Cytochrome P450 Products Research Articles

Abstract 224: 20-hydroxyeicosatetraenoic Acid Mediated Amp Activated Protein Kinase Suppression Induces Endothelial Nitric Oxide Synthase Uncoupling

20-Hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450 product of arachidonic acid metabolism, is a potent vasoconstrictor thought to be involved in vascular dysfunction and the regulation of blood pressure. 20-HETE has been demonstrated to play an inhibitory role in eNOS activity and both animal and human studies have revealed strong associations between 20-HETE and endothelial dysfunction. However, the signalling mechanisms are largely unknown. Previous studies have shown that AMPK activity is involved in the Hsp90-eNOS association. Therefore we sought to investigate the effect of 20-HETE on eNOS and Hsp90 association, and whether this was mediated by AMPK. Treatment of human umbilical vein endothelial cells (HUVECs) with 10μM 20-HETE for 24h induces an increase in eNOS phosphorylation, which is not observed following acute treatment (30mins). This was accompanied by transient changes in Akt phosphorylation. Conversely, upstream AMPK activity increased rapidly following 20-HETE treatment (<1min) before decreasing by 30mins. Treatment with 20-HETE also resulted in an acute increase in the production of reactive oxygen species (ROS), which returned to control levels at >2hr post-20-HETE treatment. Immunoprecipitation of eNOS in cells treated with 20-HETE revealed a decrease in basal, VEGF and A23187 stimulated Hsp90 association with eNOS, indicating an uncoupling mechanism. Pre-treatment of the cells with AICAR (a chronic activator of AMPK) prevented the loss of Hsp90 association with eNOS following 20-HETE treatment. To further investigate the functional effect of Hsp90 dissociation, we utilised an endothelial-smooth muscle cell system to measure cGMP production. Chronic 20-HETE treatment appeared to reduce both basal and VEGF stimulated cGMP levels. Pre-treatment of the cells with AICAR protected against the 20-HETE induced decrease in cGMP levels, both basally and following VEGF stimulation. In conclusion, 20-HETE suppression of AMPK activity impairs eNOS-Hsp90 association, providing a mechanism for reduced NO bioactivity and endothelial dysfunction in diseases with elevated 20-HETE levels, such as hypertension.

Analysis ofl-NAME-dependent and -resistant responses to acetylcholine in the rat

The mechanism by which acetylcholine (ACh) decreases systemic arterial pressure and hindlimb vascular resistance was investigated in the anesthetized rat. ACh injections caused dose-dependent decreases in systemic arterial pressure and hindlimb vascular resistance. N(omega)-nitro-L-arginine methyl ester (L-NAME) had little effect on the magnitude of depressor and vasodilator responses but decreased response duration when baseline parameters were corrected by a nitric oxide (NO) donor infusion. The decrease in the duration of the ACh depressor response was prevented by the administration of excess L-arginine. The L-NAME-resistant component of the depressor response to ACh was attenuated by ebselen, a glutathione peroxidase mimic. The calcium-activated potassium (K(Ca)) antagonists charybdotoxin (ChTX) and apamin decreased the magnitude but not the duration of the hindlimb vasodilator response to ACh. The combination of L-NAME, ChTX, and apamin reduced the magnitude and duration of the vasodilator response to ACh but not to sodium nitroprusside. Vasodepressor and hindlimb vasodilator responses to ACh were not modified by cytochrome P-450 and cyclooxygenase pathway inhibitors. These results suggest that the hindlimb vasodilator response to ACh has an initial L-NAME-resistant component mediated by the activation of K(Ca) channels and a sustained L-NAME-dependent component. The results with ebselen suggest that the L-NAME-resistant component of the depressor response involves a peroxide-sensitive mechanism. The present study suggests that vasodilator responses to ACh are not mediated by cytochrome P-450 products, since miconazole and 1-aminobentriazole alone or in combination did not affect either component of the response. The present data suggest that the hindlimb vasodilator response to ACh in the rat is mediated by two mechanisms with an initial ChTX- and apamin-sensitive, L-NAME-resistant phase not mediated by cytochrome P-450 products and a secondary sustained phase mediated by NO.

Estrogenic modulation of CYP3A38, CYP3A40, and CYP19 in mature male medaka ( Oryzias latipes)

We examined cytochrome P450 production and activity and circulating hormone concentrations in male medaka exposed to 17β-estradiol (E2) or 17α-ethinylestradiol (EE2). Intraperitoneal injection of E2 at 1, 10, or 100 μg/g-fish completely suppressed CYP3A38 protein production and suppressed CYP3A40 protein levels by 89%, 52%, or 47%, respectively. CYP3A38 and CYP3A40 mRNA expression was unaltered, and CYP3A enzymatic activity initially increased and then decreased with increasing E2 dose. Males co-cultured with females were exposed to a markedly high concentration (43 ng/L) of E2 secreted by females. CYP3A protein levels in co-cultured males were suppressed. Serum testosterone (TE) and 11 keto-testosterone levels in co-cultured males were downregulated to 40% of pre-exposure levels. Serum E2 levels increased in co-cultured males or males exposed to EE2. Testicular CYP19, which converts TE to E2, increased by 9.5 times in males exposed to 50 ng/L EE2 and by 21.5 times in those exposed to 100 ng/L EE2. Male medaka exposed to EE2 showed increased serum Vtg levels. Estrogenic exposure induced Vtg production, suppressed CYP3A protein production, downregulated TE metabolism, and enhanced CYP19 activity. Serum E2 endogenously induced by CYP19 could contribute to Vtg induction in male medaka.

Isolation and Characterization of a Stem Cell Population from Adult Human Liver

Several studies suggested the presence of stem cells in the adult normal human liver; however, a population with stem cell properties has not yet been isolated. The purpose of the present study was to identify and characterize progenitor cells in normal adult human liver. By stringent conditions of liver cell cultures, we isolated and characterized a population of human liver stem cells (HLSCs). HLSCs expressed the mesenchymal stem cell markers CD29, CD73, CD44, and CD90 but not the hematopoietic stem cell markers CD34, CD45, CD117, and CD133. HLSCs were also positive for vimentin and nestin, a stem cell marker. The absence of staining for cytokeratin-19, CD117, and CD34 indicated that HLSCs were not oval stem cells. In addition, HLSCs expressed albumin, alpha-fetoprotein, and in a small percentage of cells, cytokeratin-8 and cytokeratin-18, indicating a partial commitment to hepatic cells. HLSCs differentiated in mature hepatocytes when cultured in the presence of hepatocyte growth factor and fibroblast growth factor 4, as indicated by the expression of functional cytochrome P450, albumin, and urea production. Under this condition, HLSCs downregulated alpha-fetoprotein and expressed cytokeratin-8 and cytokeratin-18. HLSCs were also able to undergo osteogenic and endothelial differentiation when cultured in the appropriated differentiation media, but they did not undergo lipogenic differentiation. Moreover, HLSCs differentiated in insulin-producing islet-like structures. In vivo, HLSCs contributed to regeneration of the liver parenchyma in severe-combined immunodeficient mice. In conclusion, we here identified a pluripotent progenitor population in adult human liver that could provide a basis for cell therapy strategies.

Opposite effects of endotoxin on mitochondrial and endoplasmic reticulum functions

In this study, we determined functional integrity and reactive oxygen species generation in mitochondria and endoplasmic reticulum in liver of rats subjected to endotoxic shock to clarify whether intracellular reactive oxygen species (ROS) destabilize cellular integrity causing necrosis in rats challenged with lipopolysaccharide (LPS). LPS caused drastically increased plasma levels of alanine aminotransferase, suggesting damage to plasma membranes of liver cells. Liver necrosis was confirmed by histological examination. LPS induced a significant increase in ROS production in rat liver mitochondria (RLM), but did not impair mitochondrial function. In contrast to mitochondria, enzymatic activity and ROS production of cytochrome P450 were lower in microsomal fraction obtained from LPS-treated animals, suggesting the dysfunction of endoplasmic reticulum. Protein patterns obtained from RLM by two-dimensional electrophoresis showed significant upregulation of mitochondrial superoxide dismutase by LPS. We hypothesize that upregulation of this enzyme protects mitochondria against mitochondrial ROS, but does not protect other cellular compartments such as endoplasmic reticulum and plasma membrane causing necrosis.

Pharmacological characteristics of endothelium-derived hyperpolarizing factor-mediated relaxation of small mesenteric arteries from db/db mice

Endothelial dysfunction is considered as a major risk factor of cardiovascular complications of type I and type II diabetes. Our previous studies have demonstrated that endothelial dysfunction in the small mesenteric arteries from 12–16 week old type II diabetic mice was associated with decreased bio-availability of nitric oxide whereas endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation was preserved. The objective of the present study was to characterize EDHF-mediated relaxations of small mesenteric arteries from db/db mice. A depolarizing concentration of KCl or tetraethylammonium (TEA, 10 mM) significantly inhibited the EDHF-mediated relaxation to acetylcholine and bradykinin in small mesenteric arteries from both db/+ and db/db mice. Charybdotoxin or iberiotoxin alone and a combination of ouabain and barium significantly reduced the maximal relaxation to acetylcholine in small mesenteric arteries from db/db mice and charybdotoxin or iberiotoxin either alone or in combination with apamin reduced the sensitivity to the EDHF-mediated component of the relaxation response to bradykinin. 17-octadecynoic acid, but not catalase, significantly reduced the sensitivity to EDHF-mediated responses to bradykinin in db/db mice; 17-octadecynoic acid had no effect on acetylcholine-mediated relaxations. No differences were, however, detected for mRNA expression levels of calcium-activated potassium channels or connexins 37, 40, 43 and 45. Collectively, these data suggest that bradykinin-induced, EDHF-dependent relaxation in small mesenteric arteries from db/db mice is mediated via cytochrome P450 product that activates the large conductance calcium-activated potassium (BK Ca or Slo) channel, whereas the acetylcholine-induced, EDHF-mediated relaxation involves neither cytochrome P450 product nor hydrogen peroxide.

Induction of Heme Oxygenase

Carbon monoxide (CO), produced within the body as a product of the catabolism of heme by the enzyme heme oxygenase (HO), has been reported to be a local vasodilator. One of its possible pathways is to stimulate guanylyl cyclase to produce cGMP as a second messenger.1 The parallel to NO, another gaseous signaling molecule that also works through cGMP-mediated pathways (although more potent), is obvious. In fact, the vasodilator actions of these 2 may be integrally linked. Both are endothelium derived.2 NO stimulation of cGMP may facilitate CO-mediated dilation of resistance vessels through calcium-activated potassium channels.3 However, the HO–CO story has not received the same interest and attention as NO, nor is it as generally well understood by those who work outside the field. In the present issue, we are presented with a provocative study by one of the well-established laboratories within the field of HO–CO. Wang et al4 focus on HO in the genetic model of hypertension, the spontaneously hypertensive rat (SHR). They report that 3 weeks of administration of hemin, the oxidation product of heme, which is both a substrate for and an inducer of HO, produced a profound 80-mmHg decrease in blood pressure that was sustained completely for 9 months after ceasing treatment (a duration in a rat’s life, which roughly translates into 17.5 human years). Although this is a remarkable, hopeful, and possibly profound observation, the study probably raises more questions than it answers. First, it is important to point out that this sustained decrease in blood pressure and reversal of hypertension is an unprecedented result. The potential importance of this observation is that a single 3-week treatment regimen induced a protracted reduction in blood pressure, long after treatment had ceased. To put this in perspective, treatment of virtually all forms of …

Multiple eicosanoid-activated nonselective cation channels regulate B-lymphocyte adhesion to integrin ligands

Arachidonic acid (AA) is a substrate for a variety of proinflammatory mediators, which are generated by cyclooxygenases (COXs), lipoxygenases (LOXs), and cytochrome P-450 (CYP450) enzymes. COX (e.g., PGs and prostacyclins) and LOX (e.g., leukotrienes) products have well-established proinflammatory roles; however, little is known about the functions of CYP450 products in leukocytes. We previously found that mechanical strain generated by subjecting lymphocytes to hypotonic challenge triggered AA production and that two CYP450 products of AA, 5,6-epoxyeicosatrienoic acid (5,6-EET) and 20-hydroxyeicosatetraenoic acid (20-HETE), as well as a product of LOX, 5-(S)-hydroperoxyeicosatetrenoic acid (5-HPETE), induced Ca(2+) entry into primary B cells. The main goal of the present studies, therefore, was to define the biophysically properties of eicosanoid-activated channels responsible for Ca(2+) entry and the physiological consequences of activating these channels, including their role in mechanical signaling. We found that 5,6-EET, 20-HETE, and 5-HPETE each activated distinct Ca(2+)-permeant nonselective cation channels (NSCCs) in primary B cells. These NSCCs each regulate plasma membrane potential and B-cell adhesion to integrin ligands ICAM-1 and VCAM-1. Thus our data demonstrate that proinflammatory mediators produced in response to osmotic and/or physical stress play a direct role in regulating the B-cell membrane potential and their adhesion to specific ECM proteins. These results not only have important implications for understanding normal mechanisms of B-cell activation, differentiation, and trafficking but also point to novel targets for modulating the pathogenesis of B-cell-mediated inflammatory diseases.

Improved performance of primary rat hepatocytes on blended natural polymers

Alginate (Alg), chitosan (Chi), collagen (Col), gelatin (Gel), and the mixtures of every two of them were screened to determine their suitability for hepatocyte culture. The test materials were fabricated as films and then evaluated on the basis of their abilities to promote the attachment and functions of rat hepatocytes cultured on them. Cellular attachment on Col and Gel was favorable. However, cellular viability, cytoskeleton organization and function, as evaluated by albumin production, ureagenesis, and enzyme activity of cytochrome P450 as well as expression levels of hepatocyte nuclear factor 4 alpha deteriorated. Reverse cellular behavior was observed on Alg and Chi. Two blends, composed of Chi and Col or Gel, were found to be superior to other materials and sustained viability and differentiated functions of hepatocyte.

Electrical coupling and release of K+ from endothelial cells co‐mediate ACh‐induced smooth muscle hyperpolarization in guinea‐pig inner ear artery

The physiological basis of ACh-elicited hyperpolarization in guinea-pig in vitro cochlear spiral modiolar artery (SMA) was investigated by intracellular recording combined with dye labelling of recorded cells and immunocytochemistry. We found the following. (1) The ACh-hyperpolarization was prominent only in cells that had a low resting potential (less negative than -60 mV). ACh-hyperpolarization was reversibly blocked by 4-DAMP, charybdotoxin or BAPTA-AM, but not by N(omega)-nitro-L-arginine methyl ester, glipizide, indomethacin or 17-octadecynoic acid. (2) Ba(2)(+) (100 microm) and ouabain (1 microm) each attenuated ACh-hyperpolarization by approximately 30% in smooth muscle cells (SMCs) but had only slight or no inhibition in endothelial cells (ECs). A combination of Ba(2)(+) and 18beta-glycyrrhetinic acid near completely blocked the ACh-hyperpolarization in SMCs. (3) High K(+) (10 mm) induced a smaller hyperpolarization in ECs than in SMCs, with an amplitude ratio of 0.49 : 1. Ba(2)(+) blocked the K(+)-induced hyperpolarization by approximately 85% in both cell types, whereas ouabain inhibited K(+)-hyperpolarization differently in SMCs (19%) and ECs (35%) and increased input resistance. 18beta-Glycyrrhetinic acid blocked the high K(+)-hyperpolarization in ECs only. (4) Weak myoendothelial dye coupling was detected by confocal microscopy in cells recorded with a propidium iodide-containing electrode for longer than 30 min. A sparse plexus of choline acetyltransferase-immunoreactive (ChAT) fibres was observed around the SMA and its up-stream arteries. (5) Evoked excitatory junction potentials (EJP) were partially blocked by 4-DAMP in half of the cells tested. We conclude that ACh-induced hyperpolarization originates from ECs via activation of Ca(2)(+)-activated potassium channels, and is independent of the release of NO, cyclo-oxygenase or cytochrome P450 products. ACh-induced hyperpolarization in smooth muscle cells involves two mechanisms: (a) electrical spread of the hyperpolarization from the endothelium, and (b) activation of inward rectifier K(+) channels (K(ir)) and Na(+)-K(+) pump current by elevated interstitial K(+) released from the endothelial cells, these being responsible for about 60% and 40% of the hyperpolarization, respectively. The role ratio of K(ir) and pump current activation is at 8 : 1 or less.

Urinary 20-hydroxyeicosatetraenoic acid excretion is associated with oxidative stress in hypertensive subjects

Oxidative stress has been implicated in the pathogenesis of hypertension. 20-Hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P450 product of arachidonic acid metabolism, thought to be involved in the regulation of blood pressure (BP). The metabolism of arachidonic acid by cytochrome P450 enzymes may be a significant source of oxidative stress. F 2-isoprostanes are reliable markers of in vivo oxidative damage. γ-Glutamyl transpeptidase (γ-GT) has traditionally been associated with alcohol intake or liver dysfunction and may be an early marker of oxidative stress. The objective of the present study was to investigate relationships between 20-HETE excretion and markers of oxidative stress (F 2-isoprostanes and γ-GT). Sixty-nine treated hypertensive subjects underwent measurement of 24-h ambulatory BP, serum γ-GT, and urinary F 2-isoprostane and 20-HETE excretion. 20-HETE excretion was positively associated with 24-h diastolic BP ( p = 0.005), alcohol intake ( p = 0.008), γ-GT ( p = 0.007), and F 2-isoprostanes ( p = 0.005). F 2-isoprostanes were positively associated with alcohol intake ( p = 0.018) and γ-GT ( p = 0.01). In a multivariate regression, γ-GT remained an independent predictor of 20-HETE excretion, after adjustment for age, gender, BMI, and alcohol intake. In conclusion, the study highlights the positive association observed between 20-HETE excretion and markers of oxidative damage. The study also provides evidence that γ-GT may be a useful marker of oxidative stress.

Cytochrome P450 Products and Arachidonic Acid–Induced, Non–Store-Operated, Ca2+Entry in Cultured Bovine Endothelial Cells

Endothelial cells possess multiple mechanisms for the control of Ca2+ influx during agonist and mechanical stimulation. Increased intracellular Ca2+ during such events is important in the production of vasoactive substances including NO, prostacyclin, and, possibly, endothelium-derived hyperpolarizing factor(s). The present studies examined the effect of arachidonic acid on cellular Ca2+ entry and the underlying mechanisms by which this fatty acid regulates entry. Studies were conducted in cultured bovine aortic endothelial cells (passages 3 to 6) with changes in intracellular Ca2+ determined using the fluorescent Ca2+-sensitive indicator fura 2. Arachidonic acid (1 to 50 microM) stimulated Ca2+ entry from the superfusate without affecting Ca2+ release from intracellular stores. 2-aminoethoxydiphenyl borate (2APB) (100 microM) added at the peak of Ca2+ entry did not inhibit arachidonic acid-induced Ca2+ entry but, in contrast, significantly inhibited entry stimulated by ATP (1 microM). Arachidonic acid-induced Ca2+ entry was inhibited by econazole (1 microM), but not indomethacin (10 microM) or nordihydroguairetic acid (10 microM), suggesting the involvement of cytochrome P450 monooxygenase metabolite of arachidonic acid. Oleic acid (10 microM) was ineffective in inducing Ca2+ entry, whereas linoleic acid (10 microM) stimulated Ca2+ entry but by a mechanism insensitive to econazole. Collectively the data demonstrate that primary cultured aortic endothelial cells possess a Ca2+ entry mechanism modulated by arachidonic acid. This mode of Ca2+ entry appears to operate independently of store depletion-mediated mechanisms.

High-level expression of human cytochrome P450 1A2 by co-expression with human molecular chaperone HDJ-1(Hsp40)

Human cytochrome P450 (CYP) 1A2 is of great interest because of its important roles in the oxidation of numerous drugs and carcinogens. HDJ-1, a molecular chaperone in human, is known to assist the correct folding of unfolded proteins. To achieve high yield of recombinant human CYP1A2 in Escherichia coli, the CYP1A2 encoding gene was co-expressed with the chaperone HDJ-1 under the control of an inducible tac promoter in bicistronic format. Expression level of CYP1A2 in the bicistronic construct reached up to 520 nmol/liter culture within 16 h at 37 °C, which is 3.4-fold increase compared to the expression yield of CYP1A2 alone without HDJ-1. By co-expression with HDJ-1, the catalytic activity of CYP1A2 was also increased 5.5-fold. The activity increase seems to be associated with the increase of CYP production at whole cell level. The present over-expression system may be useful for rapid production of large amounts of active human CYP1A2 in E. coli.

High-level expression of human cytochrome P450 3A4 by co-expression with human molecular chaperone HDJ-1 (Hsp40).

Cytochrome P450 (CYP) 3A4 is of great interest because of its important roles in the oxidation of numerous drugs and xenobiotics. HDJ-1, a molecular chaperone in human, is known to assist the correct folding of unfolded proteins. To achieve a high yield of recombinant human CYP3A4 in Escherichia coli, the CYP3A4 encoding gene was co-expressed with the chaperone HDJ-1, under the control of an inducible tac promoter in a bicistronic format. The levels of expression of the CYP3A4 in the bicistronic construct reached up to 715 nmol (liter culture)(-1) within 16 h at 37 degrees C, which was about a 3.3-fold increase compared to that of the CYP3A4 alone without the HDJ-1. By co-expression with HDJ-1, the catalytic activity of CYP3A4 was also increased by approximately 15-fold. The amount of activity increase was similar to that of the CYP production at the whole cell level. The present over-expression system may be useful for the rapid production of large amounts of active CYP3A4 in E. coli.

The mechanism of EDHF-mediated responses in subcutaneous small arteries from healthy pregnant women.

We studied the importance of endothelium-derived hyperpolarizing factor (EDHF) vs. nitric oxide (NO) and prostacyclin (PGI(2)) in bradykinin (BK)-induced relaxation in isolated small subcutaneous arteries from normal pregnant women. We also explored the contribution of cytochrome P-450 (CYP450) product of arachidonic acid (AA) metabolism, hydrogen peroxide (H(2)O(2)), and gap junctions that have been suggested to be involved in EDHF-mediated responses. Isolated arteries obtained from subcutaneous fat biopsies of normal pregnant women (n = 30) undergoing planned cesarean section were mounted in a wire-myography system. In norepinephrine-constricted vessels, incubation with N(G)-nitro-L-arginine methyl ester (L-NAME) resulted in a significant reduction in relaxation to BK. Simultaneous incubation with L-NAME and indomethacin failed to modify this response further. BK-mediated dilatation in the presence of K(+)-modified solution was decreased to similar level as obtained after incubation with L-NAME. Incubation with L-NAME abolished BK-induced responses in K(+)-modified solution. Sulfaphenazole, a specific inhibitor of CYP450 epoxygenase, and catalase (an enzyme that decomposes H(2)O(2)) did not affect the EDHF-mediated relaxation because concentration-response curves to BK were similar in arteries after incubation with L-NAME vs. L-NAME + sulfaphenazole and L-NAME + catalase. The inhibitor of gap junctions, 18 alpha-glycyrrhetinic acid, significantly reduced BK-mediated relaxation both without and with incubation with L-NAME. We found that both NO and EDHF, but not PGI(2), are involved in the endothelium-dependent dilatation to BK. BK-induced relaxation is almost equally mediated by NO and EDHF. CYP450 epoxygenase metabolites of AA or H(2)O(2) do not account for EDHF-mediated response; however, gap junctions are involved in the EDHF-mediated responses to BK in subcutaneous small arteries in normal pregnancy.

Modeling of human cytochrome p450-mediated drug metabolism using unsupervised machine learning approach.

We developed a computational algorithm for evaluating the possibility of cytochrome P450-mediated metabolic transformations that xenobiotics molecules undergo in the human body. First, we compiled a database of known human cytochrome P-450 substrates, products, and nonsubstrates for 38 enzyme-specific groups (total of 2200 compounds). Second, we determined the cytochrome-mediated metabolic reactions most typical for each group and examined the substrates and products of these reactions. To assess the probability of P450 transformations of novel compounds, we built a nonlinear quantitative structure-metabolism relationships (QSMR) model based on Kohonen self-organizing maps (SOM). This neural network QSMR model incorporated a predefined set of physicochemical descriptors encoding the key molecular properties that define the metabolic fate of individual molecules. Isozyme-specific groups of substrate molecules were visualized, thus facilitating prediction of tissue-specific metabolism. The developed algorithm can be used in early stages of drug discovery as an efficient tool for the assessment of human metabolism and toxicity of novel compounds in designing discovery libraries and in lead optimization.

Cytochrome P450 products of arachidonate contribute to the differential control of renal cortical and medullary perfusion

Cytochrome P450 products of arachidonate contribute to the differential control of renal cortical and medullary perfusion

Dose-response modeling of cytochrome p450 induction in rats by octamethylcyclotetrasiloxane.

Inhalation of octamethylcyclotetrasiloxane (D4) induces CYP2B1/2 protein and causes liver enlargement. We have developed a pharmacodynamic (PD) extension to a physiologically based pharmacokinetic (PBPK) model to characterize these dose-response behaviors. The PD model simulates interactions of D4 with a putative receptor, leading to increased production of cytochrome P450 2B1/2. Induction was modeled with a Hill equation with dissociation constant, Kd, and Hill coefficient, N. Both a 1- and a 5-compartment liver model were evaluated. The PBPK model provided excellent simulations of tissue D4 and hepatic CYP2B1/2 protein concentrations following 6 h/day, 5-day inhalation exposures to 0, 1, 7, 30, 70, 150, 300, 500, 700, or 900 ppm D4. Either the 1- or 5-compartment liver model could accurately simulate increases in CYP2B1/2 protein in the liver. With a 1-compartment liver, Kd and N were 0.67 microM (free liver concentration) and 1.9, respectively. The 5-compartment model used higher N-values (approximately 4.0) and varied Kd between compartments. The fitted 5-compartment model parameters were Kd = 0.67 microM in the midzonal compartment with geometric differences in Kd between compartments of 2.9. On the basis of unbound (free) plasma concentrations, D4 appeared to be a higher potency inducer than phenobarbital (PB). Dose-response curves for increased liver weights had N/mS 1.0 and Kd/mS 3.4 microM, very different values from those for enzyme induction. Exposure concentration leading to a 0.1% increase in CYP2B1/2 protein predicted by the 1- and 5-compartment models were 2.1 ppm and 5.1 ppm, respectively. The 1- and 5-compartment liver models provided very similar fits to the whole liver induction data, excluding the lowest dose, but the 5-compartment liver model had the additional advantage of simultaneously describing the regional induction of CYP2B1/2.

Arachidonic acid directly activates members of the mitogen-activated protein kinase superfamily in rabbit proximal tubule cells

To explore the roles of eicosanoids in arachidonic acid-induced mitogen-activated protein kinase (MAPK) signal transduction, we have shown that exposure of proximal tubular cells to arachidonic acid induces phosphorylation of c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), two members of the MAPK superfamily. We observed that ketoconazole, an inhibitor of the cytochrome P450 pathway, blocked ERK but not JNK activation. Direct regulation of arachidonic acid on mitogen-activated protein kinase (MAPK) signaling pathways was evaluated more directly by utilizing specific enzyme inhibitors of the cytochrome P450 metabolic pathway and by comparing the relative efficacy of arachidonic acid versus its cytochrome P450 metabolites (exogenous and endogenous), eicosatetraynoic acid (ETYA), and other fatty acids on the phosphorylation of members of the MAPK superfamily (ERKs, JNK, and p38(MAPK)), by utilizing early passage rabbit proximal tubular epithelial cells. Arachidonic acid activated p38(MAPK), a third member of the MAPK superfamily, in a time- and concentration-dependent manner. Studies designed to evaluate the ability of arachidonic acid and its cytochrome P450 metabolites (endogenously and exogenously) to stimulate ERKs, JNK, and p38(MAPK) found four conclusions. First, the metabolites of arachidonic acid generated endogenously by cytochrome P450 2C1 significantly augmented basal ERK activity, whereas the metabolites generated by the 2C2 isozyme significantly augmented basal p38(MAPK) activity. However, their effects were less profound than arachidonic acid itself. In contrast, there were no significant effects with transfection of either isozyme on basal JNK activity. Second, a variety of exogenous cytochrome P450 products were less potent than arachidonic acid on a molar basis in stimulating the activity of all three MAPKs. Third, ketoconazole and 17-octadecynoic acid, inhibitors of the cytochrome P450 pathway, as well as PPOH and DDMS, inhibitors of the epoxygenase and omega-hydroxylase pathways, respectively, failed to significantly reduce the effects of arachidonic acid to activate ERK and p38(MAPK) (JNK was not evaluated). Finally, arachidonic acid, its inactive analog ETYA, and other fatty acids with differing chain lengths and degrees of saturation stimulated the activity of all three MAPKs. These observations substantiate a role for arachidonic acid and other fatty acids in signaling linked to the MAPK superfamily in rabbit proximal tubular epithelium without the necessity of conversion to cytochrome P450 metabolites.

Cytochrome P-450 metabolite of arachidonic acid mediates bradykinin-induced negative inotropic effect.

This study focused on the mechanisms of the negative inotropic response to bradykinin (BK) in isolated rat hearts perfused at constant flow. BK (100 nM) significantly reduced developed left ventricular pressure (LVP) and the maximal derivative of systolic LVP by 20-22%. The cytochrome P-450 (CYP) inhibitors 1-aminobenzotriazole (1 mM and 100 microM) or proadifen (5 microM) abolished the cardiodepression by BK, which was not affected by nitric oxide and cyclooxygenase inhibitors (35 microM NG-nitro-L-arginine methyl ester and 10 microM indomethacin, respectively). The CYP metabolite 14,15-epoxyeicosatrienoic acid (14,15-EET; 50 ng/ml) produced effects similar to those of BK in terms of the reduction in contractility. After the coronary endothelium was made dysfunctional by Triton X-100 (0.5 microl), the BK-induced negative inotropic effect was completely abolished, whereas the 14,15-EET-induced cardiodepression was not affected. In hearts with normal endothelium, after recovery from 14,15-EET effects, BK reduced developed LVP to a 35% greater extent than BK in the control. In conclusion, CYP inhibition or endothelial dysfunction prevents BK from causing cardiodepression, suggesting that, in the rat heart, endothelial CYP products mediate the negative inotropic effect of BK. One of these mediators appears to be 14,15-EET.