Masanori Takaoka

Phosphoramidon, a metalloproteinase inhibitor, suppresses the hypertensive effect of big endothelin-1

Intravenous (i.v.) injection of big endothelin-1 (1-39, 0.05-1.0 nmol/kg) to anaesthetized rats produced a dose-dependent and long-lasting hypertensive effect, the magnitude of which was similar to that evoked by ET-1 (1-21). In animals given phosphoramidon (0.25 mg/kg per min i.v.), a metalloproteinase inhibitor, the hypertensive effect of big endothelin-1 was markedly attenuated. The same dose of phosphoramidon did not influence the endothelin-1-induced hypertensive effect. The possibility that big endothelin-1 is converted to endothelin-1 by a phosphoramidon-sensitive metalloproteinase in vivo warrants further attention.

Phosphoramidon, a metalloproteinase inhibitor, suppresses the secretion of endothelin-1 from cultured endothelial cells by inhibiting a big endothelin-1 converting enzyme

Time-dependent secretion of immunoreactive-endothelin (IR-ET) from cultured porcine aortic endothelial cells was markedly suppressed by phosphoramidon is due to proteinase inhibitor. Analysis of the culture supernatant with or without phosphoramidon by reverse-phase high performance liquid chromatography confirmed that the suppression of IR-ET secretion by phosphoramidon is due to a decreae in secretion of endothelin-1-like materials. The secretion of the C-terminal fragment (CTF, 22-39)-like materials of big ET-1 was also decreased by phosphoramidon, whereas there was an increased secretion of big ET-1-like materials. These data strongly suggest that phosphoramidon suppresses the secretion of ET-1 from cultured endothelial cells by inhibiting the conversion of big ET-1 to ET-1. It is most likely that phosphoramidon-sensitive metalloproteinase is responsible for the processing of big ET-1 in vascular endothelial cells.

Phosphoramidon prevents cerebral vasospasm following subarachnoid hemorrhage in dogs: the relationship to endothelin-1 levels in the cerebrospinal fluid.

There is increasing evidence that the conversion of big endothelin-1 (big ET-1) to endothelin-1 (ET-1) is specifically inhibited by the metalloproteinase inhibitor phosphoramidon. We investigated the effect of phosphoramidon on delayed cerebral vasospasm from subarachnoid hemorrhage (SAH) using a two-hemorrhage canine model. The magnitude of the vasospasm and the drug effect were determined angiographically. On SAH Day 7, diameter of the basilar artery decreased to about 55% of the control value obtained before SAH (on Day 0). Immunoreactive ET (IR-ET) in the cerebrospinal fluid (CSF) significantly increased after SAH (on Day 7). The intracisternal pretreatment of phosphoramidon potently suppressed the decrease in diameter of the basilar artery after SAH, i.e., observed decrease was only about 20%, compared with the value before SAH. In the phosphoramidon group, IR-ET in CSF markedly increased (on SAH Day 2), but the increased levels of IR-ET significantly declined on SAH Day 7. These results clearly indicate that phosphoramidon effectively prevents delayed cerebral vasospasm. Whether the prevention is due to the inhibition of conversion of big ET-1 to ET-1 is now under study.

Different Contributions of Endothelin-A and Endothelin-B Receptors in the Pathogenesis of Deoxycorticosterone Acetate–Salt–Induced Hypertension in Rats

We investigated the involvement of actions mediated by endothelin-A (ETA) and endothelin-B (ETB) receptors in the pathogenesis of deoxycorticosterone acetate (DOCA)-salt-induced hypertension in rats. Two weeks after the start of DOCA-salt treatment, rats were given ABT-627 (10 [mg/kg]/d), a selective ETA receptor antagonist; A-192621 (30 [mg/kg]/d), a selective ETB receptor antagonist; or their vehicle for 2 weeks. Uninephrectomized rats without DOCA-salt treatment served as controls. Treatment with DOCA and salt for 2 weeks led to a mild but significant hypertension; in vehicle-treated DOCA-salt rats, systolic blood pressure increased markedly after 3 to 4 weeks. Daily administration of ABT-627 for 2 weeks almost abolished any further increases in blood pressure, whereas A-192621 did not affect the development of DOCA-salt-induced hypertension. When the degree of vascular hypertrophy of the aorta was histochemically evaluated at 4 weeks, there were significant increases in wall thickness, wall area, and wall-to-lumen ratio in vehicle-treated DOCA-salt rats compared with uninephrectomized control rats. The development of vascular hypertrophy was markedly suppressed by ABT-627. In contrast, treatment with A-192621 significantly exaggerated these vascular changes. In vehicle-treated DOCA-salt rats, renal blood flow and creatinine clearance decreased, and urinary excretion of protein, blood urea nitrogen, fractional excretion of sodium, and urinary N-acetyl-beta-glucosaminidase activity increased. Such damage was overcome by treatment with ABT-627 but not with A-192621; indeed, the latter agent led to worsening of the renal dysfunction. Histopathologic examination of the kidney in vehicle-treated DOCA-salt rats revealed tubular dilatation and atrophy as well as thickening of small arteries. Such damage was reduced in animals given ABT-627, whereas more severe histopathologic changes were observed in A-192621-treated animals. These results strongly support the view that ETA receptor-mediated action plays an important role in the pathogenesis of DOCA-salt-induced hypertension. On the other hand, it seems likely that the ETB receptor-mediated action protects against vascular and renal injuries in this model of hypertension. A selective ETA receptor antagonist is likely to be useful for treatment of subjects with mineralocorticoid-dependent hypertension, whereas ETB-selective antagonism alone is detrimental to such cases.

Conversion of big endothelin-1 to endothelin-1 by two types of metalloproteinases derived from porcine aortic endothelial cells

Incubation of big endothelin‐1 (big ET‐11–39) with either the cytosolic or membrane fraction obtained from cultured endothelial cells, resulted in an increase in immunoreactive‐endothelin (IR‐ET), which was markedly inhibited by metal chelators. Phosphoramidon, a metalloproteinase inhibitor, specifically suppressed the membrane fraction‐induced increase in IR‐ET, whereas the increase in IR‐ET observed with the cytosolic fraction was not influenced by phosphoramidon. Reverse‐phase (RP)‐HPLC of the incubation mixture of big ET‐1 with the cytosolic or membrane fraction revealed one major IR‐ET component corresponding to the elution position of synthetic ET‐11–21. Simultaneously, immunoreactivities like the C‐terminal fragment (CTF22–39) of big ET‐1 were present, as deduced from the RP‐HPLC coupled with the radioimmunoassay for CTF. Our results indicate the presence of two types of metalloproteinases, which convert big ET‐1 to ET‐1 via a single cleavage between Trp21 and Val22, in vascular endothelial cells.

Conversion of porcine big endothelin to endothelin by an extract from the porcine aortic endothelial cells

Conversion of porcine big endothelin (big ET) to endothelin (ET) by an extract from cultured porcine aortic endothelial cells was investigated using a radioimmunoassay (RIA) specific for ET and reverse-phase high performance liquid chromatography (RP-HPLC). When big ET was incubated with the extract at an acid pH in the presence of E-64, a cysteine protease inhibitor, the amount of immunoreactive-ET (IR-ET) in the incubation mixture was greatly increased and the optimum pH for this increased reaction was 4.0. The extract-induced increase in IR-ET was completely inhibited by pepstatin-A. These immunoreactive alterations correlated with those in the vasoconstrictor activity. When the incubation mixture of big ET with the cell extract was applied to the RP-HPLC, the IR-ET eluted at the same retention time as seen with synthetic porcine ET. We suggest that a pepstatin-sensitive aspartic protease is involved in the conversion of big ET to ET in vascular endothelial cells.

Role of endothelin-1 and the ETA receptor in the maintenance of deoxycorticosterone acetate-salt-induced hypertension.

1 To search for a possible role for endothelin‐1 (ET‐1) in deoxycorticosterone acetate (DOCA)‐salt‐induced hypertension, we examined changes in concentration of ET‐1 in vascular and renal tissue in DOCA‐salt hypertensive rats and evaluated the antihypertensive effect of the ETA receptor antagonist, FR139317. 2 There was an increase in aortic immunoreactive‐ET (IR‐ET) concentrations in association with hypertension‐induced treatment. There were no significant changes in ET‐1 levels in the kidney with DOCA‐salt treatment. 3 In DOCA‐salt hypertensive rats, a significant correlation (r = 0.83, P <0.01) was found between aortic IR‐ET concentrations and systolic blood pressure. 4 High‐performance liquid chromatography analysis of the aortic extract from DOCA‐salt rats revealed one major component corresponding to the elution position of synthetic ET‐1. 5 The intravenous bolus injection of FR139317 (10 mg kg−1) produced a slight decrease in blood pressure in the control rats and in the DOCA‐salt hypertensive rat, FR139317 had a more pronounced hypotensive effect. 6 We propose that ET‐1 production in vascular tissues is increased in DOCA‐salt hypertensive rats. In addition, our study indicates the pathophysiological importance of increased endogenous ET‐1 in the maintenance of DOCA‐salt‐induced hypertension, through interaction of the peptide with ETA receptors.

Antihypertensive Effect of Angiotensin I-Converting Enzyme Inhibitory Peptides from a Sesame Protein Hydrolysate in Spontaneously Hypertensive Rats

Sesame peptide powder (SPP) exhibited angiotensin I-converting enzyme (ACE) inhibitory activity, and significantly and temporarily decreased the systolic blood pressure (SBP) in spontaneously hypertensive rats (SHRs) by a single administration (1 and 10 mg/kg). Six peptide ACE inhibitors were isolated and identified from SPP. The representative peptides, Leu-Val-Tyr, Leu-Gln-Pro and Leu-Lys-Tyr, could competitively inhibit ACE activity at respective Ki values of 0.92 μM, 0.50 μM, and 0.48 μM. A reconstituted sesame peptide mixture of Leu-Ser-Ala, Leu-Gln-Pro, Leu-Lys-Tyr, Ile-Val-Tyr, Val-Ile-Tyr, Leu-Val-Tyr, and Met-Leu-Pro-Ala-Tyr according to their content ratio in SPP showed a strong antihypertensive effect on SHR at doses of 3.63 and 36.3 μg/kg, which accounted for more than 70% of the corresponding dosage for the SPP-induced hypotensive effect. Repeated oral administration of SPP also lowered both SBP and the aortic ACE activity in SHR. These results demonstrate that SPP would be a beneficial ingredient for preventing and providing therapy against hypertension and its related diseases.

Exaggerated Vascular and Renal Pathology in Endothelin-B Receptor–Deficient Rats With Deoxycorticosterone Acetate–Salt Hypertension

BackgroundEndothelin (ET)-1 plays an important role in the pathogenesis of deoxycorticosterone acetate (DOCA)-salt–induced hypertension. We evaluated the pathological role of ETB receptors in DOCA-salt–induced hypertension, cardiovascular hypertrophy, and renal damage by using the spotting-lethal (sl) rat, which carries a naturally occurring deletion in the ETB receptor gene. Methods and ResultsHomozygous (sl/ sl) rats exhibit abnormal development of neural crest–derived epidermal melanocytes and the enteric nervous system, and they do not live beyond 1 month because of intestinal aganglionosis and intestinal obstruction. The dopamine &bgr;-hydroxylase (D&bgr;H) promoter was used to direct ETB transgene expression in sl/ sl rats to support normal enteric nervous system development. D&bgr;H-ETBsl/sl rats live into adulthood and are healthy, expressing ETB receptors in adrenal glands and other adrenergic neurons. When homozygous (sl/ sl) and wild-type (+/+) rats, all of which were transgenic, were treated with DOCA-salt, homozygous rats exhibited earlier and higher increases in systolic blood pressure than did wild-type rats. Chronic treatment with ABT-627, an ETA receptor antagonist, completely suppressed DOCA-salt–induced hypertension in both groups. Renal dysfunction and histological damage were more severe in homozygous than in wild-type rats. Marked vascular hypertrophy was observed in homozygous rats than in wild-type rats. Renal and vascular injuries were significantly improved by ABT-627. In DOCA-salt–treated homozygous rats, there were notable increases in renal, urinary, and aortic ET-1, all of which were normalized by ABT-627. ConclusionsETB-mediated actions are protective in the pathogenesis of DOCA-salt–induced hypertension. Enhanced ET-1 production and ETA-mediated actions are responsible for the increased susceptibility to DOCA-salt hypertension and tissue injuries in ETB receptor–deficient rats.

Role of nitric oxide in the renal protective effects of ischemic preconditioning.

&NA; Possible involvement of nitric oxide (NO) in the protective effect of ischemic preconditioning against the ischemia/reperfusion‐induced acute renal failure was investigated. Ischemic preconditioning, which consists of three cycles of 2‐minute ischemia followed by 5‐minute reperfusion, was performed prior to 45‐minute ischemia. Ischemic preconditioning significantly improved the renal dysfunction induced by 45‐minute ischemia followed by 24‐hour reperfusion. Histopathological examination of the kidney of ischemia/reperfusion rats revealed severe renal damage, and suppression of the damage was seen with the ischemic preconditioning treatment. NO metabolites (NOx) production in the kidney after 45‐minute ischemia and reperfusion was markedly increased in ischemia/reperfusion rats with ischemic preconditioning, compared with animals not subjected to ischemic preconditioning, and these increases correlated with changes in endothelial NO synthase (eNOS) protein expression in renal tissues. The improvement of renal dysfunction in ischemic preconditioning rats was abolished by the pretreatment with NG‐nitro‐L‐arginine, a nonselective NOS inhibitor, but not with aminoguanidine, an inducible NOS inhibitor. In addition, increment of endothelin‐1 (ET‐1) content in the kidney after the reperfusion was markedly suppressed by ischemic preconditioning treatment. These findings suggest that the protective effect of ischemic preconditioning on ischemia/reperfusion ‐induced acute renal failure is closely related to the renal nitric oxide production following the increase in eNOS expression after the reperfusion and that the suppressive effect of ischemic preconditioning on the ischemia/reperfusion ‐induced renal ET‐1 overproduction may be partly involved in the ameliorating effect of ischemic preconditioning.