A role for N-arachidonylethanolamine (anandamide) as the mediator of sensory nerve-dependent Ca2+-induced relaxation

We tested the hypothesis that an endogenous cannabinoid (CB) receptor agonist, such as N-arachidonylethanolamine (anandamide), is the transmitter that mediates perivascular sensory nerve-dependent Ca2+-induced relaxation. Rat mesenteric branch arteries were studied using wire myography; relaxation was determined after inducing contraction with norepinephrine. Cumulative addition of Ca2+ caused dose-dependent relaxation (ED50 = 2.2 +/- 0.09 mM). The relaxation was inhibited by 10 mM TEA and 100 nM iberiotoxin, a blocker of large conductance Ca2+-activated K+ channels, but not by 5 microM glibenclamide, 1 mM 4-aminopyridine, or 30 nM apamin. Ca2+-induced relaxation was also blocked by the selective CB receptor antagonist SR141716A and was enhanced by pretreatment with 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (pefabloc; 30 microM), an inhibitor of anandamide metabolism. Anandamide also caused dose-dependent relaxation (ED50 =.72 +/- 0.3 microM). The relaxation was not inhibited by endothelial denudation, 10 microM indomethacin, or 1 microM miconazole, but was blocked by 3 microM SR141716A, 10 mM TEA, precontraction with 100 mM K+, and 100 nM iberiotoxin, and was enhanced by treatment with 30 microM pefabloc. Mesenteric branch arteries were 200-fold more sensitive to the relaxing action of anandamide than arachidonic acid (ED50 = 160 +/- 7 microM). These data show that: 1) Ca2+ and anandamide cause hyperpolarization-mediated relaxation of mesenteric branch arteries, which is dependent on an iberiotoxin-sensitive Ca2+-activated K+ channel, 2) relaxation induced by both Ca2+ and anandamide is inhibited by CB receptor blockade, and 3) relaxation induced by anandamide is not dependent on its breakdown to arachidonic acid and subsequent metabolism. These findings support the hypothesis that anandamide, or a similar cannabinoid receptor agonist, mediates nerve-dependent Ca2+-induced relaxation in the rat.     

Role of Ca2+-independent phospholipase A2gamma in Ca2+-induced mitochondrial permeability transition

Our laboratory previously demonstrated Ca2+-independent phospholipase A2gamma (iPLA2gamma) is localized to mitochondria and that iPLA2 inhibition blocks cisplatin-induced caspase-mediated apoptosis. Whereas the mitochondrial permeability transition (MPT) is a key control point for apoptosis, the role of mitochondrial iPLA2gamma in MPT has not been established. In the present study, we addressed this issue. Ca2+-induced renal cortex mitochondrial (RCM) swelling was blocked by the MPT inhibitor cyclosporine A. The R-isomer of bromoenol lactone (R-BEL), which enantiospecifically inhibits iPLA2gamma, inhibited Ca2+-induced RCM MPT, whereas S-BEL (negative control) had no effect. Ca2+ treatment resulted in a significant increase in free arachidonic acid (AA) (>50 microM) in the RCM suspension that was blocked by pretreatment with BEL. No increases in free myristic, palmitic, stearic, oleic, linoleic, or docosahexaenoic acid were detected after Ca2+ treatment. The addition of AA (18 microM) to Ca2+-treated RCM with inhibited iPLA2gamma activity restored MPT. We also determined that RCM iPLA2gamma displays higher activity against plasmenylcholine with AA in the sn-2 position than oleic acid. Ca2+ exposure significantly increased RCM iPLA2gamma activity; however, the Ca2+-induced activation of iPLA2gamma was not the result of mitochondrial membrane potential dissipation, opening of the MPT pore, or mitochondrial swelling. Taken together these findings provide strong evidence that Ca2+-induced RCM MPT is mediated by iPLA2gamma-catalyzed AA liberation.     

Contribution of K+ channels to arachidonic acid-induced endothelium-dependent vasodilation in rat isolated perfused mesenteric arteries.

The contribution of K+ channels and cytochrome P450 generated arachidonic acid (AA) metabolites to the endothelium-dependent vasodilation produced by this fatty acid in the perfused rat isolated mesenteric arteries was examined using a variety of compounds known to inhibit transmembrane K+ channels and cytochrome P450 enzymes. AA (1-1000 nmol) caused dose- and endothelium-dependent vasodilation in the presence of indomethacin and the effect was neither altered by lipoxygenase (AA 861) nor cytochrome P450 monooxygenase (alpha-naphthoflavone, ketoconazole and metyrapone) inhibitors indicating that AA-induced, endothelium-dependent vasodilation in this vascular bed was not mediated by product(s) of AA metabolism. The vasodilator effect of AA was also not altered by L-NG-nitro-arginine, methylene blue (50 microM), oxyhemoglobin (5 microM) or superoxide dismutase (50 U/ml), thus ruling out nitric oxide being its mediator. Conversely, arterial perfusion with K(+)-free or excess (50 mM) K+ Krebs' solution, but not ouabain infusion, minimized the vasodilator effect of AA, suggesting that this action of the fatty acid is due to changes in membrane K+ conductance that is independent of Na+/K(+)-adenosine triphosphatase activity. The vasodilator action of BRL 34915 (a K+ channel activator) was also minimized by extracellular K+ depletion or excess K+ (50 mM), but not by ouabain. Apamin (0.5 microM) and crude scorpion venom (2.5 micrograms/ml) attenuated AA- but not BRL 34915-induced vasodilation. Glyburide (inhibitor of ATP-activated K+ channel) abolished the vasodilator action of AA and BRL 34915. Procaine, a nonspecific K+ channel blocker did not affect AA-induced vasodilation even though it attenuated that caused by BRL 34915.(ABSTRACT TRUNCATED AT 250 WORDS)     

Augmented endothelium-derived hyperpolarizing factor-mediated relaxations attenuate endothelial dysfunction in femoral and mesenteric, but not in carotid arteries from type I diabetic rats

Individual vascular beds exhibit differences in vascular reactivity. The present study investigates the effects of streptozotocin-induced type I diabetes on endothelium-dependent responses of rat carotid, femoral, and mesenteric arteries. Rings with and without endothelium, suspended in organ chambers for isometric tension recording, were contracted with phenylephrine and exposed to increasing concentrations of acetylcholine. In carotid and femoral arteries, acetylcholine produced concentration- and endothelium-dependent relaxations that were abolished by Nomega-nitro-L-arginine methyl ester (L-NAME; specific nitric-oxide synthase inhibitor) and were impaired slightly in preparations from streptozotocin-treated rats (STZ-rats). This impairment could be prevented by L-arginine. In femoral arteries incubated with L-NAME, acetylcholine caused endothelium-dependent contractions that were abolished by 3-[(6-amino-(4-chlorobenzensulfonyl)-2-methyl-5,6,7,8-tetrahydronapht]-1-yl) propionic acid (S18886) (antagonist of thromboxane A2/prostaglandins H2-receptors) and reversed to relaxation by indomethacin (inhibitor of cyclooxygenase). The latter relaxation was inhibited by charybdotoxin plus apamin, suggesting a role of endothelium-dependent hyperpolarizing factor (EDHF). This EDHF-mediated component was augmented slightly in arteries from STZ-rats. In mesenteric arteries, relaxations to acetylcholine were only partially inhibited by L-NAME, and the L-NAME-resistant component was abolished by charybdotoxin plus apamin. In the mesenteric arteries from STZ-rats, L-NAME-sensitive relaxations to acetylcholine were reduced and the EDHF-component was augmented. These findings demonstrate a marked heterogeneity in endothelium-dependent responses in rat arteries and their differential adaptation in the course of type I diabetes. In particular, the EDHF-mediated component not only compensates for the reduced bioavailability of nitric oxide in the femoral and mesenteric artery but also counteracts the augmented endothelium-dependent contractions in the former.     

Inhibition of Ca2+-independent phospholipase A2 decreases prostate cancer cell growth by p53-dependent and independent mechanisms

The mechanisms by which Ca(2+)-independent phospholipase A(2) (iPLA(2)) mediates cell growth in p53-positive LNCaP and p53-negative PC-3 prostate cancer cell lines were studied. Exposure of cells to the iPLA(2) selective inhibitor bromoenol lactone (BEL; 0-20 microM) induced concentration- and time-dependent decreases in cell growth based on 3-(4, dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide staining and cell number. Decreased cell growth was not caused by cell death as BEL exposure did not alter nuclear morphology or increase annexin V (apoptotic cell marker) or propidium iodide (necrotic cell marker) staining after 48 h. Decreased growth correlated to a G(1)/G(0) arrest in LNCaP cells and aG(2)/M arrest in PC-3 cells. In LNCaP cells, G(1) arrest was preceded by time- (0-48 h) and concentration-dependent (0-10 microM) increases in the expression of the tumor suppressor protein p53 and the cyclin-dependent kinase inhibitor p21. Increases in p53 expression preceded increases in p21 expression by 8 h. In LNCaP cells, BEL treatment decreased the expression of the p53 antagonist Mdm2, while increasing Akt phosphorylation. BEL treatment also increased Akt phosphorylation in PC-3 cells, but Mdm2 was not detected. The ability of BEL to increase Akt phosphorylation was inhibited by the phosphoinositide 3-kinase inhibitor LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one]. BEL treatment also decreased agonist-induced activation of the epidermal growth factor receptor. These data suggest that inhibition of iPLA(2) decreases prostate cancer cell growth by p53-dependent and independent mechanisms. Furthermore, alterations in Mdm2 and epidermal growth factor receptor activation following BEL exposure suggest novel roles for iPLA(2) in prostate cancer cell signaling.     

Increased superoxide anion production by interleukin-1beta impairs nitric oxide-mediated relaxation in resistance arteries

The present study was designed to analyze the effect of long-term incubation with interleukin-1beta (IL-1beta) on endothelium-dependent relaxation in rat mesenteric resistance arteries. Vessels were incubated in culture medium with or without IL-1beta (10 ng/ml, 14 h). Changes in lumen diameter were recorded in a pressure myograph. Protein expression, nitrite, and superoxide anion (O(2)(.)) production were evaluated by either Western blot or immunofluorescence, Griess reaction, and ethidium fluorescence, respectively. IL-1beta impaired acetylcholine (ACh) and sodium nitroprusside (SNP) vasodilation and increased nitrite and O(2)(.) production and inducible nitric-oxide synthase (iNOS), xanthine oxidase, and p22(phox) expression. However, neither endothelial nitric-oxide synthase (NOS) nor soluble guanylate cyclase protein expression were affected by IL-1beta treatment. Polyethylene glycol superoxide dismutase partially reversed the impairment of ACh relaxation and abolished the O(2)(.) production observed in IL-1beta-treated arteries. The impairment of ACh relaxation induced by IL-1beta was also partially reversed by the xanthine oxidase inhibitor allopurinol (1 mM) but not by either the NADPH oxidase inhibitor apocynin (0.3 mM) or the inducible NOS inhibitor N-3-aminomethylbenzylacetamidine (1 microM). However, all these inhibitors improved the impaired SNP response. The results of the present study demonstrate that long-term incubation with IL-1beta induces an impairment of the nitric oxide-mediated relaxation in mesenteric resistance arteries through the production of O(2)(.), mainly from xanthine oxidase.     

Differential roles for cytosolic and microsomal Ca2+-independent phospholipase A2 in cell growth and maintenance of phospholipids

Physiological roles of microsomal (iPLA(2)gamma) and cytosolic (iPLA(2)beta)Ca(2+)-independent phospholipase A(2) were determined in two different epithelial cell models. R- and S-enantiomers of the iPLA(2) inhibitor bromoenol lactone (BEL) were isolated and shown to selectively inhibit iPLA(2gamma) and iPLA(2beta), respectively. The effect of these enantiomers on cell growth was assessed in human embryonic kidney 293 and Caki-1 cells using 3-(4-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT). S-BEL (0-5.0 microM) decreased MTT staining 35% after 24 h compared with control cells, whereas treatment with either R-BEL or R/S-BEL induced 15% decreases. Neither R-BEL nor S-BEL induced cell death as determined by annexin V and propidium iodide staining. Transfection of cells with iPLA(2)beta siRNA reduced MTT staining approximately 35%, whereas transfection of cells with iPLA(2)gamma siRNA only decreased MTT staining 10 to 15% compared with control cells. The effect of iPLA(2)beta and iPLA(2)gamma siRNA on cell number and protein was also determined, and iPLA(2)beta siRNA decreased cell number and protein 25% compared with control cells. In contrast, iPLA(2)gamma siRNA decreased cell number, but not cellular protein, compared with control cells. Selective inhibition of iPLA(2)beta, but not iPLA(2)gamma, decreased several arachidonic acid-containing phospholipids, including 16:1-20:4, 16:0-20:4, 18:1-20:4, and 18:0-20:4 phosphatidylcholine, showing that the ability of iPLA(2)beta inhibitors to decrease cell growth correlates with their ability to decrease arachidonic acid-containing phospholipids. These data show that iPLA(2)beta inhibition results in greater decreases in cell growth and proliferation than iPLA(2)gamma, identifies specific phospholipids whose expressions are differentially regulated by iPLA(2)beta and iPLA(2)gamma, and suggests novel roles for iPLA(2)beta in cell growth.     

Differences in responsiveness of intrapulmonary artery and vein to arachidonic acid: mechanism of arterial relaxation involves cyclic guanosine 3':5'-monophosphate and cyclic adenosine 3':5'-monophosphate

The objective of this study was to examine the relationship between responses of bovine intrapulmonary artery and vein to arachidonic acid and cyclic nucleotide levels in order to better understand the mechanism of relaxation elicited by arachidonic acid and acetylcholine. Arachidonic acid relaxed phenylephrine-precontracted arterial rings and elevated both cyclic GMP and cyclic AMP levels in arteries with intact endothelium. In contrast, endothelium-damaged arterial rings contracted to arachidonic acid without demonstrating significant changes in cyclic nucleotide levels. Indomethacin partially inhibited endothelium-dependent relaxation and abolished cyclic AMP accumulation whereas methylene blue, a guanylate cyclase inhibitor, partially inhibited relaxation and abolished cyclic GMP accumulation in response to arachidonic acid. All vessel responses were blocked by a combination of the two inhibitors. Prostaglandin (PG) I2 relaxed arterial rings and elevated cyclic AMP levels whereas PGE2 and PGF2 alpha caused contraction, suggesting that the indomethacin-sensitive component of arachidonic acid-elicited relaxation is due to PGI2 formation and cyclic AMP accumulation. The methylene blue-sensitive component is attributed to an endothelium-dependent but cyclooxygenase-independent generation of a substance causing cyclic GMP accumulation. Intrapulmonary veins contracted to arachidonic acid with no changes in cyclic nucleotide levels and PGI2 was without effect. Homogenates of intrapulmonary artery and vein formed 6-keto-PGF1 alpha, PGF2 alpha and PGE2 from [14C]arachidonic acid, which was inhibited by indomethacin. Thus, bovine intrapulmonary vein may not possess receptors for PGI2. The failure of endothelium-intact vein to relax to acetylcholine may be related to the lack of a relaxant effect by arachidonic acid, perhaps attributed to the absence of generation of an endothelium-derived relaxing factor.     

Urocortin induces endothelium-dependent vasodilatation and hyperpolarization of rat mesenteric arteries by activating Ca2+-activated K+ channels

Urocortin, a member of corticotropin releasing factor (CRF) peptide family, has positive chronotropic and inotropic effects on heart and also shows a vasodilatory effect. However, the mechanism underlying its vasodilatory effect has yet to be elucidated. Endothelium-dependent relaxation of resistance arteries is mainly achieved by activation of K+ channels. Therefore, we investigated possible role of K+ channels and hyperpolarization for the vasodilatory effect of urocortin using the isolated perfused rat mesenteric arteries. Urocortin (0.2 nM) produced a slow-onset decrease in the perfusion pressure of the mesenteric vascular bed, which was elevated by an alpha1-adrenoceptor agonist, phenylephrine (2-4 microM). Urocortin also hyperpolarized the main mesenteric artery. Removal of endothelium with saponin treatment considerably inhibited the relaxation and hyperpolarization induced by urocortin. In contrast, the hyperpolarization was not significantly changed by cyclooxygenase inhibitor, indomethacin (1 microM) and/or nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine (100 microM). Urocortin-induced relaxation was not affected by the combination of a guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1 microM), indomethacin and N(omega)-nitro-L-arginine. However, the relaxation and hyperpolarization were abolished by high extracellular potassium concentration (40 mM) or by a large conductance Ca(2+)-activated K+ channel blocker, charybdotoxin (1 nM). Glibenclamide (1 microM), an ATP-dependent K+ channel inhibitor, did not affect the relaxation and hyperpolarization. These results suggest that urocortin causes endothelium-dependent relaxation and hyperpolarization of rat mesenteric arteries, probably through the activation of charybdotoxin sensitive Ca2+-activated K+ channels. These findings also indicate an essential role of the endothelium for the urocortin-elicited vascular relaxation and hyperpolarization.     

Heterogeneity of endothelium-dependent vasodilation in pressurized cerebral and small mesenteric resistance arteries of the rat.

We compared endothelial responses to calcium-mobilizing agents in mesenteric and cerebral resistance arteries of the rat. Middle cerebral and small mesenteric arteries were mounted in a pressure myograph, and myogenic responses were recorded. The effects of acetylcholine (ACh), bradykinin, substance P, histamine, A23187, cyclopiazonic acid (CPA), and sodium nitroprusside were investigated in both arteries with myogenic tone in the absence and presence of nitric oxide synthase and cyclooxygenase inhibitors. The effects of raised potassium, K(+) channel blockers, and arachidonic metabolism inhibition were examined on the nitric oxide (NO) synthase/cyclooxygenase inhibitor-resistant dilation induced by ACh and CPA. Cerebral arteries display a high level of myogenic reactivity compared with mesenteric arteries. In cerebral arteries, only bradykinin and substance P induced endothelium-dependent dilation. The observed dilation was solely related to the activation of the NO pathway. However, in mesenteric arteries, all of the vasoactive agents induced endothelium-dependent dilation. A combination of NO, cyclooxygenase-derived prostanoids, and a factor with endothelium-derived hyperpolarizing factor-like properties was responsible for the observed vasodilation. NO and cyclooxygenase derivatives were able to compensate for each other in the CPA-induced endothelium-dependent vasodilation when one of the two pathways was blocked. Moreover, small Ca(2+)-activated K(+) channels and a combination of both large and small Ca(2+)-activated K(+) channels were implicated in the endothelium-derived hyperpolarizing factor-like component of dilation to ACh and CPA, respectively. Finally, the results suggest that the pathway by which agonists raise intracellular calcium concentration may determine the nature of the endothelial secretory product.     

Phorbol dibutyrate inhibits release and action of endothelium-derived relaxing factor(s) in canine blood vessels

The effects of phorbol esters on endothelium-dependent relaxations evoked by ACh and the calcium ionophore A23187 were analyzed in isolated canine femoral and coronary arteries mounted in organ chambers or in a bioassay system. In rings of femoral and coronary arteries, phorbol 12,13-dibutyrate (PDBu) (10(-7) M) evoked contraction (ED50 2.9 x 10(-8) M) and depressed endothelium-dependent relaxations to ACh (4-fold increase in ED50 and 72% depression of maximal response). PDBu depressed maximal relaxations to A23187 by 50% but had no effect on ED50. The inactive phorbol ester 4-alpha-phorbol didecanoate (10(-7) M) did not evoke contractions and had no effect on endothelium-dependent relaxations to ACh or A23187. Endothelium-independent relaxations to sodium nitroprusside were not effected by PDBu (10(-7) M) in femoral or coronary arteries. In bioassay experiments, selective treatment of perfused femoral artery segments with PDBu (10(-8)-10(-6) M) caused concentration-dependent inhibition of basal and ACh- (10(-6) M) and A23187-(10(-6) M) induced release of endothelium-derived relaxing factor (EDRF) (as assessed by relaxation of superfused bioassay coronary artery rings without endothelium). PDBu inhibited these responses with different potency: basal (10(-8) M) greater than ACh (10(-7) M) much greater than A23187 (only partial inhibition at 10(-6) M).(ABSTRACT TRUNCATED AT 250 WORDS)     

Nitric oxide-dependent and -independent mechanisms account for gender differences in vasodilation to acetylcholine

The purpose of this study was to examine the mechanism of enhanced endothelium-dependent dilation in arteries from female rats compared with arteries from males. Isolated mesenteric resistance arteries ( approximately 250 microm) from sexually mature male and female Sprague-Dawley rats were pressurized and outer diameter was measured. Arteries from females were more sensitive to the endothelium-dependent vasodilator acetylcholine (Ach) compared with those from males (-log EC(50): male = 6.74 +/- 0.06; female = 6.96 +/- 0.06; P =.037). After incubation with N(omega)-nitro-L-arginine (100 microM) or apamin (30 nM), there was no longer a gender difference in midrange sensitivity to ACh. In contrast, at higher concentrations of ACh, N(omega)-nitro-L-arginine had a greater inhibitory effect in the males than in the females. Indomethacin (10 microM) decreased sensitivity to ACh in arteries from both males and females, but did not alter the maximal response or eliminate the gender difference. Finally, there was no gender difference in vasodilation to the nitric oxide (NO) donor spermine-NO complex, nor did apamin alter the spermine-NO complex response. In conclusion, mesenteric arteries from female rats are more sensitive to ACh than those from males. An enhanced contribution of an apamin-sensitive K(Ca) channel on the endothelium of female arteries appears to be responsible for the augmented ACh-stimulated NO production compared with that of males. In addition, ACh stimulates the production of a non-NO, noncyclooxygenase, endothelium-derived hyperpolarizing factor to a greater extent in females compared with males.     

Predominant role for nitric oxide in the relaxation induced by acetylcholine in cat cerebral arteries.

The possible role of nitric oxide (NO) and other endothelial relaxant factors in the vasodilation induced by acetylcholine (ACh) in isolated segments of cat cerebral arteries was analyzed by using the following treatments: 1) the blockers of cyclo- and lipoxygenase, indomethacin and 5,8,11,14-eicosatetraynoic acid; 2) the NO inactivators, phenidone, hydroquinone and oxyhemoglobin; 3) the inhibitors of NO synthesis, NG-nitro-L-arginine methyl ester and NG-monomethyl-L-arginine; 4) the blockers of sodium pump activity, ouabain and K(+)-free medium; and 5) the antagonist of K+ channels, 4-aminopyridine (4-AP). A comparative study between the relaxant actions of exogenous NO and ACh was also performed. The most relevant results obtained were: 1) cat cerebral arteries are very sensitive to the endothelial effects of ACh, as well as to the exogenous NO; 2) ACh-induced endothelium-dependent dilatation is not affected by indomethacin and 4-AP, partially inhibited by 5,8,11,14-eicosatetraynoic acid, phenidone, hydroquinone, oxyhemoglobin and NG-nitro-L-arginine methyl ester and abolished by NG-monomethyl-L-arginine; 3) this latter effect is selectively antagonized by L-arginine, suggesting that the inhibition of NO synthase may be enough to abolish relaxation to ACh; and 4) sodium pump blockade abolished endothelial but not exogenous NO effects. From these results, we conclude that ACh-induced relaxation in these vessels can be entirely mediated by the release of endothelial NO. Although other endothelial factors cannot be discarded, their possible contribution to ACh-evoked relaxation is likely negligible.     

Pardaxin stimulation of phospholipases A2 and their involvement in exocytosis in PC-12 cells

Pardaxin (PX) is a voltage-dependent ionophore that stimulates catecholamine exocytosis from PC-12 pheochromocytoma cells both in the presence and absence of extracellular calcium. Using a battery of phospholipase A(2) inhibitors we show that PX stimulation of phospholipase A(2) (PLA(2)) enzymes is coupled with induction of exocytosis. We investigated the relationship between PX-induced PLA(2) activity and neurotransmitter release by measuring the levels of arachidonic acid (AA), prostaglandin E(2) (PGE(2)), and dopamine release. In the presence of extracellular calcium, the cytosolic PLA(2) inhibitor arachidonyl trifluoromethyl ketone (AACOCF(3)) inhibited by 100, 70, and 73%, respectively, the release of AA, PGE(2), and dopamine induced by PX. The mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor 2'-amino-3'-methoxyflavone (PD98059) reduced by 100 and 82%, respectively, the release of AA and PGE(2) induced by PX. In the absence of extracellular calcium, the calcium-independent PLA(2) (iPLA(2)) inhibitors methyl arachidonyl fluorophosphonate, AACOCF(3), and bromoenol lactone (BEL) inhibited by 80 to 90% PX stimulation of AA release, by 65 to 85% PX stimulation of PGE(2) release, and by 80 to 90% PX-induced dopamine release. Using vesicle fusion-based enzyme-linked immunosorbent assay we found similar levels of inhibition of PX-induced exocytosis by these inhibitors. Also, PX induced the formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor complexes, an effect that was augmented by N-methylmaleimide. This complex formation was completely inhibited by BEL. Botulinum toxins type C1 and F significantly inhibited the release of AA, PGE(2), and dopamine induced by PX. Our data suggest that PX stimulates exocytosis by activating cystolic PLA(2) and iPLA(2), leading to the generation of AA and eicosanoids, which, in turn, stimulate vesicle competence for fusion and neurotransmitter release.     

Enhanced prostanoid-mediated vasorelaxation in pulmonary arteries isolated during experimental endotoxemia.

Endotoxemia secondary to gram-negative sepsis has been shown to inhibit endothelium-dependent vasomotion in numerous vascular beds, including guinea pig aortae and coronary arteries. We tested the hypothesis that in vivo endotoxin impairs endothelium-dependent nitric oxide-mediated relaxation responses of pulmonary arteries isolated from guinea pigs given intraperitoneal injections of Escherichia coli endotoxin lipopolysaccharide (LPS) or saline (control) 16 h before sacrifice. Pulmonary rings from the main artery and primary branches were isolated and studied in vitro using conventional isometric techniques. Interestingly, endotoxemia resulted in enhanced pulmonary artery relaxation in response to the endothelium-dependent receptor agonists acetylcholine (10(-10) -10(-5) M) and adenosine diphosphate (ADP; 10(-9) -10(-5) M), as compared with control responses (p < .05). Nitric oxide synthase inhibitors N-monomethyl-L-arginine (300 microM) and N-nitro-L-arginine methyl ester (100 microm) reduced acetylcholine- and adenosine diphosphate-mediated relaxation in both groups (p < .05); however, vasodilation responses in arteries from LPS animals remained enhanced relative to those of control arteries. In contrast to nitric oxide synthase inhibitors, the cyclooxygenase inhibitor indomethacin markedly inhibited acetylcholine- and adenosine diphosphate-mediated relaxation responses of pulmonary arteries isolated from LPS-treated animals (p < .05) but not control arteries; indomethacin effectively reversed LPS-induced enhanced vasodilation of pulmonary arteries. Relaxation responses to the receptor-independent calcium ionophore (A23187) and to the direct smooth muscle vasodilator sodium nitroprusside (+ N-nitro-L-arginine methyl ester) were not significantly altered by LPS treatment (p > .05). These data suggest that in pulmonary arteries, unlike aortae and coronary arteries isolated from the same model, in vivo LPS enhances agonist-mediated endothelium-dependent vasodilation responses to acetylcholine and adenosine diphosphate. Underlying mechanisms appear to involve increased dependency upon vasodilator prostanoids and decreased dependency on nitric oxide synthesis/release for LPS-induced alterations in pulmonary relaxation responses.     

Effect of NG-monomethyl-L-arginine on endothelium-dependent relaxation of human subcutaneous resistance arteries

1. Using a myograph to measure isometric tension, we have looked at the action of NG-monomethyl-L-arginine on the endothelium-dependent relaxation of human subcutaneous resistance arteries. 2. NG-Monomethyl-L-arginine, the novel inhibitor of endothelium-derived relaxing factor synthesis, caused concentration-dependent but only partial inhibition of maximal relaxation induced by acetylcholine in human subcutaneous resistance arteries. 3. The inhibitory action of NG-monomethyl-L-arginine on acetylcholine-induced maximal relaxation was partially reversed by incubation of the arteries in equimolar concentrations of L-arginine and NG-monomethyl-L-arginine. Subsequent incubation in L-arginine led to further reversal, but this was no greater than with incubation in physiological saline. 4. A component of acetylcholine-induced relaxation was sensitive to indomethacin, suggesting that this response is mediated by prostanoids as well as by endothelium-derived relaxing factor. 5. NG-Monomethyl-L-arginine did not increase the tension of resting human subcutaneous resistance arteries. NG-Monomethyl-L-arginine did enhance the contractile response to noradrenaline, possibly due to inhibition of release of endothelium-derived relaxing factor resulting from stimulation of alpha 2-adrenoreceptors on the endothelial cells.     

Substituted adamantyl-urea inhibitors of the soluble epoxide hydrolase dilate mesenteric resistance vessels

The epoxyeicosatrienoic acids (EETs) have been identified as endothelium-derived hyperpolarizing factors. Metabolism of the EETs to the dihydroxyeicosatrienoic acids is catalyzed by soluble epoxide hydrolase (sEH). Administration of urea-based sEH inhibitors provides protection from hypertension-induced renal injury at least in part by lowering blood pressure. Here, we investigated the hypothesis that a mechanism by which sEH inhibitors elicit their cardiovascular protective effects is via their action on the vasculature. Mesenteric resistance arteries were isolated from Sprague-Dawley rats, pressurized, and constricted with the thromboxane A2 agonist U46619 (9,11-dideoxy-11,9-epoxymethano-prostaglandin F2alpha). Mesenteric arteries were then incubated with increasing concentrations of the sEH inhibitor 12-(3-adamantan-1-yl-ureido)dodecanoic acid (AUDA). AUDA resulted in a concentration-dependent relaxation of mesenteric arteries, with 10 microM resulting in a 48 +/- 7% relaxation. Chain-shortened analogs of AUDA had an attenuated vasodilatory response. Interestingly, at 10 microM, the sEH inhibitors 1-cyclohexyl-3-dodecylurea, 12-(3-cyclohexylureido)dodecanoic acid, and 950 [adamantan-1-yl-3-{5-[2-(2-ethoxyethoxy)ethoxy]pentyl}urea] were significantly less active, resulting in a 25 +/- 8%, 10 +/- 9%, and -8 +/- 3% relaxation, respectively. Treatment of mesenteric arteries with tetraethylammonium, iberiotoxin, ouabain, or glibenclamide did not alter AUDA-induced relaxation. The AUDA-induced relaxation was completely inhibited when constricted with KCl. In separate experiments, denuding mesenteric resistance vessels did not alter AUDA-induced relaxation. Taken together, these data demonstrate that adamantyl-urea inhibitors have unique dilator actions on vascular smooth muscle compared with other sEH inhibitors and that these dilator actions depend on the adamantyl group and carbon chain length.     

Impaired endothelium-dependent relaxation in mesenteric arteries of reduced renal mass hypertensive rats.

Endothelium-dependent relaxation is not fully understood in volume-dependent models of hypertension. This study investigated the relaxation mediated by endothelium-derived nitric oxide (EDNO) and hyperpolarizing factor (EDHF) in superior mesenteric arteries from reduced renal mass hypertensive rats, an experimental model for volume-dependent hypertension. Hypertension was induced in male Wistar rats by subtotal nephrectomy and salt-loading (hypertensive group). The control group comprised rats that drank tap water after subtotal nephrectomy. Relaxation of isolated superior mesenteric arterial rings was investigated at the end of the 2-week study. In high K+-precontracted arterial rings, relaxation caused by acetylcholine (ACh) was markedly reduced in the hypertensive group compared with the findings for the control group (34+/-4% vs. 54+/-5% decrease in tension). In both groups, the relaxation was abolished by N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase. In phenylephrine-precontracted arterial rings, relaxation caused by ACh was also small in the hypertensive group, while it was large in the control group (49 +/- 5% vs. 96 +/- 2%). Superfusion of L-NAME inhibited most of the relaxation caused by ACh, but the arteries still exhibited relaxation. Apamin, a blocker of Ca-dependent K+ channel, together with L-NAME further inhibited the residual relaxation. The relaxation inhibited by apamin was also reduced in the hypertensive group. We conclude that the relaxation inhibited by L-NAME was mediated by EDNO, while that inhibited by apamin was mediated by EDHF. Endothelium-independent relaxation caused by nitroprusside and diazoxide was normal in the hypertensive group. The relaxation mediated by both EDNO and EDHF was depressed in the arteries of reduced renal mass hypertensive rats as the result of an arterial endothelial abnormality.     

Tumor necrosis factor-alpha impairs endothelium-dependent relaxation of rat renal arteries,independent of tyrosine kinase

We hypothesized that tumor necrosis factor-alpha (TNF-alpha) mimics endotoxin in attenuating endothelium-dependent vasodilation and smooth muscle constriction of rat renal arteries, and that tyrosine kinase is involved. Isolated rat renal arteries (n =6 per group), pretreated for 2 h by genistein (4',5,7-trihydroxyisoflavone, 10 microg/mL, a tyrosine kinase inhibitor) or vehicle, were exposed for 2 h to recombinant human (rh) TNF-alpha (100 ng/mL) or vehicle. rhTNF-alpha attenuated (P < 0.05) the constriction response to depolarizing 125 mM KCl (952.6+/-125.3 mg/mm vs. 1191.4+/-136.8 mg/mm in rhTNF-alpha-exposed and control segments, respectively), but did not affect the constriction response to norepinephrine (NE, 0.01-10 microM). Genistein did not affect the constriction response to KCl. The concentration-response relation to NE in genistein-pretreated control segments showed (P < 0.05) a rightward shift, while the maximum constriction was not affected. Genistein did not prevent a reduction (P < 0.05) by rhTNF-alpha in the maximum response to NE (721.7+/-42.4 mg/mm vs. 999.8+/-84.4 mg/mm in controls). The endothelium-dependent relaxation induced by (acetyl choline) ACh (0.001-1.0 microM) was attenuated (P < 0.05) by rhTNF-alpha (39.4%+/-6.7% and 77.4%+/-10.0% in rhTNF-alpha-exposed and control segments, respectively). The reduction (P < 0.05) in maximum ACh-induced relaxation after exposure to rhTNF-alpha was not affected by genistein (44.6%+/-3.4% and 70.8% x 2.2% in genistein-pretreated rhTNF-alpha-exposed and control segments, respectively). Hence, the attenuated endothelium-dependent relaxation and smooth muscle constriction of rat renal arteries following short-term rhTNF-alpha exposure, mimicking the effect of endotoxin, does not involve the activity of tyrosine kinase. The latter may be involved in pharmacomechanical coupling, by increasing Ca2+ sensitivity, but less in the electromechanical coupling of smooth muscle constriction.