Smooth muscle of the male pelvic floor: An anatomic study.

Knowledge of the anatomy of the male pelvic floor is important to avoid damaging the pelvic floor muscles during surgery. We set out to explore the structure and innervation of the smooth muscle (SM) of the whole pelvic floor using male fetuses. We removed en-bloc the entire pelvis of three male fetuses. The specimens were serially sectioned before being stained with Masson's trichrome and hematoxylin and eosin, and immunostained for SMs, and somatic, adrenergic, sensory and nitrergic nerve fibers. Slides were digitized for three-dimensional reconstruction. We individualized a middle compartment that contains SM cells. This compartment is in close relation with the levator ani muscle (LAM), rectum, and urethra. We describe a posterior part of the middle compartment posterior to the rectal wall and an anterior part anterior to the rectal wall. The anterior part is split into (1) a centro-levator area of SM cells localized between the right and left LAM, (2) an endo-levator area that upholsters the internal aspect of the LAM, and (3) an infra-levator area below the LAM. All these areas are innervated by autonomic nerves coming from the inferior hypogastric plexus. The core and the infra-levator area receive the cavernous nerve and nerves supplying the urethra. We thus demonstrate that these muscular structures are smooth and under autonomic influence. These findings are relevant for the pelvic surgeon, and especially the urologist, during radical prostatectomy, abdominoperineal resection and intersphincteric resection. Clin. Anat., 2019. © 2019 Wiley Periodicals, Inc.

Prognostic and predictive values of oncogenic BRAF, NRAS, c-KIT and MITF in cutaneous and mucous melanoma.

BACKGROUND: Mutations of BRAF, NRAS and c-KIT oncogenes are preferentially described in certain histological subtypes of melanoma and linked to specific histopathological features. BRAF-, MEK- and KIT-inhibitors led to improvement in overall survival of patients harbouring mutated metastatic melanoma. OBJECTIVES: To assess the prevalence and types of BRAF, NRAS, c-KIT and MITF mutations in cutaneous and mucous melanoma and to correlate mutation status with clinicopathological features and outcome. METHODS: Clinicopathological features and mutation status of 108 samples and of 98 consecutive patients were, respectively, assessed in one retrospective and one prospective study. Clinicopathological features were correlated with mutation status and the predictive value of these mutations was studied. RESULTS: This work identified significant correlations between BRAF mutations and melanoma occurring on non-chronic sun-damaged skin and superficial spreading melanoma (P < 0.05) on one hand, and between NRAS mutations and nodular melanoma (P < 0.05) on the other hand. Younger age (P < 0.05), microscopic (P < 0.05) and macroscopic (P < 0.05) lymphatic involvement at diagnosis of primary melanoma were significantly linked to BRAF mutations. A mutated status was a positive predictive factor of a response to BRAF inhibitors (OR = 3.44). Mutated melanoma showed a significantly (P = 0.038) higher objective response rate to cytotoxic chemotherapy (26.3%) than wild-type tumours (6.7%). CONCLUSION: Clinical and pathological characteristics of the primary melanoma differed between wild-type and BRAF- or NRAS-mutated tumours. Patients with BRAF-mutated tumours were younger at diagnosis of primary melanoma. Patients carrying mutations showed better responses better to specific kinase inhibitors and interestingly also to systemic cytotoxic chemotherapy.

Interleukin-1 beta antagonizes phenobarbital induction of several major cytochromes P450 in adult rat hepatocytes in primary culture.

We have investigated the effects of interleukin (IL)-1 beta and IL6 on expression and phenobarbital (PB) induction of ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-deethylase (PROD) activities, as well as on mRNA levels of cytochromes P450 (CYP) 1A, 2B, 2C, 2E and 3A, in rat hepatocytes in primary culture. IL6 slightly antagonized PB-induced PROD activity. Strikingly, IL1 beta strongly inhibited basal EROD and PROD activities, and fully blocked their induction by PB in a dose-dependent fashion. Furthermore IL1 beta completely suppressed PB induction of all CYP mRNAs analyzed. Our results demonstrate that IL1 beta can suppress basal CYP activities, as well as PB-inducible expression of five CYP mRNAs in rat hepatocytes in primary culture.

Phenobarbital induces cytochrome P4501A2 hnRNA, mRNA and protein in the liver of C57BL/6J wild type and aryl hydrocarbon receptor knock-out mice.

The aryl hydrocarbon receptor mediates the transcriptional response to a variety of hydrocarbons of members of the aryl hydrocarbon gene battery. Phenobarbital does not bind the aryl hydrocarbon receptor with high affinity but induces, in liver cells, expression of cytochrome P4501A. Using both wild type and aryl hydrocarbon receptor knock out C57BL/6J mice, we demonstrate that phenobarbital induced hnRNA, mRNA and protein for the cytochrome P-4501A2 gene in the presence or absence of the aryl hydrocarbon receptor. Using the DNA binding site for the aryl hydrocarbon receptor as a probe, gel retardation analyses showed that phenobarbital treatment induced protein binding, regardless of the presence of the aryl hydrocarbon receptor.

Survival and function of isolated hepatocytes after cryopreservation.

Cryopreservation in liquid nitrogen is presently the only way for long-term storage of isolated hepatocytes. Freeze-thaw conditions are not well defined yet; the most critical parameters appear to be the choice of the cryoprotectant, composition of the freezing medium, and cooling and thawing rates. Comparable results have been obtained with hepatocytes from various species, including man. Cryopreservation usually results in low cell recovery and early alterations of functional activities. However, both phase I and phase II xenobiotic metabolism is still active after thawing, at least during a short period. Moreover, survival and function of cryopreserved hepatocytes can be improved when these cells have a high energy status, are cryopreserved after immobilization in a gel, separated from dead cells on a Percoll gradient or placed in more favorable culture conditions (e.g. in coculture with liver non parenchymal cells). Additional studies are needed to improve freeze-thaw protocols and to better characterize liver parenchymal cells after storage, including evaluation of their responsiveness to specific inducers.

A multi-laboratory evaluation of cryopreserved monkey hepatocyte functions for use in pharmaco-toxicology.

Ethical, economic and technical reasons hinder regular supply of freshly isolated hepatocytes from higher mammals such as monkey for preclinical evaluation of drugs. Hence, we aimed at developing optimal and reproducible protocols to cryopreserve and thaw parenchymal liver cells from this major toxicological species. Before the routine use of these protocols, we validated them through a multi-laboratory study. Dissociation of the whole animal liver resulted in obtaining 1-5 billion parenchymal cells with a viability of about 86%. An appropriate fraction (around 20%) of the freshly isolated cells was immediately set in primary culture and various hepato-specific tests were performed to examine their metabolic, biochemical and toxicological functions as well as their ultrastructural characteristics. The major part of the hepatocytes was frozen and their functionality checked using the same parameters after thawing. The characterization of fresh and thawed monkey hepatocytes demonstrated the maintenance of various hepato-specific functions. Indeed, cryopreserved hepatocytes were able to survive and to function in culture as well as their fresh counterparts. The ability for synthesis (proteins, ATP, GSH) and conjugation and secretion of biliary acids was preserved after deep freeze storage. A better stability of drug metabolizing activities than in rodent hepatocytes was observed in monkey. After thawing, Phase I and Phase II activities (cytochrome P450, ethoxycoumarin-O-deethylase, aldrin epoxidase, epoxide hydrolase, glutathione transferase, glutathione reductase and glutathione peroxidase) were well preserved. The metabolic patterns of several drugs were qualitatively and quantitatively similar before and after cryopreservation. Lastly, cytotoxicity tests suggested that the freezing/thawing steps did not change cell sensitivity to toxic compounds.

Use of cryopreserved animal and human hepatocytes for cytotoxicity studies.

To evaluate the potential use of cryopreserved hepatocytes for toxicological studies, rat, dog and human hepatocytes were frozen in Leibovitz medium containing 20% foetal calf serum and various concentrations of dimethylsulphoxide and stored in liquid nitrogen. 50% or more of the hepatocytes that attached and survived immediately after isolation still possessed these properties after freezing/thawing. Thawed hepatocytes from the three species showed only a slight reduction in their ability to metabolize phenacetin or to conjugate paracetamol with glucuronic acid. Sensitivity to the toxic effects of erythromycin was not affected by the MTT and neutral red assays. Similar observations were made with rat hepatocytes for five other toxic agents-chloramphenicol, chlorpromazine, acrylamide, chloroquine sulphate and p-chloromercuribenzoic acid. These results suggest that, after cryopreservation, isolated hepatocytes represent a suitable model for drug metabolism and toxicity screening studies.

A multicentre study of acute in vitro cytotoxicity in rat liver cells.

A multicentre validation study of the acute in vitro cytotoxicity of drugs involving six French laboratories from INSERM or pharmaceutical companies has been carried out. Thirty liquid or solid chemicals such as antibiotics, anticancer drugs and solvents were selected and incubated for 20 hr with normal rat hepatocytes and FaO hepatoma cells. Miniaturized and automated methods were defined for the evaluation of cytotoxic effects. Four endpoints were evaluated: the ratio of extracellular lactate dehydrogenase to total lactate dehydrogenase, total cellular protein content, reduction of a tetrazolium salt, and neutral red uptake. For each test IC(50) values were calculated. A good interlaboratory reproducibility was demonstrated. The neutral red assay was found to be the most sensitive and the least reproducible endpoint. More compounds were shown to be cytotoxic to hepatocytes than to hepatoma cells (18 v. 12). On the basis of the IC(50) values a few compounds were found to be much less cytotoxic than predicted from in vivo data, suggesting that a simple experimental protocol and non-specific cytotoxicity parameters are not sufficient to test certain drug families. However, such methods appear to provide a useful means of defining the concentration range of the drug that will be selected for further analysis using more specific tests.

Pentoxifylline aggravates fatty liver in obese and diabetic ob/ob mice by increasing intestinal glucose absorption and activating hepatic lipogenesis.

BACKGROUND AND PURPOSE: Pentoxifylline is in clinical trials for non-alcoholic fatty liver disease and diabetic nephropathy. Metabolic and hepatic effects of pentoxifylline were assessed in a murine model of obesity and type 2 diabetes. EXPERIMENTAL APPROACH: Pentoxifylline (100 mg·kg(-1) ·day(-1)) was administered for 4 days or 3 weeks in lean and obese/diabetic ob/ob mice. Plasma lipids, glucose, other metabolites and relevant enzymes were measured by standard assays. Hepatic lipids in vivo were assessed with magnetic resonance spectroscopy and by histology. Hepatic extracts were also analysed with RT-PCR and Western blotting. KEY RESULTS: Four days of pentoxifylline treatment slightly increased liver lipids in ob/ob mice. After 3 weeks, pentoxifylline exacerbated fatty liver and plasma transaminases in ob/ob mice but did not induce liver steatosis in lean mice. Plasma glucose was highest in fed, but not fasted, ob/ob mice treated with pentoxifylline. During the first 10 min of an oral glucose tolerance test, blood glucose increased more rapidly in pentoxifylline-treated mice. Jejunal expression of glucose transporter 2 isoform was increased in pentoxifylline-treated obese mice. Hepatic activity of carbohydrate response element binding protein (ChREBP) increased after pentoxifylline in ob/ob, but not lean, mice. Hepatic expression of lipogenic enzymes was highest in pentoxifylline-treated ob/ob mice. However, pentoxifylline reduced markers of oxidative stress and inflammation in ob/ob liver. CONCLUSION AND IMPLICATIONS: Pentoxifylline exacerbated fatty liver in ob/ob mice through enhanced intestinal glucose absorption, increased postprandial glycaemia and activation of hepatic lipogenesis. Long-term treatment with pentoxifylline could worsen fatty liver in some patients with pre-existing hyperglycaemia.

RNAi-mediated ERK2 knockdown inhibits growth of tumor cells in vitro and in vivo.

The MAPK MEK/ERK pathway is often upregulated in cancer cells and represents an attractive target for development of anticancer drugs. Only few data concerning the specific functions of ERK1 and 2 are reported in the literature. In this report, we investigated the specific role of ERK1 and 2 in liver tumor growth both in vitro and in vivo. DNA synthesis and cells in S phase analysed by flow cytometry, correlated with strong inhibition of Cdk1 and cyclin E levels, are strongly reduced after exposure to the MEK inhibitor, U0126. We obtained a significant reduction of colony formation in soft agar assays and a reduction in the size of tumor xenografts in nude mice treated with U0126. Then, we could specifically abolished ERK1 or 2 expression by small-interfering RNA (siRNA) and demonstrated that ERK2 knockdown but not ERK1 interferes with the process of replication. Moreover, we found that colony formation and tumor growth in vivo were significantly inhibited by targeting ERK2 using stable chemically modified siRNA. Taken together, our results emphasize the importance of the MEK/ERK pathway in liver cancer cell growth in vitro and in vivo and argue for a crucial role of ERK2 in this regulation.

Genetic analysis of the phenobarbital regulation of the cytochrome P-450 2b-9 and aldehyde dehydrogenase type 2 mRNAs in mouse liver.

The aim of this study was to investigate the effect of the genetic background on the phenobarbital inducibility of cytochrome P-450 2b-9, cytochrome P-450 2b-10 and aldehyde dehydrogenase type 2 mRNAs in mice. We analysed the basal expression and the phenobarbital inducibility of both cytochrome P-450 mRNAs by semi-quantitative specific reverse transcription-PCR analyses in five inbred mouse strains (A/J,BALB/cByJ,C57BL/6J, DBA/2J and SWR/J). Male mice constitutively expressed cytochrome P-450 2b-9 and cytochrome P-450 2b-10 mRNAs, but a number of differences in their response to phenobarbital were observed. In all these mouse strains, phenobarbital induced cytochrome P-450 2b-10 mRNA whereas it could have either a positive or a negative effect on cytochrome P-450 2b-9 expression, depending on the strain and the sex of the mice. Specifically, phenobarbital increased cytochrome P-450 2b-9 expression in C57BL/6J males while it decreased it in DBA/2J mice. Interestingly, dexamethasone was able to mimic the phenobarbital effect on both cytochromes P-450 in these two strains. Aldehyde dehydrogenase type 2 mRNA was always induced by phenobarbital, except in the C57BL/6J strain. Genetic analysis revealed that the phenobarbital-inducible phenotype was either a semi-dominant or a recessive trait in F1 animals from a C57BL/6J x DBA/2J cross for the cytochrome P-450 2b-9 and the aldehyde dehydrogenase type 2 genes, respectively. This study suggests that the genetic basis for phenobarbital induction in mice depends on the target gene, and that more than one regulatory step would by involved in this response pathway.

Long-term maintenance of drug-metabolizing enzyme activities in rat hepatocytes after cryopreservation.

Recent studies have demonstrated that freshly isolated adult hepatocytes from various species can be hypothermically preserved for a short period or cryopreserved for a prolonged period before seeding in primary culture. This study was designed to determine whether rat hepatocytes could be maintained functional for a prolonged period after either hypothermic preservation or cryopreservation. Cold storage was carried out in University of Wisconsin solution (UW) and freezing in Leibovitz medium added with 10% fetal calf serum and 16% dimethyl sulfoxide. Rat hepatocytes were then set up either in pure conventional culture or in coculture with rat liver epithelial cells. Various functions were measured over 4- and 15-day periods, i.e., albumin secretion rate, deethylation of ethoxyresorufin and phenacetin, dealkylation of pentoxyresorufin, glucuronidation and sulfoconjugation of paracetamol, and N-acetylation of procainamide. No major differences were observed between unfrozen, frozen, and UW-preserved cells. While in pure culture all the functions tested were markedly decreased after 3 or 4 days, they remained high over the 15-day period in coculture, being either maintained or increased after 7-12 days compared to initial values. These results clearly demonstrate that when maintained under suitable culture conditions, rat hepatocytes can fully recover after hypothermic preservation or cryopreservation and therefore represent a suitable in vitro model system for pharmacotoxicological studies.

Phenobarbital induction of aldehyde dehydrogenase type 2 mRNA in mouse liver: a candidate region on chromosome 7 for a putative regulatory gene.

Phenobarbital (PB) strongly induces in the liver the expression of many genes encoding detoxication enzymes, such as the aldehyde dehydrogenase type 2 in the mouse (Aldh2). With the aim of identifying genes involved in this response, we have undertaken an approach based on a genetic analysis in mice. In a previous report, the genetic analysis of both the C57BL/6J (B6) x DBA/2J (D2) F1 and the (F1 x F1) F2 led us to the hypothesis that Aldh2 responsiveness to PB was under the control of one major locus independent of the structural gene. In the present study, the genetic analysis of the inducibility by PB of Aldh2 in the backcross population B6D2F1 x D2 has allowed us to confirm the involvement of a major regulatory gene in this mechanism. By searching for genetic linkage between this locus and a series of microsatellites DNA markers, we obtained indicative evidence for a region on chromosome 7, which may carry this gene.

Transforming growth factor-beta 1 down-regulates basal and polycyclic aromatic hydrocarbon-induced cytochromes P-450 1A1 and 1A2 in adult human hepatocytes in primary culture.

The effects of interleukin (IL)-1 beta, IL-4, IL-6, tumor necrosis factor (TNF)-alpha, interferon (IFN)-alpha, IFN-gamma, and transforming growth factor (TGF)-beta 1 on cytochrome P-450 (CYP) 1A expression and polycyclic aromatic hydrocarbon (PAH)-mediated induction in primary human hepatocyte cultures were determined. Most cytokines that were previously found to decrease basal CYP expression could counteract PAH induction of CYP1A mRNA and its associated ethoxyresorufin-O-deethylation (EROD) activity. IL-1 beta and TNF-alpha blocked 3-methylcholanthrene (3-MC)-induced EROD activity by up to 25 and 44%, respectively. IFN-alpha and IFN-gamma antagonized EROD induction by up to 61 and 70%, respectively. TGF-beta 1 proved to be the most effective cytokine, because 72 hr of treatment with 2 ng/ml TGF-beta 1 produced nearly 100% inhibition of 3-MC- and benzo(a)pyrene-induced CYP1A1 and CYP1A2 mRNAs and EROD activity. Treatment with cycloheximide in combination with 3-MC led to superinduction of CYP1A mRNA, under which conditions TGF-beta 1 did not block induction, suggesting the requirement for protein synthesis for the suppressive effect of the cytokine. In addition, TGF-beta 1 augmented AP-1-binding activity, suggesting that fos and/or jun protooncogene products could be implicated in the response. Our results demonstrate that IL-1 beta, TNF-alpha, and IFNs antagonized PAH-mediated induction of CYP1A gene expression in human hepatocytes. In addition, we report the finding of a novel effect of TGF-beta 1, which was able to prevent CYP1A1 and -1A2 induction by two different PAHs.

Halothane-induced cytotoxicity to rat centrilobular hepatocytes in primary culture is not increased under low oxygen concentration.

BACKGROUND: Halothane can be metabolized by both oxidative and reductive pathways in the liver. This anesthetic can induce direct liver injury preferentially localized in centrilobular areas, probably in relation with lower oxygen tension. The reductive pathway has been related to liver damage; however, a correlation between lower oxygen concentration in centrilobular areas, the extent of reductive metabolism of halothane, and the degree of liver injury has not yet been demonstrated. This study was designed to better evaluate the toxicity of the reduced metabolites by using centrilobular and periportal rat hepatocyte subpopulations. METHODS: Adult rat hepatocytes, either as whole cell preparations or after separation in centrilobular and periportal cell subpopulations, were placed in primary culture and exposed to either 2% or 4% halothane under various oxygen concentrations. The enriched centrilobular hepatocyte subpopulations isolated by the digitonin-collagenase method were characterized by immunolocalization of glutamine synthetase. Three oxygen concentrations were tested: 5%, 20%, and 95%, and the main parameters measured were cell viability and fluoride ion formation. RESULTS: Viability of centrilobular hepatocytes was similar under 5% and 20% O2, but the unpurified hepatocyte population was more susceptible to 5% O2 (P < 0.01). Significantly higher cytochrome P-450 content was found in whole hepatocyte populations under 5% versus 20% oxygen, indicating that centrilobular hepatocytes that contained higher cytochrome P-450 monooxygenase activities were less sensitive to low oxygen concentrations. Halothane toxicity to centrilobular hepatocytes was enhanced under 95% versus 20% O2 (P < 0.05). By contrast, no significant difference was observed when the cells were maintained under 5% O2, although fluoride ions, indicative of reductive metabolism of halothane, were found in much higher amounts in the culture medium. Moreover, under 20% O2, halothane toxicity was significantly greater in centrilobular versus unpurified hepatocytes (P < 0.05). CONCLUSIONS: Isolated centrilobular hepatocytes appear to be more sensitive to halothane than their periportal counterparts in vitro. However, the authors' results support the conclusion that increased reductive metabolism of halothane induced by decreasing oxygen concentration is not a critical parameter for the occurrence of liver damage in these cells.

Viability and primary culture of rat hepatocytes after hypothermic preservation: the superiority of the Leibovitz medium over the University of Wisconsin solution for cold storage.

Hepatocytes isolated from adult rat livers were hypothermically preserved for 24 or 48 hr before being plated under conventional culture conditions. They were stored either in the Leibovitz medium, a cell culture medium with and without polyethylene glycol (PEG), a compound known to suppress ischemia-induced cell swelling, or in the University of Wisconsin solution, the most effective solution for cold organ preservation. After 24 or 48 hr of storage at 4.5 degrees C in Leibovitz medium, cell viability and adherence efficiency to plastic dish, were only slightly reduced, whereas University of Wisconsin hepatocytes had a decreased viability and (especially after 48-hr storage) lost their adhesion ability; they did not survive in vitro. The metabolic competence of hepatocytes maintained in Leibovitz medium was retained over the 3 days of culture, as shown by low extracellular levels of the membrane-bound and cytosolic hepatic enzymes, as well as by intracellular glutathione content, albumin secretion rate and several phase I and phase II drug metabolic reactions very close to those found with fresh hepatocytes maintained under similar culture conditions. Addition of polyethylene glycol to the Leibovitz medium resulted in slightly higher viability and function of hepatocytes after cold storage. These results clearly demonstrate that viability of a transplanted liver does not correlate with long-term in vitro viability of isolated hepatocytes after hypothermic preservation in University of Wisconsin solution. They also suggest that nutritional and energy substrates as found in the Leibovitz medium are probably required to define a suitable solution for cold preservation of isolated parenchymal cells. The findings with Leibovitz medium favor the conclusion that hypothermically preserved hepatocytes could be used for various metabolic studies and for the treatment of liver insufficiency.

Isolation of a new mouse cDNA clone: hybrid form of cytochrome P450 2b10 and NADPH-cytochrome P450 oxidoreductase.

We have isolated a novel cDNA sequence from a male mouse liver library. It consists of an open reading frame containing no stop codon of 1348 bp which share 98% similarity with the cytochrome P450 2b10 followed by 95 nucleotides sharing 88% identity with the mouse NADPH cytochrome P450 oxidoreductase. Proteins are encoded by the opposite DNA strands. Genomic amplification excludes a library artefact or a de novo recombination event. RT-PCR analyses revealed expression of this clone in the liver of male and female mice as well as in kidney, lung and intestine. This cDNA was not, however, induced by phenobarbital. Preliminary results indicate that this cDNA could span more than 2.5 kb. This study is the first description of a cytochrome P450 cDNA sequence encoding part of both a cytochrome P450 and the NADPH cytochrome P450 oxidoreductase in mammalian liver.

Involvement of cyclic nucleotide- and calcium-regulated pathways in phenobarbital-induced cytochrome P-450 3A expression in mouse primary hepatocytes.

Several of the hepatic microsomal cytochromes P-450 (CYP) including CYP3A are inducible by phenobarbital (PB). However, the intracellular pathways involved in the action of PB on CYP3A remain poorly known. With the aim to unravel some of the main aspects of PB signaling, we first devised a simple model of mouse cultured primary hepatocytes in which CYP3A mRNA and protein were strongly induced by PB in the absence of dexamethasone and were at maximum levels after a 48-h treatment with a 2-mM dose of PB. Under these culture conditions, we studied the effects of inhibitors and activators of different protein kinases or phosphatases on CYP3A mRNA and protein induction by PB. CYP3A-induced expression was inhibited by activators of cyclic AMP-dependent protein kinase (PKA) (dibutyryl-cyclic AMP and forskolin) whereas inhibition of PKA by PKA inhibitor enhanced induction. 8-br-cGMP produced effects similar to the activators of PKA, and so did the specific inhibitor of cGMP-dependent protein kinase, beta-phenyl-1, N(2)-etheno-8-bromoguanosine-3,5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-Br-PET-cGMPS). Inhibition of Ca(2+)/calmodulin-dependent protein kinase by KN-62 or the intracellular Ca(2+) chelator BAPTA-AM produced an inhibition of CYP3A induction by PB. Specific inhibitors of protein kinase C, mitogen-activated protein kinase kinase, phosphatidylinositol-3-kinase, or serine/threonine phosphatase did not produce any effect. Taken together, our results suggest that CYP3A induction by PB is regulated positively by calmodulin-dependent protein kinase and cGMP-dependent protein kinase, and negatively by PKA in mouse hepatocytes in primary culture.