In mammals, natural-resistance-associated macrophage protein 1 (Nramp1) regulates macrophage activation and is associated with infectious and autoimmune diseases. Nramp2 is associated with anaemia. Both belong to a highly conserved eukaryote/prokaryote protein family. We used Xenopus oocytes to demonstrate that, like Nramp2, Nramp1 is a bivalent cation (Fe2+, Zn2+ and Mn2+) transporter. Strikingly, however, where Nramp2 is a symporter of H+ and metal ions, Nramp1 is a highly pH-dependent antiporter that fluxes metal ions in either direction against a proton gradient. At pH 9.0, oocytes injected with cRNA from wild-type murine Nramp1 with a glycine residue at position 169 (Nramp1(G169); P=3.22x10(-6)) and human NRAMP1 (P=3.87x10(-5)) showed significantly enhanced uptake of radiolabelled Zn2+ compared with water-injected controls. At pH 5.5, Nramp1(G169) (P=1.34x10(-13)) and NRAMP1 (P=1.09x10(-6)) oocytes showed significant efflux of Zn2+. Zn2+ transport was abolished when the proton gradient was dissipated using carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Using pre-acidified oocytes, currents of 130+/-57 nA were evoked by 100 microM Zn2+ at pH 7.5, and 139+/-47 nA by 100 microM Fe2+ at pH 7.0, in Nramp1(G169) oocytes; currents of 254+/-49 nA and 242+/-26 nA were evoked, respectively, in NRAMP1 oocytes. Steady-state currents evoked by increasing concentrations of Zn2+ were saturable, with apparent affinity constants of approx. 614 nM for Nramp1(G169) and approx. 562 nM for NRAMP1 oocytes, and a curvilinear voltage dependence of transporter activity (i.e. the data points approximate to a curve that approaches a linear asymptote). In the present study we propose a new model for metal ion homoeostasis in macrophages. Under normal physiological conditions, Nramp2, localized to early endosomal membranes, delivers extracellularly acquired bivalent cations into the cytosol. Nramp1, localized to late endosomal/lysosomal membranes, delivers bivalent cations from the cytosol into this acidic compartment where they may directly affect antimicrobial activity.

Objective Red wine polyphenols have been reported to possess beneficial properties for preventing cardiovascular diseases but their effects on hemodynamic and functional cardiovascular changes during inhibition of nitric oxide (NO) synthesis have not been elucidated. Design The effects of the red wine polyphenols, Provinols, on arterial hypertension as well as left ventricular (LV) hypertrophy, myocardial fibrosis and vascular remodeling were investigated in rats during chronic inhibition of nitric oxide synthase (NOS) activity. Rats were divided into four groups: a control group, a group treated with N^G-nitro-l-arginine methyl ester (L-NAME) (40 mg/kg per day), a group receiving Provinols (40 mg/kg per day) alone or Provinols plus L-NAME. Results Provinols markedly reduced the increase in both blood pressure and protein synthesis in the heart and aorta caused by chronic inhibition of NO synthesis. Provinols reduced myocardial fibrosis even though it did not affect LV hypertrophy. In addition, Provinols prevented aortic thickening and corrected the augmented reactivity of the aorta to norepinephrine and the attenuated endothelium-dependent relaxation to acetylcholine in NO-deficient rats. These alterations were associated with an increase of NOS activity, a moderate enhancement of endothelial NOS expression and a reduction of oxidative stress in the LV and aorta. Conclusion Our results provide evidence that Provinols partially prevents L-NAME-induced hypertension, cardiovascular remodeling and vascular dysfunction via the increase of NO-synthase activity and prevention of oxidative stress. Thus, the beneficial effects of plant polyphenols in prevention of hypertension may result from their complex influence on the NO balance in the cardiovascular system.

This study used an inexpensive and versatile environmental exposure system to test the hypothesis that hypoxia promoted free radical production in primary cultures of rat main pulmonary artery smooth muscle cells (PASMCs). Production of reactive species was detected by fluorescence microscopy with the probe 2′,7′-dichlorodihydrofluorescein, which is converted to the fluorescent dichlorofluorescein (DCF) in the presence of various oxidants. Flushing the airspace above the PASMC cultures with normoxic gas (20% O₂, 75% N₂, and 5% CO₂) resulted in stable Po₂values of ∼150 Torr, whereas perfusion of the airspace with hypoxic gas (0% O₂, 95% N₂, and 5% CO₂) was associated with a reduction in Po₂values to stable levels of ∼25 Torr. Hypoxic PASMCs became increasingly fluorescent at ∼500% above the normoxic baseline after 60 min. Hypoxia-induced DCF fluorescence was attenuated by the addition of the antioxidants dimethylthiourea and catalase. These findings show that PASMCs acutely exposed to hypoxia exhibit a marked increase in intracellular DCF fluorescence, suggestive of reactive oxygen or nitrogen species production.

Activation of Ca²⁺entry is known to produce endothelial cell shape change, leading to increased permeability, leukocyte migration, and initiation of angiogenesis in conduit-vessel endothelial cells. The mode of Ca²⁺entry regulating cell shape is unknown. We hypothesized that activation of store-operated Ca²⁺channels (SOCs) is sufficient to promote cell shape change necessary for these processes. SOC activation in rat pulmonary arterial endothelial cells increased free cytosolic Ca²⁺that was dependent on a membrane current having a net inward component of 5.45 ± 0.90 pA/pF at −80 mV. Changes in endothelial cell shape accompanied SOC activation and were dependent on Ca²⁺entry-induced reconfiguration of peripheral (cortical) filamentous actin (F-actin). Because the identity of pulmonary endothelial SOCs is unknown, but mammalian homologues of the Drosophila melanogaster transient receptor potential ( trp) gene have been proposed to form Ca²⁺entry channels in nonexcitable cells, we performed RT-PCR using Trp oligonucleotide primers in both rat and human pulmonary arterial endothelial cells. Both cell types were found to express Trp1, but neither expressed Trp3 nor Trp6. Our study indicates that 1) Ca²⁺entry in pulmonary endothelial cells through SOCs produces cell shape change that is dependent on site-specific rearrangement of the microfilamentous cytoskeleton and 2) Trp1 may be a component of pulmonary endothelial SOCs.

Background: Increase of blood pressure is accompanied by functional and morphological changes in the vascular wall. The presented study explored the effects of curcuma and black pepper compounds on increased blood pressure and remodeling of aorta in the rat model of experimental NO-deficient hypertension. Methods: Wistar rats were administered for 6 weeks clear water or L-NAME (40 mg/kg/day) dissolved in water, piperine (20 mg/kg/day), curcumin (100 mg/kg/day) or their combination in corn oil by oral gavage. The systolic blood pressure was measured weekly. Histological slices of thoracic aorta were stained with hematoxylin and eosin, Mallory's phosphotungstic acid hematoxylin (PTAH), orcein, picrosirius red and van Gieson staining and with antibodies against smooth muscle cells actin. Microscopic pictures were digitally processed and morphometrically evaluated. Results: The increase of blood pressure caused by L-NAME was partially prevented by piperine and curcumin, but the effect of their combination was less significant. Animals with hypertension had increased wall thickness and cross-sectional area of the aorta, accompanied by relative increase of PTAH positive myofibrils and decrease of elastin, collagen and actin content. Piperine was able to decrease the content of myofibrils and slightly increase actin, while curcumin also prevented elastin decrease. The combination of spices had similar effects on aortic morphology as curcumin itself. Conclusions: Administration of piperine or curcumin, less their combination, is able to partially prevent the increase of blood pressure caused by chronic L-NAME administration. The spices modify the remodeling of the wall of the aorta induced by hypertension. Our results show that independent administration of curcumin is more effective in preventing negative changes in blood vessel morphology accompanying hypertensive disease.