Jin-ichi Inokuchi

Antihypertensive substance in seeds of Areca catechu L.

Among various tannins tested, Areca II-5-C, a fraction isolated from seeds of Areca catechu L., showed the most potent angiotensin-converting enzyme (ACE) inhibitory activity in vitro. Its antihypertensive activity was therefore investigated in normotensive and spontaneous hypertensive rats (SHR) after both oral and intravenous (i.v.) administration. The activity was compared with that of captopril (D-3-mercapto-2-methylpropanoyl-L-proline), a potent ACE inhibitor. Oral administration of Areca II-5-C to SHR produced a lasting, dose-related antihypertensive effect, and the responses obtained with doses of 100 and 200 mg/kg were comparable to those of captopril at doses of 30 and 100 mg/kg. Intravenous administration of Areca II-5-C to SHR produced a rapid and marked reduction in blood pressure at doses of 10 and 15 mg/kg. The maximum antihypertensive effect of Areca II-5-C in SHR, at an i.v. dose of 15 mg/kg, was about 5 times as large as that of captopril at the same dose. Although the vasopressor response to norepinephrine and vasodepressor responses to bradykinin and acetylcholine were not appreciably changed by i.v. treatment with Areca II-5-C at a dose of 5 mg/kg, it did produce dose-related inhibition of the pressor responses to angiotensin I and II. It is suggested that Areca II-5-C has favorable properties as a hypotensive drug through its ability to inhibit the pressor responses to both angiotensin I and II.

Tripeptidyl carboxypeptidase activity of kininase II (angiotensin-converting enzyme)

The degradation of des-Arg9-brady kinin and its analogues by highly purified preparations of hog lung and kidney kininase II (angiotensin-converting enzyme; peptidyldipeptide hydrolase, EC 3.4.15.1) was studied. The degradative peptides fragments were separated and isolated by high performance liquid chromatography and identified by amino acid analysis. Both enzymes released C-terminal tripeptides from des-Arg9-bradykinin, des-Arg9-(Leu8)-bradykinin, Pro-Pro-Gly-Phe-Ser-Pro-Phe, Pro-Gly-Phe-Ser-Pro-Phe, Gly-Phe-Ser-Pro-Phe, Bz-Gly-Ser-pro-Phe and Bz-Gly-Ala-Pro-Phe. Hydrolysis of Phe-Ser-Pro-Phe, Bz-Gly-His-Pro-Phe, Bz-Gly-Phe-Pro-Phe and Bz-Gly-Gly-Pro-Phe by both enzymes was negligible. These data indicate that kininase II can release C-terminal tripeptides of substrates having a proline residue in the penultimate position such as des-Arg9-bradykinin and its analogues, and that this enzyme is able not only to act as a dipeptidyl carboxypeptidase but also acts as a tripeptidyl carboxy-peptidase. The tripeptidyl carboxypeptidase enzyme was sensitive to inhibition by kininase II inhibitors.

Rapid kidney changes resulting from glycosphingolipid depletion by treatment with a glucosyltransferase inhibitor

The ceramide analog, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, inhibits the glucosylation of ceramide and thus, by virtue of the normal catabolism of the higher glucosphingolipids, leads to a general depletion of cellular glucolipids. In a previous study with chronic administration of this inhibitor in mice, it was found that the kidneys and liver, particularly the former, grew more poorly than the organs of control mice. This study shows that the inhibitor produces rapid decreases in glucolipid concentration in kidney which are maintained for at least 5 days without noticeable harm. The changes were enhanced by inclusion of L-cycloserine in the injection scheme. Cycloserine blocks ketosphinganine synthase and thus slows the synthesis of all sphingolipids. However, sphingomyelin levels did not drop significantly in this study. The glucosyltransferase inhibitor also produced a small decrease in kidney beta-D-glucuronidase and distinct increases in the levels of glucocerebrosidase, galactocerebrosidase and sphingomyelinase. It also produced a small but distinct decrease in the level of glucosyltransferase, after a delay of a few hours, possibly because the inhibitor was metabolized to a covalently inactivating product. Comparison with kidney, liver and brain showed that the kidney was more sensitive to the action of the morpholino inhibitor.

Glucosylceramide synthetase inhibitor, d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol exhibits a novel decarcinogenic activity against Shope carcinoma cells

The glucosylceramide synthetase inhibitor, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) was tested to determine whether it could exhibit anti-tumor activity against two different Shope carcinoma cell lines. The cell growth was suppressed in a dose-dependent manner in the presence of D-PDMP. This supression seem to be accounted for by prolongation of the lag phase and this phenomenon was especially marked in the undifferentiated cell line. The growth suppression was also partly explained by direct inhibition of cell proliferation because the suppression was released by removing the agent from the medium. The treated cells became morphologically differentiated with lower density at confluence and regained contact inhibition in flask culture. Colony-forming ability in soft agar, which has been reported to be closely correlated with tumorigenicity, was also inhibited dose-dependently in the presence of D-PDMP. These results suggested that D-PDMP could exhibit a novel decarcinogenic activity against Shope carcinoma cells.

Expression of ganglioside GM3 and H-2 antigens in clones with different metastatic and growth potentials isolated from Lewis lung carcinoma (3LL) cell line

In view of the evidence that cell expression of gangliosides in several tumors is positively involved in the metastatic phenotype, Lewis lung carcinoma (3LL) cell line, expressing GM3 as the major ganglioside, was analysed for the cell surface expression of GM3. An indirect immunofluorescence assay, using a M2590 monoclonal antibody recognizing GM3, was used for this purpose. Since the parental 3LL cells consist of heterogenous subpopulations differing in the degrees of GM3 expression, we have developed clones of this cell line with different degrees of metastatic potentials by using anin vitro non-selective procedure in order to investigate whether the expression of GM3 is associated with metastatic potential. The degree of cell surface expression of GM3 among the clones correlated well with their total cellular content of this ganglioside. However, we were unable to confirm the report of increased level of GM3 in high metastatic 3LL clones, nor did a decreased level correlate with weak metastatic ability. In our recent work, an inhibitor of glucosylceramide synthase,d-threo-l-phenyl-2-decanoylamino-3-morpholino-l-propanol (DPDMP), was found to decrease the levels of all cellular glucosphingolipids and cause the accumulation of the precursors of glucosylceramide. The present study does not, however, rule out the possible involvement of this lipid family in metastatic dissemination, since treatment of 3LL cells with D-PDMP resulted in significant inhibition of their experimental metastatic potential. Clones expressing very low GM3 grew slowly in culture dishes, suggesting that GM3 may have a regulatory role in cell proliferation. The low metastatic clones expressed high levels of H-2Kb antigen, while the expression of the same antigen on the high metastatic clones was relatively low, confirming the previous observation of this tumor system. Moreover, a clone showing the lowest tumorigenic potency revealed both a high cell surface expression of H-2Kb and a high H-2Kb/H-2Db ratio.

Tripeptidyl Carboxypeptidase Activity of Angiotensin-Converting Enzyme in Human Tissues of the Urogenital Tract

The tripeptidyl carboxypeptidase activity of angiotensin-converting enzyme (EC 3.4.15.1) has been determined in several human tissues of the urogenital tract. Benzoyl-glycyl-L-seryl-L-prolyl-L-phenylalanine was used as substrate. The cleaved benzoyl-glycine was measured by means of high-performance liquid chromatography. Results obtained showed a high enzymatic activity for seminal plasma, vesicula seminalis and benign prostatic hyperplasia. Normal prostate and in particular prostatic adenocarcinoma have low enzymatic activity. The activity of the enzyme is sensitive to inhibition by the angiotensin-converting enzyme inhibitor captopril.

Sphingosine inhibits attachment of murine Lewis lung carcinoma cells to laminin and type IV collagen

The effect of sphingosine (SPH) on the adhesive properties of Lewis lung carcinoma (3LL) cells was investigated using plastic precoated with the extracellular matrix proteins, laminin, fibronectin, or type IV collagen. Treatment of 3LL cells with SPH (0.5–10 μM) resulted in a dose‐dependent decrease in the ability to bind to laminin and type IV collagen but had little or no effect on atachment to fibronectin. Phorbol 12‐myristate 13‐acetate (PMA) selectively enhanced attachment of 3LL cells to laminin and collagen. The inhibitory effect of SPH on attachment to both proteins was competitively antagonized by PMA. These results suggest that SPH acts as a negative effector for cell attachment to laminin and collagen, and that the cell attachment process to both proteins might be regulated in part by protein kinase C.

Tripeptidylcarboxypeptidase activity of angiotensin I converting enzyme in human serum.

Fig. 2 Correlation between serum viscosity and the decrease in serum sodium when measured by filme photometry. sodium measurement. When direct potentiometric methods are used, the presence of hyperviscosity does not significantly influence the serum sodium value. With measurements using flame photometry and indirect potentiometry, pseudohyponatremia occurs. With indirect potentiometric measurements, a correction factor which can be used is a 2 mmolI decrease in serum sodium for every centistoke increase in serum viscosity. This is linear, at least in our study to a serum viscosity of approximately 20 centistokes. With flame photometric measurements of serum sodium, no such correction factor can be meaningfully applied. For example,