaced with alanine to assess the role of SNO-Hb in red cell mediated hypoxic vasodilation. Substitution of this residue, precluding hemoglobin S-nitrosation, did not change total red cell S-nitrosothiol levels but shifted S-nitrosothiol distribution to lower MWt species, consistent with the loss of SNO-Hb. Loss of β93cys resulted in no deficits in systemic nor pulmonary hemodynamics under basal conditions and, importantly, did not affect isolated red cell dependent hypoxic vasodilation. These results demonstrate that SNO-Hb is not essential for the physiologic coupling of erythrocyte deoxygenation with increased NO-bioactivity in vivo. *Co corresponding Authors: Rakesh P Patel, PhD, Department of Pathology, University of Alabama at Birmingham, 901 19 th street south, BMR 2, room 302, Birmingham, AL 35294, E mail: E-mail: rakeshp@uab.edu. Tim M Townes, PhD, Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Kaul Human Genetics Bldg, room 502, 720 20 th street south, Birmingham, AL 35294, E mail: E-mail: ttownes@uab.edu. # these authors contributed equally to this work Author Contributions TSI, CWS, LCW, XT, DAV and KMP were responsible for performing experiments. TSI, CWS, DAV, RPP and TMT were responsible for planning all experiments, analyzing data and writing manuscript. MBR contributed to mass spectrometry assays, LS was responsible for exercise related studies, CGK and BGB for capillary density measurements, NP and JW contributed to blood pressure measurements and NA for assessment of pulmonary hemodynamics. JR did the ES cell injections to generate the chimeras. In addition to hemoglobin oxygen affinity, blood flow is a key component of the processes that match oxygen delivery to demand. Increased blood flow in response to hypoxia is a critical physiological response which does not correlate with dissolved oxygen tensions but does correlate with hemoglobin oxygen fractional saturation 4 . These observations have led to the concept that the red blood cell (RBC) itself is a regulator of flow and to the general paradigm that RBC/hemoglobin deoxygenation is coupled to the stimulation of vasodilation 1,5,6 . Three mechanisms for this coupling have been proposed (...

Proanthocyanidin rich plant extracts derived from grape seed extract (GSE), hawthorn and cranberry are on markets for their preventive effects against cardiovascular diseases and uroinfections in woman. However, the importance of these health beneficial effects of these botanicals remains elusive due to incomplete understanding of uptake, metabolism and bioavailability of proanthocyanidins in vivo. In the present study rats were given GSE orally (300 mg/kg, twice a day) and blood and urine were collected over a 24 h period. Monomeric catechins and their methylated metabolites, and proanthocyanidins up to trimers were detected in blood samples treated with GSE using LC-MS/MS operating in the multiple reaction monitoring (MRM) mode. A new tetramethylated metabolite of dimeric proanthocyanidin (m/z 633) in GSE-treated urine was tentatively identified. Using LC-MS/MS, (+)-catechin and (−)-epicatechin were identified in the brain conclusively. These data suggested that GSE catechins cross the blood brain barrier and may be responsible for the neuroprotective effects of GSE.

The authors wish to add the following information. Table 1: The doses of antagonists used to treat wild-type and RGS2-knockout mice were hexamethonium at 5 mg/kg i.v., prazosin at 200 µg/kg i.v., and candesartan at 100 µg/kg i.v. Effective ganglionic blockade by hexamethonium was established as described previously, whereas blockade by prazosin or candesartan was demonstrated by the inability of a subsequent infusion of phenylephrine (10 µg/kg i.v.) or angiotensin II (1 µg/kg i.v.) to increase blood pressure of wild-type or RGS2-knockout mice. Figure 3:Blood pressure responses were determined using anesthetized mice as described in Methods. In Figure 3a, the doses of vasoconstrictors used to treat wild-type and RGS2-knockout mice were 1 µg/kg i.v. for angiotensin II and 10 µg/kg i.v. for phenylephrine. These doses were determined empirically, as were those that elicited a near-maximal (>75%) increase in blood pressure (determined by dose-response experiments such as those shown in Figure 4a for phenylephrine). In Figure 3b, candesartan (100 µg/kg i.v.) was infused into wild-type and RGS2-knockout mice over a period of 10 seconds, after which blood pressure was recorded continuously over the time period indicated. In Figure 3c, the same dose of angiotensin II (1 µg/kg iv) was used to treat wild-type and RGS2-knockout mice in order to increase systolic blood pressure to similar absolute levels (160-170 mmHg) prior to antagonist infusion (candesartan, 100 µg/kg i.v.). This approach was established by the results shown in Figure 3a, in which MAP of wild-type mice increased from a resting value (prior to agonist infusion) of ∼85 mmHg to a value of ∼135 mmHg after angiotensin II infusion, and the MAP of RGS2-knockout mice increased from a resting value of ∼135 mmHg to ∼140 mmHg by the same treatment. After a maximal effect of angiotensin II on blood pressure was achieved (∼1 minute), candesartan (100 µg/kg i.v.) was infused over 10 seconds, and decreases in blood pressure were recorded continuously over the time period indicated.

Signaling by hormones and neurotransmitters that activate G protein–coupled receptors (GPCRs) maintains blood pressure within the normal range despite large changes in cardiac output that can occur within seconds. This implies that blood pressure regulation requires precise kinetic control of GPCR signaling. To test this hypothesis, we analyzed mice deficient in RGS2, a GTPase-activating protein that greatly accelerates the deactivation rate of heterotrimeric G proteins in vitro. Both rgs2+/– and rgs2–/– mice exhibited a strong hypertensive phenotype, renovascular abnormalities, persistent constriction of the resistance vasculature, and prolonged response of the vasculature to vasoconstrictors in vivo. Analysis of P2Y receptor–mediated Ca2+ signaling in vascular smooth muscle cells in vitro indicated that loss of RGS2 increased agonist potency and efficacy and slowed the kinetics of signal termination. These results establish that abnormally prolonged signaling by G protein–coupled vasoconstrictor receptors can contribute to the onset of hypertension, and they suggest that genetic defects affecting the function or expression of RGS2 may be novel risk factors for development of hypertension in humans

Premature termination codons (PTCs) prevent translation of a full-length protein and trigger nonsense-mediated mRNA decay (NMD). Nonsense suppression (also termed readthrough) therapy restores protein function by selectively suppressing translation termination at PTCs. Poor efficacy of current readthrough agents prompted us to search for better compounds. An NMD-sensitive NanoLuc readthrough reporter was used to screen 771,345 compounds. Among the 180 compounds identified with readthrough activity, SRI-37240 and its more potent derivative SRI-41315, induce a prolonged pause at stop codons and suppress PTCs associated with cystic fibrosis in immortalized and primary human bronchial epithelial cells, restoring CFTR expression and function. SRI-41315 suppresses PTCs by reducing the abundance of the termination factor eRF1. SRI-41315 also potentiates aminoglycoside-mediated readthrough, leading to synergistic increases in CFTR activity. Combining readthrough agents that target distinct components of the translation machinery is a promising treatment strategy for diseases caused by PTCs.

The incidence of type 2 diabetes and metabolic disease is rapidly increasing, but effective therapies for their prevention and treatment have been poorly tolerated or minimally effective. In this study, chronic administration of kudzu root extract (8 months, 0.2% w/w in diet) decreased baseline fasting plasma glucose (183±14 vs 148±11 mg/dl) and improved glucose and insulin tolerance in C57BL/6J ob/ob mice (1.67±0.17 ng/ml [kudzu treated] vs. 2.35±0.63 ng/ml [control]), but such treatment did not alter these parameters in lean control mice. Among the mice on the kudzu supplementation, plasma levels of isoflavone metabolites were significantly higher in ob/ob versus lean control mice, and unmetabolized puerarin (11.50±5.63 ng/gram) was found in adipose tissue only in the treated mice. Together, these data demonstrate that a puerarin containing kudzu diet improves glucose and insulin responsiveness in ob/ob mice, suggesting that puerarin may be a beneficial adjuvant for treating metabolic disease.

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Both endogenous and dietary estrogens reduce hypertension and enhance cognitive abilities in estrogen-depleted female spontaneously hypertensive rats (SHR). Many of the beneficial effects of estrogens/phytoestrogens also appear to be provided by other polyphenols (e.g., proanthocyanidins) in grape seed, which lack appreciable estrogenic receptor binding. The present study tested the hypothesis that similar to phytoestrogens, proanthrocyanidins in grape seed polyphenols reduce salt-sensitive hypertension in young, estrogen-depleted SHR. SHR were ovariectomized at 4 wk of age and placed on phytoestrogen-free diets with or without 0.5% grape seed extract added and with high (8.0%) or basal (0.6%) NaCl. After 10 wk on the diets, grape proanthrocyanidin supplementation significantly reduced arterial pressure in the rats fed the basal (10 mmHg) and high (26 mmHg)-NaCl diet, compared with the nonsupplemented controls. In vitro superoxide production was significantly reduced (23%) by the grape seed polyphenols. Spatial learning (8-arm-radial maze) in the SHR on the basal NaCl diets was improved by dietary grape seed polyphenols. These results indicate that grape seed polyphenols decrease arterial pressure in SHR, probably via an antioxidant mechanism.