Kazuhiro Hisaki

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.

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.

Effects of endothelin on renal function and renin secretion in anesthetized rats

The infusion of endothelin into the renal artery of anesthetized rats at 1 ng/kg per min, a dose with no influence on renal function, had no effect on the basal levels of plasma renin activity (PRA) and renin secretory rate (RSR), but significantly inhibited the isoproterenol-induced increases in PRA and RSR. Endothelin, 2 ng/kg per min, elicited significant decreases in renal hemodynamics and in urine formation but the mean arterial pressure was not affected. The possibility that endothelin may participate in controlling renal function and renin secretion in vivo warrants further attention.

Endothelium-independent pressor effect of big endothelin-1 and its inhibition by phosphoramidon in rat mesenteric artery.

We asked whether or not the endothelium plays a functional role in the conversion of big endothelin-1 to endothelin-1 in the perfused rat mesenteric artery. In endothelium-denuded preparations, big endothelin-1 produced a much more potent pressor effect than in intact preparations. Phosphoramidon suppressed the big endothelin-1-induced pressor action without affecting the action of endothelin-1, irrespective of the presence or absence of the endothelium. The amounts of immunoreactive-endothelin in the perfusate during perfusion of endothelium-denuded preparations with big endothelin-1 were extremely low compared with those observed in intact preparations and were not significantly suppressed by the metalloproteinase inhibitor, phosphoramidon, in contrast to the case with intact preparations. When synthetic endothelin-1 was perfused in the endothelium-denuded mesentery, the peptide disappeared from the perfusate more rapidly than with intact preparations, suggesting that endothelin-1 generated from big endothelin-1 is effectively trapped by vascular smooth muscle cells in the endothelium-denuded preparation. Our results suggest that the endothelium is not essential for the conversion of big endothelin-1 to endothelin-1, in rat mesenteric artery.

Evidence for phosphoramidon-sensitive conversion of big endothelin-1 to endothelin-1 in isolated rat mesenteric artery.

The effect of phosphoramidon, a metalloproteinase inhibitor, on the pressor response to big endothelin-1 (big ET-1) in the isolated perfused rat mesenteric artery was examined. Big ET-1 (10(-9)-10(-7) M) caused a concentration-dependent increase in the perfusion pressure. The pressor response was markedly suppressed by phosphoramidon (10(-5) M). The inhibitor did not influence the ET-1 (5 X 10(-11)-10(-8) M)-induced pressor action. Big ET-1 (5 X 10(-8) M)-induced pressor action was accompanied by an increase in immunoreactive (IR)-ET in the perfusate, and this increase was suppressed by the addition of phosphoramidon. IR-ET in the perfusate was confirmed to be ET-1, as determined by reverse-phase HPLC. These findings strongly suggest that phosphoramidon-sensitive metalloproteinase contributes to the conversion of big ET-1 to ET-1, in vivo.

Phosphoramidon-sensitive conversion of big endothelin-1 and degradation of endothelin-1 in rat kidney.

We investigated the intrarenal conversion of big endothelin-1 (ET-1) to ET-1 in the isolated perfused rat kidney. Big ET-1 caused a concentration-dependent increase in perfusion pressure, and the pressor molar potency of the peptide was 50-fold less than that of ET-1. The big ET-1 (2 x 10(-8) mol/L)-induced pressor action was accompanied by increases in immunoreactive endothelin levels in both the perfusate and renal tissues. Phosphoramidon (10(-4) mol/L), a metalloproteinase inhibitor, significantly suppressed the big ET-1-induced pressor action and the accumulation of immunoreactive endothelin in renal tissues. On the other hand, phosphoramidon slightly but significantly sustained the ET-1-induced pressor effect. The effect of kelatorphan (10(-4) mol/L), a specific inhibitor of neutral endopeptidase 24.11, on the ET-1-induced pressor effect was the same as that seen with phosphoramidon. When ET-1 was exogenously added to the perfusate, phosphoramidon or kelatorphan significantly increased the immunoreactive endothelin levels in renal tissues after perfusion, without affecting the disappearance rate of immunoreactive endothelin from the perfusate. Therefore, the phosphoramidon-sensitive ET-1-converting enzyme in the kidney seems to contribute to the functional local conversion of big ET-1 to ET-1, and neutral endopeptidase 24.11 may be responsible for the proteolytic degradation of ET-1 in the kidney. In addition, immunoreactive endothelin levels in renal tissues but not in the perfusate can account for the functional conversion of big ET-1 to ET-1 and for the local proteolytic degradation of ET-1 in the kidney.

Differences in potency of big endothelin-1-induced pressor action in rat isolated perfused mesenteric artery, hindquarter and lung

We compared the pressor response to endothelin-1 (ET-1) with that of big endothelin-1 (big ET-1) in mesenteric arteries, hindquarters and lungs of rats. In these three preparations, both peptides caused a concentration-dependent increase in the perfusion pressure. The ratio of big ET-1 concentration to ET-1 concentration needed for causing the same pressor action is different between organs; i.e., a mesentery >> a hindquarter > or = a lung. Exposure to phosphoramidon, a metalloproteinase inhibitor, significantly suppressed the pressor response to big ET-1, in a similar fashion. This suppression is likely to be due to the inhibition of phosphoramidon-sensitive endothelin converting enzyme, since the inhibitor does not suppress an action of ET-1. Apparently there is a difference in potency for phosphoramidon-sensitive vasoconstriction of big ET-1 between organs and presumably regional differences in the functional phosphoramidon-sensitive conversion of big ET-1 in vasculatures.

Conversion of big endothelin-1 to endothelin-1 by phosphoramidon-sensitive metalloproteinase derived from aortic endothelial cells.

When big endothelin-1 (big ET-1, 1-39) was incubated with the membrane fraction obtained from cultured endothelial cells (ECs) at pH 7.0 for 6 h, the immunoreactive (ir) ET in the reaction mixture was markedly increased. Phosphoramidon, a metalloproteinase inhibitor, as well as metal chelators specifically suppressed the above increase. Using reverse-phase high-performance liquid chromatography, ir-ET was confirmed to be ET-1[1-21]. In addition, we noted that the alterations in ET-1 correlated with those in the C-terminal fragment (CTF, 22-39) of big ET-1. When cultured ECs were incubated with phosphoramidon, time-dependent secretion of ET-1 and CTF from the cells was markedly suppressed. In contrast, the secretion of big ET-1 was increased by phosphoramidon. Thiorphan, a specific inhibitor of neutral endopeptidase 24.11, was without effect on the secretion of ET-related peptides. Moreover, phosphoramidon potently inhibited the hypertensive effect of big ET-1 without affecting the ET-1-induced hypertension in anesthetized rats. From these findings, it seems reasonable to consider that phosphoramidon-sensitive and membrane-bound metalloproteinase, which is not a neutral endopeptidase 24.11, is the most plausible candidate for big ET-1-converting enzyme in vivo.

Intrarenal conversion of big endothelin-1 to endothelin-1 in the rat

Intravenous (i.v.) or intrarenal arterial (i.r.a.) injection of big endothelin-1 (big ET-1) (1.6 nmol/kg) in anesthetized rats produced a significant increase in mean arterial pressure (MAP), a decrease in renal blood flow (RBF) and an increase in renal vascular resistance (RVR). Although the pressor responses to big ET-1 of i.v. and i.r.a. injection were not significantly different, i.r.a. injection of big ET-1 caused a significantly greater reduction in RBF than that seen with i.v. injection of big ET-1. The effects of i.r.a. injection of the peptide on MAP and RBF were markedly suppressed by phosphoramidon. Big ET-1 caused dose-dependent pressor effects in isolated perfused rat kidney and these pressor effects were significantly suppressed by phosphoramidon. Thus, renal vasculature possesses a phosphoramidon-sensitive endothelin converting enzyme, which may play an important role for the renal hemodynamic regulation.