Novel dynamic flow cytoenzymological determination of intracellular esterase inhibition by BCNU and related isocyanates

We present a novel dynamic flow cytoenzymological demonstration of the potent inhibition by the antitumour chloroethylnitrosourea BCNU of the intracellular hydrolysis of fluorescein diacetate by esterases of viable, intact murine and human tumour cells in vitro. The BCNU metabonate chloroethyl isocyanate and the related compound n-butyl isocyanate were also potent inhibitors. I50 values were in the range 4.2 X 10(-5)-2.0 X 10(-4) M. Generally similar quantitative results were obtained for intact cells and sonicates by conventional spectrofluorimetry, and inhibition of purified porcine liver carboxyl esterase (EC 3.1.1.1) was demonstrated. Little or no inhibitory activity was seen with the alkylating agents methyl methane-sulphonate, melphalan and nitrogen mustard. The results are consistent with carbamoylation of the esterase molecules by isocyanates, and this may provide a basis for the flow cytometric determination of intracellular carbamoylation in discrete subpopulations of heterogeneous samples.     

Differential inhibition of cellular glutathione reductase activity by isocyanates generated from the antitumor prodrugs Cloretazine and BCNU

The antitumor, DNA-alkylating agent 1,3-bis[2-chloroethyl]-2-nitrosourea (BCNU; Carmustine), which generates 2-chloroethyl isocyanate upon decomposition in situ, inhibits cellular glutathione reductase (GR; EC 1.8.1.7) activity by up to 90% at pharmacological doses. GR is susceptible to attack from exogenous electrophiles, particularly carbamoylation from alkyl isocyanates, rendering the enzyme unable to catalyze the reduction of oxidized glutathione. Evidence implicates inhibition of GR as a cause of the pulmonary toxicity often seen in high-dose BCNU-treated animals and human cancer patients. Herein we demonstrate that the prodrug Cloretazine (1,2-bis[methylsulfonyl]-1-[2-chloroethyl]-2-[(methylamino)carbonyl]hydrazine; VNP40101M), which yields methyl isocyanate and chloroethylating species upon activation, did not produce similar inhibition of cellular GR activity, despite BCNU and Cloretazine being equally potent inhibitors of purified human GR (IC(50) values of 55.5 microM and 54.6 microM, respectively). Human erythrocytes, following exposure to 50 microM BCNU for 1h at 37 degrees C, had an 84% decrease in GR activity, whereas 50 microM Cloretazine caused less than 1% inhibition under the same conditions. Similar results were found using L1210 murine leukemia cells. The disparity between these compounds remained when cells were lysed prior to drug exposure and were partially recapitulated using purified enzyme when 1mM reduced glutathione was included during the drug exposure. The superior antineoplastic potential of Cloretazine compared to BCNU in animal models could be attributed in part to the contribution of the methyl isocyanate, which is synergistic with the co-generated cytotoxic alkylating species, while at the same time unable to significantly inhibit cellular GR.     

Antitumour imidazotetrazines--IV. An investigation into the mechanism of antitumour activity of a novel and potent antitumour agent, mitozolomide (CCRG 81010, M & B 39565; NSC 353451)

8-Carbamoyl-3-(2-chloroethyl)imidazo[5,1-d]-1,2,3,5-tetrazin-4-(3H )-one- mitozolomide (CCRG 81010, M & B 39565, NSC 353451) is a potent inhibitor of the growth of a number of experimental tumours and can potentially decompose to give either an isocyanate or the monochloroethyltriazene (MCTIC). In vitro CCRG 81010 is not cross-resistant with the bifunctional alkylating agents against the Walker carcinoma. To investigate the mechanism of the antitumour activity of CCRG 81010 a comparison has been made with BCNU and MCTIC on precursor incorporation into macromolecules in TLX5 mouse lymphoma cells. Whereas BCNU produces a rapid and extensive inhibition of both (methyl 3H) thymidine and [5-3H]uridine incorporation into acid-insoluble material, neither CCRG 81010 or MCTIC have an early effect on precursor incorporation. Inhibition of precursor uptake is also not produced by concentrations of 2-chloroethylisocyanate that inhibit intracellular glutathione reductase activity. The potential carbamoylating activity of CCRG 81010 has also been assessed by comparing its effect with that of BCNU and 2-chloroethyl isocyanate on enzymes known to be inhibited by carbamoylation. Such enzymes, glutathione reductase, chymotrypsin and gamma-glutamyltranspepidase are not inhibited by CCRG 81010 under conditions where BCNU and 2-chloroethyl isocyanate show complete inhibition of enzyme activity, suggesting an absence of carbamoylating species. The results suggest that the most likely antitumour metabonate produced from CCRG 81010 is the triazene MCTIC.     

The role of isocyanates in the toxicity of antitumour haloalkylnitrosoureas

The cytotoxicity of the antitumour nitrosoureas BCNU and CCNU and the isocyanates which they liberate (chloroethylisocyanate and cyclohexylisocyanate respectively) has been measured utilising an in vitro-in vivo bioassay. Lines of the TLX5 lymphoma and L1210 leukaemia were used which were either sensitive or resistant to nitrosoureas in vivo. An estimated logarithmic cell kill produced by each compound in vitro (before injecting the cells into animals) was calculated by reference to assays of the survival time of animals given from 2 × 10⁵ to 2 × 10⁰ cells of each line. Resistance to both BCNU and CCNU was observed in vitro in the cell lines of the TLX5 lymphoma made resistant to either BCNU or a dimethyltriazene in vivo. The latter tumour was cross-resistant in vivo to nitrosoureas. Resistance in vitro to nitrosoureas was also observed in a line of L1210 leukaemia which had had resistance to BCNU induced in vivo. The nitrosourea resistant TLX5 lymphomas were cross-resistant in vitro to both cyclohexylisocyanate and chloroethylisocyanate whereas the nitrosourea resistant L1210 line showed no cross-resistance to cyclohexylisocyanate and marginal cross-resistance to chloroethylisocyanate. The results suggest that the TLX5 lymphoma, which is naturally resistant to alkylating agents of the 2-chloroethylamine type, may be sensitive in vivo to nitrosoureas as a consequence of the intracellular release of isocyanates. This hypothesis was supported by the finding that the resistant TLX5 lymphoma showed no cross-resistance to other electrophilic agents, for example formaldehyde, monomethyltriazene or HN2. The transport of nitrosoureas into the sensitive and resistant cell lines was similar in profile and there was no difference in the concentration of non-protein thiols.     

Correlation of nitrosourea murine bone marrow toxicity with deoxyribonucleic acid alkylation and chromatin binding sites

All of the clinically available nitrosourea antitumor agents produce serious treatment-limiting bone marrow toxicity. A reduction in this toxicity can be achieved by attaching the chloroethylnitrosourea cytotoxic group to C2 (chlorozotocin) or C1 (1-(2-chloroethyl)-3-(β-d-glucopyranosyl)-1-nitrosourea, GANU) of glucose. Both glucose analogs are less myelotoxic in mice than 1-(2-chloroethyl)-3-cyclohepyl-1-nitrosourea (CCNU) or 1-(4-amino-2-methylpyrimidin-5-yl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), while retaining comparable antitumor activity against the murine L121o leukemia. To define the nuclear mechanisms for this reduced myelotoxicity, alkylation of L1210 and murine bone marrow DNA was quantitated. With the use of the endonucleases micrococcal nuclease and DNase I, the sites of alkylation within the chromatin substructure were determined. Experiments were performed on L1210 leukemia or bone marrow cells that had been incubated in vitro for 2 hr with 0.1 mM [¹⁴C]chloroethyl drug. The quantitative alkylation of DNA by GANU was 1.3-fold greater in L1210, as compared to bone marrow, cells. This ratio of DNA alkylation is comparable to the 1.3 ratio we previously reported for chlorozotocin [L. C. Panasci, D. Green and P. S. Schein, J. clin. Invest.64, 1103 (1979)]. In contrast, the ratio of alkylation (L1210: bone marrow DNA) for the myelotoxic ACNU was 0.66, similar to 0.59 for CCNU. Nuclease digestion experiments demonstrated that chlorozotocin and GANU preferentially alkylated internucleosomal linker regions of bone marrow chromatin, while nucleosome core particles were the preferred targets of CCNU and ACNU. The reduced myelotoxicity of chlorozotocin and GANU may be correlated with the advantageous ratio of L1210: bone marrow DNA alkylation and preferential alkylation of internucleosomal regions of bone marrow chromatin.     

Alkylating agent interactions with the nuclear matrix

The interrelationship of DNA to the nuclear matrix is integral to the organization of chromatin within the nucleus and to the DNA replication process. The influence of nitrosourea and nitrogen mustard interactions with the nuclear matrix were studied in log phase HeLa cells. Alkylation of the nuclear matrix by chlorozotocin (CLZ) or 1-(2-chloroethyl-3-cyclohexyl)-1-nitrosourea (CCNU) was 1.58 and 1.27 pmoles drug/micrograms protein, respectively, whereas carbamoylation by CCNU was 32.5 pmoles/micrograms. These constituted approximately 30% of the total (nuclear) drug modifications. The structural matricin fibrillar components of the matrix were alkylated and carbamoylated twice as much as the ribonuclear protein elements (RNP). However, when alkylations are measured per microgram of protein, the ratio of covalently bound drug to RNP:matricin was 1.2 for both CLZ and CCNU. The RNP:matricin carbamoylation ratio for CCNU was 0.9. The importance of DNA and matrix protein alkylations to the process of reassociation was studied. Under control conditions, in vitro, approximately 80% of the DNA was associated with the matrix at a protein:DNA ratio (micrograms for micrograms) of 50:1. Direct alkylation or carbamoylation of the matrix proteins did not affect these DNA-protein interactions. However, using in vitro alkylated DNA (1 alkylation/10(2) base pairs), there was a 60% reduction of the alkylated nucleic acid bound to the matrix at the same protein: DNA ratio. The reduced binding of DNA to matrix may be a function of interference with the DNA recognition sites by alkylation of specific bases. The interference of DNA-matrix association by DNA alkylation may contribute to the cytotoxic activity of these antineoplastic agents.     

Effects of carbamoylation with alkyl isocyanates on the assay of proteins by dye binding

The effect of carbamoylation with alkyl isocyanate was used both to monitor the stability of the isocyanates and to study the influence of charge modification on protein assay. Carbamoylation of poly (L-lysine) with methyl isocyanate, ethyl isocyanate and 2-chloroethyl isocyanate was observed to decrease binding of methyl orange. The data emphasized the lability of alkyl isocyanates and indicated the importance of preparing aqueous solutions at low temperatures for studies on protein carbamoylation. After carbamoylation of several proteins, there was decreased metachromasia on binding to Coomassie Blue G. Poly (L-lysine) and H1 histone showed anomalous behavior in that with low concentrations of Coomassie Blue G the metachromasia was increased by carbamoylation, but at high concentrations of the dye the metachromasia was decreased by carbamoylation. In contrast to some reports in the literature, the data indicated that there is not always a simple relationship between the positive charge on a protein and the interaction with anionic dyes.     

Capillary Gas Chromatography and Thermionic N-P-Specific Detection of 13-Bis(2-chloroethyl)-l-nitrosourea (BCNU) or l-(2-Chloroethyl)-3-cyclohexyl-l-nitrosourea (CCNU) in Stability and Pharmacokinetic Studies

An expedient, rapid, and sensitive capillary gas chromatographic method for the analysis of l,3-bis(2-chloroethyl)-l-nitrosourea (BCNU) or l-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea (CCNU) in plasma is described. Separation of the underivatized nitrosourea compounds was performed on a 0.33-mm-i.d., 25-m fused-silica, SE-30 capillary column, and detection was carried out using a thermionic N–P-specific detector. The compounds were extracted from plasma with benzene with a yield of >87%. The assay was linear in the ranges of 0.001 to 0.5 and 0.5 to 25 µg/ml for CCNU or 0.003 to 0.50 and 0.5 to 25 µg/ml for BCNU, with correlation coefficients from 0.9914 to 0.9999 and coefficients of variation (CV) of <3.3%. Other antineoplastic agents did not interfere in the assay. The method was employed to study the pharmacokinetics of BCNU in rabbits. The plasma concentration-time curves were fit to a two-compartment model with a mean (SE) , , and total-body clearance of 2.898 (0.913) hr^–1, 0.1228 (0.0179) hr^–1, and 7.211 (2.862) liters/hr · kg, respectively. Further, the stability of BCNU and CCNU in solution was examined at different temperatures. Both compounds were stable in benzene or acetone (4 to 37°C) but labile in plasma even if refrigerated. The apparent rate constants for degradation of BCNU and CCNU were 0.09921 and 0.02853 hr^–1 at 4°C and 5.998 and 2.553 hr^–1 at 37°C, respectively.     

Evaluation of the role of isocyanates in the action of therapeutic nitrosoureas

The half-lives of chloroethyl and cyclohexyl isocyanate have been determined in tissue culture medium, and the isocyanate concentration produced during the breakdown of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) has been calculated. L1210 or HeLa cells exposed either to the parent nitrosourea or to an equivalent constant isocyanate concentration show no deficiency in the repair of gamma-irradiation damage as measured by DNA strand separation in alkali. Viability studies indicate that the isocyanates play a minor role in the overall cytotoxicity of the nitrosoureas.     

Angiotensin converting enzyme inhibitors: modifications of a tripeptide analogue

Modified nonhydrolyzable tripeptide analogues of (S)-1-[5-(benzoylamino)-1,4-dioxo-6-phenylhexyl]-L-proline (1), designed to impart oral angiotensin converting enzyme (ACE) inhibitory activity, were made and evaluated in vivo and in vitro. The N-methyl and C5-methyl analogues of 1 were inactive. Insertion of heteroatoms (O, S, NH) into the C--C chain of 1 gave a series of compounds with high in vitro activity in the guinea pig serum ACE assay. The O-analogue was the most potent with an IC50 = 4.4 x 10(-9) M compared to 1 with an IC50 = 3.2 x 10(-9) M. The structure-activity relationships in this series of compounds lead one to speculate that the heteroatom provides an additional binding site to the surface of the enzyme; however, these compounds were inactive when tested for antihypertensive activity in the renal hypertensive rat at 30 mg/kg by the oral route (captopril is active at 1.0 mg/kg po).     

亚硝脲类化合物的急性毒性及对耐药艾氏腹水癌小鼠的疗效研究及与构效关系初探

测定了亚硝脲类化合物ＣＮＵ、ＧＡＮＵ、ＡＣＮＵ、ＧＣＮＵ、ＣＬＺ、ＰＣＮＵ、ＢＣＮＵ、ＣＣＮＵ、ＳＴＺＮ、ＭｅＣＣＮＵ、ＦＣＮＵ的小鼠ＬＤ５０分别为５．８、１８．０、２６．０、２７．０、３１．５、３８．０、４８．５、５８．０、１５０．０、１８５．０、５００．０ｍｇ·ｋｇ－１．毒性大小和治疗存活率的高低无正相关。化学结构在１－Ｎ－有脂环或杂环基团则不易产生耐药性．且毒性低疗效高；ＣＮＵ因３－Ｎ－无－ＣＨ２ＣＨ２Ｃｌ基因则毒性增加；１－Ｎ－侧链有氟原子取代其毒性可明显降低。     

Pathways and kinetics of aqueous decomposition and carbamoylating activity of new anticancer nitroimidazole-linked 2-chloroethylnitrosoureas

The products of decomposition in anaerobic aqueous solution at pH 7.1 and 37 degrees were determined for two series of novel anticancer agents incorporating both nitroimidazole and 2-chloroethylnitrosourea moieties (NI-CENUs) and examples of which exhibit preferential hypoxic toxicity against HeLa-MR cells. The decomposition products identified were vinyl chloride, acetaldehyde, 2-chloroethanol, ethylene glycol and imidazole-bearing compounds of the type including oxazolidinone, ethylamine or urea moieties. Series A NI-CENUs, which contain a 2-hydroxypropyl unit, gave rise to the oxazolidinone intramolecularly compared with the series B agents which gave rise to the imidazole-ethylamine and ureas. The half-lives of the B series agents were comparable with those of 1,3-bis(2-chloroethyl)nitrosourea (BCNU), 2-cyclohexyl-1-(2-chloroethyl)-1-nitrosourea (CCNU) and streptozotocin. The carbamoylation activity of the series B agents was approximately ten times that of series A compounds. This latter property may be related to the greater potency of series B than series A NI-CENUs against Mer+ HeLa-S3 cells via inhibition of relevant repair enzymes.     

Spin labeled antioxidants protect bacteria against the toxicity of alkylating antitumor drug CCNU

We have studied the toxic effect of the alkylating antitumor drug N'-cyclohexyl-N-(2-chloroethyl)-N-nitrosourea (lomustine, CCNU) on Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) strains, alone and in presence of oxygen radical-scavenging substances [Vitamin E, stable nitroxyl radical 2,2,6,6-tetramethylpiperidine-N-oxyl (TMPO), and spin labeled (nitroxyl free radical moiety containing) analogues of CCNU] and compared with that of the alkylating antitumor drug 5-(3,3-dimethyltriazene-1-yl)-imidazole-4-carboxamide (dacarbazine, DTIC). All spin labeled compounds tested were almost no toxic at doses of 50-500 microM/ml, whereas the alkylating antitumor drug CCNU showed toxicity in a dose dependent manner. Even low doses of spin labeled nitrosoureas provided protection against the toxicity caused by the antitumor drug CCNU alone. The lowest toxicity against E. coli and S. aureus were achieved when 500 microM/ml of CCNU was combined with 200 microM/ml of spin labeled nitrosourea N-[N'-(2-chloroethyl)-N'-nitrosocarbamoyl]-glycine amid of 2,2,6,6-tetramethyl-4-aminopiperidine-1-oxyl (SLCNUgly). A combination of TMPO with vitamin E completely abolished the toxicity of CCNU. Endogenous formation of oxygen radicals and their possible involvement in CCNU toxicity towards the bacteria strains tested have been also discussed.     

Thenoyltrifluoroacetone, a potent inhibitor of carboxylesterase activity

Thenoyltrifluoroacetone (TTFA), a conventional mitochondrial complex II inhibitor, was found to inhibit purified porcine liver carboxylesterase non-competitively with a K(i) of 0.61x10(-6)M and an IC(50) of 0.54x10(-6)M. Both rat plasma and liver mitochondrial esterases were inhibited in a concentration-dependent fashion. Results indicate that TTFA is a potent inhibitor of carboxylesterase activity, in addition to its ability to inhibit mitochondrial complex II activity. Therefore, caution is warranted in using TTFA as a mitochondrial complex inhibitor in combination with esterase substrates, such as fluorescence probes or vitamin E esters.     

Characterization of the inhibition of glutathione reductase and the recovery of enzyme activity in exponentially growing murine leukemia (L1210) cells treated with 1,3-bis(2-chloroethyl)-1-nitrosourea

The inactivation of the enzyme glutathione reductase by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) was studied in exponentially growing murine leukemia cells. A 1-hr incubation with 1.6 +/- 0.2 microM BCNU resulted in a 50% inhibition of glutathione reductase, while 10 microM BCNU caused total inhibition of the enzyme. The time required for 50% inhibition of glutathione reductase by 10 microM BCNU was 7 min. The recovery of glutathione reductase activity was studied by incubating cells with 10 microM BCNU for 30 min to inhibit all glutathione reductase activity, washing the cells free of drug, and continuing the incubation in fresh medium. Fifty percent of the activity returned within 12 hr. Glutathione reductase activity recovered normally when cell growth and DNA synthesis were inhibited in the cells, but it failed to recover when protein synthesis was inhibited. Therefore, the inactivation of glutathione reductase appears irreversible, and the recovery of enzymatic activity is dependent on the synthesis of new protein. Continuous incubation with 19.8 +/- 0.4 microM BCNU resulted in a 50% inhibition of cell growth. A 1-hr incubation with 7.3 +/- 0.8 microM BCNU resulted in a 50% loss of viability as measured by a soft agar clonogenic assay. These experiments quantify the inhibition of glutathione reductase by BCNU and the recovery of enzyme activity in the context of the toxic effects of the compound. A clinically useful inhibitor of glutathione reductase will require a wider difference between the concentrations required for enzyme inhibition and cytotoxicity than BCNU provides.     

Cardiovascular effects induced by reticuline in normotensive rats

The cardiovascular effects of reticuline, isolated in a pure form from the stem of Ocotea duckei Vattimo, were studied in rats by using a combined in vivo and in vitro approach. In normotensive rats, reticuline (5, 10 and 20 mg/kg, i. v., randomly) injections produced an intense hypotension. This hypotensive response was attenuated after either, L-NAME (20 mg/kg, i. v.), a nitric oxide (NO) synthase inhibitor, or atropine (2 mg/kg, i. v.), a muscarinic receptor antagonist. In isolated rat aortic rings with intact endothelium, reticuline (3 x 10 ( - 6), 3 x 10 ( - 5), 3 x 10 ( - 4), 9 x 10 ( - 4) and 1.5 x 10 ( - 3) M) inhibited in a concentration-dependent manner the contractions induced by phenylephrine (1 microM), KCl (80 mM) and KCl (30 mM), [IC (50) = (0.4 +/- 0.1, 2.4 +/- 0.4 and 3 +/- 0.4) x 10 ( - 4) M, respectively). The effect of reticuline on phenylephrine-induced contractions was attenuated by removal of the vascular endothelium [IC (50) = (2.5 +/- 0.7) x 10 ( - 4) M]. Similar results were obtained after pretreatment of the rings with L-NAME 100 microM [IC (50) = (1.3 +/- 0.1) x 10 ( - 4) M], L-NAME 300 microM [IC (50) = (3 +/- 0.3) x 10 ( - 4) M] or atropine 1 microM [IC (50) = (1.2 +/- 0.2) x 10 ( - 4) M]. On the other hand, the effect of reticuline on phenylephrine-induced contractions was not affected by indomethacin 1 microM [IC (50) = (0.7 +/- 0.3) x 10 ( - 4) M]. Reticuline (3 x 10 ( - 6), 3 x 10 ( - 5), 3 x 10 ( - 4), 9 x 10 ( - 4) and 1.5 x 10 ( - 3) M) antagonized CaCl (2)-induced contractions, and also inhibited the intracellular calcium dependent transient contractions induced by norepinephrine (1 microM), but not those induced by caffeine (20 mM). These results suggest that the hypotensive effect of reticuline is probably due to a peripheral vasodilation in consequence of: 1) muscarinic stimulation and NOS activation in the vascular endothelium, 2) voltage-dependent Ca (2+) channel blockade and/or 3) inhibition of Ca (2+) release from norepinephrine-sensitive intracellular stores.     

Two glycine containing 2-chloroethylnitrosoureas--a comparative study on some physicochemical properties, in vivo antimelanomic effects and immunomodulatory properties.

Physicochemical properties such as alkylating and carbamoylating activity and in vivo antimelanomic effects against B16 melanoma of the spin labeled (nitroxyl free radical containing) glycine nitrosourea (SLCNUgly) and its nonlabeled analogue (ChCNUgly), synthesized in our laboratory are studied and compared to those of antitumour drug 3-cyclohexyl-1-(2-chloroethyl)-1-nitrosourea (CCNU). We have demonstrated that introducing of glycine moiety in the nitrosourea structure in practice does not affect either alkylating or carbamoylating activity. On the other hand replacement of cyclohexyl moiety in ChCNUgly structure with nitroxyl free radical leads to a decrease in carbamoylating activity and an increase in alkylating activity. Compound ChCNUgly showed in vivo a higher antimelanomic activity against B16 melanoma in comparison with CCNU and SLCNUgly. It completely inhibited B16 melanoma growth (TGI=100%) at a dose 64.0 mg/kg. Moreover, we established that joint i.p. application in normal mice of SLCNUgly plus a new immunostimulator (C3bgp) formerly isolated in our laboratory led to a 75% restoration in immune function with respect to antibody production measured by Jerne hemolytic plaque assay. In contrast, no immunostimulation was found after joint application of C3bgp plus ChCNUgly or CCNU at the same experimental conditions. Based on these preliminary results, a possibility for developing of new combination immunochemotherapy schemes for treatment of human cancers is discussed.     

Effects of oxodipine on 45Ca movements and contractile responses in vascular smooth muscle.

1. The inhibitory effects of oxodipine, a new dihydropyridine, were compared with those of nifedipine on contractile responses in rat isolated aortic strips and on spontaneous mechanical activity in portal vein segments. 2. In rat isolated aorta oxodipine (IC50 = 7.8 +/- 1.8 x 10(-9)M) and nifedipine (IC50 = 8.5 +/- 2.5 x 10(-9)M) dose-dependently inhibited the contractile responses induced by high K (80 mM), whereas responses to noradrenaline (NA, 10(-6)M) were only slightly affected (IC50 greater than 10(-7)M). These inhibitory actions were observed with both drugs added either before or after the induced contractions. 3. Contractile responses induced by addition of Ca to 0Ca high-K solution were also dose-dependently inhibited by oxodipine (IC50 = 4.5 +/- 2.5 x 10(-9)M). However, oxodipine up to 10(-6)M did not modify the contractile responses obtained in strips incubated in 0Ca when Ca was added in the presence of NA. 4. Oxodipine and nifedipine also inhibited the development of spontaneous mechanical activity in portal vein segments. 5. Oxodipine inhibited 45Ca uptake stimulated by high K (I50 = 8.7 +/- 2.5 x 10(-9)M) or by NA (I50 greater than 10(-7)M). However, it did not modify 45Ca uptake and 45Ca efflux in resting strips or 45Ca efflux stimulated by NA. 6. These results indicate that the effects of oxodipine on vascular smooth muscle may be due to the blockade of Ca entry through potential- and receptor-operated channels; it was at least 10 times more selective for potential-operated channels. Oxodipine did not modify Ca entry through passive leak channels and NA-induced intracellular Ca release.