Shiro Morimoto

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.

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.

Phosphoramidon inhibits the generation of endothelin-1 from exogenously applied big endothelin-1 in cultured vascular endothelial cells and smooth muscle cells

When cultured porcine aortic endothelial cells (ECs) were incubated with porcine big endothelin‐1 (bit ET‐I1–39), there was a time‐dependent increase in immunoreactive (IR)‐ET in the culture supernatant, in addition to an endogenous IR‐ET release fron the cells. Reverse‐phase HPLC of the culture supernatant revealed one major IR‐ET component corresponding to the elution position of synthetic ET‐1, thereby indicating that the additional increase in IR‐ET was due to the conversion of big ET‐1 to mature ET‐11–21. Phosphoramidon, a metalloproteinase inhibitor, strongly suppressed this increase in IR‐ET as well as the endogenous IR‐ET release. Cultured vascular smooth muscle cells (VSMCs) also released IR‐ET. The apparent conversion of exogenously applied big ET‐1 to ET‐1 and its inhibition by phosphoramidon were observed using cultured VSMCs, although the enzyme inhibitor did not influence the basal secretion of IR‐ET from VSMCs. These results suggest that both cultured ECs and VSMCs can generate ET‐1 from exogenously applied big ET‐1 via action of the same type of phosphoramidon‐sensitive metalloproteinase, which is also involved in the endogenous ET‐1 generation in ECs.

The endothelium-derived vasoconstrictor peptide endothelin inhibits renin release in vitro

The effect of endothelin, a newly identified endothelium-derived vasoconstrictor peptide, on renin release from rat kidney cortical slices was examined. Endothelin produced a concentration-dependent inhibition of renin release and this inhibitory effect was dependent on extracellular calcium. The dihydropyridine calcium channel blockers nifedipine and nicardipine did not antagonize the inhibitory effect induced by endothelin. On the other hand, nifedipine completely antagonized the extracellular high potassium- or Bay K 8644-induced inhibition of renin release. The endothelin-induced inhibition of the release was markedly blocked by the addition of Co2+. Similar blocking effects of Co2+ were also observed with extracellular high potassium or Bay K 8644. Thus, endothelin exerts an inhibitory action on renin release in vitro, in a calcium-dependent manner. This inhibition may be mediated by the increased calcium influx through dihydropyridine-insensitive calcium channels.

Evidence for pepstatin-sensitive conversion of porcine big endothelin-1 to endothelin-1 by the endothelial cell extract

We designed a radioimmunoassay (RIA) specific for the C-terminal fragment (CTF, 22-39) of porcine big endothelin-1 (big ET-1, 1-39), and investigated whether the generation of ET-1 (1-21) is concomitant with that of the CTF during incubation of big ET-1 with the endothelial cell (EC)-extract. When the incubation mixture was applied to reverse-phase high performance liquid chromatography coupled with the RIAs for CTF and ET, immunoreactive (IR)-CTF eluted as one major and one minor peak, and IR-ET as one major peak. The retention times of each peak corresponded to those of synthetic CTF, big ET-1 and ET-1, respectively. An aspartic protease inhibitor pepstatin-A completely inhibited the generation of CTF- and ET-1-like materials. Both materials were also detected in culture medium of porcine aortic ECs. These findings strongly suggest that ET-1 is generated from big ET-1 via a single cleavage between Trp21 and Val22 in vascular ECs and that a pepstatin-sensitive aspartic protease is a possible candidate for big ET-1 converting enzyme.

Conversion of big endothelin-1 to endothelin-1 by two-types of metalloproteinases of cultured porcine vascular smooth muscle cells

Incubation of big endothelin-1 (big ET-1, 1-39) with the membrane fraction obtained from cultured vascular smooth muscle cells (VSMCs) resulted in an increase in immunoreactive-ET (IR-ET), which was inhibited by EDTA but not by phosphoramidon, a metalloproteinase inhibitor. When the incubation was performed in the presence of N-ethylmaleimide (NEM), the generation of IR-ET was markedly augmented and this augmentation was abolished by phosphoramidon. The pH profile for IR-ET generation in the presence of NEM was apparently distinct from that observed in the absence of NEM. Reverse-phase HPLC of the incubation mixture with or without NEM revealed one major IR-ET component corresponding to the elution position of synthetic ET-1 (1-21). When the cultured VSMCs were incubated with big ET-1, a conversion to the mature ET-1 was observed. This ET-1 generation from exogenously applied big ET-1 was markedly inhibited by the addition of phosphoramidon, although the inhibitor did not influence the basal secretion of ET-1-like materials. These results suggest the presence of two types of metalloproteinases, which can generate ET-1, in VSMCs. The possibility that ET-1 functions in an autocrine manner to control the cardiovascular system warrants further attention.