Inhibition of experimental metastasis of murine Lewis lung carcinoma by an inhibitor of glucosylceramide synthase and its possible mechanism of action.

In view of the increasing evidence that glucosphingolipids (GSLs) on tumor cell surfaces play an important role in tumor metastasis, an inhibitor of glucosylceramide synthase, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) was used to evaluate the role of GSLs in this respect. Treatment of Lewis lung carcinoma cells with 5 microM D-PDMP resulted in a time-dependent marked decrease in levels of all cellular GSLs (glucosylceramide, lactosylceramide, ceramide trihexoside, globoside, and ganglioside GM3). By 6 days, the total GSL content was reduced to approximately 20% of the level in the untreated control cells and at the same time the lung-colonizing capacity of the PDMP-treated cells in inoculated mice was greatly reduced. Closely associated with the degree of GSL depletion, the ability of the cells to invade reconstituted basement membranes in vitro was also reduced, suggesting that GSLs in tumor cell membranes modulate the cell surface interaction with basement membrane components. In order to assess a possible contribution of the defective capacities to drug-induced suppression of experimental metastasis and invasion, we tested the effect of D-PDMP on attachment and migration to laminin and fibronectin and found that the inhibitor specifically reduced the laminin-mediated attachment and migration, whereas it had no effect on fibronectin-mediated attachment and migration. These effects of the inhibitor on lung colonizing capacity in vivo and the invasion, adhesion, and migration properties of the cells in vitro were reversible within 24 h after removal of the drug. By contrast, L-PDMP (the enantiomeric form of D-PDMP), which has no inhibitory activity on glucosylceramide synthesis, did not cause any of the changes produced by D-PDMP. Together, these results suggest that GSLs in tumor cell membranes are essential for the metastatic spread of tumor cells through basement membranes, modulating the interaction of laminin and its receptors.

An aminopeptidase activity from porcine kidney that hydrolyzes oxytocin and vasopressin: purification and partial characterization.

An aminopeptidase from porcine kidney, hydrolyzing oxytocin and vasopressin in vitro, was purified by chromatography on hydroxyapatite, DEAE-cellulose and nickel ion chelate gel and gel filtration on Sephadex G-100. The enzyme appeared to be a high molecular mass (M(r) 105,000) monomeric protein. It was sensitive to inhibition by metal chelator, o-phenanthroline. Cobalt ion and sulfhydryl activator, 2-mercaptoethanol, had activating effects, while p-chloromercuribenzoate, amino acids with large hydrophobic side chains, L-cystine and aminopeptidase inhibitors, bestatin and amastatin, had inhibitory effects on the enzyme activity. The enzyme hydrolyzed several aminoacyl p-nitroanilides, and had the highest specificity against S-benzyl-L-cysteine p-nitroanilide. The properties of the enzyme were distinct from those of well-characterized leucyl aminopeptidase (EC 3.4.11.1), membrane alanyl aminopeptidase (EC 3.4.11.2) and primate placental cystinyl aminopeptidase (EC 3.4.11.3).

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.

Cytochrome P-450 in liver microsomes of streptozotocin-induced diabetic rats: purification and characterization.

Two diabetes-inducible forms of cytochrome P-450, named P-450ST-1 and -ST-2, were purified from the liver microsomes of streptozotocin-diabetic male rats by sodium cholate solubilization, octylamino-Sepharose 4B chromatography and high-performance liquid chromatography with DEAE-5PW and hydroxyapatite columns. The purified P-450 forms gave a single band each on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular weight of 48,500 for P-450ST-1 or 48,000 for P-450ST-2. The CO-reduced spectral maxima of P-450ST-1 and -ST-2 were at 451 nm. The two cytochromes had the low-spin state of heme in the oxidized form. Both P-450ST-1 and -ST-2 catalyzed the metabolism of aniline, benzphetamine, p-nitroanisole, testosterone and aminopyrine. However, the catalytic activity of P-450ST-2 for these substrates was apparently higher than that of ST-1. Analyses of the NH2-terminal amino-acid sequence and Western immunoblot showed that P-450ST-1 and -ST-2 differed structurally from each other. The catalytic activities, molecular weights, NH2-terminal sequences and/or immunochemical properties of P-450ST-1 and -ST-2 did not agree with those of the other cytochrome P-450 forms purified from diabetic rats previously.

Oversulfated fucoidan and heparin suppress endotoxin induction of plasminogen activator inhibitor-1 in cultured human endothelial cells: their possible mechanism of action.

Plasminogen activator inhibitor-1 (PAI-1) is a primary endogenous inhibitor of tissue-type plasminogen activator (t-PA). In this study, we examined the effects of oversulfated fucoidan (OSF) derivatives and heparin on lipopolysaccharide (LPS)-induced release of PAI-1 antigen from cultured human umbilical vein endothelial cells (HUVEC). Addition of LPS (10 micrograms/ml) enhanced the release of PAI-1 by HUVEC but not of t-PA antigen. At 18 h, a 2.4-fold increase in the extracellular PAI-1 level was observed. The increased PAI-1 level was reduced to control level by the simultaneous addition of 10 micrograms/ml of OSF or heparin. The suppressive effect of native fucoidan was negligible. We also examined the molecular size effect of OSF, using 10-20, 20-40, and 40-60 kDa fragments. The result indicated that these fragments were effective as well as the 100-130 kDa form of OSF, hence suggesting an important role of the degree of sulfation. Interleukin-1 beta (IL-1 beta) is a potent inducer of PAI-1 in cultured HUVEC. Heparin, OSF, and its fragments did not suppress the IL-1 beta-induced release of PAI-1 antigen. Treatment of HUVEC with heparitinase or monoclonal antibody against heparin sulfate proteoglycan (HSPG) resulted in a complete loss of its ability to enhance PAI-1 release in response to LPS stimulation, while the chondroitinase ABC treatment hardly affected the PAI-1 production. These results suggest that HSPG is involved in the initial binding of LPS to HUVEC. The suppressive effects of OSF and heparin on LPS-induced PAI-1 release may result from the inhibition of LPS binding to the cell surface HSPG.

Further characterization of the binding of plasminogen to heparin: evidence for the involvement of lysine residues.

We have previously demonstrated that the heparin-binding site of plasminogen is located in Val442-plasminogen region (kringle 5 domain plus light (B) chain) (Soeda, S., Kakiki, M., Shimeno, H., and Nagamatsu, A. (1987) Biochim. Biophys. Acta 916, 279-287). The chemical modification of Val442-plasminogen with a lysine reagent, pyridoxal 5'-phosphate (PLP), and sodium borohydride resulted in the incorporation of 8-10 PLP moieties per molecule of the zymogen. This PLP-labeled zymogen had no affinity for a heparin-Sepharose column, whereas the non-labeled one bound to the column. Modification in the presence of heparin decreased the extent of labeling by 1-2 mol of PLP per mol of Val442-plasminogen. To further examine the binding site of plasminogen to heparin, functionally active A and B chains were separated from Lys-plasmin after mild reduction and S-carboxymethylation. Only B chain possessed affinity for heparin-Sepharose. Furthermore, plasmin(ogen) bound to heparin was protected from alpha 2-antiplasmin inhibition. These results indicate that one or two lysine residues located in the catalytic region (B chain) of plasmin(ogen) are essential to heparin binding, and that the binding of plasminogen to heparin or heparin-like substance in extracellular matrix environments may be important for the localization and activation of plasminogen and for the prolongation of the resultant plasmin activity.

Localization of the binding sites of porcine tissue-type plasminogen activator and plasminogen to heparin.

To localize the binding region of porcine tissue-type plasminogen activator (EC 3.4.21.31) (t-plasminogen activator) to heparin, functionally active A and B chains (molecular mass of each 33 kDa) were separated from the two-chain t-plasminogen activator after mild reduction and alkylation. The A chain bound to fibrin-Sepharose, but not to heparin-Sepharose. In contrast, the B chain showed amidase activity toward HD-Ile-Pro-Arg-p-nitroanilide (S-2288) and a high affinity for heparin-Sepharose, but no affinity for fibrin-Sepharose. Plasminogen activator activity of the B chain was stimulated by heparin (about 3-fold), but not by fibrin. On the other hand, the elastase digestion fragments of plasminogen, kringle 1-3 and kringle 4, had no affinity for a heparin-Sepharose column, whereas the other fragment, Val442-plasminogen, efficiently bound to the column and was eluted with 1.6 M KSCN-containing buffer. The stimulatory effect of fibrin on two-chain t-plasminogen activator-catalyzed Val442-plasminogen activation was clearly diminished by heparin. These results suggest that heparin can form a complex with both t-plasminogen activator and plasminogen molecules through their catalytic regions located in each B chain, and that the heparin connection between t-plasminogen activator and plasminogen may improve the plasminogen activation kinetics by making a situation in which t-plasminogen activator is easily approachable to plasminogen.

Inhibition of proline endopeptidase activity by acyl-coenzyme A esters.

Coenzyme A (CoA), its related compounds and acylcarnitine non-competitively inhibited the activity of proline endopeptidase (PEPase) purified from rat liver cytosol. The degree of inhibition was in the order of acyl-CoA greater than CoA greater than dephospho-CoA greater than or equal to acylcarnitine. However, carnitine did not inhibit the enzyme activity. Among the compounds examined, n-decanoyl-CoA showed the highest inhibitory activity (Ki = 9 microM). These results suggest that both the acyl group and CoA contribute to the inhibition of PEPase by acyl-CoA. The abilities of n-decanoyl-CoA and its related compounds to quench the intrinsic fluorescence at 332 nm from PEPase excited at 280 nm, was used as a probe for the binding affinity of the enzyme for these compounds. The quenching of fluorescence by CoA was nearly equal to that by n-decanoyl-CoA. n-Decanoylcarnitine and carnitine were unable to quench the fluorescence. These results indicate that n-decanoyl-CoA at least binds to PEPase through its CoA portion.

Regulation of prolyl oligopeptidase activity in regenerating rat liver.

We have previously shown that the naturally occurring polyamines, spermidine and spermine, reverse effectively the in vitro inhibition of prolyl oligopeptidase (POPase) by its endogenous inhibitor by forming a kinetically significant complex (Soeda et al., J. Neurochem. (1986) 46, 1304-1307). In this study, we examined changes in the activities of POPase and its endogenous inhibitor and in the concentrations of polyamines during the regeneration of rat liver. POPase activity in the liver cytosol peaked 2 days after partial hepatectomy and then decreased near to control activity by 9 days, without its altered synthetic levels. Total polyamine concentrations also peaked at 2 days and remained elevated by 9 days, while cytosolic POPase inhibitor activity was minimal (56% of control) at 2 days. Treatment of the animals with a synthetic POPase inhibitor, Z-Gly-Pro-CHN2 (4 mg/kg), resulted in an obvious suppression of the liver regeneration. These results imply that the activity of POPase involved in nonlysosomal proteolytic pathway is exquisitely regulated by changes not only in its endogenous inhibitor levels but also in intracellular cationic potentials such as polyamines, and that POPase plays a crucial role for the growth and differentiation of liver cell.

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 microM) resulted in a dose-dependent decrease in the ability to bind to laminin and type IV collagen but had little or no effect on attachment 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.

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 an in 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-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), 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.

Aminated fucoidan promotes the invasion of 3 LL cells through reconstituted basement membrane: its possible mechanism of action.

Fucoidan is reported to have an antimetastatic activity. In the present study, we prepared an amino group-introduced derivative of fucoidan and examined its effect on the invasion of 3 LL cells through a reconstituted basement membrane (MatrigelTM). Unlike native fucoidan, the aminated derivative promoted the tumor cell invasion: maximal promotion (240% of control invasion) was obtained with 5 micrograms/ml. However, with higher concentrations (10-30 micrograms/ml) of the fucoidan derivative, the promotion was gradually reduced to 130% of control. Both native and aminated fucoidans inhibited specifically the attachment of 3 LL cells to laminin. Interestingly, aminated fucoidan, unlike the native one, promoted the tumor cell adhesion to immobilized synthetic laminin B 1 chain peptide, YIGSR, over a concentration range of 0.5-5 micrograms/ml. Higher concentrations (7-20 micrograms/ml) of the aminated derivative suppressed the adhesive ability of 3 LL cells to YIGSR. 3 LL cells secreted a 50-kDa form of urokinase-type plasminogen activator (u-PA) in the culture medium. Addition of aminated fucoidan (5 micrograms/ml) or YIGSR (10 micrograms/ml) resulted in a 1.7-fold increase in u-PA activity. This effect was enhanced up to 3.5-fold when both substances were simultaneously added. The addition of native fucoidan had no effect. The present results suggest that the 67-kDa receptor-mediated binding of 3 LL cells to laminin activates their invasiveness, especially by enhancing the extracellular u-PA levels. Aminated, but not native, fucoidan may act to enhance the laminin-receptor interaction at the limited concentration range.

Fibrinolytic and anticoagulant activities of highly sulfated fucoidan.

A series of fucoidan [sulfated poly(L-fucopyranose)] derivatives were prepared by chemical sulfation and desulfation, and they were tested for their abilities to stimulate tissue plasminogen activator (t-PA)-catalyzed plasminogen activation, clot lysis, and the inhibition of fibrin polymer formation. The magnitude of their activities was dependent upon the degree of sulfation. A striking feature of the sulfated fucoidan was that, unlike heparin, it stimulated t-PA-induced plasma clot lysis by protecting plasmin activity from alpha 2-plasmin inhibitor and decreased the rate of fibrin polymer formation. The inhibition of hyaluronic acid-mediated enhancement of fibrin clot formation was also observed with the fucoidan derivative. We also showed that highly sulfated fucoidan prevents significantly endotoxin-induced hepatic vein thrombosis in the hyperlipemic rat model. The present results are the first to describe the fibrinolytic and anticoagulant activities of fucoidan, and thus may provide useful clues for the development of an ideal thrombolytic agent.

Improved inhibitors of glucosylceramide synthase.

An inhibitor of glucosylceramide (GlcCer) synthase, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), has been reported to deplete cells and mice of their glucosphingolipids. This inhibitor has proved useful for the elucidation of the many functions of this lipid family [reviewed by Radin, N.S. & Inokuchi, J. (1991) Trends Glycosci. Glycotechnol. 3, 200-213]. In the present study, we have synthesized homologs of PDMP having different acyl chains (C6-C18) and compared their effectiveness for the inhibition of GlcCer synthase in vitro and their inhibition of GlcCer, protein, and DNA synthesis in cultured MDCK (Madin-Darby canine kidney) cells. Using MDCK homogenates and mouse brain and liver microsomes, we found that the C6 compound was relatively inactive and that the longer chain compounds did not differ much in inhibitory power. However, the use of intact MDCK cells showed that the longer chain homologs were much more effective in inhibiting GlcCer synthesis, cell growth, and incorporation of [3H]thymidine. Tests with two radioactive homologs showed that the inhibitor with a longer acyl chain was taken up much more effectively by MDCK cells and that this difference explains the much greater effectiveness of this homolog in intact cells. The inhibitors were effective when solubilized either with a nonionic detergent or with bovine serum albumin. The extent of decrease in DNA synthesis was not directly proportional to the decrease in cellular glucosylceramide, possibly because only a low level of the glycolipid is needed for DNA synthesis.

Preparation of oversulfated fucoidan fragments and evaluation of their antithrombotic activities.

Oversulfated fucoidan fragments (20-40 and 40-60 kDa) were prepared, and their fibrinolytic and anticoagulant activities were compared with those of oversulfated fucoidan (100-130 kDa) reported previously [Soeda et al., Biochem. Pharmacol. 43, 1853-1858, 1992]. The results of these experiments indicated that the in vitro abilities of oversulfated fucoidan to stimulate tissue plasminogen activator (t-PA)-catalyzed plasminogen activation and to potentiate thrombin inhibition by antithrombin III or heparin cofactor II decreased with a decrease in its molecular size. However, the preventive effects of both fucoidan fragments on endotoxin-induced hepatic vein thrombosis in hyperlipemic rats were almost the same as that of oversulfated fucoidan (100-130 kDa). We also found that, unlike heparin treatment, the concentrations of serum and vascular endothelium t-PA in rats treated with oversulfated fucoidan or its fragments (1 mg each/kg/week) were maintained at normal levels. The 20-40 and 40-60 kDa fragments had an ability to decrease the elevated levels of serum cholesterol in hyperlipemic rats, whereas the 100-130 kDa fucoidan derivative did not. These results suggest that oversulfated fucoidan and its fragments have another function(s), besides the regulation of blood coagulation and fibrinolysis, and are of therapeutic benefit for the prevention of thrombus formation in hyperlipemia.

Synthesis of taurine- and/or acetyl group-conjugated poly(D-Glu, D-Lys) derivatives and their effects on fibrinolysis.

A series of C-sulfoethylated and/or N-acetylated poly(D-Glu, D-Lys) (polyGL) derivatives was synthesized and their effects on tissue-type plasminogen activator (t-PA)-induced fibrinolysis were investigated. These derivatives accelerated t-PA-induced plasma (fibrin) clot lysis and t-PA-catalyzed plasminogen activation with the following order of potency: C-sulfoethylated and N-acetylated polyGL greater than N-acetylated polyGL greater than C-sulfoethylated polyGL greater than polyGL. The most potent stimulator C-sulfoethylated and N-acetylated polyGL associated with both t-PA and plasminogen under low ionic conditions, whereas it did not prevent the bindings of t-PA and plasminogen to fibrin. These results suggest that t-PA and plasminogen preferentially bind to sulfated or carboxylated polyanions, which may be required to possess certain neutral groups, and such complex formation may improve the plasminogen activation kinetics in a different manner from that of fibrin.

Sphingomyelinase and cell-permeable ceramide analogs increase the release of plasminogen activator inhibitor-1 from cultured endothelial cells.

We investigated the effect of exogenous staphylococcal sphingomyelinase (SMase) on the release of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) from cultured human umbilical vein endothelial cells (HUVEC). Addition of SMase (2 units/ml) to the culture medium induced an approx. 15-fold increase in the extracellular level of PAI-1 antigen at 3 h. No significant increase in the level of t-PA antigen was detected. Treatment of HUVEC with SMASE (2 units/ml) for 3 h resulted in a significant decrease in the cellular sphingomyelin (SM) level, accompanied by a corresponding increase in the ceramide level. Cell-permeable ceramide analogs also enhanced the release of PAI-1 from cultured HUVEC in concentration- and time-dependent manners. A 6-fold increase in PAI-1 antigen level was observed after incubation for 3 h with 10 microM N-acetylsphingosine. Similar effect was noted as early as 2 h with 10 microM N-hexyanoylsphingosine. Addition of sphingosine failed to affect the release of PAI-1 from cultured HUVEC, indicating that the effects of ceramide analogs were independent of sphingosine generation. Pretreatment with cycloheximide or actinomycin D abated the response of HUVEC to N-acetylsphingosine in the increased levels of both extracellular and intracellular PAI-1. These results suggest that ceramide, generated via "SM cycle", acts as a lipid mediator of PAI-1 release from vascular endothelial cells, and may contribute to a better understanding of the pathogenesis of the PAI-1-associated thrombotic disorders.

Rapid and high-yield purification of porcine heart tissue-type plasminogen activator by heparin-sepharose choromatography.

A rapid and high-yield procedure for the purification of single polypeptide tissue-type plasminogen activator (t-PA) from porcine heart tissue has been developed. Delipidated heart tissue was extracted with 0.45 M potassium acetate. The extract was fractionated with ammonium sulfate and purified by a combination of affinity chromatography on heparin-Sepharose CL-6B and gel filtration on Toyopearl HW-55S. The final product had a specific activity of 220,000 IU/mg protein and gave a single protein band (apparent molecular weight; 67,000) in SDS-polyacrylamide gels in the presence or absence of a reducing agent. The increase in specific activity was 3,200-fold, most of which was achieved in the step of heparin-Sepharose chromatography. The yield calculated from the active ammonium sulfate precipitate was about 90% and 500 micrograms or more of the purified enzyme was obtained from 1 kg wet tissue. This procedure may also be useful for the large-scale production of highly purified t-PA from other tissues or tissue culture cells.

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.

Partial resection for leiomyoblastoma of stomach.

We herein report the case of a 51-year-old male with a submucosal tumor of the stomach which was histopathologically demonstrated to have features compatible with leiomyoblastoma. As the submucosal tumor of the posterior wall of the antrum, which was initially found in the upper gastrointestinal series done during a health examination, had grown 3.8 cm in diameter, the partial resection of the full thickness of the gastric wall containing the tumor with a sufficient tumor margin was performed.