Newly developed Learning and Verbal Memory Test (TAMV-I): Normative data for Spanish-speaking pediatric population.

OBJECTIVE: To generate normative data for the Learning and Verbal Memory Test (TAMV-I) in Spanish-speaking pediatric populations. METHOD: The sample consisted of 4,373 healthy children from nine countries in Latin America (Chile, Cuba, Ecuador, Guatemala, Honduras, Mexico, Paraguay, Peru, and Puerto Rico) and Spain. Each participant was administered the TAMV-I as part of a larger neuropsychological battery. Free recall, memory delay and recognition scores were normed using multiple linear regressions and standard deviations of residual values. Age, age2, sex, and mean level of parental education (MLPE) were included as predictors in the analyses. RESULTS: The final multiple linear regression models indicated main effects for age on all scores, such that scores increased linearly as a function of age. Age2 had a significant effect in all countries except Cuba, and Puerto Rico for free recall score; a significant effect for memory delay score in all countries except Cuba and Puerto Rico; and a significant effect for recognition score in in all countries except Guatemala, Honduras, and Puerto Rico. Models showed an effect for MLPE in Chile (free recall), Honduras (free recall), Mexico (free recall), Puerto Rico (free recall, memory delay, and recognition), and Spain (free recall and memory delay). Sex affected free recall score for Cuba, Ecuador, Guatemala, Mexico, Paraguay, Peru, and Spain, memory delay score for all countries except Chile, Paraguay, and Puerto Rico, and recognition score for Ecuador, Mexico, Peru, and Spain, with girls scoring higher than boys. CONCLUSIONS: This is the largest Spanish-speaking pediatric normative study in the world, and it will allow neuropsychologists from these countries to have a more accurate way to interpret the TAMV-I with pediatric populations.

Symbol Digit Modalities Test: Normative data for Spanish-speaking pediatric population.

OBJECTIVE: To generate normative data for the Symbol Digit Modalities Test (SDMT) in Spanish-speaking pediatric populations. METHOD: The sample consisted of 4,373 healthy children from nine countries in Latin America (Chile, Cuba, Ecuador, Guatemala, Honduras, Mexico, Paraguay, Peru, and Puerto Rico) and Spain. Each participant was administered the SDMT as part of a larger neuropsychological battery. SDMT scores were normed using multiple linear regressions and standard deviations of residual values. Age, age2, sex, and mean level of parental education (MLPE) were included as predictors in the analyses. RESULTS: The final multiple linear regression models showed main effects for age in all countries, such that score increased linearly as a function of age. In addition, age2 had a significant effect in all countries, except in Honduras and Puerto Rico. Models indicated that children whose parent(s) had a MLPE >12 years of education obtained higher score compared to children whose parent(s) had a MLPE ≤12 years for Chile, Guatemala, Mexico, and Spain. Sex affected SDMT score for Paraguay and Spain. CONCLUSIONS: This is the largest Spanish-speaking pediatric normative study in the world, and it will allow neuropsychologists from these countries to have a more accurate interpretation of the SDMT with pediatric populations.

Shortened Version of the Token Test: Normative data for Spanish-speaking pediatric population.

OBJECTIVE: To generate normative data for the Shortened Version of the Token Test in Spanish-speaking pediatric populations. METHOD: The sample consisted of 4,373 healthy children from nine countries in Latin America (Chile, Cuba, Ecuador, Guatemala, Honduras, Mexico, Paraguay, Peru, and Puerto Rico) and Spain. Each participant was administered the Shortened Version of the Token Test as part of a larger neuropsychological battery. Shortened Version of the Token Test total scores were normed using multiple linear regressions and standard deviations of residual values. Age, age2, sex, and mean level of parental education (MLPE) were included as predictors in the analyses. RESULTS: The final multiple linear regression models showed main effects for age in all countries, such that score increased linearly as a function of age. In addition, age2 had a significant effect in all countries, except Guatemala and Puerto Rico. Models showed that children whose parent(s) had a MLPE >12 years obtained higher score compared to children whose parents had a MLPE ≤12 years in Ecuador, Guatemala, Honduras, Mexico, Paraguay, Peru, Puerto Rico, and Spain. The child's sex did not have an effect in the Shortened Version of the Token Test total score for any of the countries. CONCLUSIONS: This is the largest Spanish-speaking pediatric normative study in the world, and it will allow neuropsychologists from these countries to have a more accurate interpretation of the Shortened Version of the Token Test when used in pediatric populations.

Stroop Color-Word Interference Test: Normative data for Spanish-speaking pediatric population.

OBJECTIVE: To generate normative data for the Stroop Word-Color Interference test in Spanish-speaking pediatric populations. METHOD: The sample consisted of 4,373 healthy children from nine countries in Latin America (Chile, Cuba, Ecuador, Guatemala, Honduras, Mexico, Paraguay, Peru, and Puerto Rico) and Spain. Each participant was administered the Stroop Word-Color Interference test as part of a larger neuropsychological battery. The Stroop Word, Stroop Color, Stroop Word-Color, and Stroop Interference scores were normed using multiple linear regressions and standard deviations of residual values. Age, age2, sex, and mean level of parental education (MLPE) were included as predictors in the analyses. RESULTS: The final multiple linear regression models showed main effects for age on all scores, except on Stroop Interference for Guatemala, such that scores increased linearly as a function of age. Age2 affected Stroop Word scores for all countries, Stroop Color scores for Ecuador, Mexico, Peru, and Spain; Stroop Word-Color scores for Ecuador, Mexico, and Paraguay; and Stroop Interference scores for Cuba, Guatemala, and Spain. MLPE affected Stroop Word scores for Chile, Mexico, and Puerto Rico; Stroop Color scores for Mexico, Puerto Rico, and Spain; Stroop Word-Color scores for Ecuador, Guatemala, Mexico, Puerto Rico and Spain; and Stroop-Interference scores for Ecuador, Mexico, and Spain. Sex affected Stroop Word scores for Spain, Stroop Color scores for Mexico, and Stroop Interference for Honduras. CONCLUSIONS: This is the largest Spanish-speaking pediatric normative study in the world, and it will allow neuropsychologists from these countries to have a more accurate approach to interpret the Stroop Word-Color Interference test in pediatric populations.

Rey-Osterrieth Complex Figure - copy and immediate recall (3 minutes): Normative data for Spanish-speaking pediatric populations.

OBJECTIVE: To generate normative data for the Rey-Osterrieth Complex Figure (ROCF) in Spanish-speaking pediatric populations. METHOD: The sample consisted of 4,373 healthy children from nine countries in Latin America (Chile, Cuba, Ecuador, Guatemala, Honduras, Mexico, Paraguay, Peru, and Puerto Rico) and Spain. Each participant was administered the ROCF as part of a larger neuropsychological battery. The ROCF copy and immediate recall (3 minutes) scores were normed using multiple linear regressions and standard deviations of residual values. Age, age2, sex, and mean level of parental education (MLPE) were included as predictors in the analyses. RESULTS: The final multiple linear regression models showed main effect for age on copy and immediate recall scores, such that scores increased linearly as a function of age. Age2 affected ROCF copy score for all countries, except Puerto Rico; and ROCF immediate recall scores for all countries, except Chile, Guatemala, Honduras, Paraguay, and Puerto Rico. Models indicated that children whose parent(s) had a MLPE >12 years obtained higher scores compared to children whose parent(s) had a MLPE≤12 years for Chile, Puerto Rico, and Spain in the ROCF copy, and Paraguay and Spain for the ROCF immediate recall. Sex affected ROCF copy and immediate recall score for Chile and Puerto Rico with girls scoring higher than boys. CONCLUSIONS: This is the largest Spanish-speaking pediatric normative study in the world, and it will allow neuropsychologists from these countries to have a more accurate approach to interpret the ROCF Test in pediatric populations.

Molecular cloning and enzymatic characterization of sheep CYP2J.

Cytochrome P450 (CYP) 2Js have been studied in various mammals, but not in sheep, as an animal model used to test veterinary drug metabolism. Sheep CYP2J was cloned from liver messenger RNA (mRNA) by RACE. The cDNA, after modification at its N- and C-terminals, was expressed in Escherichia coli and the sheep CYP2J protein, purified by chromatography, was 80% homologous to human and monkey CYP2J2. Reverse transcriptase-polymerase chain reaction (RT-PCR) experiments showed that CYP2J mRNA was expressed in liver, cortex, respiratory and olfactory mucosa, heart, bronchi, lung, spleen, small intestine and kidney. The purified enzyme was catalytically active towards aminopyrine, all-trans-retinoic acid, and particularly arachidonic acid forming 20-HETE, 19-HETE, and 18-HETE (about 86% of the total) and 14,15-, 11,12-, 8,9-, and 5,6-EETs (cis-epoxyeicosatrienoic acids; about 14% of total), with a regioselectivity similar to that shown by the mammalian CYP2J2s.

Modulation by 20-HETE of phenylephrine-induced mesenteric artery contraction in spontaneously hypertensive and Wistar-Kyoto rats.

Small mesenteric arteries of spontaneously hypertensive (SHR) and Wistar-Kyoto rats (WKY) were compared for the production of 20-HETE and the effects of 20-HETE and N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS, 30 micromol/L), a 20-HETE synthesis inhibitor, on contractile responsiveness to phenylephrine (0.1 to 50.0 micromol/L). 20-HETE production was higher in vessels of SHR compared with WKY (1.34+/-0.16 versus 0.27+/-0.09 pmol/mg tissue, P<0.05). Phenylephrine elicited concentration-dependent vascular contraction; the R(max) was similar in vessels of SHR and WKY, but the former were more sensitive as denoted by the lower EC(50) (1.10+/-0.14 versus 1.89+/-0.33 micromol/L, P<0.05). DDMS caused a rightward shift in the concentration-response curve to phenylephrine, increasing (P<0.05) the EC(50) by 258% and 134% in vessels of SHR and WKY, respectively. In contrast, in DDMS-treated vessels, 20-HETE (0.01 to 10.0 micromol/L) caused a leftward shift in the phenylephrine concentration-response curve, decreasing (P<0.05) the EC(50) without affecting the R(max). Importantly, the minimal concentration of 20-HETE that decreased the EC(50) of phenylephrine was much smaller in vessels of SHR that of WKY (0.01 versus 1.0 micromol/L). We conclude that 20-HETE increases the sensitivity of mesenteric arterial vessels to phenylephrine, vessels of SHR are more sensitive to this action of the eicosanoid than vessels of WKY, and vessels of SHR produce more 20-HETE than do vessels of WKY. Hence, 20-HETE of vascular origin may be a determinant of the increased reactivity to constrictor agonists in the vasculature of SHR.

Human heme oxygenase-1 gene transfer lowers blood pressure and promotes growth in spontaneously hypertensive rats.

Heme oxygenase (HO) catalyzes the conversion of heme to biliverdin, with release of free iron and carbon monoxide. Both heme and carbon monoxide have been implicated in the regulation of vascular tone. A retroviral vector containing human HO-1 cDNA (LSN-HHO-1) was constructed and subjected to purification and concentration of the viral particles to achieve 5x10(9) to 1x10(10) colony-forming units per milliliter. The ability of concentrated infectious viral particles to express human HO-1 (HHO-1) in vivo was tested. A single intracardiac injection of the concentrated infectious viral particles (expressing HHO-1) to 5-day-old spontaneously hypertensive rats resulted in functional expression of the HHO-1 gene and attenuation of the development of hypertension. Rats expressing HHO-1 showed a significant decrease in urinary excretion of a vasoconstrictor arachidonic acid metabolite and a reduction in myogenic responses to increased intraluminal pressure in isolated arterioles. Unexpectedly, HHO-1 chimeric rats showed a simultaneous significant proportionate increase in somatic growth. Thus, delivery of HHO-1 gene by retroviral vector attenuates the development of hypertension and promotes body growth in spontaneously hypertensive rats.

Detection of endogenous 12-hydroxyeicosatrienoic acid in human tear film.

PURPOSE: Increased production of 12-hydroxyeicosatetraenoic acid [12(R)-HETE] and 12-hydroxyeicosatrienoic acid [12(R)-HETrE] positively correlates with the in vivo progression of ocular surface inflammation in rabbits. Tear film was collected from human subjects with inflamed eyes to determine whether these eicosanoids could be detected from endogenous sources. METHODS: Control and inflamed eyes were assessed and assigned a subjective inflammatory score. Tears were collected and extracted with an internal standard. Single-ion-monitoring gas chromatography-mass spectrometry (SIM-GC-MS) was performed to quantitate endogenous levels of 12-HETE and 12-HETrE. RESULTS: 12-HETrE was detected in the tear film of both control and inflamed eyes, with the mean level being seven times higher in inflamed tears. 12-HETE was not detected in control tears and was detected in only 6 of 38 inflamed-eye tear samples. CONCLUSIONS: The current findings demonstrate that the human eye produces detectable amounts of 12-HETrE, which is released into the tear flow. The increased levels of 12-HETrE associated with ocular surface inflammation suggest that this eicosanoid may contribute to inflammation of the ocular surface in humans.

Corneal epithelial VEGF and cytochrome P450 4B1 expression in a rabbit model of closed eye contact lens wear.

PURPOSE: The similar and overlapping activity of VEGF and the potent corneal-derived angiogenic eicosanoid 12(R)-HETrE calls for a study of the temporal relationship in the expression of these two autocoids. Since recent evidence suggests that hypoxia induces the expression of a CYP4B1 mRNA which might be involved in the conversion of arachidonic acid to 12(R)-HETrE, we determined its time-dependent expression and correlated it to that of VEGF mRNA in the rabbit model of closed eye contact lens-induced injury. METHODS: Rabbit eyes were fitted with contact lenses followed by a silk suture tarsorrhaphy. The anterior surface was analyzed at 2-, 4- and 7-days by slit lamp biomicroscopy, subjective inflammatory scoring and corneal pachymetry. Corneal epithelium was scraped and CYP4B1 and VEGF mRNA levels were measured by Southern hybridization of RT-PCR products amplified from a single cornea with specific primers. RESULTS: Corneal thickness and inflammatory scores increased in a time dependent manner in the model of closed eye contact lens induced hypoxic injury. Corneal epithelial CYP4B1 and VEGF mRNAs, as well as the production of the angiogenic eicosanoid, 12-HETrE, increased in a time-dependent manner and correlated with the in situ inflammatory response. CONCLUSIONS: The present study documents the increased expression of CYP4B1 isoform in the corneal epithelium during hypoxic injury in vivo. It also demonstrates the presence of VEGF mRNA in the corneal epithelium and its increased expression in this model of hypoxic injury. All together, the results of this study raise the possibility of interaction between these autocoids, VEGF and CYP4B1-12(R)-HETrE, in mediating the neovascularization response induced by the prolonged hypoxic state brought about by closed eye contact lens wear.

CYP4A1 antisense oligonucleotide reduces mesenteric vascular reactivity and blood pressure in SHR.

The cytochrome P-450 4A (CYP4A)-derived arachidonic acid metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) affects renal tubular and vascular functions and has been implicated in the control of arterial pressure. We examined the effect of antisense oligonucleotide (ODN) to CYP4A1, the low K(m) arachidonic acid omega-hydroxylating isoform, on vascular 20-HETE synthesis, vascular reactivity, and blood pressure in the spontaneously hypertensive rat (SHR). Administration of CYP4A1 antisense ODN decreased mean arterial blood pressure from 137 +/- 3 to 121 +/- 4 mmHg (P < 0.05) after 5 days of treatment, whereas treatment with scrambled antisense ODN had no effect. Treatment with CYP4A1 antisense ODN reduced the level of CYP4A-immunoreactive proteins along with 20-HETE synthesis in mesenteric arterial vessels. Mesenteric arteries from rats treated with antisense ODN exhibited decreased sensitivity to the constrictor action of phenylephrine (EC(50) 0.69 +/- 0.17 vs. 1.77 +/- 0.40 microM). Likewise, mesenteric arterioles from antisense ODN-treated rats revealed attenuation of myogenic constrictor responses to increases of transmural pressure. The decreased vascular reactivity and myogenic responses were reversible with the addition of 20-HETE. These data suggest that CYP4A1-derived 20-HETE facilitates myogenic constrictor responses in the mesenteric microcirculation and contributes to pressor mechanisms in SHR.

The effect of hypoxia on endogenous corneal epithelial eicosanoids.

PURPOSE: Injury to the corneal epithelium increases arachidonic acid (AA) metabolism through the cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 pathways. The authors used the rabbit corneal organ culture model to demonstrate the effect of hypoxia on the endogenous formation of 12-hydroxy-5,8,11,14-eicosatetraenoic acid (12-HETE), 12-hydroxy-5,8,14-eicosatrienoic acid (12-HETrE), and prostaglandin (PG) E2 by the intact cornea in the absence of exogenously added cofactors or substrate. METHODS: Rabbit corneas were isolated and cultured for 24 hours in normoxia or hypoxia. After culture, PGE2 in media was quantitated by enzyme immunoassay. 12-HETE and 12-HETrE were extracted from culture media and corneal epithelium and quantitated by negative chemical ionization-gas chromatography-mass spectrometry. COX-1 and -2 protein expression in corneal epithelium was determined by Western blot. Acute (2 hours) COX activity in normoxia and hypoxia was determined as the conversion rate of [14C]AA to [14C]PGE2, quantitated through reverse-phase-high-performance liquid chromatography and radiodetection. RESULTS: In the media of cultured rabbit corneas, both 12-HETE and 12-HETrE were detected, with 12-HETrE levels being four times higher. Hypoxia did not significantly increase extracellular 12-HETE or 12-HETrE; however, it caused more than 90% inhibition of PGE2 synthesis. Intracellular 12-HETE and 12-HETrE were undetectable in normal corneas but increased to 7.7+/-1.3 and 2.2+/-0.4 ng/mg protein, respectively, after 24 hours in culture. Culture in hypoxia further increased intracellular 12-HETE threefold but had no additional effect on 12-HETrE. CONCLUSIONS: Hypoxia creates an environment in which epithelial COX activity is severely suppressed, whereas cytochrome P450-AA and/or 12-LOX metabolizing activity is maintained or enhanced. Additionally, the findings suggest that 12-HETE produced by the corneal epithelium acts intracellularly to promote corneal edema, whereas 12-HETrE acts in a paracrine manner to initiate an inflammatory cascade that can elicit neutrophil chemotaxis and neovascularization of the cornea.

Adenoviral vector-mediated transfer of human heme oxygenase in rats decreases renal heme-dependent arachidonic acid epoxygenase activity.

Intravenous administration of an adenovirus human heme oxygenase (HO)-1 gene construct to rats resulted in functional expression of human HO-1 in brain, heart, lung, liver, and kidney. Because accurate assessment of human HO-1 mRNA in various tissues by Northern analysis is not sufficiently sensitive, we developed a method for quantifying human HO-1 mRNA copies with quantitative reverse transcription- polymerase chain reaction techniques; this allowed us to use the same primers for both the sample and internal standard. Administration of the adenovirus human HO-1 gene resulted in the detection of human HO-1 mRNA in various tissues with the highest levels seen in the kidney followed, in order, by lung > liver > brain > heart. Human HO-1 was detectable for up to 4 weeks in all tissues studied. Administration of adenovirus human HO-1 resulted in maximal increase of HO activity after 1 to 2 weeks in rats. The increase in HO activity due to gene transfer also was associated with a parallel decrease (approximately 25%) in cytochrome P-450 (CYP) content and in CYP-dependent arachidonic acid metabolism. In addition, we investigated the possibility that the human HO-1 gene altered the expression of the endogenous rat enzyme after administration of cobalt chloride s.c. Cobalt chloride administration resulted in increased HO activity in all tissues examined in rats transduced with the human HO-1 gene to the same degree as in nontransduced rats. The metal was a more potent inducer of renal HO activity than was the adenoviral-mediated human HO-1 vector. The increase in HO activity after adenoviral-mediated human HO-1 transfer was associated with a decrease in microsomal heme-CYP and CYP activity. The increase in HO-1 activity after adenovirus-mediated human HO-1 gene transfer may prove useful as a means of selectively increasing enzyme activity in a specific organ and regulating homeostasis by modulation of vasoactive molecules such as carbon monoxide and bilirubin and, in addition, providing a means of delivering the human HO-1 gene for experimental purposes.

Regulation of cyclooxygenase-2 by hypoxia and peroxisome proliferators in the corneal epithelium.

Hypoxic injury provokes inflammation of many tissues including the ocular surface. In rabbit corneal epithelial cells, both peroxisome proliferator-activated receptor (PPAR)-inducible cytochrome P450 4B1 and cyclooxygenase-2 (COX-2) mRNAs were increased by hypoxia. PPAR alpha and beta but not gamma mRNAs were detected in these cells. The PPAR activator, WY-14,643 increased COX-2 expression. Similarly, non-steroidal anti-inflammatory drugs with the ability to activate PPARs induced COX-2 independently of prostaglandin synthesis inhibition. COX-2 protein overexpression by hypoxia and PPAR activation was not associated with a parallel increase in prostaglandin E(2) accumulation. However, the enzyme regained full catalytic activity when: 1) hypoxic cells were re-exposed to normoxic conditions in the presence of heme and arachidonic acid, and 2) WY-14,643-treated cells were depleted of intracellular GSH. Consistent with previous observations showing that the corneal production of cytochrome P450-derived inflammatory eicosanoids is elevated by hypoxia and inflammation, the current data suggest that hypoxic injury is a model of inflammation in which molecules other than COX-derived arachidonic acid metabolites play a major proinflammatory role. This study also suggests that increased cellular GSH may be the mechanism responsible for the characteristic dissociation of PPAR-induced COX-2 expression and activity. Moreover, we provide new insights into the commonly observed lack of efficacy of classical non-steroidal anti-inflammatory drugs in the treatment of hypoxia-related ocular surface inflammation.

Selective inhibition of arachidonic acid epoxidation in vivo.

Cytochrome P450 (CYP)-derived arachidonic acid metabolites, including epoxyeicosatrienoic acids (EETS) and 20-HETE, have been implicated in the regulation of renal function and vascular tone. Studying the function of specific CYP arachidonate metabolites has been hampered due to lack of selective inhibitors and difficulty in their solubilization. We have identified MS-PPOH as a potent and selective inhibitor of CYP-catalyzed arachidonate epoxidation in vitro. We used 2-hydroxypropyl-beta-cyclodextrin as a vehicle in order to administer MS-PPOH in vivo. One hour after administration, MS-PPOH (5 mg, IV bolus) significantly inhibited arachidonic acid epoxidation in rat renal cortical microsomes (vehicle-282 +/- 12 pmol/mg/min, MS-PPOH-206 +/- 10 pmol/mg/min, p < 0.05) but had no effect on 20-HETE formation (vehicle-383 32 pmol/mg/min, MS-PPOH-367 +/- 9 pmol/mg/min). The inhibitory effect lasts at least for 6 hours. There was no inhibition of 20-HETE synthesis at any time point. We also examined the effect of MS-PPOH on renal excretiry function. Three hours after MS-PPOH administration to anesthetized rats, urine flow rate became significantly higher (vehicle-275 +/- 16 microl/hour, MS-PPOH-406 +/- 44 microl/hour, p < 0.05). Sodium excretion rate was also significantly higher (vehicle-28.7 +/- 4 micromol/hour, MS-PPOH-63.3 +/- 10 micromol/hour, p < 0.05) but potassium excretion rate was not affected (vehicle-65.5 +/- 5 micromol/hour, MS-PPOH-79.2 +/- 2 micromol/hour). These results suggest that MS-PPOH may be useful as a selective inhibitor of CYP-catalyzed arachidonic acid epoxidation in vivo, and implicate EETs and anti-diuretic and anti-natriuretic in the regulation of renal function.

Children with cystic fibrosis benefit from massage therapy.

OBJECTIVE: To measure the effects of parents giving massage therapy to their children with cystic fibrosis to reduce anxiety in parents and their children and to improve the children's mood and peak air flow readings. METHODS: Twenty children (5-12 years old) with cystic fibrosis and their parents were randomly assigned to a massage therapy or a reading control group. Parents in the treatment group were instructed and asked to conduct a 20-minute child massage every night at bedtime for one month. Parents in the reading control group were instructed to read for 20 minutes a night with their child for one month. On days 1 and 30, parents and children answered questions relating to present anxiety levels and children answered questions relating to mood, and their peak air flow was measured. RESULTS: Following the first and last massage session, children and parents reported reduced anxiety. Mood and peak air flow readings also improved for children in the massage therapy group. CONCLUSIONS: These findings suggest that parents may reduce anxiety levels by massaging their children with cystic fibrosis and their children may benefit from receiving massage by having less anxiety and improved mood, which in turn may facilitate breathing.

Kinetic profile of the rat CYP4A isoforms: arachidonic acid metabolism and isoform-specific inhibitors.

20-Hydroxyeicosatetraenoic acid (HETE), the cytochrome P-450 (CYP) 4A omega-hydroxylation product of arachidonic acid, has potent biological effects on renal tubular and vascular functions and on the control of arterial pressure. We have expressed high levels of the rat CYP4A1, -4A2, -4A3, and -4A8 cDNAs, using baculovirus and Sf 9 insect cells. Arachidonic acid omega- and omega-1-hydroxylations were catalyzed by three of the CYP4A isoforms; the highest catalytic efficiency of 947 nM-1. min-1 for CYP4A1 was followed by 72 and 22 nM-1. min-1 for CYP4A2 and CYP4A3, respectively. CYP4A2 and CYP4A3 exhibited an additional arachidonate 11,12-epoxidation activity, whereas CYP4A1 operated solely as an omega-hydroxylase. CYP4A8 did not catalyze arachidonic or linoleic acid but did have a detectable lauric acid omega-hydroxylation activity. The inhibitory activity of various acetylenic and olefinic fatty acid analogs revealed differences and indicated isoform-specific inhibition. These studies suggest that CYP4A1, despite its low expression in extrahepatic tissues, may constitute the major source of 20-HETE synthesis. Moreover, the ability of CYP4A2 and -4A3 to catalyze the formation of two opposing biologically active metabolites, 20-HETE and 11, 12-epoxyeicosatrienoic acid, may be of great significance to the regulation of vascular tone.

Hypoxia-induced production of 12-hydroxyeicosanoids in the corneal epithelium: involvement of a cytochrome P-4504B1 isoform.

The corneal epithelium metabolizes arachidonic acid by a cytochrome P-450 (CYP)-mediated activity to 12-hydroxy-5,8,11, 14-eicosatetraenoic acid (12(R)-HETE) and 12-hydroxy-5,8, 14-eicosatrienoic acid (12(R)-HETrE ). Both metabolites possess potent inflammatory properties, with 12(R)-HETrE being a powerful angiogenic factor, and they assume the role of inflammatory mediators in hypoxia- and chemical-induced injury in the cornea in vivo and in vitro. We used a model of corneal organ culture that exhibits hypoxia-induced epithelial CYP-dependent 12(R)-HETE and 12(R)-HETrE synthesis for isolating, identifying, and characterizing the CYP protein responsible for these eicosanoid syntheses. Northern analysis revealed the presence of a CYP4A-hybridizable mRNA, the levels of which were increased after hypoxia. Reverse transcription-polymerase chain reaction analysis with primers specific for the CYP4A family led to the isolation of a 671-base pair fragment with a 98.8% sequence homology to the rabbit lung CYP4B1 isoform, of which the levels in the corneal epithelium were greatly increased under hypoxic conditions. Moreover, phenobarbital, an inducer of hepatic CYP4B1 in the rabbit, also induced 12-HETE and 12-HETrE synthesis. Antibodies against CYP4B1, but not against CYP4A1, inhibited hypoxia-, clofibrate-, and phenobarbital-induced 12-HETE and 12-HETrE synthesis. These results suggest the involvement of a CYP4B1 isoform in the corneal epithelial synthesis of these eicosanoids in response to hypoxia.