Efficacy of BAY 60-2770, a soluble guanylate cyclase activator, for coronary spasm in animal models.

Ischemia with non-obstructive coronary arteries (INOCA), caused by coronary artery spasm, has gained increasing attention owing to the poor quality of life of impacted patients. Therapeutic options to address INOCA remain limited, and developing new therapeutic agents is desirable. Herein, we examined whether soluble guanylate cyclase (sGC) activators could be beneficial in preventing coronary spasms. In organ chamber experiments with isolated canine coronary arteries, prostaglandin F2α-, endothelin-1-, 5-hydroxytryptamine-, and potassium chloride-induced contractions were suppressed by the sGC activator BAY 60-2770 (0.1, 1, and 10 nM). In isolated pig coronary arteries, BAY 60-2770 (0.1, 1, and 10 nM) could prolong the cycle length of phasic contractions induced by 3,4-diaminopyridine, as well as lower the peak and bottom tension of the contraction in a concentration-dependent manner. Additionally, BAY 60-2770 (1 pM‒0.1 µM) evoked a concentration-related relaxation to a greater extent in small (first diagonal branch) coronary arteries than in large (left anterior descending) coronary arteries. In vasopressin-induced angina model rats, pretreatment with BAY 60-2770 (3 µg/kg) suppressed electrocardiogram S-wave depression induced by arginine vasopressin without affecting changes in mean blood pressure and heart rate. These findings suggest that BAY 60-2770 could be valuable in preventing both large and small coronary spasms. Therefore, sGC activators could represent a novel and efficacious therapeutic option for INOCA. Significance Statement The sGC activator BAY 60-2770 exerted antispastic effects on the coronary arteries in animal vasospasm models as proof-of-concept studies. These data can help to support potential clinical development with sGC activators, suitable for human use in patients with vasospastic angina.

Acute Kynurenine Exposure of Rat Thoracic Aorta Induces Vascular Dysfunction via Superoxide Anion Production.

The accumulation of uremic toxins is known to be one of the causes of cardiovascular disorder related to renal disease. Among the many uremic toxins, we focused on kynurenine (kyn), whose levels have been shown to be positively correlated with vascular endothelial dysfunction markers, and directly evaluated the influence of kyn on the rat thoracic aorta. Exposure of the endothelium-intact aorta to kyn markedly attenuated the acetylcholine (ACh)-induced relaxation and significantly increased superoxide anion (O2·-) production. These effects were ameliorated by pretreatment with ascorbic acid, an antioxidant, and CH223191, an aryl hydrocarbon receptor (AhR) inhibitor, but not by apocynin, a reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor. In the endothelium-denuded aorta, kyn significantly attenuated the nitric oxide (NO) donor sodium nitroprusside (SNP)-induced vasorelaxation and increased the O2·- production. Ascorbic acid treatment significantly ameliorated these effects, whereas CH223191 and apocynin treatments did not. Kyn had no influence on the vasorelaxant response to BAY 41-2272, a soluble guanylate cyclase stimulator. This suggested that kyn attenuates the NO-mediated vasorelaxation response by promoting O2·- production in thoracic aorta to inactivate NO. O2·- production is likely stimulated in both vascular endothelium and smooth muscle, the former of which may be mediated by AhR activation.

Soluble Guanylate Cyclase-Mediated Relaxation in Aortas from Rats with Renovascular Hypertension.

Soluble guanylate cyclase (sGC) plays an important role in nitric oxide (NO)-mediated regulation of vascular tone; however, NO bioavailability is often reduced in diseased blood vessels. Accumulating evidence suggests that a shift of sGC from the NO-sensitive form to the NO-insensitive form could be an underlying cause contributing to this reduction. Herein, we investigated the impact of renovascular hypertension on NO-sensitive and NO-insensitive sGC-mediated relaxation in rat aortas. Renovascular hypertension was induced by partially clipping the left renal artery (2-kidneys, 1-clip; 2K1C) for 10 weeks. Systolic, diastolic, and mean arterial pressures were significantly increased in the 2K1C group when compared with the sham group. In addition, plasma thiobarbituric acid reactive substances and aortic superoxide generation were significantly enhanced in the 2K1C group when compared with those in the sham group. The vasorelaxant response of isolated aortas to the sGC stimulator BAY 41-2272 (NO-sensitive sGC agonist) was comparable between the sham and 2K1C groups. Likewise, the sGC activator BAY 60-2770 (NO-insensitive sGC agonist)-induced relaxation did not differ between the sham and 2K1C groups. In addition, the cGMP mimetic 8-Br-cGMP (protein kinase G agonist) induced similar relaxation in both groups. Furthermore, there were no differences in BAY 41-2272-stimulated and BAY 60-2770-stimulated cGMP generation between the groups. These findings suggest that the balance between NO-sensitive and NO-insensitive forms of sGC is maintained during renovascular hypertension. Therefore, sGC might not be responsible for the reduced NO bioavailability observed during renovascular hypertension.

Essential Role of NADPH Oxidase-Dependent Production of Reactive Oxygen Species in Maintenance of Sustained B Cell Receptor Signaling and B Cell Proliferation.

Reactive oxygen species (ROS) are not only toxic substances inducing oxidative stress but also play a role as a second messenger in signal transduction through various receptors. Previously, B cell activation was shown to involve prolonged ROS production induced by ligation of BCR. However, the mechanisms for ROS production and ROS-mediated activation in B cells are still poorly understood. In this study, we demonstrate that BCR ligation induces biphasic ROS production in both mouse spleen B cells and the mouse B cell line BAL17; transient and modest ROS production is followed by sustained and robust ROS production at 2-6 h after BCR ligation. ROS production in the late phase but not in the early phase augments activation of signaling pathways, such as the NF-κB and PI3K pathways, and is essential for B cell proliferation. ROS production in the late phase appears to be mediated by NADPH oxidases (NOXes) because prolonged ROS production is inhibited by various NOX inhibitors, including the specific inhibitor VAS2870. BCR ligation-induced ROS production is also inhibited by CRISPR/Cas9-mediated deletion of either the Cyba gene encoding p22phox, the regulator of NOX1-4 required for their activation, or NOX3, whereas ROS production is not affected by double deficiency of the DUOXA1 and DUOXA2 genes essential for the activation of the NOX isoforms DUOX1 and DUOX2. These results indicate that NOXes play a crucial role in sustained but not early BCR signaling and suggest an essential role of NOX-dependent sustained BCR signaling in B cell activation.

Grape Extract from Chardonnay Seeds Restores Deoxycorticosterone Acetate-Salt-Induced Endothelial Dysfunction and Hypertension in Rats.

Grape extract (GE), which contains various polyphenolic compounds, exerts protective effects against lifestyle-related diseases, such as diabetes and hypertension. We pharmacologically investigated whether dietary supplements with an extract from Chardonnay exerted antihypertensive effects in deoxycorticosterone acetate (DOCA)-salt-induced hypertensive rats. GE increased nitric oxide (NO) production by activating the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in cultured endothelial cells and induced vasorelaxation in the aorta and mesenteric artery via the same pathway. The development and progression of hypertension by the DOCA-salt treatment was significantly inhibited in GE-fed rats. Reduced vasoreactive responses to acetylcholine in the aorta of DOCA-salt rats were significantly ameliorated by the GE diet. Dietary GE supplements slightly diminished vascular superoxide anion production induced by the DOCA-salt treatment. On the other hand, dietary GE supplements had no effect on the progression of hypertension in rats in which NO synthase was pharmacologically and chronically suppressed. In addition, the oral administration of GE for 5 d in healthy rats enhanced endothelial NO synthase (eNOS) gene expression and vascular reactivity to acetylcholine in the aorta. Thus, GE has endothelium-dependent vasorelaxant properties that are mediated by the activation of endothelial NO synthase via the PI3K/Akt pathway, and this mechanism is conducive to the antihypertensive effects of GE observed in DOCA-salt-treated rats.

Preventive Effects of Grape Extract on Ischemia/Reperfusion-Induced Acute Kidney Injury in Mice.

Since grape extract (GE) contains oligomeric proanthocyanidins and numerous polyphenols, dietary GE supplements may exert protective effects against various diseases. The present study investigated the pharmacological effects of GE derived from Chardonnay in vitro and in vivo. GE (100 µg/mL) completely inhibited tumor necrosis factor-α-induced endothelin-1, monocyte chemoattractant protein-1, interleukin-1ß, and intercellular adhesion molecule-1 gene expression in cultured endothelial cells. GE also strongly stimulated the phosphatidylinositol 3-kinase (PI3K)/Akt/endothelial nitric oxide synthase (eNOS) pathway. In the in vivo study, the effects of GE on ischemic acute kidney injury (AKI) were examined using male C57bl/6J wild-type and eNOS-/- mice. Right nephrectomized mice were exposed to 45 min of ischemia in the left kidney and this was followed by reperfusion. Although renal functional parameters in AKI mice significantly increased 48 h after reperfusion, the administration of GE (0.1 and 1 mg/kg, intravenous (i.v.)) 5 min before ischemia dose-dependently improved post-ischemic renal dysfunction in wild-type mice. Renal histopathological studies on AKI mice revealed tubular necrosis, proteinaceous casts in tubuli, and medullary congestion. The administration of GE ameliorated this damage in wild-type mice, but not in eNOS-/- mice. Furthermore, GE significantly restored decreases in the renal nitric oxide metabolite content due to ischemia in wild-type mice, but not in eNOS-/- mice. Thus, eNOS is closely involved in the renoprotective effects of GE, strongly suggesting that GE supplements are useful as a prophylactic treatment for the development of ischemic AKI.

Involvement of γ-Glutamyl Transpeptidase in Ischemia/Reperfusion-Induced Cardiac Dysfunction in Isolated Rat Hearts.

GGsTop is a highly potent and specific, and irreversible γ-glutamyl transpeptidase (GGT) inhibitor without any influence on glutamine amidotransferases. The aim of the present study was to investigate the involvement of GGT in ischemia/reperfusion-induced cardiac dysfunction by assessing the effects of a treatment with GGsTop. Using a Langendorff apparatus, excised rat hearts underwent 40 min of global ischemia without irrigation and then 30 min of reperfusion. GGT activity was markedly increased in cardiac tissues exposed to ischemia, and was inhibited by the treatment with GGsTop. Exacerbation of cardiac functional parameters caused by ischemia and reperfusion, namely the reduction of left ventricular (LV) developed pressure and the maximum and negative minimum values of the first derivative of LV pressure, and the increment in LV end-diastolic pressure was significantly attenuated by GGsTop treatment. The treatment with GGsTop suppressed excessive norepinephrine release in the coronary perfusate, a marker for myocardial dysfunction, after ischemia/reperfusion. In addition, oxidative stress indicators in myocardium, including superoxide and malondialdehyde, after ischemia/reperfusion were significantly low in the presence of GGsTop. These observations demonstrate that enhanced GGT activity contributes to cardiac damage after myocardial ischemia/reperfusion, possibly via increased oxidative stress and subsequent norepinephrine overflow. GGT inhibitors have potential as a therapeutic strategy to prevent myocardial ischemia/reperfusion injury in vivo.

Sex differences in ischaemia/reperfusion-induced acute kidney injury depends on the degradation of noradrenaline by monoamine oxidase.

Ischaemic acute kidney injury (AKI) is a leading killer of both sexes; however, resistance to this injury is higher among women than men. We found that renal venous noradrenaline (NAd) overflow after reperfusion played important roles in the development of ischaemic AKI, and that the attenuation of AKI observed in female rats may be dependent on depressing the renal sympathetic nervous system with endogenous oestrogen. In the present study, we used male and female Sprague-Dawley rats to investigate whether sex differences in the pathogenesis of ischaemic AKI are related to the degradation of NAd by monoamine oxidase (MAO) in the kidney. Ischaemic AKI was achieved by clamping the left renal artery and vein for 45 minutes followed by reperfusion 2 weeks after contralateral nephrectomy. Renal injury was more severe in male rats than in female rats and renal venous plasma NAd levels after reperfusion were markedly elevated in males, but not in females. These sex differences were eliminated by a treatment with isatin, a non-selective MAO inhibitor, and moclobemide, a selective MAOA inhibitor, but not by selegiline, a selective MAOB inhibitor. Ischaemia decreased the mRNA expression levels of both MAOs in the kidney 1 day after reperfusion; however, MAOA mRNA expression levels were higher in female rats than in male rats. These results suggest that the degradation of NAd by MAOA in the kidney contributes to sex differences in the pathogenesis of ischaemia/reperfusion-induced AKI.

Mechanisms underlying the renoprotective effect of GABA against ischaemia/reperfusion-induced renal injury in rats.

Excitation of the renal sympathetic nervous system is important for the development of ischaemic acute kidney injury (AKI) in rats. We reported that intravenous treatment with GABA has preventive effects against ischaemia/reperfusion (I/R)-induced renal dysfunction with histological damage in rats; however, the mechanisms underlying these effects on renal injury remain unknown. Thus, the aim of the present study was to clarify how GABA mechanistically affects ischaemic AKI in rats. Ischaemic AKI was induced in rats by clamping the left renal artery and vein for 45 min and then reperfusing the kidney to produce I/R-induced injury. Treatment with the GABAB receptor antagonist CGP52432 (100 nmol/kg, i.v., or 1 nmol/kg, i.c.v.) abolished the suppressive effects of 50 µmol/kg, i.v., GABA on enhanced renal sympathetic nerve activity (RSNA) during ischaemia, leading to elimination of the renoprotective effects of GABA. Intracerebroventricular treatment with 0.5 µmol/kg GABA or i.v. treatment with 1 µmol/kg baclofen, a selective GABAB receptor agonist, prevented the I/R-induced renal injury equivalent to i.v. treatment with GABA. Conversely, i.v. treatment with 10 µmol/kg bicuculline, a GABAA receptor antagonist, failed to affect the preventive effects of GABA against ischaemic AKI. We therefore concluded that GABAB receptor stimulation in the central nervous system, rather than peripheral GABAB receptor stimulation, mediates the preventive effect of GABA against ischaemic AKI by suppressing the enhanced RSNA induced by renal ischaemia.

Involvement of renal sympathetic nerve overactivation in the progression of chronic kidney disease in rats.

Time-dependent changes in the renal sympathetic nerve activity (RSNA) in the progression of chronic kidney disease (CKD) have not been investigated, despite the fact that renal sympathetic nervous system is augmented in the condition of CKD. In the present study, we examined time-dependent changes in RSNA and renal venous norepinephrine concentrations for 12 weeks using 5 of 6 nephrectomized CKD rats. Both RSNA and norepinephrine concentrations were increased during the early phase in the progression of CKD. Urinary protein excretion and systolic blood pressure (SBP) were gradually increased during 12 weeks after 5 of 6 nephrectomy. Treatment with γ-aminobutyric acid or the combination of prazosin and propranolol in the early phase (0-4 weeks) after 5 of 6 nephrectomy significantly attenuated the increases in urinary protein excretion and SBP in 5 of 6 nephrectomized rats. On the other hand, the same treatment in the late phase (8-12 weeks) after 5 of 6 nephrectomy failed to suppress the proteinuria and increase in SBP. Treatment with hydralazine at hypotensive dose for 12 weeks also failed to affect the proteinuria in 5 of 6 nephrectomized CKD rats. In conclusion, the augmentation of renal sympathetic nervous system in early phase after 5 of 6 nephrectomy is closely related to the development of partial ablation-induced CKD in rats.

Oligomycin, an F1Fo-ATPase inhibitor, protects against ischemic acute kidney injury in male but not in female rats.

We investigated the effects of oligomycin, an F1Fo-ATPase inhibitor, on ischemic acute kidney injury in male and female rats. Ischemic acute kidney injury was induced by clamping the left renal artery and vein for 45 or 60 min followed by reperfusion, 2 weeks after contralateral nephrectomy. Renal dysfunction and histological renal damage were observed 1 day after reperfusion in both male and female rats, although these renal injuries were more marked in male rats than in female rats. Intravenous bolus injection of oligomycin (0.5 mg/kg) 5 min before ischemia markedly attenuated the ischemia/reperfusion-induced renal injury in male rats. However, oligomycin did not show the protective effect in female rats subjected to ischemia/reperfusion-induced renal injury. Pre-ischemic treatment with oligomycin suppressed partly but significantly ischemia-induced renal ATP depletion only in male rats. These results indicate that oligomycin prevents the onset of ischemic acute kidney injury in male but not in female rats, and the effect is accompanied by suppression of the ATP depletion only in the male rat kidney during ischemia, thereby suggesting that the ATP hydrolysis pathway by mitochondrial F1Fo-ATPase induces a sex difference in ischemic acute kidney injury.

Effects of beetroot juice supplementation on vascular functional and structural changes in aged mice.

This study investigated whether beetroot juice (BRJ) ingestion ameliorates aging-induced functional and structural changes in vasculature. Aged mice (98-100 weeks old) were supplemented with BRJ (nitrate: 3.5 mmol/L) or drinking water for 4 weeks and compared with young mice (12-15 weeks old). The vasorelaxant response of isolated aortas to acetylcholine was markedly weaker in aged mice than in young mice, but the attenuated relaxation was significantly improved in BRJ-supplemented aged mice. The acetylcholine-induced relaxation was completely abolished by Nω -nitro-l-arginine methyl ester in all groups. Additionally, the response to sodium nitroprusside was comparable among the three groups. The aortic medial thickness was significantly greater in aged mice than in young mice, and BRJ supplementation did not suppress this thickening. Plasma nitrate levels were significantly higher in BRJ-supplemented aged mice than in non-supplemented aged mice. Conversely, non-supplemented aged mice had high plasma levels of thiobarbituric acid-reactive substances, but the levels were suppressed in BRJ-supplemented aged mice. These findings suggest that BRJ ingestion improves vascular endothelial dysfunction associated with aging, at least in part, by enhancing nitric oxide bioavailability and reducing oxidative stress. Therefore, beetroot ingestion may be a highly useful self-medication option to prevent vascular aging.

Sex differences in postischemic cardiac dysfunction and norepinephrine overflow in rat heart: the role of estrogen against myocardial ischemia-reperfusion damage via an NO-mediated mechanism.

The purpose of this study is to elucidate the relationship between sex difference and norepinephrine (NE) release in the pathogenesis of myocardial ischemia/reperfusion (I/R) injury. Isolated male and female rat hearts were subjected to 40-minute global ischemia followed by 30-minute reperfusion. Compared with male hearts, I/R-induced cardiac dysfunction, such as decreased left ventricular developed pressure and dP/dtmax and increased left ventricular end diastolic pressure, was significantly attenuated in female hearts. An excessive NE overflow in the coronary effluent from the postischemic heart in females was much less than that in males. These sex differences were abolished by ovariectomy, but in vivo treatment with 17ß-estradiol recovered it. This ameliorating effect of 17ß-estradiol was not observed in the presence of nitric oxide (NO) synthase inhibitor N-nitro-L-arginine. When NOx (NO2/NO3) levels in the coronary effluent after onset of reperfusion were measured, reversed correlated relationships between NOx production and I/R-induced cardiac dysfunction, and NE overflow, were observed. These findings suggest that sex differences in the postischemic cardiac dysfunction are closely related to the NE overflow from the postischemic heart and that estrogen plays a key role in the cardioprotective effect against I/R injury in female rats, by suppressing NE release via the enhancement of NO production.

Different effects of AT1 receptor antagonist and ET(A) receptor antagonist on ischemia-induced norepinephrine release in rat hearts.

Angiotensin type 1 receptor (AT1R) antagonist and endothelin type A receptor (ET(A)R) antagonist were compared as regards their effects on ischemia-induced exocytotic or carrier-mediated norepinephrine (NE) release from cardiac sympathetic nerve endings. According to the Langendorff technique, isolated rat hearts were subjected to 20-minute or 40-minute global ischemia followed by 30-minute reperfusion. Candesartan (selective AT1R antagonist) and ABT-627 (selective ET(A)R antagonist) were perfused, beginning 15 minutes before ischemia. Candesartan (10 and 100 nM) and ABT-627 (3 µM) suppressed excessive NE overflow (exocytotic release) in the coronary effluent from the heart exposed to 20-minute ischemia. In addition, these agents improved postischemic cardiac dysfunction. On the other hand, the beneficial effects of ABT-627 (1 and 3 µM) on NE overflow (carrier-mediated release) and cardiac dysfunction were also observed in 40-minute ischemia, whereas those were not improved by treatment with candesartan (10 and 100 nM and 1 µM). These findings suggest that both AT1R antagonist and ET(A)R antagonist have ability to inhibit the exocytotic NE release, but the carrier-mediated NE release induced by prolonged ischemia cannot be avoided by AT1R antagonist. Thus, ET(A)R antagonist may be more useful than AT1R antagonist in the clinical settings of ischemic heart disease.

Pathophysiological roles of endothelin receptors in cardiovascular diseases.

Endothelin (ET)-1 derived from endothelial cells has a much more important role in cardiovascular system regulation than the ET-2 and ET-3 isoforms. Numerous lines of evidence indicate that ET-1 possesses a number of biological activities leading to cardiovascular diseases (CVD) including hypertension and atherosclerosis. Physiological and pathophysiological responses to ET-1 in various tissues are mediated by interactions with ET(A)- and ET(B)-receptor subtypes. Both subtypes on vascular smooth muscle cells mediate vasoconstriction, whereas the ET(B)-receptor subtype on endothelial cells contributes to vasodilatation and ET-1 clearance. Although selective ET(A)- or nonselective ET(A)/ET(B)-receptor antagonisms have been assumed as potential strategies for the treatment of several CVD based on clinical and animal experiments, it remains unclear which antagonisms are suitable for individuals with CVD because upregulation of the nitric oxide system via the ET(B) receptor is responsible for vasoprotective effects such as vasodilatation and anti-cell proliferation. In this review, we have summarized the current understanding regarding the role of ET receptors, especially the ET(B) receptor, in CVD.

Vascular Endothelial Dysfunction in the Thoracic Aorta of Rats with Ischemic Acute Kidney Injury: Contribution of Indoxyl Sulfate.

Chronic kidney disease (CKD) and cardiovascular disease are known to be linked, and the involvement of indoxyl sulfate (IS), a type of uremic toxin, has been suggested as one of the causes. It is known that IS induces vascular dysfunction through overproduction of reactive oxygen species (ROS). On the other hand, the involvement of IS in the vascular dysfunction associated with acute kidney injury (AKI) is not fully understood. Therefore, we investigated this issue using the thoracic aorta of rats with ischemic AKI. Ischemic AKI was induced by occlusion of the left renal artery and vein for 45 min, followed by reperfusion 2 weeks after contralateral nephrectomy. One day after reperfusion, there was marked deterioration in renal function evidenced by an increase in plasma creatinine. Furthermore, blood IS levels increased markedly due to worsening renal function. Seven days and 28 days after reperfusion, blood IS levels decreased with the improvement in renal function. Of note, acetylcholine-induced vasorelaxation deteriorated over time after reperfusion, contradicting the recovery of renal function. In addition, 28 days after reperfusion, we observed a significant increase in ROS production in the vascular tissue. Next, we administered AST-120, a spherical adsorbent charcoal, after reperfusion to assess whether the vascular endothelial dysfunction associated with the ischemic AKI was due to a temporary increase in blood IS levels. AST-120 reduced the temporary increase in blood IS levels after reperfusion without influencing renal function, but did not restore the impaired vascular reactivity. Thus, in ischemic AKI, we confirmed that the vascular endothelial function of the thoracic aorta is impaired even after the recovery of kidney injury, probably with excessive ROS production. IS, which increases from ischemia to early after reperfusion, may not be a major contributor to the vascular dysfunction associated with ischemic AKI.

Preventive effect of GGsTop, a novel and selective γ-glutamyl transpeptidase inhibitor, on ischemia/reperfusion-induced renal injury in rats.

GGsTop [2-amino-4-{[3-(carboxymethyl)phenyl](methyl)phosphono}butanoic acid], is a novel, highly selective, and irreversible γ-glutamyl transpeptidase (GGT) inhibitor with no inhibitory activity on glutamine amidotransferases. In this study, we investigated the effects of treatment with GGsTop on ischemia/reperfusion-induced renal injury in uninephrectomized rats. Ischemic acute kidney injury (AKI) was induced by occlusion of the left renal artery and vein for 45 min followed by reperfusion 2 weeks after contralateral nephrectomy. Renal function in vehicle-treated AKI rats markedly decreased at 1 day after reperfusion. Treatment with GGsTop (1 and 10 mg/kg i.v.) 5 min before ischemia attenuated the ischemia/reperfusion-induced renal dysfunction in a dose-dependent manner. Histopathological examination of the kidney of AKI rats revealed severe renal damage, which was significantly suppressed by the GGsTop treatment. In renal tissues exposed to ischemia/reperfusion, GGT activity was markedly increased immediately after reperfusion, whereas renal superoxide production and malondialdehyde level were significantly increased 6 h after reperfusion. These alterations were abolished by the treatment with GGsTop. In addition, renal glutathione content was decreased by the 45-min ischemia, but its level was markedly elevated by the GGsTop treatment. Our results demonstrate that the novel and highly selective GGT inhibitor GGsTop prevents ischemia/reperfusion-induced AKI. The renoprotective effect of GGsTop seems to be attributed to the suppression of oxidative stress by inhibiting GGT activation, thereby preventing the degradation of glutathione.

Mechanisms underlying the renoprotective effect of GABA against ischemia/reperfusion-induced renal injury in rats.

The excitation of the renal sympathetic nervous system plays an important role in the development of ischemic acute kidney injury (AKI) in rats. We have reported that intravenous treatment with GABA has preventive effects on ischemia/reperfusion (I/R)-induced renal dysfunction with histological damage in rats. However, detailed mechanisms of the action of GABA on the renal injury were still unknown. Therefore, in the present study, we aimed to clarify the detailed mechanisms of GABA in ischemic AKI in rats. Ischemic AKI was induced by clamping the left renal artery and vein for 45 min. Thereafter, the kidney was reperfused to produce I/R-induced injury. Intravenous or intracerebroventricular treatment with 3-[[[(3,4-dichlorophenyl)methyl]amino]propyl] diethoxymethyl) phosphinic acid (CGP52432), a GABA(B) receptor antagonist, abolished the suppressive effects of intravenously applied GABA on enhanced renal sympathetic nerve activity during ischemia, leading to the elimination of the renoprotective effects of GABA. Intracerebroventricular treatment with GABA or intravenous treatment with baclofen, a selective GABA(B) receptor agonist, prevented I/R-induced renal injury equivalent to intravenous treatment with GABA. However, intravenous treatment with bicuculline, a GABA(A) receptor antagonist, failed to affect the preventive effects of GABA on ischemic AKI. Therefore, we demonstrated the novel finding that the preventive effect of GABA on ischemic AKI through the suppression of enhanced renal sympathetic nerve activity induced by renal ischemia is presumably mediated via GABA(B) receptor stimulation in the central nervous system rather than peripheral GABA(B) receptor.

Endothelin ETB receptor is involved in sex differences in the development of balloon injury-induced neointimal formation.

The purpose of this study was to evaluate the involvement of endothelin (ET)(B) receptor-mediated action in the sex differences in balloon injury-induced neointimal formation using the spotting-lethal rat, which carries a naturally occurring deletion in its ET(B) receptor gene. Male and female ET(B)-deficient and wild-type rats underwent balloon injury of the carotid artery. In the wild-type rats, the neointima/media ratio was significantly lower in females than in males, but this sex difference was attenuated by ovariectomy and restored by treatment with 17ß-estradiol (20 µg/kg/day). In the ET(B)-deficient rats, the neointima/media ratio of the male and female rats was markedly increased to the same level, and this increase was not affected by ovariectomy or 17ß-estradiol treatment. Treatment with (+)-(5S,6R,7R)-2-butyl-7-[2-((2S)-2-carboxypropyl)-4-methoxyphenyl]-5-(3,4-methylenedioxyphenyl)cyclopenteno[1,2-b]pyridine-6-carboxylic acid (J-104132) (10 mg/kg/day), an ET(A)/ET(B) dual receptor antagonist, markedly decreased the neointima/media ratio of the male wild-type rats and the male and female ET(B)-deficient rats, but not the female wild-type rats. In addition, 2R-(4-propoxyphenyl)-4S-(1,3-benzodioxol-5-yl)-1-(N-(2,6-diethylphenyl)aminocarbonyl-methyl)-pyrrolidine-3R-carboxylic acid (A-192621) (30 mg/kg/day), a selective ET(B) receptor antagonist, abolished the sex difference of balloon injury-induced neointimal formation. 2R-(4-methoxyphenyl)-4S-(1,3-benzodioxol-5-yl)-1-(N,N-di(n-butyl)aminocarbonyl-methyl)-pyrrolidine-3R-carboxylic acid (ABT-627) (10 mg/kg/day), a selective ET(A) receptor antagonist, and J-104132 (10 mg/kg/day) markedly decreased the neointima/media ratio to the same extent in males but not intact females. These results indicate that the sex difference in balloon injury-induced neointimal formation was abolished by genetic ET(B) receptor deficiency or its pharmacological blockade. The lack of a vasoprotective effect of estrogen and the augmentation of ET(A) receptor-mediated action seem to be responsible for the abolition of sex differences in the ET(B) receptor-inhibited condition.

Contribution of nitric oxide in big endothelin-1-induced cardioprotective effects on ischemia/reperfusion injury in rat hearts.

We have recently shown that an appropriate amount of exogenous big endothelin-1 (ET-1) has beneficial effects on ischemia-/reperfusion-induced norepinephrine overflow and cardiac dysfunction in rat hearts and that these effects occur through a conversion to ET-1 by endothelin-converting enzyme and following stimulation of ETB receptor. In this study, we examined the possible involvement of nitric oxide (NO) in the big ET-1-induced cardioprotective effects. According to the Langendorff technique, isolated rat hearts were subjected to 40-minute global ischemia followed by 30-minute reperfusion. Exogenous big ET-1 (0.3 nM) significantly increased NOx (NO2/NO3) level in the coronary effluent after onset of reperfusion. This effect was markedly attenuated by treatment with SM-19712 (selective endothelin-converting enzyme inhibitor), A-192621 (selective ETB receptor antagonist), or NG-nitro-l-arginine (nonselective NO synthase inhibitor), respectively. In addition, N-nitro-l-arginine blunted big ET-1-induced suppression of norepinephrine overflow and improvement of cardiac dysfunction after ischemia/reperfusion. These findings suggest that NO produced by ETB receptor activation plays an important role in exogenous big ET-1-induced actions.