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Kupffer cells are a key source of mediators of alcohol-induced liver damage such as reactive oxygen species, chemokines, growth factors, and eicosanoids. Since diets rich in polyunsaturated fatty acids are a requirement for the development of alcoholic liver disease, we hypothesized that polyunsaturated fatty acids could synergize with ethanol to promote Kupffer cell activation and TNFα production, hence, contributing to liver injury. Primary Kupffer cells from control and from ethanol-fed rats incubated with arachidonic acid showed similar proliferation rates than nontreated cells; however, arachidonic acid induced phenotypic changes, lipid peroxidation, hydroperoxides, and superoxide radical generation. Similar effects occurred in human Kupffer cells. These events were greater in Kupffer cells from ethanol-fed rats, and antioxidants and inhibitors of arachidonic acid metabolism prevented them. Arachidonic acid treatment increased NADPH oxidase activity. Inhibitors of NADPH oxidase and of arachidonic acid metabolism partially prevented the increase in oxidant stress. Upon arachidonic acid stimulation, there was a rapid and sustained increase in TNFα, which was greater in Kupffer cells from ethanol-fed rats than in Kupffer cells from control rats. Arachidonic acid induced ERK1/2 phosphorylation and nuclear translocation of early growth response-1 (Egr1), and ethanol synergized with arachidonic acid to promote this effect. PD98059, a mitogen extracellular kinase 1/2 inhibitor, and curcumin, an Egr1 inhibitor, blocked the arachidonic acid-mediated upregulation of TNFα in Kupffer cells. This study unveils the mechanism whereby arachidonic acid and ethanol increase TNFα production in Kupffer cells, thus contributing to alcoholic liver disease.

Intracellular recordings were made from hippocampal CA1 neurons in rat slice preparations. Superfusion with oxygen- and glucose-deprived medium (in vitro ischemia) produced a rapid depolarization approximately 5 min after the onset of the superfusion. Even when oxygen and glucose were reintroduced immediately after rapid depolarization, the membrane depolarized further (persistent depolarization) and reached 0 mV (irreversible depolarization) after 5 min from the reintroduction. The pretreatment of the slice preparation with a phospholipase A2 (PLA2) inhibitor, para-bromophenacyl bromide, or a cytochrome p-450 inhibitor, 17-octadecynoic acid, significantly restored the membrane to the preexposure potential level after the reintroduction of oxygen and glucose. The administration of 14,15-epoxyeicosatrienoic acid or 20-hydroxyeicosatetraenoic acid did not change the latency of the rapid depolarization and did not allow the membrane potential to recover after the ischemic exposure. In contrast, after pretreatment with cyclooxygenase or lipoxygenase inhibitors, such as indomethacin, resveratrol, Dup-697, nordihydroguaiaretic acid, and 3,4-dihydrophenyl ethanol, a minority of neurons tested showed postischemic recovery from the persistent depolarization. Improved recovery was also seen after treatment with the free radical scavengers, edaravone and alpha-tocopherol. These results suggest that the activation of the arachidonic acid cascade via PLA2 and the free radicals produced by arachidonic acid metabolism contribute to the irreversible depolarization produced by in vitro ischemia.

A single intraperitoneal injection of endotoxin (40 microg/kg) significantly delayed gastric emptying of a solid nutrient meal. Blockade of nitric oxide synthase (NOS) with 30 mg/kg ip N(G)-nitro-L-arginine methyl ester or 20 mg/kg ip 7-nitroindazole [neuronal NOS (nNOS) inhibitor] significantly delayed gastric emptying in control animals but failed to modify gastric emptying in endotoxin-treated rats. Administration of 2.5, 5, and 10 mg/kg ip N(6)-iminoethyl-L-lysine [inducible NOS (iNOS) inhibitor] had no effect in either experimental group. Indomethacin (5 mg/kg sc), NS-398 (cyclooxygenase-2 inhibitor; 10 mg/kg ip), and dexamethasone (10 mg/kg sc) but not quinacrine (20 mg/kg ip) significantly prevented delay in gastric emptying induced by endotoxin but failed to modify gastric emptying in vehicle-treated animals. Ca(2+)-dependent NOS activity in the antrum pylorus of the stomach was diminished by endotoxin, whereas Ca(2+)-independent NOS activity was not changed. In addition, decreased nNOS mRNA and protein were observed in the antrum pylorus of endotoxin-treated rats. Our results suggest that downregulation of nNOS in the antrum pylorus of the stomach and synthesis of prostaglandins mediate the delay in gastric emptying of a solid nutrient meal induced by endotoxin.

The cellular mechanisms that determine specificity of synaptic connections between mutually connected neurons in the nervous system have not yet been fully examined in vertebrate and invertebrate species. Here we report on a novel form of synaptic interaction during early stages of synapse formation between reciprocally connected Lymnaea neurons. Specifically, using soma-soma synapses between an identified dopaminergic neuron (also known as the giant dopamine cell), right pedal dorsal 1 (RPeD1), and a FMRFamidergic neuron, visceral dorsal 4 (VD4), we demonstrate that although reciprocal inhibitory synapses re-form between the somata after 24-36 h of pairing, VD4 is, however, the first cell to establish synaptic contacts with RPeD1 (within 12-18 h). We show that VD4 "captures" RPeD1 first as a postsynaptic cell by suppressing its transmitter secretory machinery during early stages of cell-cell pairing. The VD4-induced suppression of transmitter release from RPeD1 was transient, and it required transcription and de novo protein synthesis dependent step in VD4 but not in RPeD1. The VD4-induced effects on RPeD1 were mimicked by a FMRFamide-like peptide. Perturbation of FMRFamide-activated metabolites of the arachidonic acid pathway in RPeD1 not only prevented FMRFamide-induced suppression of transmitter release from the giant dopamine cell but also shifted the synaptic balance in favor of RPeD1, thus making it the first cell to begin synaptic transmission with VD4 within 12-18 h. A single RPeD1 that had developed dopamine secretory capabilities overnight and was subsequently paired with VD4 for 12-18 h was, however, immune to VD4-induced suppression of transmitter release. Under these experimental conditions, both cells developed mutual inhibitory synapses concurrently. Taken together, our data provide evidence for novel synaptic interaction between reciprocally connected neurons and underscore the importance of transmitter-receptor interplay in regulating the timing of synapse formation in the nervous system.

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.

The mechanism by which bradykinin (BK) decreases vascular resistance was investigated in the hindquarters vascular bed of the rat. Under conditions of controlled blood flow, BK produced dose-related decreases in hindquarters perfusion pressure when injected into the perfusion circuit in doses of 0.1-1.0 pg. Responses to BK were reproducible with respect to time, and HOE-140 (D-Arg, [Hyp3,Thi5,D-Tic7,Oic8] -BK), a kinin B2 receptor antagonist, decreased hindquarters vasodilator responses to the peptide. HOE-140 had no significant effect on responses to vasodilator agents, which act by a variety of pharmacologically distinct mechanisms. Nitric oxide synthase inhibitors N(omega)-nitro-L-arginine methyl ester or N(omega)-nitro-L-arginine benzyl ester did not decrease responses to BK in doses that decreased hindquarters vasodilator responses to acetylcholine. The cyclooxygenase inhibitors meclofenamate and indomethacin had no effect on responses to BK in doses that attenuated vasodilator responses to the prostaglandin precursor, arachidonic acid. Clotrimazole or 5,8,11,14-eicosatetraynoic acid (ETYA), cytochrome P-450 arachidonic acid metabolism inhibitors, in doses that attenuated vasodilator responses to arachidonic acid, had no effect on responses to BK. Glybenclamide or U-37883A (4-morpholinecarboximidine-N-1-adamantyl-N'-cyclohexyl), K+-ATP channel antagonists, in doses that attenuated responses to lemakalim, had no significant effect on responses to BK. Finally, des-Arg9-BK, a reported kinin B1 receptor agonist, decreased hindquarters perfusion pressure when injected in doses of 3-30 microg, and responses to the B1 agonist were attenuated by HOE-140. The observation that des-Arg9-BK, in high doses, induces modest HOE-140-sensitive responses suggests that kinin B1 receptors are not normally expressed in the hindquarters vascular bed of the rat. The present results indicate that BK dilates the hindquarters vascular bed by a kinin B2 receptor mechanism that is independent of the release of nitric oxide, cyclooxygenase, or P-450 metabolites of arachidonic acid or the activation of K+-ATP channels.

Arachidonic acid (AA) is metabolized by the cytochrome P-450 (P-450) epoxygenase pathway to epoxyeicosatrienoic acids (EETs) in the brain parenchymal tissue and perivascular astrocytes. EETs dilate cerebral microvessels and enhance K+ current in cerebrovascular smooth muscle cells. In the current study, the effect of a subdural administration of miconazole, an inhibitor of P-450 epoxygenase, on microvascular perfusion of rat cerebral cortex was evaluated using laser-Doppler flowmetry (LDF) Baseline cerebral blood flow (CBF) decreased by 29.7 +/- 7.3% (n = 5) after administration of 20 microM miconazole into the subdural space for 30 min. Responses of CBF to sodium nitroprusside and 5-hydroxytryptamine were unaltered by miconazole treatment. Administration of vehicle alone in time-control experiments had no effect on CBF. In other experiments, the effects of miconazole on the metabolism of [14C]AA by cultured rat astrocytes and on nitric oxide synthase activity in homogenates of rat brain were examined. Miconazole inhibited conversion of AA to EETs by cultured astrocytes but had no effect on the conversion of L-arginine to L-citrulline by homogenates of rat brain. These results implicate endogenous P-450 epoxides of AA in the regulation of basal blood flow in cerebral microcirculation.

Arachidonic acid (AA) has been shown to inhibit the activity of the low-conductance ATP-sensitive K+ channel in the apical membrane of the cortical collecting duct [W. Wang, A. Cassola, and G. Giebisch. Am. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F554-F559, 1992]. ROMK1, a K+ channel derived from the rat renal outer medulla, shares many biophysical properties of the native low-conductance K+ channel, which is localized to the apical membranes of the cortical collecting duct and thick ascending limb. This study was designed to determine whether the ROMK channel maintains the property of AA sensitivity of the native low-conductance K+ channel. Experiments were conducted in Xenopus oocytes injected with cRNA encoding the ROMK1 channel by use of patch-clamp techniques. We have confirmed previous reports that the cloned ROMK1 has similar channel kinetics, high open probability, and inward slope conductance as the native low-conductance K+ channel, respectively. Addition of 5 microM AA to an inside-out patch resulted in reversible inhibition of channel activity at a concentration similar to the inhibitor constant for AA on the native K+ channel. The effect of AA on channel activity was preserved in the presence of 10 microM indomethacin, a cyclooxygenase inhibitor, 4 microM cinnamyl-3,4-dihydroxycyanocinnamate, a lipoxygenase inhibitor, and 4 microM 17-octadecynoic acid, an inhibitor of cytochrome P-450 monooxygenases, thus indicating that the effect of AA was not mediated by metabolites of AA. The effect did not appear to be the result of changes in membrane fluidity, since 5 microM eicosatetraynoic acid, an AA analogue that is a potent modulator of membrane fluidity, had no effect. Furthermore, the addition of AA to the outside of the patch also had no effect on channel activity. These results indicate that, like the native low-conductance channel, AA is able to directly inhibit ROMK1 channel activity.

ATP-sensitive K-channels (K(ATP)) play a major role in controlling blood vessel tone, particularly in coronary vasculature. Arachidonic acid (AA) exhibits potent vasorelaxant effects on coronary arteries with unclear mechanisms. We examined the activity of AA on the K(ATP) channel of dog coronary artery smooth muscle cells using the whole cell voltage-clamp technique. AA was found to have dual effects or the K(ATP) current. In cells dialyzed with 1 mM ATP pipette solution, external application of AA activated the K(ATP) current with a bell-shaped concentration-response curve. K(ATP) current (-50 mV) increased from 1.0 +/- 1.1 to 4.6 +/- 3.0, 25 +/- 9, 43 +/- 13, 26 +/- 10, and 23 +/- 16 pA by 1, 2, 4, 8, and 16 microM AA, respectively. If K(ATP) current was activated by dialyzing the cell with ATP-free pipette solution, external application of AA (from 2 to 16 microM) inhibited the K(ATP) current (half-maximal inhibitory concentration = 3.8 microM). Pretreatment of cells with 10 microM indomethacin or 10 microM nordihydroguaiaretic acid, which respectively blocked the cyclooxygenase or lipoxygenase pathway of AA metabolism, did not prevent the dual effects of AA. Eicosatetraynoic acid (ETYA), the nonmetabolizable analogue of AA, also produced dual effects on the K(ATP) current. ETYA (4 microM) increased the K(ATP) current (-50 mV) from 2.9 +/- 1.2 to 37 +/- 7 pA in 1 mM intracellular ATP, whereas 8 microM ETYA inhibited the K(ATP) current in 0 mM intracellular ATP by 71 +/- 3%. These results suggest that the dual effects of AA on the K(ATP) current of dog coronary smooth muscle cells do not require AA metabolism. Activation of K(ATP) channels in smooth muscle cells may contribute to the AA-induced coronary vasodilation.

Condensed tannin, isolated from cotton bracts extract (CBE), increases arachidonic acid (AA) release from rabbit alveolar macrophages and inhibits its subsequent reacylation. We determined whether tannin from CBE had any effect upon AA release in bovine tracheal epithelial cells (BTE). [14C] AA release was measured at timed intervals after addition of various concentrations of tannin to BTE cells grown to confluence in the presence of [14C] AA. Tannin caused a time- and dose-dependent release of AA from airway cells, with a maximum release occurring at 1 min in the presence of 100 micrograms/ml of tannin, and was confirmed by high-pressure liquid chromatography. The pattern of release was similar to that observed with bradykinin (2 x 10(-6) M). AA release by tannin was partially inhibited by indomethacin (10(-5) M) but not by 5,8,11,14-eicosatetraynoic acid (ETYA; 10(-5) M. Both of these drugs were effective in inhibiting bradykinin-induced AA release. In addition, AA release was not inhibited by cycloheximide. Endotoxin at 100 pg/ml and higher also caused a time-dependent release of AA that was not inhibitable by indomethacin or ETYA. Tannin-induced AA release was inhibited by pretreatment with pertussis toxin but not by neomycin, an inhibitor of phospholipase C (PLC). Neither pertussis toxin nor neomycin had any effect upon endotoxin-induced AA release. In other experiments, neither tannin nor endotoxin had any effect on [14C]AA uptake by BTE. These data demonstrate that tannin at low concentrations and endotoxin at high concentrations increase AA release by BTE cells. The AA release by tannin is partially metabolized by the cyclooxygenase pathway. We hypothesize that tannin-induced AA release is not mediated by PLC but may be mediated by other phospholipases, including PLA2.

General anesthetics inhibit Ca(2+)-activated potassium (BK) channels at clinically relevant concentrations. This study examined the possibility that general anesthetics produce their effect on BK channels by disrupting the phospholipase A2 (PLA2)-arachidonic acid signal transduction pathway. Treatment of excised patches with exogenous arachidonic acid (2.5 microM) resulted in a 3.6 +/- 1.3-fold increase in BK channel activity. Subsequent exposure of these patches to concentrations of halothane (0.6 mM), ketamine (100 microM), or etomidate (10 microM) that would normally block the channel by approximately 60-80% in the absence of arachidonic acid did not reduce the channel activity. Arachidonic acid resulted in a significant increase in the 50% effective concentration for the ketamine dose-response curve from 3.4 +/- 0.4 to 693 +/- 379 microM (P < 0.001) as well as a significant decrease in slope from 1.40 +/- 0.21 to 0.59 +/- 0.05 (P < 0.001). The PLA2 inhibitors quinacrine (1 microM), aristolochic acid (250 microM), and octadecylbenzoylacrylic acid (7 microM) inhibited BK channels by 61 +/- 6, 47 +/- 2, and 30 +/- 9%, respectively, and in a manner indistinguishable from general anesthetics inhibition. Aristolochic acid and ketamine significantly inhibit the PLA2-mediated production of arachidonic acid in GH3 cells.

The present study examined the effects of 20-hydroxyeicosatetraenoic acid (20-HETE) and 17-octadecynoic acid (17-ODYA), an inhibitor of the metabolism of arachidonic acid by P-450, on K(+)-channel activity in vascular smooth muscle cells (VSM) isolated from renal arterioles of the rat. Two types of K+ channels were characterized using inside-out excised membrane patches. One channel exhibited a large conductance (250.3 +/- 5 pS), was activated by membrane depolarization and elevations in cytoplasmic Ca2+ concentration, and was blocked by low concentrations (< 1 mM) of tetraethylammonium (TEA). The other K+ channel exhibited an intermediate conductance (46.3 +/- pS), was activated by membrane depolarization but not by changes in intracellular Ca2+ concentration, and was blocked by 4-aminopyridine (5 mM). Addition of 20-HETE to the bath (1-100 nM), reduced the frequency of opening of the large-conductance Ca(2+)-activated K+ channel recorded using cell-attached patches on VSM. It had no effect on the intermediate-conductance K+ channel: 17-ODYA (1 microM) increased the activity of the large-conductance Ca(2+)-activated K+ channel, and this effect was reversed by 20-HETE (10 nM). 20-HETE (1-1000 nM) reduced the diameter of isolated perfused small renal arteries of the rat by approximately 15% TEA (1 mM) blocked the vasoconstrictor response to 20-HETE (100 nM). These studies suggest that 20-HETE is an endogenously formed vasoconstrictor that acts in part by inhibiting the opening of the large-conductance Ca(2+)-activated K+ channel in renal arteriolar VSM.

The polyunsaturated fatty acids, arachidonic, linoleic, and linolenic acids, were potent blockers of regulatory volume decrease (RVD) and of the swelling-activated efflux of [3H]taurine, D-[3H]aspartate, [3H]inositol, and 125I (used as marker of Cl) from rat cerebellar astrocytes in culture. The monounsaturated oleic and ricinoleic acids and saturated fatty acids were ineffective. The amino acid and 125I fluxes were similarly inhibited by fatty acids, whereas inositol release was less sensitive. Polyunsaturated fatty acids appear to directly affect RVD in trypsinized astrocytes as the inhibition was immediate and fully reversible. Blockers of the arachidonic acid metabolic pathways, indomethacin (cyclooxygenase), esculetin (lipoxygenases), and metyrapone (P-450 monooxygenases), did not prevent the effect of arachidonic acid, suggesting that further metabolism is not required for displaying the effects of arachidonic acid on RVD and osmolyte fluxes. Some blockers of arachidonic acid metabolic pathways, such as nordihydroguaiaretic acid (lipoxygenases) and naphthoflavone (P-450 monooxygenases), also exhibited marked inhibitory effects on RVD and on osmolyte fluxes. The predominant arachidonic acid metabolite in astrocytes, 12-hydroxyeicosatetraenoic acid, did not affect RVD or osmolyte fluxes. These results suggest that arachidonic acid and other polyunsaturated fatty acids directly inhibit the permeability pathways correcting cell volume after swelling in cultured astrocytes.

We have shown that acetylcholine (ACh)-induced contraction of esophageal circular muscle cells is mediated by activation of protein kinase C (PKC). We now examine the role of phospholipase A2 (PLA2). ACh increases [3H]arachidonic acid release in esophageal but not in lower esophageal sphincter (LES) muscle. In addition, ACh-induced contraction of esophageal but not of LES cells was reduced by the PLA2 antagonist dimethyleicosadienoic acid and by antiserum to a 100-kDa cytosolic PLA2 (cPLA2). These data suggest that the 100-kDa cPLA2 plays a role in ACh-induced contraction of esophageal but not of LES muscle. In esophageal cells, arachidonic acid produced by PLA2 caused little contraction by itself but potentiated contraction induced by the PKC agonist diacylglycerol (DAG). The free fatty acids linoleic acid and linolenic acid also potentiated DAG-induced contraction. Indomethacin and nordihydroguaiaretic acid had no effect on arachidonic acid-induced potentiation of DAG. The potentiation of DAG-induced contraction by arachidonic acid was inhibited by the PKC inhibitor H-7, but it was not affected by the calmodulin inhibitor CGS-9343B. We conclude that a 100-kDa cPLA2 participates in ACh-induced esophageal contraction by producing arachidonic acid and potentiating DAG-induced activation of a PKC-dependent pathway.

Rat C6 glioma cells chronically acclimated to hypertonic media accumulate large quantities of inositol. When returned to isotonic conditions, the cells swell and lose inositol slowly via a four- to fivefold increase in the rate of passive inositol efflux. The inositol efflux pathway is a Na(+)-independent transport mechanism with low affinity for inositol and is inhibited by quinidine, quinine, various anion transport blockers, and cis-unsaturated fatty acids. Ionomycin-induced elevation of intracellular Ca2+ (Ca2+i) had no effect on basal or swelling-induced inositol efflux. Inositol efflux was not inhibited by chelation of Ca2+i with 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid. In addition, Ca2+i measured with fura 2 did not change during cell swelling, indicating that increases in Ca2+i do not regulate inositol efflux. Exposure of C6 cells to 20 nM phorbol 12-myristate 13-acetate, 0.5 mM adenosine 3',5'-cyclic monophosphate (cAMP), or 50 microM forskolin had no effect on basal inositol efflux but stimulated swelling-induced inositol loss by 2.6-, 2.2-, and 3.4-fold, respectively. Exposure to the protein kinase inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine or staurosporine or downregulation of protein kinase C (PKC) activity, however, had no inhibitory effect on inositol efflux, and cellular cAMP levels were not altered by cell swelling. Taken together, these results indicate that stimulation of PKC and protein kinase A modulates the activity of the efflux pathway but is not required for swelling-induced activation. Ketoconazole, cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate, and gossypol, inhibitors of lipoxygenase enzymes, blocked both basal and swelling-induced inositol efflux, suggesting indirectly that lipoxygenase metabolites may be responsible for swelling-induced activation of the efflux mechanism. The characteristics of inositol efflux in C6 cells are similar to those described for volume regulatory sorbitol and taurine efflux in a number of cell types, suggesting the existence of a common transport mechanism.

To test the hypothesis that an endothelial cell membrane-associated serine esterase is involved in regulating phospholipase A2 (PLA2), we studied the effect of the calcium ionophore A23187 on intracellular PLA2 activity and arachidonic acid (AA) release in bovine pulmonary arterial endothelial cells. Exposure of these cells to A23187 causes a concentration-dependent increase in PLA2 activity and [14C]AA release. In addition to increasing PLA2 activity and AA release, A23187 enhances the activity of endothelial cell membrane-associated serine esterase that acts on the synthetic substrate N alpha-p-tosyl-L-arginine methyl ester. Serine esterase inhibitors, such as phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate, prevent the A23187-mediated increase in serine esterase activity, PLA2 activity, and AA release. Pretreatment of the cells with actinomycin D or cycloheximide does not prevent the A23187-mediated increase in AA release, serine esterase activity, or PLA2 activity. The membrane-associated serine esterase activity directly correlates with membrane PLA2 activity. These results suggest that a membrane-associated serine esterase plays a pivotal role in regulating PLA2 activity after exposure to A23187.

The effects of arachidonic acid (AA) and its metabolites on the conductance (gj) of the gap junctions between neonatal rat myocardial cells was investigated. AA reduced gj in a dose- (2, 5, and 20 microM) and time-dependent fashion. Pretreatment of the cells with an inhibitor of the 5-lipoxygenase pathway, U-70344A, shifted the dose-response curve to the right; pretreatment with indomethacin, an inhibitor of the cyclooxygenase pathway, had no effect. The mean time to uncoupling was 3.7 +/- 0.3, 3.8 +/- 0.9, and 4.6 +/- 0.6 min (means +/- SE, P less than 0.05) for 5 microM AA, 5 microM AA + indomethacin, and 5 microM AA + U-70344A, respectively. Incorporation of AA into membrane phospholipids was not affected by the inhibitor. These studies suggest that complete uncoupling of the cells occurred at membrane concentrations of 3-4 mol%. The data indicate that AA and a 5-lipoxygenase metabolite uncouple neonatal rat heart cells. The data are discussed with respect to the possible underlying mechanism of uncoupling and the potential role of gap junctions in arrhythmia formation in ischemic heart disease.

Nephrotoxic nephritis (NTN) is characterized by glomerular inflammation, an increase in glomerular eicosanoid synthesis, and renal dysfunction. Data further suggest that eicosanoids may play a critical role in the inflammatory response. In the current study, we examined the effects of in vivo manipulation of arachidonate metabolism on the cellular component of the inflammatory response in NTN. We found that inhibition of cyclooxygenase with indomethacin in mild NTN caused a two- to fourfold increase in the leukocyte influx into glomeruli with a change histologically from a focal to a more diffuse lesion. Both the accompanying proteinuria and the increase in ex vivo glomerular eicosanoid production were also augmented by the administration of indomethacin. The effect of indomethacin was reversible and not limited to the acute phase of NTN. The administration of aspirin, like indomethacin, augmented the glomerular inflammation of NTN. Neither OKY-046 (a thromboxane synthase inhibitor) nor MK-886 (a 5-lipoxygenase inhibitor) altered the glomerular inflammation of NTN. Administration of exogenous prostaglandin E (in the form of misoprostol) did diminish the proteinuria accompanying NTN; however, glomerular inflammation was not significantly affected. Incubation of glomeruli with [14C]arachidonate demonstrated the presence of noncyclooxygenase pathways of arachidonate metabolism (11-, 12-, and 15-lipoxygenases) with increased activity in NTN. These data demonstrate that cyclooxygenase inhibition may paradoxically worsen glomerular inflammation and suggest a potential role for noncyclooxygenase/non-5-lipoxygenase pathways of arachidonate metabolism.

Epoxygenase and omega- and omega-1-hydroxylases are the major cytochrome P-450-arachidonate (P-450-AA) metabolizing enzymes in renal tissues. We measured P-450-AA metabolism in single nephron segments and determined the tubular localization of this activity in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Formation of 20-hydroxyeicosatetraenoic acid (20-HETE), the product of AA omega-hydroxylase was specifically localized in the entire proximal tubules (S1, S2, and S3 segments), whereas formation of 19-HETE, the product of omega-1-hydroxylase and epoxyeicosatrienoic acids (EETs), products of AA epoxygenase, was demonstrable throughout the tubule. Although distribution patterns were similar in SHR and WKY, formation of 19- and 20-HETE in the proximal tubules was higher in SHR, whereas the formation of EETs was not different between the two strains. In the proximal tubules, angiotensin II (ANG II) significantly stimulated epoxygenase activity (EETs formation), whereas parathyroid hormone (PTH) and epidermal growth factor (EGF) had no effect on epoxygenase but significantly stimulated omega-hydroxylase activity (20-HETE formation). Because P-450-AA metabolites have a wide and contrasting spectrum of biological and renal effects, from vasodilation to vasoconstriction and from inhibition to stimulation of Na(+)-K(+)-adenosinetriphosphatase, their localization to the specific nephron segments and differential stimulation of their formation by ANG II, PTH, and EGF may contribute not only to renal hemodynamics and blood pressure regulation but also to the regulation of renal sodium and water balance.

We studied the role of the guinea pig tracheal epithelium in modulating tracheal smooth muscle responses to the relaxant agonists albuterol, sodium nitroprusside, and theophylline. We used an in vitro preparation that allowed separation of the fluids bathing the luminal (internal) and serosal (external) surfaces of the trachea, and bronchodilators were administered to either surface of carbachol-contracted tracheae. All three drugs produced dose-dependent relaxation. However, albuterol and nitroprusside were less potent (concentration that produced half-maximal effect increased by 100- and 32-fold, respectively) when given to the epithelial side with the epithelium intact compared with the epithelium denuded or compared with serosal administration with the epithelium intact. These differences were not observed for theophylline, where smooth muscle responses were independent of either the side of stimulation or of the presence or absence of the epithelium. Direct measurements of the diffusion of theophylline across the tracheal wall in the presence or absence of epithelium showed that after 5 h of incubation with a fixed luminal concentration of theophylline, only 1.7% had diffused across the tracheal wall with the epithelium intact. This increased to only approximately 3.3% when the epithelium was denuded. These results suggest that the epithelial is a relatively weak barrier for lipophilic agents but has a major role as a diffusion barrier to hydrophilic substances.