Hepatic and renal cytochrome P-450s in spontaneously hypertensive rats.

The differences in the levels of cytochrome P-450s in hepatic and renal microsomes between spontaneously hypertensive rats (SHR) and normotensive control rats (Wistar Kyoto rats, WKY) were investigated by Western blotting with a specific antibody. Differences in the metabolic activity of the microsomes were also studied. In hepatic microsomes, the content of P450 PB-1 (IIIA2) was 140% higher in SHR than in WKY and the content of P450 IF-3 (IIA1) in SHR was one-seventh that in WKY. The differences reflected the increase in testosterone 6 beta-hydroxylation activity and decrease in testosterone 7 alpha-hydroxylation activity in hepatic microsomes of SHR. The level of P450 K-5 (IVA2) in hepatic microsomes of SHR was 4-times that in microsomes of WKY. The levels of other cytochrome P-450s in SHR were not very different from those in WKY. In renal microsomes, the levels of three renal cytochrome P-450s, P450 K-2, K-4, and K-5, were measured. The level of P450 K-5 (fatty acid omega-hydroxylase) in SHR was 50% higher than that in WKY and the difference reflected the increase in lauric acid omega- and (omega-1)-hydroxylation activities of the renal microsomes of SHR. The levels of P450 K-2 and K-4 did not differ in both rats.

Purification and characterization of liver microsomal cytochrome P-450 from untreated male rats.

Different forms of cytochrome P-450 from untreated male rats were simultaneously purified to homogeneity using the HPLC technique. The absorption maximum, molecular weight, NH2-terminal sequence and catalytic activity of them were determined. The NH2-terminal sequences of six forms of cytochrome P-450 (designated P450 UT-1, UT-2, UT-4, UT-5, UT-7 and UT-8) indicate that these cytochrome P-450 isozymes are of different molecular species. The hydrophobicity values of the NH2-terminal sequences of P450 UT-1 and P450 UT-8 were lower than that of other forms. P450 UT-8 has the highest molecular weight, 54,000, of the six forms of P-450. P450 UT-2 was active in demethylation of benzphetamine, P450 UT-4 was active in the metabolism of 7-ethoxycoumarin and p-nitroanisole. P450 UT-1 and P450 UT-2 were active in the 2 alpha- and 16 alpha-hydroxylation of testosterone, whereas P450 UT-4 was active in the 6 beta-, 7 alpha- and 15 alpha-hydroxylation of the same steroid. We believe that P450 UT-1, P450 UT-7 and P450 UT-8 are as yet unrecognized forms of cytochrome P-450.

Simultaneous purification of multiple forms of rat liver microsomal cytochrome P-450 by high-performance liquid chromatography.

14 microsomal cytochromes P-450 were purified from the liver of untreated and phenobarbital- or 3-methylcholanthrene-treated male rats. Following solubilization of microsomes with sodium cholate, poly(ethylene glycol) fractionation and aminohexyl-Sepharose 4B chromatography, cytochromes P-450 were purified by high-performance liquid chromatography (HPLC), using a preparative DEAE-anion-exchange column. The pass-through fraction was further purified by HPLC using a cation-exchange column. Other fractions eluted on preparative DEAE-HPLC were further applied onto an HPLC using a DEAE-column. Five kinds (P-450UT-2-6), four kinds (P-450PB-1,2,4 and 5) and five kinds (P-450MC-1-5) of cytochromes P-450 were purified from untreated rats or rats treated with phenobarbital or 3-methylcholanthrene, respectively. HPLC profiles of tryptic peptides of cytochromes P-450UT-2 and P-450MC-2 were identical and the other profiles obtained from seven purified cytochromes P-450 were distinct from each other. Amino-terminal sequences of eight forms of cytochrome P-450 (UT-2, UT-5, PB-1, PB-2, PB-4, PB-5, MC-1 and MC-5) were distinct except for cytochromes P-450PB-4 and P-450PB-5.

Age-dependent expression of cytochrome P-450s in rat liver.

Age-related changes in the levels of multiple forms of cytochrome P-450 as well as in the testosterone hydroxylation activities of hepatic microsomes of male and female rats of different ages from 1 week to 104 weeks (24 months) were investigated. The total cytochrome P-450 measured photometrically did not change much with age in either male and female rats. Testosterone 2 alpha-, 2 beta-, 6 beta-, 15 alpha-, 16 beta-hydroxylation activities of male rats were much higher than those in female rats and were induced developmentally. These activities in male rats declined with aging to the very low level in female rats by 104 weeks of age. Testosterone 7 alpha-hydroxylation activity was maximum at 3 weeks of age in rats of both sexes. The levels of individual cytochrome P-450s were measured by immunoblotting. P450IA1 and IA2 (3-methylcholanthrene-inducible forms) and P450IIB1 and IIB2 (phenobarbital-inducible forms) were detected at low levels in rats of both sexes at all ages. P450IIA2, IIC11 and IVA2 were detected in male rats only and were induced developmentally. These male-specific forms disappeared in male rat liver at 104 weeks of age. P450IIC12, a typical female-specific form, was induced developmentally in female rats and was also detected in male rats at 3 and 104 weeks of age. P450IIIA2 (testosterone 6 beta-hydroxylase) was induced developmentally in male rats, but disappeared when the rats were 104 weeks of age. In female rats, P450IIIA2 was detected only at 1 and 3 weeks of age. P450IIA1, IIC6, IIE1 and IVA3 were detected in rats of both sexes at any age. P450IIC6 and IVA3 were induced developmentally and detected at a similar level in rats of both sexes. The level of P450IIA1 was maximum at 3 weeks of age in rats of both sexes. The changes in the level of P450IIE1 during aging were small compared with the changes in other cytochrome P-450s used in this study. These observations provide concrete evidence to our earlier hypothesis that each of the forms of cytochrome P-450 in male rats alter with aging in different patterns resulting in a practical feminization of over-all cytochrome P-450 composition at old age.

Purification and characterization of two constitutive cytochromes P-450 (F-1 and F-2) from adult female rats: identification of P-450F-1 as the phenobarbital-inducible cytochrome P-450 in male rat liver.

Two hepatic microsomal cytochromes P-450, P-450F-1 and P-450F-2 were purified to electrophoretic homogeneity from untreated adult female rats by high-performance liquid chromatography (HPLC) with anion-exchange, cation-exchange, and hydroxyapatite columns. Cytochromes P-450F-1 and P-450F-2 were not adsorbed with the anion-exchange column, but were retained on a cation-exchange column and were separated poorly. These forms separated on hydroxyapatite HPLC. The molecular weights of cytochromes P-450F-1 and P-450F-2 were 50,000 and 49,000, respectively. The absolute spectrum of the oxidized forms indicated that they had the low-spin state of heme, and the CO-reduced spectral maxima of cytochromes P-450F-1 and P-450F-2 were at 450 and 448 nm, respectively. Both forms catalyzed the N-demethylation of benzphetamine and had low catalytic activity for 7-ethoxycoumarin. Cytochrome P-450F-1 had low 2 alpha-hydroxylation activity toward testosterone. Cytochrome P-450F-2 had low 15 alpha-hydroxylation activity. On the basis of these results and those of NH2-terminal sequence analysis, cytochrome P-450F-2 seemed to be the typical female-specific cytochrome P-450. The NH2-terminal sequence of cytochrome P-450F-1 was identical to that of cytochrome P-450PB-2 purified from hepatic microsomes of male rats treated with phenobarbital. Cytochromes P-450F-1 and P-450PB-2 had identical chromatographic properties, minimum molecular weight, spectral properties, and peptide maps. Furthermore, the antibody to phenobarbital-inducible cytochrome P-450PB-2 gave a single immunoprecipitin band with cytochrome P-450F-1 by Ouchterlony double-diffusion analysis.

Maintenance and activation of Cyp2e-1 gene expression in mouse hepatocytes in primary culture.

The expression of Cyp2e-1 mRNA and protein was investigated in the C57BL/6NCrj mouse hepatocytes in primary culture, as well as liver and kidney. The mRNA and protein expression in the liver was in the same range in both sexes and was not affected by orchiectomy or ovariectomy. The mRNA expression was enhanced in the kidney of ovariectomized mice, in which the protein contents were not influenced. Orchiectomy decreased the expression of both mRNA and protein. When the hepatocytes were transferred to primary culture, the amounts of the mRNA were not changed within 24 h and about half remained by day 3. However, the expression was low thereafter. The expression of the protein gradually decreased after the start of culture. Dexamethasone showed a potential as an inducer at more than 10(-8) M. Sex hormones increased the expression of this P-450 species a little in culture, but growth hormone did not. These observations indicated that glucocorticoid hormone plays a role in modifying expression of Cyp2e-1 and that the mouse hepatocyte culture is useful for examining its regulation mechanism.

Induction and regulation of cytochrome P450 K-5 (lauric acid hydroxylase) in rat renal microsomes by starvation.

The effects of starvation on rat renal cytochrome P-450s were studied. The content of spectrally measured cytochrome P-450 in the renal microsomes of male rats increased 2-fold with 72 h starvation, but cytochrome b5 and NADPH-cytochrome P-450 reductase were not induced. 7-Ethoxycoumarin O-dealkylation and aniline hydroxylation activities of the renal microsomes of control male rats were very low but were induced 2.5-3-fold by 72 h starvation. Aminopyrine N-demethylation and lauric acid hydroxylation activities were induced 1.5-2-fold by 72 h starvation. The changes in catalytic activities suggested that the contents of individual cytochrome P-450s in the renal microsomes were altered by starvation. The contents of some cytochrome P-450s were measured by Western blotting. P450 DM (P450IIE1), a typical form of cytochrome P-450 induced by starvation in rat liver, was barely detected in rat kidney and was induced 2-fold by 72 h starvation. P450 K-5, a typical renal cytochrome P-450 and lauric acid hydroxylase, accounted for 81% of the spectrally measured cytochrome P-450 in the renal microsomes of control male rats and was induced 2-fold by 72 h starvation. P450 K-5 was not induced in rat kidney by treatment with chemicals such as acetone or clofibrate. The renal microsomes of male rats contained 6-times as much P450 K-5 as those of female rats. These results suggest that P450 K-5 is regulated by an endocrine factor.

Characterization of two P-450 isozymes placed in the rat CYP2D subfamily.

Two P-450s with debrisoquine 4-hydroxylation activity, designated P-450 UT-7 and UT-7b, were purified and partially purified, respectively, from hepatic microsomes of untreated male rats. Both purified P-450s with an apparent molecular weight of 49,000, were associated with another protein with an apparent molecular weight of 29,000 which was designated 29 k-protein. The CO-reduced spectra of both P-450 UT-7 and UT-7b showed a peak at 448 nm. The NH2-terminal amino acid sequences of P-450 UT-7 and UT-7b were the same as the amino acid sequences of CYP2D1 and CYP2D2 deduced from the cDNA, respectively, except for the lack of a terminal methionine for P-450 UT-7b. In a reconstituted systems, P-450 UT-7 and UT-7b catalyzed lidocaine 3-hydroxylation and N-deethylation in the presence of the 29 k-protein. The Km and Vmax values for lidocaine 3-hydroxylation were 3.6 microM and 0.50 nmol/min/nmol of P-450 for P-450 UT-7, and 3.6 microM and 0.93 nmol/min/nmol of P-450 for P-450 UT-7b, respectively. Antibody against P-450 UT-7, which also cross-reacted with P-450 UT-7b, inhibited lidocaine 3-hydroxylation in liver microsomes from untreated male rats, but had little effect on lidocaine N-deethylation. These findings suggested that lidocaine 3-hydroxylation in hepatic microsomes from untreated male rats was catalyzed by P-450 UT-7 and/or UT-7b.P-450 UT-7 not containing 29 k-protein was obtained as the non-absorbed fraction from hydroxylapatite HPLC. The activities of debrisoquine 4-hydroxylation as well as lidocaine 3-hydroxylation and N-deethylation in a reconstituted system with P-450 UT-7 without 29 k-protein were one-fifth of those of P-450 UT-7 containing 29 k-protein at the same substrate concentration. These findings suggested that the 29 k-protein was essential to express the maximal metabolic activities. However, the lidocaine metabolic activity in a reconstituted system with P-450 UT-7 containing 29 k-protein and in hepatic microsomes were not inhibited by 29 k-protein antibody.

Tissue distributions of CYP2D1, 2D2, 2D3 and 2D4 mRNA in rats detected by RT-PCR.

The tissue distributions of four isoforms (CYP2D1/5, 2D2, 2D3 and 2D4/18) in rat CYP2D subfamily were investigated. Twelve kinds of tissue (liver, kidney, brain, lung, heart, spleen, adrenal gland, small intestine mucosa, bladder, testis, ovary and gonecystis) were removed from Sprague-Dawley male and female rats. The expression of CYP2D mRNA in these tissues was detected by RT-PCR. Specific primers were designed to recognize the four isoforms individually. In liver, kidney and small intestine mucosa, the mRNA expression of all four CYP2D isoforms was detected as high-intensity PCR products. mRNA of CYP2D1/5 was expressed in all tissues used in this study except the brain, although the intensity of PCR products varied among tissues. mRNAs of CYP2D2 and CYP2D3 were mainly expressed in liver, kidney and small intestine mucosa, which were exposed to xenobiotics such as drugs, food components and environmental contaminations. mRNA of CYP2D4/18 was expressed in liver, kidney, small intestine mucosa and brain. In brain, only mRNA of CYP2D4/18 was expressed. CYP2D4/18 mRNA was also expressed in ovary, testis and gonecystis. The tissue distributions help to clarify the differences in physiological and pharmacological functions between CYP2D isoforms.

P450 isoforms in a murine macrophage cell line, RAW264.7, and changes in the levels of P450 isoforms by treatment of cells with lipopolysaccharide and interferon-gamma.

The presence of P450 in a murine macrophage cell line, RAW264.7, was investigated to clarify the biological role and regulation of P450. Microsomes of RAW264.7 cells were isolated and subjected to immunoblotting with anti-rat CYP2A1, 2B1, and 4A2 antibodies. The microsomes gave staining bands with all these antibodies, suggesting the presence of mouse Cyp2a, 2b, and 4a isoforms in RAW264.7. RAW264. 7 cells were treated with typical inducers of P450 (phenobarbital, clofibrate, beta-naphthoflavone and 3-methylcholanthrene). None of these chemicals induced these P450s. Stimulation of RAW264.7 cells with lipopolysaccharide (LPS) and interferon-gamma (INF-gamma) which increase inducible nitric oxide synthase (iNOS) and cytokines in cells decreased Cyp4a protein but not Cyp2a and 2b proteins. To identify P450 isoforms in RAW264.7, we used polymerase chain reaction (PCR) primers for mouse Cyp2a4, 2a12, 2b9/10, 4a10, and 4a12. Total RNA was isolated from these cells and converted to cDNA by reverse transcriptase. PCR was done with these primers and the amplified nucleotides were analyzed by a DNA sequencer. Only Cyp2b9/10 and 4a12 primers gave clear bands, although all primers gave clear bands from liver total RNA. Nucleotide sequences of these products amplified by PCR were identical with Cyp2b9 and 4a12. These findings indicate that Cyp2b9 and 4a12 were present in a macrophage cell line, RAW264.7, and the regulation of P450 by inducers and cytokine differed from that in liver.

Omega- and (omega-1)-hydroxylation of arachidonic acid, lauric acid and prostaglandin A1 by multiple forms of cytochrome P-450 purified from rat hepatic microsomes.

The metabolism of arachidonic acid, lauric acid and prostaglandin A1 by rat hepatic microsomes and multiple forms of cytochrome P-450 purified from rat hepatic microsomes was studied. Arachidonic acid was hydroxylated by hepatic microsomes of male rats by omega- and (omega-1)-hydroxylation. Phenobarbital treatment of rats decreased the hydroxylation activity slightly, but 3-methylcholanthrene treatment increased the hydroxylation activity 2-fold. However, lauric acid and prostaglandin A1 omega- and omega-1)-hydroxylation activities decreased after treatment with phenobarbital and 3-methylcholanthrene. Arachidonic acid and lauric acid were metabolized with similar ratios of omega- and (omega-1)-hydroxylation, but prostaglandin A1 was efficiently metabolized at the omega-position by hepatic microsomes of untreated male rats. In a reconstituted system with purified cytochromes P-450, P450 UT-1, UT-2 (P-450h), MC-1 (P-450d) and MC-5 (P-450c) effectively hydroxylated arachidonic acid at both the omega- and (omega-1)-position. P450 UT-8 hydroxylated arachidonic acid only at the omega-position. P450 DM (P-450j) hydroxylated arachidonic acid at the (omega-1)-position efficiently. Lauric acid was also hydroxylated by P450 UT-1, UT-2, PB-1, PB-2, MC-1, IF-3 (P-450a) and DM, at the (omega - 1)-position only. Only P450 UT-8 could hydroxylate laruic acid at the omega-position. Prostaglandin A1 was efficiently and specifically metabolized by P450 UT-8 with omega-hydroxylation. P450 UT-2 and PB-1 could hydroxylate prostaglandin A1 by (omega-1)-hydroxylation, but with low activity.

Progesterone oxidation by cytochrome P450 2D isoforms in the brain.

The existence of cytochrome P450 2D isoforms in the brain has been demonstrated, although their physiological functions remain to be elucidated. In this study we demonstrated that recombinant rat cytochrome P450 2D1 and 2D4 and human cytochrome P450 2D6 possess progesterone 6 beta- and 16 alpha- hydroxylation activities; 2 beta- and 21-hydroxylation activities; and 2 beta-, 6 beta-, 16 alpha- and 21-hydroxylation activities, respectively. Cytochrome P450 2D4 had the lowest K(m) value and the highest maximum velocity value toward these activities. Progesterone 2 beta- and 21-hydroxylation activities were also detected in rat brain microsomes, and these activities were completely inhibited by anticytochrome P450 2D antibodies. The presence of endogenous 2 beta- and 21-hydroxyprogesterones in rat brain tissues was also demonstrated. The mRNAs of cytochrome P450 2D4, CYP11A, and 3 beta-hydroxysteroid dehydrogenase were detected in the rat brain, suggesting that progesterone was generated from cholesterol by CYP11A and 3 beta-hydroxysteroid dehydrogenase and then underwent hydroxylation to hydroxyprogesterones by cytochrome P450 2D4 in rat brain. Collectively, our findings support the idea that cytochrome P450 2D may be involved in the regulation (metabolism and/or synthesis) of endogenous neuroactive steroids, such as progesterone and its derivatives, in brain tissues.

Differential effects of adrenocorticotropin in vivo on cytochromes P4502D16 and P450c17 in the guinea pig adrenal cortex.

Studies were performed to compare the effects of ACTH treatment in vivo on cytochromes P4502D16 and P450c17 in the guinea pig adrenal cortex. In untreated animals, CYP2D16 protein and messenger RNA (mRNA) expression as well as xenobiotic-metabolizing activities (bufuralol 1'-hydroxylase, benzphetamine N-demethylase, and benzo(a)pyrene hydroxylase) were far greater in the inner (zona reticularis) than the outer (zona fasciculata plus zona glomerulosa) zones of the cortex. ACTH treatment for 3 or 7 days significantly decreased the rates of xenobiotic metabolism in both the inner and outer adrenal zones. Western and Northern blot analyses revealed that adrenal CYP2D16 protein and mRNA concentrations were significantly decreased by ACTH. In contrast to its inhibitory effects on CYP2D16, ACTH treatment increased steroid 17 alpha-hydroxylase activity in the adrenal inner zone, but did not affect outer zone activity. Microsomal CYP17 protein concentrations were not affected by ACTH despite increases in CYP17 mRNA levels in both zones. The results indicate that ACTH causes down-regulation of adrenal CYP2D16, probably at the transcriptional level. Thus, modulation of CYP2D16 by ACTH is opposite that for the steroidogenic P450 isozymes, suggesting unique regulatory mechanisms. In addition, the data suggest that posttranscriptional mechanisms contribute to ACTH regulation of 17 alpha-hydroxylase activity in the guinea pig adrenal cortex.

A new deleted allele in the human cytochrome P450 2A6 (CYP2A6) gene found in individuals showing poor metabolic capacity to coumarin and (+)-cis-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one hydrochloride (SM-12502).

The S-oxidation of (4)-cis-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one hydrochloride (SM-12502) and the 7-hydroxylation of coumarin are primarily catalyzed by cytochrome P450 2A6 (CYP2A6). The activities of SM-12502 S-oxidase and coumarin 7-hydroxylase were investigated with liver microsomes from 20 human individuals. Liver microsomes from individual H16 showed the lowest activities of both enzymes. The expression of CYP2A6 protein was not detectable in liver microsomes from individuals H4, H5, H7, H8, H12 and H16. CYP2A6 mRNA was hardly detectable in the liver of the individual H16. A new SacI-restriction fragment length polymorphism showing the lack of a 2.6 kb fragment was found in two of forty genomic DNA preparations from individuals H16 and No. 594, using CYP2A6 cDNA as a probe. This deletional 2.6 kb fragment was isolated from a genomic library prepared from one individuals showing normal coumarin 7-hydroxylase activity and was sequenced. This fragment contained a CYP2A6 gene region from 319 bp upstream of a putative exon 6 to a SacI site in exon 9, indicating that this region was deleted in the two individuals in this study. We also demonstrated by polymerase chain reaction analysis that the exon 8 of CYP2A6 gene was deleted in individuals H16 and No. 594. These results indicate that the reduced activity of SM-12502 S-oxidase and no activity of coumarin 7-hydroxylase are caused by the lack of CYP2A6 mRNA and CYP2A6 protein caused by the CYP2A6 gene deletion.

Functional evaluation of cytochrome P450 2D6 with Gly42Arg substitution expressed in Saccharomyces cerevisiae.

A single amino acid-substituted mutant protein, CYP2D6 (G42R) was expressed in Saccharomyces cerevisiae and its enzymatic properties were compared with those of other single (P34S, R296C and S486T) and double amino acid-substituted mutant proteins (P34S/S486T and R296C/S486T) expressed in yeast cells, all of which were known to occur in the CYP2D6 gene as single nucleotide polymorphisms. The protein levels of G42R, P34S and P34S/S486T in microsomal fractions and their oxidation capacities towards debrisoquine as a prototypic substrate and bunitrolol as a chiral substrate were different from those of wild-type CYP2D6, while the R296C, S486T and R296C/S486T behaved similarly to the wild-type in these indices. The CYP contents both in yeast microsomal and in whole cell fractions indicated that some part of G42R protein was localized in the endoplasmic reticulum membrane fraction, whereas most of G42R protein was in some subcellular fractions other than endoplasmic reticulum. In kinetic analysis, the G42R substitution increased apparent Km and decreased Vmax for debrisoquine 4-hydroxylation, while it increased both Km and Vmax for bunitrolol 4-hydroxylation. The P34S substitution did not drastically change Km but decreased Vmax for debrisoquine 4-hydroxylation, whereas Km was increased and Vmax unchanged or decreased for bunitrolol 4-hydroxylation by P34S substitution. These results suggest that the G42R substitution causes a change in the CYP2D6 conformation, which may be different from the change produced by the P34S substitution.

Isoforms of cytochrome P450 on organic nitrate-derived nitric oxide release in human heart vessels.

Glutathione S-transferases and the cytochrome P450 system have been proposed for the vascular biotransformation systems in the metabolic activation of organic nitrates. The present study was designed to elucidate the role of human cytochrome P450 isoforms on nitric oxide formation from organic nitrates using lymphoblast microsomes transfected with human CYP isoforms cDNA. CYP3A4-transfected microsomes had the most effective potential of nitric oxide formation from isosorbide dinitrate. Anti-CYP3A2 antibody (which cross-reacts with CYP3A4) or ketoconazole (an inhibitor of the CYP3A superfamily) inhibited nitric oxide formation from isosorbide dinitrate in rat heart microsomes. Immunohistochemistry of human heart also showed intense bindings of CYP3A4 antibody in the endothelium of the endocardium and coronary vessels. These results suggest that the CYP3A4-NADPH-cytochrome P450 reductase system specifically participates in nitric oxide formation from isosorbide dinitrate.

Escape from tolerance of organic nitrate by induction of cytochrome P450.

The mechanism of organic nitrate tolerance is poorly defined. We studied the rat P450-catalyzed conversion of organic nitrate to nitric oxide (NO) by purified P450 isoforms relationship between P450 expression and nitrate tolerance following continuous infusion of organic nitrates in rats. The hypotensive effect of an nitroglycerin (NTG) bolus injection was abolished in rats that had been previously provided a continuous 48 h infusion of NTG. This effect was accompanied by a gradual but marked decrease in plasma and urinary nitrate levels following a peak at 18-24 h. Nitrate tolerance was reversible; the decline in the hypotensive effect and P450 levels observed after 2 d of continuous infusion was followed by restoration to control levels 2 d after cessation of the infusion. Similarly, the hypotensive action disappeared in P450-depleted, and -inhibited rats. At 48 h after infusion, NTG-induced NO generation of the vessels increased in acetone (a P450 inducer) -pretreated rats. The appearance and disappearance of P450 paralleled the conversion of organic nitrates to NO. Our observations indicate that nitrate tolerance is in large part the result of decreased P450 expression and activity. Interventions that maintain or increase P450 activity may be a strategy to provide relief from ischemic conditions in humans.

Identification and characterization of motility stimulating factor secreted from pancreatic cancer cells: role in tumor invasion and metastasis.

Cell motility is an important factor in the process of invasion and metastasis of tumor. In this study, the relationship between cell motility and experimental metastatic potential was examined using two human pancreatic cancer cell lines, SW1990 and PANC-1. Serum-free conditioned medium from the highly metastatic cell line SW1990 was found to contain a factor that stimulated the migration of and induced a fibroblast-like morphological change in the weakly metastatic cell line PANC-1. Preincubation of PANC-1 cells with SW1990 conditioned medium (SW-C.M.) induced liver metastasis following splenic injection of PANC-1 cells in nude mice, although no liver metastasis was observed without pretreatment of SW-C.M. This factor, temporarily termed PDMF (pancreatic cancer-derived motility factor) is a heparin non-binding protein having a molecular weight of 40 kDa calculated by gel-filtration HPLC which acts not only chemotactically but also chemokinetically, and also acts mainly in a paracrine fashion. However, this factor had no effect on the proliferation of PANC-1 cells; it therefore appears to be a so-called motility factor. Only TGF-beta1 and IL-6 were recognized in the SW-C.M. among cytokines thought to stimulate cell motility. These cytokines stimulated the motility of PANC-1 cells, but differed from PDMF in the neutralizing test with antibody against these cytokines. Results of characterization and preliminary purification suggest that this factor may be a novel motility factor. The above findings suggest that this motility factor may play an important role in the invasion and metastasis of pancreatic cancer, and complete purification of it will be useful in elucidating the mechanism of progression of cancer and designing a strategy for inhibition of invasion and metastasis of pancreatic cancer.

Prenatal and neonatal exposure to bisphenol-A enhances the central dopamine D1 receptor-mediated action in mice: enhancement of the methamphetamine-induced abuse state.

Bisphenol-A (BPA), one of the most common environmental endocrine disrupters, has been extensively evaluated for toxicity in a variety of tests in rodents, including developmental and reproductive toxicity, and carcinogenicity. However, little is known about its action on the CNS. In this report, we show that prenatal and neonatal exposure to BPA in mice leads to the enhancement of the dopamine D1 receptor-dependent rewarding effect induced by a psychostimulant methamphetamine. Furthermore, this treatment with BPA markedly enhanced hyperlocomotion and its sensitization induced by methamphetamine, which reflects extensive abuse associated with sociological and psychiatric problems. We also demonstrated that chronic exposure to BPA produced an up-regulation of dopamine D1 receptor function to activate G-protein in the mouse limbic forebrain, which is thought to be a critical site for the expression of rewarding effects by abuse drugs. Additionally, chronic BPA exposure produced a significant increase in levels of the dopamine D1 receptor mRNA in the whole brain. In contrast, no change in protein levels of methamphetamine-targeted proteins, dopamine transporter or the type 2 vesicle monoamine transporter in the brain was observed by prenatal and neonatal exposure to BPA. The present data provide the first evidence that prenatal and neonatal exposure to BPA can potentiate the central dopamine D1 receptor-dependent neurotransmission, resulting in supersensitivity of methamphetamine-induced pharmacological actions related to psychological dependence on psychostimulants.

Involvement of CYP3A-derived arachidonic acid metabolite(s) in responses to endothelium-derived K+ channel opening substance in monkey lingual artery.

1. In monkey lingual artery strips partially contracted with prostaglandin F2alpha, acetylcholine-induced, concentration-related relaxations were abolished by removal of the endothelium. The response was not significantly influenced by indomethacin but attenuated by NG-nitro-L-arginine (L-NOARG); the effect of the nitric oxide (NO) synthase inhibitor was reversed by L-arginine. 2. The response to acetylcholine resistant to L-NOARG was suppressed in the strips exposed to high K+ media. Charybdotoxin partially inhibited the relaxation, and the remaining relaxation was abolished by additional treatment with apamin, whereas glibenclamide, iberiotoxin or apamin alone was without effect. Relaxations induced by sodium nitroprusside were not influenced by charybdotoxin. 3. The L-NOARG-resistant acetylcholine-induced relaxation was inhibited by metyrapone, proadifen and 17-octadecynoic acid, non-selective cytochrome P450 mono-oxygenase (CYP) inhibitors, and progesterone and ketoconazole, inhibitors selective to CYP3A. The inhibitors did not affect the nitroprusside-induced relaxation. Selective inhibitors of other CYP isoforms, such as debrisoquine and lauric acid, did not reduce the response to acetylcholine. 4. Reaction mixture containing human liver microsome rich in CYPs, arachidonic acid and NADPH incubated at 37 degrees C and filtrated relaxed endothelium-denuded monkey lingual artery strips, used as bioassay tissues. This response was abolished in the strips exposed to high K+ media. The response was also suppressed by combined treatment of the assay tissue with charybdotoxin plus apamin, but was not affected by treatment with iberiotoxin. The reaction mixture co-incubated with ketoconazole failed to relax the strips. 5. It is concluded that the monkey lingual arterial relaxation dependent on the endothelium is mediated by NO and also by a charybdotoxin plus apamin-sensitive but iberiotoxin-insensitive Ca2+-activated K+ channel opening substance(s) that may be a CYP3A-derived arachidonic acid metabolite(s).