Heme oxygenase 2: endothelial and neuronal localization and role in endothelium-dependent relaxation.

Heme oxygenase 2 (HO-2), which synthesizes carbon monoxide (CO), has been localized by immunohistochemistry to endothelial cells and adventitial nerves of blood vessels. HO-2 is also localized to neurons in autonomic ganglia, including the petrosal, superior cervical, and nodose ganglia, as well as ganglia in the myenteric plexus of the intestine. Enzyme studies demonstrated that tin protoporphyrin-9 is a selective inhibitor of HO with approximately 10-fold selectivity for HO over endothelial nitric oxide synthase (NOS) and soluble guanylyl cyclase. Inhibition of HO activity by tin protoporphyrin 9 reverses the component of endothelial-derived relaxation of porcine distal pulmonary arteries not reversed by an inhibitor of NOS. Thus, CO, like NO, may have endothelial-derived relaxing activity. The similarity of NOS and HO-2 localizations and functions in blood vessels and the autonomic nervous system implies complementary and possibly coordinated physiologic roles for these two mediators.

Endothelin ETA receptor blockade restores NO-mediated endothelial function and inhibits atherosclerosis in apolipoprotein E-deficient mice

This study investigated whether endothelin-1 (ET-1), a potent vasoconstrictor, which also stimulates cell proliferation, contributes to endothelial dysfunction and atherosclerosis. Apolipoprotein E (apoE)-deficient mice and C57BL/6 control mice were treated with a Western-type diet to accelerate atherosclerosis with or without ETA receptor antagonist LU135252 (50 mg/kg/d) for 30 wk. Systolic blood pressure, plasma lipid profile, and plasma nitrate levels were determined. In the aorta, NO-mediated endothelium-dependent relaxation, atheroma formation, ET receptor-binding capacity, and vascular ET-1 protein content were assessed. In apoE-deficient but not C57BL/6 mice, severe atherosclerosis developed within 30 wk. Aortic ET-1 protein content (P < 0.0001) and binding capacity for ETA receptors was increased as compared with C57BL/6 mice. In contrast, NO-mediated, endothelium-dependent relaxation to acetylcholine (56 ± 3 vs. 99 ± 2%, P < 0.0001) and plasma nitrate were reduced (57.9 ± 4 vs. 93 ± 10 μmol/liter, P < 0.01). Treatment with the ETA receptor antagonist LU135252 for 30 wk had no effect on the lipid profile or systolic blood pressure in apoE-deficient mice, but increased NO-mediated endothelium-dependent relaxation (from 56 ± 3 to 93 ± 2%, P < 0.0001 vs. untreated) as well as circulating nitrate levels (from 57.9 ± 4 to 80 ± 8.3 μmol/liter, P < 0.05). Chronic ETA receptor blockade reduced elevated tissue ET-1 levels comparable with those found in C57BL/6 mice and inhibited atherosclerosis in the aorta by 31% without affecting plaque morphology or ET receptor-binding capacity. Thus, chronic ETA receptor blockade normalizes NO-mediated endothelial dysfunction and reduces atheroma formation independent of plasma cholesterol and blood pressure in a mouse model of human atherosclerosis. ETA receptor blockade may have therapeutic potential in patients with atherosclerosis.

Down-regulation of the expression of endothelial NO synthase is likely to contribute to glucocorticoid-mediated hypertension

Hypertension is a side effect of systemically administered glucocorticoids, but the underlying molecular mechanism remains poorly understood. Ingestion of dexamethasone by rats telemetrically instrumented increased blood pressure progressively over 7 days. Plasma concentrations of Na+ and K+ and urinary Na+ and K+ excretion remained constant, excluding a mineralocorticoid-mediated mechanism. Plasma NO2−/NO3− (the oxidation products of NO) decreased to 40%, and the expression of endothelial NO synthase (NOS III) was found down-regulated in the aorta and several other tissues of glucocorticoid-treated rats. The vasodilator response of resistance arterioles was tested by intravital microscopy in the mouse dorsal skinfold chamber model. Dexamethasone treatment significantly attenuated the relaxation to the endothelium-dependent vasodilator acetylcholine, but not to the endothelium-independent vasodilator S-nitroso-N-acetyl-d,l-penicillamine. Incubation of human umbilical vein endothelial cells, EA.hy 926 cells, or bovine aortic endothelial cells with several glucocorticoids reduced NOS III mRNA and protein expression to 60–70% of control, an effect that was prevented by the glucocorticoid receptor antagonist mifepristone. Glucocorticoids decreased NOS III mRNA stability and reduced the activity of the human NOS III promoter (3.5 kilobases) to ≈70% by decreasing the binding activity of the essential transcription factor GATA. The expressional down-regulation of endothelial NOS III may contribute to the hypertension caused by glucocorticoids.

Circulating plasma xanthine oxidase contributes to vascular dysfunction in hypercholesterolemic rabbits.

Reactive oxygen species play a central role in vascular inflammation and atherogenesis, with enhanced superoxide (O2.-) production contributing significantly to impairment of nitric oxide (.NO)-dependent relaxation of vessels from cholesterol-fed rabbits. We investigated potential sources of O2.- production, which contribute to this loss of endothelium-dependent vascular responses. The vasorelaxation elicited by acetylcholine (ACh) in phenylephrine-contracted, aortic ring segments was impaired by cholesterol feeding. Pretreatment of aortic vessels with either heparin, which competes with xanthine oxidase (XO) for binding to sulfated glycosaminoglycans, or the XO inhibitor allopurinol resulted in a partial restoration (36-40% at 1 muM ACh) of ACh-dependent relaxation. Furthermore, O2.(-)-dependent lucigenin chemiluminescence, measured in intact ring segments from hypercholesterolemic rabbits, was decreased by addition of heparin, allopurinol or a chimeric, heparin-binding superoxide dismutase. XO activity was elevated more than two-fold in plasma of hypercholesterolemic rabbits. Incubation of vascular rings from rabbits on a normal diet with purified XO (10 milliunits/ml) also impaired .NO-dependent relaxation but only in the presence of purine substrate. As with vessels from hypercholesterolemic rabbits, this effect was prevented by heparin and allopurinol treatment. We hypothesize that increases in plasma cholesterol induce the release of XO into the circulation, where it binds to endothelial cell glycosaminoglycans. Only in hypercholesterolemic vessels is sufficient substrate available to sustain the production of O2.- and impair NO-dependent vasorelaxation. Chronically, the continued production of peroxynitrite, (ONOO-) which the simultaneous generation of NO and O2.- implies, may irreversibly impair vessel function.

Discovery of adrenomedullin in rat ischemic cortex and evidence for its role in exacerbating focal brain ischemic damage.

Focal brain ischemia is the most common event leading to stroke in humans. To understand the molecular mechanisms associated with brain ischemia, we applied the technique of mRNA differential display and isolated a gene that encodes a recently discovered peptide, adrenomedullin (AM), which is a member of the calcitonin gene-related peptide (CGRP) family. Using the rat focal stroke model of middle cerebral artery occlusion (MCAO), we determined that AM mRNA expression was significantly increased in the ischemic cortex up to 17.4-fold at 3 h post-MCAO (P < 0.05) and 21.7-fold at 6 h post-MCAO (P < 0.05) and remained elevated for up to 15 days (9.6-fold increase; P < 0.05). Immunohistochemical studies localized AM to ischemic neuronal processes, and radioligand (125I-labeled CGRP) displacement revealed high-affinity (IC50 = 80.3 nmol) binding of AM to CGRP receptors in brain cortex. The cerebrovascular function of AM was studied using synthetic AM microinjected onto rat pial vessels using a cranial window or applied to canine basilar arteries in vitro. AM, applied abluminally, produced dose-dependent relaxation of preconstricted pial vessels (P < 0.05). Intracerebroventricular (but not systemic) AM administration at a high dose (8 nmol), prior to and after MCAO, increased the degree of focal ischemic injury (P < 0.05). The ischemia-induced expression of both AM mRNA and peptide in ischemic cortical neurons, the demonstration of the direct vasodilating effects of the peptide on cerebral vessels, and the ability of AM to exacerbate ischemic brain damage suggests that AM plays a significant role in focal ischemic brain injury.

Overexpression of MYC causes p53-dependent G2 arrest of normal fibroblasts

Overexpression of the proto-oncogene MYC has been implicated in the genesis of diverse human cancers. One explanation for the role of MYC in tumorigenesis has been that this gene might drive cells inappropriately through the division cycle, leading to the relentless proliferation characteristic of the neoplastic phenotype. Herein, we report that the overexpression of MYC alone cannot sustain the division cycle of normal cells but instead leads to their arrest in G2. We used an inducible form of the MYC protein to stimulate normal human and rodent fibroblasts. The stimulated cells passed through G1 and S but arrested in G2 and frequently became aneuploid, presumably as a result of inappropriate reinitiation of DNA synthesis. Absence of the tumor suppressor gene p53 or its downstream effector p21 reduced the frequency of both G2 arrest and aneuploidy, apparently by compromising the G2 checkpoint control. Thus, relaxation of the G2 checkpoint may be an essential early event in tumorigenesis by MYC. The loss of p53 function seems to be one mechanism by which this relaxation commonly occurs. These findings dramatize how multiple genetic events can collaborate to produce neoplastic cells.

ΔpH-Dependent Photosystem II Fluorescence Quenching Induced by Saturating, Multiturnover Pulses in Red Algae1

We have previously shown that in the red alga Rhodella violacea, exposure to continuous low intensities of light 2 (green light) or near-saturating intensities of white light induces a ΔpH-dependent PSII fluorescence quenching. In this article we further characterize this fluorescence quenching by using white, saturating, multiturnover pulses. Even though the pulses are necessary to induce the ΔpH and the quenching, the development of the latter occurred in darkness and required several tens of seconds. In darkness or in the light in the presence of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, the dissipation of the quenching was very slow (more than 15 min) due to a low consumption of the ΔpH, which corresponds to an inactive ATP synthase. In contrast, under far-red illumination or in the presence of 3-(3,4-dichlorophenyl)-1,1′-dimethylurea (only in light), the fluorescence quenching relaxed in a few seconds. The presence of N,N′-dicyclohexyl carbodiimide hindered this relaxation. We propose that the quenching relaxation is related to the consumption of ΔpH by ATP synthase, which remains active under conditions favoring pseudolinear and cyclic electron transfer.

Up-regulation of pressure-activated Ca(2+)-permeable cation channel in intact vascular endothelium of hypertensive rats.

In endothelial cells, stretch-activated cation channels have been proposed to act as mechanosensors for changes in hemodynamic forces. We have identified a novel mechanosensitive pressure-activated channel in intact endothelium from rat aorta and mesenteric artery. The 18-pS cation channel responded with a multifold increase in channel activity when positive pressure was applied to the luminal cell surface with the patch pipette and inactivated at negative pipette pressure. Channel permeability ratio for K+, Na+, and Ca2+ ions was 1:0.98:0.23. Ca2+ influx through the channel was sufficient to activate a neighboring Ca2(+)-dependent K+ channel. Hemodynamic forces are chronically disturbed in arterial hypertension. Endothelial cell dysfunction has been implicated in the pathogenesis of arterial hypertension. In two comparative studies, density of the pressure-activated channel was found to be significantly higher in spontaneously hypertensive rats and renovascular hypertensive rats compared with their respective normotensive controls. Channel activity presumably leads to mechanosensitive Ca2+ influx and induces cell hyperpolarization by K+ channel activity. Both Ca2+ influx and hyperpolarization are known to induce a vasodilatory endothelial response by stimulating endothelial nitric oxide (NO) production. Up-regulation of channel density in hypertension could, therefore, represent a counterregulatory mechanism of vascular endothelium.

Platelets roll on stimulated endothelium in vivo: an interaction mediated by endothelial P-selectin.

P-selectin, found in storage granules of platelets and endothelial cells, can be rapidly expressed upon stimulation. Mice lacking this membrane receptor exhibit a severe impairment of leukocyte rolling. We observed that, in addition to leukocytes, platelets were rolling in mesenteric venules of wild-type mice. To investigate the role of P-selectin in this process, resting or activated platelets from wild-type or P-selectin-deficient mice were fluorescently labeled and transfused into recipients of either genotype. Platelet-endothelial interactions were monitored by intravital microscopy. We observed rolling of either wild-type or P-selectin-deficient resting platelets on wild-type endothelium. Endothelial stimulation with the calcium ionophore A23187 increased the number of platelets rolling 4-fold. Activated P-selectin-deficient platelets behaved similarly, whereas activated wild-type platelets bound to leukocytes and were seen rolling together. Platelets of either genotype, resting or activated, interacted minimally with mutant endothelium even after A23187 treatment. The velocity of platelet rolling was 6- to 9-fold greater than that of leukocytes. Our results demonstrate that (i) platelets roll on endothelium in vivo, (ii) this interaction requires endothelial but not platelet P-selectin, and (iii) platelet rolling appears to be independent of platelet activation, indicating constitutive expression of a P-selectin ligand(s) on platelets. We have therefore observed an interesting parallel between platelets and leukocytes in that both of these blood cell types roll on stimulated vessel wall and that this process is dependent on the expression of endothelial P-selectin.

Increase of skeletal muscle relaxation speed by direct injection of parvalbumin cDNA.

Parvalbumin (PV) is a high affinity Ca(2+)-binding protein found at high concentration in fast-contracting/relaxing skeletal muscle fibers of vertebrates. It has been proposed that PV acts in the process of muscle relaxation by facilitating Ca2+ transport from the myofibrils to the sarcoplasmic reticulum. However, on the basis of metal-binding kinetics of PV in vitro, this hypothesis has been challenged. To investigate the function of PV in skeletal muscle fibers, direct gene transfer was applied in normal and regenerating rat soleus muscles which do not synthesize detectable amounts of PV. Two weeks after in vivo transfection with PV cDNA, considerable levels of PV mRNA and protein were detected in normal muscle, and even higher amounts were detected in regenerating muscle. Twitch half-relaxation time was significantly shortened in a dose-dependent way in transfected muscles, while contraction time remained unaltered. The observed shortening of half-relaxation time is due to PV and its ability to bind Ca2+, because a mutant protein lacking Ca(2+)-binding capacity did not promote any change in physiology. These results directly demonstrate the physiological function of PV as a relaxing factor in mammalian skeletal muscle.

Oxygen-sensitive calcium channels in vascular smooth muscle and their possible role in hypoxic arterial relaxation.

We have investigated the modifications of cytosolic [Ca2+] and the activity of Ca2+ channels in freshly dispersed arterial myocytes to test whether lowering O2 tension (PO2) directly influences Ca2+ homeostasis in these cells. Unclamped cells loaded with fura-2 AM exhibit oscillations of cytosolic Ca2+ whose frequency depends on extracellular Ca2+ influx. Switching from a PO2 of 150 to 20 mmHg leads to a reversible attenuation of the Ca2+ oscillations. In voltage-clamped cells, hypoxia reversibly reduces the influx of Ca2+ through voltage-dependent channels, which can account for the inhibition of the Ca2+ oscillations. Low PO2 selectively inhibits L-type Ca2+ channel activity, whereas the current mediated by T-type channels is unaltered by hypoxia. The effect of low PO2 on the L-type channels is markedly voltage dependent, being more apparent with moderate depolarizations. These findings demonstrate the existence of O2-sensitive, voltage-dependent, Ca2+ channels in vascular smooth muscle that may critically contribute to the local regulation of circulation.