<p>The human cytochrome P450 (CYP) family of monooxygenases is important for metabolism of drugs and endogenous compounds, e.g., vitamin A and D, cholesterol, steroids, fatty acids, and eicosanoids. This thesis describes the tissue distribution, catalytic properties, and possible function of CYP4F8 and CYP4F12. To this respect, methods for immunohistological analysis, and real-time PCR for analysis of their transcripts, were developed.</p><p>CYP4F8 was originally cloned from human seminal vesicles and proposed to catalyze 19-hydroxylation of prostaglandin H₂(PGH₂). This notion could now be supported, as cyclooxygenase-2, CYP4F8, and microsomal prostaglandin E synthase-1 were found to be co-localized in the epithelial linings of seminal vesicles. The three enzymes were also co-localized in the suprabasal layers of epidermis, suggesting a similar function of CYP4F8 in skin. Real-time PCR showed that CYP4F8 mRNA was more than 10-fold increased in psoriatic lesions compared to non-lesional skin. CYP4F8 immunoreactivity was also found in kidney cortex, transitional epithelium, corneal epithelium, and retina. Although transcripts of all three enzymes were detectable in retina, no co-localization was found. Pro inflammatory stimuli were found to increase CYP4F8 mRNA expression in cultured epidermal and corneal keratinocytes. In these tissues CYP4F8 might oxidize fatty acids or other eicosanoids than PGH₂.</p><p>CYP4F12 was originally cloned from the liver and small intestine, and found to oxidize arachidonic acid and two anti-histamines. Immunohistological studies showed that CYP4F12 immunoreactivity was present mainly in the gastrointestinal tract, e.g., stomach, ilium, and colon, but also in placenta. Although CYP4F8 and CYP4F12 have catalytic properties in common, there are important differences. CYP4F12 does not oxidize PGH₂, certain eicosanoids, and fatty acids. The prominent expression in the gut suggests that CYP4F12 might be involved in oxidation of drugs.</p>

The human cytochrome P450 (CYP) family of monooxygenases is important for metabolism of drugs and endogenous compounds, e.g., vitamin A and D, cholesterol, steroids, fatty acids, and eicosanoids. This thesis describes the tissue distribution, catalytic properties, and possible function of CYP4F8 and CYP4F12. To this respect, methods for immunohistological analysis, and real-time PCR for analysis of their transcripts, were developed. CYP4F8 was originally cloned from human seminal vesicles and proposed to catalyze 19-hydroxylation of prostaglandin H2 (PGH2). This notion could now be supported, as cyclooxygenase-2, CYP4F8, and microsomal prostaglandin E synthase-1 were found to be co-localized in the epithelial linings of seminal vesicles. The three enzymes were also co-localized in the suprabasal layers of epidermis, suggesting a similar function of CYP4F8 in skin. Real-time PCR showed that CYP4F8 mRNA was more than 10-fold increased in psoriatic lesions compared to non-lesional skin. CYP4F8 immunoreactivity was also found in kidney cortex, transitional epithelium, corneal epithelium, and retina. Although transcripts of all three enzymes were detectable in retina, no co-localization was found. Pro inflammatory stimuli were found to increase CYP4F8 mRNA expression in cultured epidermal and corneal keratinocytes. In these tissues CYP4F8 might oxidize fatty acids or other eicosanoids than PGH2. CYP4F12 was originally cloned from the liver and small intestine, and found to oxidize arachidonic acid and two anti-histamines. Immunohistological studies showed that CYP4F12 immunoreactivity was present mainly in the gastrointestinal tract, e.g., stomach, ilium, and colon, but also in placenta. Although CYP4F8 and CYP4F12 have catalytic properties in common, there are important differences. CYP4F12 does not oxidize PGH2, certain eicosanoids, and fatty acids. The prominent expression in the gut suggests that CYP4F12 might be involved in oxidation of drugs.

The enzyme phospholipase A2 (PLA2) specifically hydrolyzes the 2-acyl ester bond of 1,2-diacyl-3-sn-phosphoglycerides releasing fatty acids and lysophospholipids in the presence of Ca2+. Both products represent precursors for signaling molecules that can exert a multitude of biological functions including phospholipid metabolism, exocytosis and inflammation. Consequently, PLA2 not only plays a role in regulating physiological processes, but also exhibits pharmacological effects in inflammatory diseases. Nevertheless, the signaling pathway leading to cell death still remains elusive. In the present study, the cytotoxicity of Naja naja atra PLA2 toward human neuroblastoma SK-N-SH cells and leukemia K562 cells were respectively evaluated to explore the signaling pathway of PLA2-induced cell death. Upon exposure to PLA2, p38 mitogen-activated protein kinase (p38 MAPK) or c-Jun N-terminal kinase (JNK) activation, extracellularsignal-regulated protein kinase (ERK) inactivation, reactive oxygen species (ROS) generation, increase in intracellular Ca2+ concentration, the loss of mitochondrial membrane potential (£G£Zm), cytochrome c release and upregulation of Fas/FasL were found in SK-N-SH or K562 cells. N-Acetylcysteine (ROS scavenger), BAPTA-AM (Ca2+ chelator), SB202190 (p38 MAPK inhibitor) or SP600125 (JNK inhibitor) abrogated p38 MAPK or JNK activation and rescued cell viability, £G£Zm, cytochrome c release and suppressed Fas/FasL upregulation of PLA2-treated cells, but restored phosphorylation of ERK. Activated ERK was found to attenuate p38 MAPK-mediated upregulation of Fas/FasL. Besides, sustained JNK activation was also observed in SB202190/PLA2-treated K562 cells after exterminating p38 MAPK activation, but also retained the cytotoxicity of PLA2. Knockdown of p38 MAPK or JNK1 by siRNA proved that PLA2 induced Fas/FasL upregulation through p38 MAPK/ATF-2 or JNK1/c-Jun pathways in K562 cells. Furthermore, deprivation of catalytic activity could not diminish PLA2-induced cell death and Fas/FasL upregulation. The cytotoxicity of arachidonic acid (AA) and lysophosphatidylcholine (LPC) was not related to the expression of Fas/FasL. The results showed that the cytotoxicity of AA is mediated through mitochondria-dependent death pathway, eliciting by AA-induced ROS generation and Ca2+-evoked activation of p38 MAPK and JNK. Besides, ERK activation abrogated by U0126 improved the ability of AA-mediated Fas/FasL upregulation in K562 cells. Taken together, our results indicate that PLA2-induced cell death is through Ca2+- and ROS evoked p38 MAPK or JNK activation. Upregulation of Fas/FasL partially involves in cytotoxicity of PLA2.

Proper autonomic regulation of mammalian cardiac function is dependent upon very complex and precise communication among the intracardiac ganglia and individual neurons within the ganglia. An array of neuromodulators is found within the ganglia that direct neuronal activity by modulating the movement of calcium. The current study determines that opioidergic agonists, which have been found to contribute to severe cardiac disease states and intracellular calcium mobilization, are also responsible for changes in the function of the intracardiac neuron via their effects on store-operated calcium channels (SOCs). Previous studies suggest that phosphorylation plays a role in SOC regulation. Using Fura-2 calcium fluorometry, we determined that protein kinase A (PKA), protein kinase C (PKC), and cyclic adenosine monophosphate (cAMP) had no effect on store-operated calcium entry in the presence of antagonists, phorbol 12, 13 dibutyrate (PDBu), forskolin, and 8-Br cAMP, respectively. We also found pharmacologically that using both electrophysiology and calcium imaging that μ-opioid agonists, met-enkephalin (ME) and endomorphin (EM) depress SOC activity in intracardiac neurons. Arachidonic acid (AA), which has been found to depress SOC function in rat liver cells and μ-opioid receptor activation (MOR), blocked both store-operated calcium entry (SOCE) and the calcium release-activated current (ICRAC) significantly. Contrastingly, AA metabolites, prostaglandin E2)(PGE2) and prostaglandin D2 (PGD2), do not significantly influence SOCE which suggests that the effects of AA may be direct. The block elicited by EM was partially reversed by pertussis toxin (PTX), indicative of activation of a PTX-sensitive G-protein following MOR activation. Similarly, PLA2 inhibitors, OBAA and AACOCF3, decreased the percent block of SOCE due to opioid agonist-induced inhibition. Using the perforated-patch method of I-clamp electrophysiology, we demonstrated that gadolinium, at low micromolar concentrations, reversibly reduced action potential firing. Importantly, these results suggest that SOCs may influence action potential firing in mammalian intracardiac neurons. Similarly, AA and EM depressed action potential firing. Taken together, these experiments suggest that a pathway involving EM and AA influences repetitive firing through SOC inhibition. The importance of SOCs in the maintenance of action potential firing and more specifically, the expression and biophysical functionality of the individual pore-forming subunits (Orai1, 2, and 3) in any neuronal cell type has previously not been explored. Quantitative RT-PCR along with I-clamp electrophysiology revealed that Orai3 was exclusive to repetitively firing neurons. As a result, we hypothesize that robust Ca2+-dependent fast inactivation, also associated Orai3, is a factor in the maintenance of repetitive action potential firing. Using Fura-2 calcium fluorometry and patch-clamp electrophysiology, we determined pharmacologically that μ-opioid receptor activation precedes an intracellular cascade that is dependent on a PTX-sensitive G-protein and AA but independent of prostaglandin and protein kinase activity. Finally, we used RT-PCR to determine the Orai subunits expressed in the intracardiac neurons and their influence on neuronal firing patterns. This study is the first to determine the role expressed subunits has in the maintenance of the electrical activity of the neuron.

The oxidation of polyunsaturated fatty acids to the corresponding hydroperoxide by plant and animal lipoxygenases is an important step for the generation of bioactive lipid mediators. Thereby fatty acid hydroperoxide represent a common intermediate, also in human innate immune cells, like neutrophil granulocytes. In these cells a further key component is the heme protein myeloperoxidase producing HOCl as a reactive oxidant. On the basis of different investigation a reaction of the fatty acid hydroperoxide and hypochlorous acid (HOCl) could be assumed. Here, chromatographic and spectrometric analysis revealed that the hydroperoxide moiety of 15S-hydroperoxy-5Z,8Z,11Z,13E-eicosatetraenoic acid (15-HpETE) and 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13-HpODE) is not affected by HOCl. No reduction of the hydroperoxide group due to a reaction with HOCl could be measured. It could be demonstrated that the double bonds of the fatty acid hydroperoxides are the major target of HOCl, present either as reagent or formed by the myeloperoxidase-hydrogen peroxide-chloride system.

No description available.

The blood-brain barrier, formed by brain microvessel endothelial cells, is the restrictive barrier between the brain parenchyma and the circulating blood. It was previously demonstrated in our laboratory that knock down of fatty acid transport proteins FATP-1 and CD36 attenuated apical to basolateral monounsaturated fatty acid transport across human brain microvessel endothelial cells (HBMEC). Arachidonic acid (AA; 5,8,11,14 - cis-eicosatetraenoic acid) is a conditionally essential, polyunsaturated fatty acid [20:4(n-6)] and a major constituent of brain lipids. We examined transport of AA across confluent monolayers of HBMEC. Control cells or HBMEC with knock down of FATP-1 or CD36 were cultured on Transwell® plates and incubated apically with [3H]AA and incorporation of [3H]AA into the basolateral medium was determined temporally. [3H]AA was rapidly incorporated into the basolateral medium with time in control cells. Surprisingly, knock down of FATP-1 or CD36 did not alter [3H]AA movement into the basolateral medium. The increased permeability mediated by AA was likely caused by a metabolite of AA produced de novo and was confirmed by an increased movement of fluorescent dextran from apical to basolateral medium. HBMECs expressed PGE2 synthase, cyclooxygenase-1 and -2, PGE2 receptors, tight junction proteins and prostaglandin transporters. The AA-mediated increase in membrane permeability was not attenuated by cyclooxygenase inhibitor drugs (NSAIDs). Incubation of the HBMEC monolayers with exogenous PGE2 resulted in attenuation of the AA-mediated permeability increases. The results indicate that AA increases the permeability of the HBMEC monolayer likely via increased production of metabolites or by-products of the lipoxygenase or epoxygenase pathways. These observations may explain the rapid influx of AA into the brain previously observed upon plasma infusion with AA.

The blood-brain barrier, formed by brain microvessel endothelial cells, is the restrictive barrier between the brain parenchyma and the circulating blood. It was previously demonstrated in our laboratory that knock down of fatty acid transport proteins FATP-1 and CD36 attenuated apical to basolateral monounsaturated fatty acid transport across human brain microvessel endothelial cells (HBMEC). Arachidonic acid (AA; 5,8,11,14 - cis-eicosatetraenoic acid) is a conditionally essential, polyunsaturated fatty acid [20:4(n-6)] and a major constituent of brain lipids. We examined transport of AA across confluent monolayers of HBMEC. Control cells or HBMEC with knock down of FATP-1 or CD36 were cultured on Transwell® plates and incubated apically with [3H]AA and incorporation of [3H]AA into the basolateral medium was determined temporally. [3H]AA was rapidly incorporated into the basolateral medium with time in control cells. Surprisingly, knock down of FATP-1 or CD36 did not alter [3H]AA movement into the basolateral medium. The increased permeability mediated by AA was likely caused by a metabolite of AA produced de novo and was confirmed by an increased movement of fluorescent dextran from apical to basolateral medium. HBMECs expressed PGE2 synthase, cyclooxygenase-1 and -2, PGE2 receptors, tight junction proteins and prostaglandin transporters. The AA-mediated increase in membrane permeability was not attenuated by cyclooxygenase inhibitor drugs (NSAIDs). Incubation of the HBMEC monolayers with exogenous PGE2 resulted in attenuation of the AA-mediated permeability increases. The results indicate that AA increases the permeability of the HBMEC monolayer likely via increased production of metabolites or by-products of the lipoxygenase or epoxygenase pathways. These observations may explain the rapid influx of AA into the brain previously observed upon plasma infusion with AA.

Effect of lipids on binding characteristics of opioid receptors in membranes prepared from rat brain were studied. Lipid concentrations causing changes in specific binding of [3H]Endomorphin-1 (ProE1), an opioid agonist highly specific to mu-type opioid, [3H]Ile5,6deltorphin II (DIDI), an agonist ligand highly specific to delta type receptor and [3H]Naloxone (Nlx), a universal opioid receptor antagonist were determined. Inhibition of [3H]ProE1, [3H]DIDI and [3H]Nlx specific binding was also examined by homologous displacement experiments in the presence and absence of lipids. In order to understand whether the changes occurring in the specific binding is due to changes in equilibrium dissociation constant (KD) or maximum number of binding sites (Bmax), the equilibrium binding experiments were performed. Arachidonic acid (AA) inhibited binding of both agonist and antagonist ligand in a dose dependent manner with IC50 values of 0.15, 0.1, and 0.6 mM for [3H]ProE1, [3H]DIDI and [3H]Nlx, respectively. Kd values were not affected while Bmax values decreased 38 % and 76 % for mu, and delta receptor subtypes, respectively. For [3H]Nlx, Bmax values decreased 20 and 56 % in the absence and presence of 100 mM NaCl, respectively. Cholesteryl hemisuccinate (CHS) enhances (100 % of control) ligand binding at mu-sites however no effect was encountered at delta sites. Furthermore, CHS also enhances (50 % of control) the binding of antagonist ligand in the absence of NaCl. Bmax values were increased by 70 % for mu sites and 40% for antagonist ligand binding site. Under similar conditions Kd values were not affected. Phosphatidic acid (PA) and phosphatidylcholine (PC) exhibited negligible effect on ligand binding. PA decreased specific binding of ProE1 and DIDI by 16 and 10 %, respectively. Specific binding of antagonist ligand Nlx decreased 11 % in the presence of NaCl whereas in the absence of NaCl specific binding is very close to control. In the presence of PC specific binding of both agonist and antagonist ligands were around control values. In this study modulatory effect of lysophospholipids, lysophosphatidic acid and lysophosphatidylcholine on opioid binding sites were evaluated for the first time. Both lysophospholipids exhibited similar effects: decreasing specific binding in receptor subtype independent manner between 0.1 to 1 mM range. Kd values were not significantly affected, while remarkable decrease (45-75 %) in Bmax values were observed.

Thesis (Ph. D.)--University of Missouri--Columbia, 2002. / Typescript. Includes bibliographical references. Also available on the Internet.