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Cerebral ischemia is a common refractory brain disease, resulting from a reduction in the blood flow to the brain. Mitochondrial dysfunction leads to ischemic stroke and brain injury. Cordyceps sinensis (CS) is an important traditional Chinese medicine, which has been linked to neuroprotection in recent studies. In this study, we investigated the role of the mitochondrial respiratory chain and the mitochondrial apoptotic pathway on the protective effect of Cordyceps sinensis extract (CSE) against cerebral ischemia injury both in vivo and in vitro. In a murine middle cerebral artery occlusion (MCAO) model, administration of CSE relieved neuronal morphological damage and attenuated the neuronal apoptosis. CSE also reduced neurobehavioral scores and oxygen free radical (OFR), while improving the levels of ATP, cytochrome c oxidase (COX), and mitochondrial complexes I-IV. Furthermore, the mRNA expression of Bax, cytochrome c (Cyt c) and caspase-3 were down-regulated. In brain microvascular endothelial cells (BMECs) exposed to oxygen and glucose deprivation (OGD), CSE prevented OGD-induced cellular apoptosis, and recovered the reduction of mitochondrial membrane potential (MMP). Moreover, CSE treatment induced an increase of Bcl-2 protein expression and a decrease of Bax, Cyt c and caspase-3 protein expression. Meanwhile, the caspase-3, -8, and -9 activities were also inhibited. The results indicate that CSE can relieve cerebral ischemia injury and exhibit protective effects via modulating the mitochondrial respiratory chain and inhibiting the mitochondrial apoptotic pathway.

Purpose: Zebularine is a DNA methyltransferase inhibitor proposed for clinical evaluation. Experimental Design: We developed a liquid chromatography/mass spectrometry assay and did i.v. and oral studies in mice, rats, and rhesus monkeys. Results: In mice, plasma zebularine concentrations declined with terminal half-lives (t1/2) of 40 and 91 minutes after 100 mg/kg i.v. and 1,000 mg/kg given orally, respectively. Zebularine plasma concentration versus time curves (area under the curve) after 100 mg/kg i.v. and 1,000 mg/kg given orally were 7,323 and 4,935 μg/mL min, respectively, corresponding to a total body clearance (CLtb) of 13.65 mL/min/kg, apparent total body clearance (CLapp) of 203 mL/min/kg, and oral bioavailability of 6.7%. In rats, plasma zebularine concentrations declined with t1/2 of 363, 110, and 126 minutes after 50 mg/kg i.v., 250 mg/kg given orally, and 500 mg/kg given orally, respectively. Zebularine areas under the curve after 50 mg/kg i.v., 250 mg/kg given orally, and 500 mg/kg given orally were 12,526, 1,969, and 7,612 μg/mL min, respectively, corresponding to a CLtb of 3.99 mL/min/kg for 50 mg/kg i.v. and CLapp of 127 and 66 mL/min/kg for 250 and 500 mg/kg given orally, respectively. Bioavailabilities of 3.1% and 6.1% were calculated for the 250 and 500 mg/kg oral doses, respectively. In monkeys, zebularine t1/2 was 70 and 150 minutes, CLtb was 3.55 and 10.85 mL/min/kg after i.v. administration, and CLapp was 886 and 39,572 mL/min/kg after oral administration of 500 and 1,000 mg/kg, respectively. Zebularine oral bioavailability was Conclusions: Zebularine has limited oral bioavailability. Interspecies scaling projects a CLtb of 296 mL/min in humans.

Recent studies have indicated that urotensin II (UII), a cyclic peptide, is vasoactive and may be involved in cardiovascular dysfunctions. It remains unknown, however, whether UII plays a role in the control of renal vascular tone and tubular function. In the present study, a continuous infusion of synthetic human UII (hUII) into the renal artery (RA) in anesthetized rats was found to increase renal blood flow (RBF) and urinary water and sodium excretion (UV and UNaV) in a dose-dependent manner. At a dose of 20 ng · kg–1 · min–1, it increased RBF by 20% and UV and UNaV by 94 and 109%, respectively. Nitric oxide (NO) synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME) completely abolished hUII-induced increases in RBF and water/sodium excretion. In isolated, pressurized, and phenylephrine-precontracted small RA with internal diameter of ∼200 μm, hUII produced a concentration-dependent vasodilation with a maximal response of 55% at 1.5 μM. l-NAME significantly blocked this hUII-induced vasodilation by 60%. In denuded RA, hUII had neither vasodilator nor vasoconstrictor effect. With the use of 4,5-diaminofluorescein diacetate-based fluorescence imaging analysis of NO levels, hUII (1 μM) was shown to double the NO levels within the endothelium of freshly dissected small RA, and l-NAME blocked this UII-induced production of endothelial NO. These results indicate that UII produces vasodilator and natriuretic effects in the kidney and that UII-induced vasodilation is associated with increased endothelial NO in the RA.

A membrane-permeable SOD mimetic, 4-hydroxytetramethyl-piperidine-1-oxyl (tempol), has been used as an antioxidant to prevent hypertension. We recently found that this SOD mimetic could not prevent development of hypertension induced by inhibition of renal medullary SOD with diethyldithiocarbamic acid. The present study tested a hypothesis that increased H2O2counteracts the effects of tempol on renal medullary blood flow (MBF) and Na+excretion (UNaV), thereby restraining the antihypertensive effect of this SOD mimetic. By in vivo microdialysis and Amplex red H2O2microassay, it was found that interstitial H2O2levels in the renal cortex and medulla in anesthetized rats averaged 55.91 ± 3.66 and 102.18 ± 5.16 nM, respectively. Renal medullary interstitial infusion of tempol (30 μmol·min-1·kg-1) significantly increased medullary H2O2levels by 46%, and coinfusion of catalase (10 mg·min-1·kg-1) completely abolished this increase. Functionally, removal of H2O2by catalase enhanced the tempol-induced increase in MBF, urine flow, and UNaV by 28, 41, and 30%, respectively. Direct delivery of H2O2by renal medullary interstitial infusion (7.5-30 nmol·min-1· kg-1) significantly decreased renal MBF, urine flow, and UNaV, and catalase reversed the effects of H2O2. We conclude that tempol produces a renal medullary vasodilator effect and results in diuresis and natriuresis. However, this SOD mimetic increases the formation of H2O2, which constricts medullary vessels and, thereby, counteracts its vasodilator actions. This counteracting effect of H2O2may limit the use of tempol as an antihypertensive agent under exaggerated oxidative stress in the kidney.

Background— Hyperhomocysteinemia (hHcys) is considered an independent risk factor of cardiovascular diseases. Recent studies in our laboratory have shown that hHcys produced glomerular dysfunction and sclerosis independently of hypertension. However, the mechanism mediating these pathogenic effects of homocysteine (Hcys) is poorly understood. Because Hcys and adenosine (Ado) are simultaneously produced via hydrolysis of S -adenosylhomocysteine (SAH), we hypothesized that hHcys may produce its pathogenic effects by decrease in plasma or tissue Ado concentrations. Methods and Results— l -Hcys (1.5 μmol/min per kilogram) was infused intravenously for 60 minutes to produce acute hHcys in Sprague-Dawley rats. Plasma Hcys levels increased from 6.7±0.4 to 14.7±0.5 μmol/L, but Ado decreased from 141.7±15.1 to 52.4±6.8 nmol/L in these rats with acute hHcys. This hHcys-induced reduction of Ado was also observed in the kidney dialysate. In rats with chronic hHcys, plasma Ado levels were also significantly decreased. By kinetic analysis of the enzyme activities, decrease in renal Ado levels in hHcys was shown to be associated with inhibition of SAH hydrolase but not 5′-nucleotidase. Functionally, intravenous infusion of Hcys was found to decrease renal blood flow, glomerular filtration rate, and sodium and water excretion, which could be blocked by the Ado receptor antagonist 8-SPT. Conclusions— These results strongly suggest that hHcys decreases plasma and tissue Ado concentrations associated with inhibition of SAH hydrolase. Decrease in plasma and tissue Ado may be an important mechanism mediating the pathogenic effects of Hcys.

Hyperhomocysteinemia (hHcys) has been recognized as a new risk factor for cardiovascular diseases independent of plasma lipid levels or other factors. However, it remains unknown whether hHcys is implicated in the target organ damages associated with hypertension. The present study first examined the possible role of hHcys in the development of glomerulosclerosis in Dahl salt-sensitive (DS) hypertensive rats. High-performance liquid chromatography showed that plasma total homocysteine (tHcys) concentration was 7.64 +/- 0.29 micromol/L in conscious DS rats on a low salt (0.4% NaCl) diet, which was higher than 5.23 +/- 0.25 micromol/L in Dahl salt-resistant normotensive rats. When these rats were exposed to a high salt (4% NaCl) diet, plasma tHcys markedly increased in DS rats (14.7 +/-1.31 micromol/L) but not in Dahl salt-resistant rats (5.34 +/- 0.54 micromol/L). An iron chelater, desferrioxamine (0.3 mg/kg IV per day), completely normalized high salt--induced elevations of plasma tHcys and significantly attenuated the sclerotic changes in the glomeruli in DS rats. To further determine whether hHcys has an independent effect in the development of glomerulosclerosis, Sprague-Dawley rats were fed drinking water containing methionine (1 g/kg per day) for 6 weeks to produce hHcys. In these rats, plasma tHcys increased to 12.5 +/- 1.9 micromol/L (versus 6.1 +/- 2.6 micromol/L in control rats), and the aorta exhibited typical sclerotic changes, but arterial pressure was not altered. Urinary protein excretion increased to 52 +/- 2 mg/24 hours (versus 17 +/- 2 mg/24 hours in control rats), and the glomerular mesangium was expanded with glomerular hypercellularity, capillary collapse, and fibrous deposition in the rats with hHcys. These results suggest that elevated plasma homocysteine may be an important pathogenic factor for glomerular damage in hypertension independent of arterial pressure.

Cyclic ADP-ribose (cADPR) is a novel Ca(2+)-mobilizing second messenger in mammalian cells including cardiomyocytes. It is unknown whether myocardial ischemia and reperfusion affect the metabolism of cADPR in the myocardium. The present study therefore examined the effects of myocardial ischemia and reperfusion on the concentrations of myocardial cADPR using high-performance liquid chromatography. Basal levels of cADPR in rat myocardium were 5.3 +/- 1.8 nmol x mg(-1) protein. Myocardial ischemia for 30 min significantly decreased cADPR concentrations to 2.1 +/- 0.4 nmol x mg(-1) protein. During reperfusion, cADPR was maintained at ischemic levels. The activity of ADP-ribosyl cyclase was expressed as the conversion rate of nicotinamide guanine dinucleotide (NGD(+)) to cyclic GDP-ribose. Myocardial ischemia and reperfusion did not alter the activity of ADP-ribosyl cyclase. However, cADPR hydrolase activity, as measured by the conversion rate of cADPR to ADP-ribose, was significantly elevated by ischemia and reperfusion. To determine the mechanism resulting in the enhancement of cADPR hydrolase activity, we examined the effects of changes in ADP, ATP, pH, and PO(2) on the conversion rate of cADPR to ADPR. Alterations of ADP, ATP, or pH in myocardial tissue had no effect on the degradation of cADPR, whereas a decrease in tissue PO(2) markedly increased the hydrolysis of cADPR. These results suggest that myocardial ischemia and reperfusion decrease cADPR in the myocardium by increasing its hydrolysis. Tissue hypoxia may be one of the important mechanisms to activate cADPR hydrolase.

Ryanodine receptors (RyR) play an important role in the regulation of intracellular Ca(2+) concentration and in the control of vascular tone. However, the mechanism regulating the activity of RyR is poorly understood. The present study determined whether protein methylation participates in the control of RyR activity. Using a planar lipid bilayer clamping system, S-adenosyl-L-methionine (SAM), a methyl donor, significantly increased the activity of a 245-pS reconstituted Ca(2+) release channel from coronary arterial smooth muscle (CASM) in a concentration-dependent manner. Addition of the protein methylation blockers, 3-deazaadenosine, S-adenosylhomocysteine or sinefungin into the cis solution markedly attenuated SAM-induced activation of RyR/Ca(2+) release channels. By Western blot analysis, arginine N-methyltransferase (PRMT1) and FK506 binding protein (FKBP) were detected in the SR used for reconstitution of RyR. In the presence of anti-PRMT1 antibody (1:100), SAM-induced activation of RyR/Ca(2+) channel was completely abolished. In addition, this SAM-induced increase in RyR/Ca(2+) channel activity was blocked by 30 microM ryanodine and by FK506 (100 microM), a ligand for the RyR accessory protein. These results suggest that protein methylation activates RyR/Ca(2+) release channels and may participate in the control of intracellular Ca(2+) mobilization in CASM cells by transferring a methyl group to the arginine moiety of the RyR accessory protein, FKBP 12.

The present study determined the role of cyclic ADP-ribose (cADPR) in mediating vasoconstriction and Ca(2+) release in response to the activation of muscarinic receptors. Endothelium-denuded small bovine coronary arteries were microperfused under transmural pressure of 60 mm Hg. Both acetylcholine (ACh; 1 nmol/L to 1 micromol/L) and oxotremorine (OXO; 2.5-80 micromol/L) produced a concentration-dependent contraction. The vasoconstrictor responses to both ACh and OXO were significantly attenuated by nicotinamide (Nicot; an ADP-ribosyl cyclase inhibitor), 8-bromo-cADPR (8-Br-cADPR; a cADPR antagonist) or ryanodine (Ry; an Ry receptor antagonist). Intracellular Ca(2+) ([Ca(2+)](i)) was determined by fluorescence spectrometry using fura-2 as a fluorescence indicator. OXO produced a rapid increase in [Ca(2+)](i) in freshly isolated single coronary arterial smooth muscle cells (CASMCs) bathed with Ca(2+)-free Hanks' solution. This OXO-induced rise in [Ca(2+)](i) was significantly reduced by pirenzepine (PIR; an M(1) receptor-specific blocker), Nicot, 8-Br-cADPR or Ry. The effects of OXO on the activity of ADP-ribosyl cyclase (cADPR synthase) were examined in cultured CASMCs by measuring the rate of cyclic GDP- ribose (cGDPR) formation from beta-nicotinamide guanine dinucleotide. It was found that OXO produced a concentration-dependent increase in the production of cGDPR. The stimulatory effect of OXO on ADP-ribosyl cyclase was inhibited by both PIR and Nicot. These results suggest that the cADPR signaling pathway participates in the contraction of small coronary arterial smooth muscle and Ca(2+) release induced by activation of M(1) muscarinic receptors.

Mitochondrial ATP-sensitive potassium (mitoK ATP ) channels have been suggested as triggers and end effectors in myocardial ischemic preconditioning. However, the intracellular mechanism regulating mitoK ATP channels remains unclear. In the present study, mitoK ATP channels from bovine ventricular myocardium were reconstituted using planar lipid bilayers, and the effect of superoxide (O 2 −· ) on the activity of these reconstituted channels was examined. After incorporation, a potassium-selective current was recorded. The mean conductance of this current was 56 pS at 150 mmol/L KCl, which was substantially inhibited by 1 mmol/L MgATP. 5-Hydroxydecanoate (5-HD, 10 to 100 μmol/L), a selective mitoK ATP antagonist, reduced the open state probability (NPo) of these channels in a concentration-dependent manner, whereas diazoxide (10 μmol/L), a selective mitoK ATP agonist, significantly increased channel activity. HMR-1098 (100 μmol/L), a selective sarcolemmal K ATP antagonist, had no effect on the activity of reconstituted channels. Addition of xanthine/xanthine oxidase (100 μmol/L per 0.038 U/mL), an O 2 −· -generating system, resulted in a marked activation of mitoK ATP channels; the NPo of the channels was increased from 0.60±0.10 to 1.94±0.02. This O 2 −· -induced channel activation was completely abolished by pretreatment with 5-HD (100 μmol/L) or a sulfhydryl alkylating compound, N -ethylmaleimide (2 mmol/L). It is concluded that myocardial mitoK ATP channels can be reconstituted into lipid bilayers and that O 2 −· activates these channels. The effect of O 2 −· may be associated with its direct action on the sulfhydryl groups of the channel protein.

The purpose of this study was to determine whether superoxide anions (O[Formula: see text]·) activate 5′-nucleotidase (5′-ND), thereby increasing the production of renal adenosine and regulating renal function. Using HPLC analysis, we found that incubation of renal tissue homogenate with the O[Formula: see text]· donor KO2doubled adenosine production and increased the maximal reaction velocity of 5′-ND from 141 to 192 nmol · min−1· mg protein−1. The O[Formula: see text]·-generating system, xanthine/xanthine oxidase increased the maximal reaction velocity of 5′-ND from 122 to 204 nmol · min−1· mg protein−1. Superoxide dismutase (SOD) with catalase produced a concentration-dependent reduction of 5′-ND activity in renal tissue homogenate, while the SOD inhibitor diethyldithiocarbamic acid significantly increased 5′-ND activity. Inhibition of disulfide bond formation by thioredoxin or thioredoxin reductase significantly decreased xanthine/xanthine oxidase-induced activation of renal 5′-ND. In in vivo experiments, inhibition of SOD by diethyldithiocarbamic acid (0.5 mg · kg−1· min−1iv) enhanced renal vasoconstriction induced by endogenously produced adenosine and increased renal tissue adenosine concentrations under control condition and in ischemia and reperfusion. We conclude that oxidative stress activates 5′-ND and increases adenosine production in the kidney and that this redox regulatory mechanism of adenosine production is important in the control of renal vascular tone and glomerular perfusion.