Characterization of the responses of equine digital veins and arteries to calcitonin gene-related peptide.

OBJECTIVE: To compare responses of equine digital arteries (EDAs) and veins (EDVs) to human-αcalcitonin gene-related peptide (hαCGRP), evaluate effect of the endothelium, and characterize receptors and sources of endogenous CGRP. SAMPLE: Palmar digital vessels (5 to 9/experiment) from healthy adult horses killed at an abattoir. PROCEDURES: Vessel rings were mounted under tension in organ baths containing Krebs-Henseleit solution at 30 °C, with relaxation responses examined in vessels preconstricted with a thromboxane-mimetic (3 × 10(-8)M). Responses of endothelium-intact (+e) and -denuded (-e) EDAs and EDVs to hαCGRP C10(-10) to 3 × 10(-7)M) were compared. Following incubation with an hαCGRP receptor antagonist (hαCGRP(8-37); 1 µM), responses of EDA(-e) and EDV(-e) to hαCGRP (10(-7)M) were obtained. Responses of endothelium-intact and -denuded arteries and veins to hαCGRP (3 × 10(-7)M) or capsaicin (10(-5)M) were evaluated as well as responses of endothelium-intact and -denuded EDA and EDV to hαCGRP (10(-10) to 10(-6)M) after incubation with endothelin-1 (ET-1; 10(-12)M). RESULTS: hαCGRP resulted in nonendothelium, concentration-dependent relaxation in EDAs and EDVs, with greater responses in EDAs. Treatment with hαCGRP(8-37) had minimal effect on responses to hαCGRP in either vessel type. Capsaicin induced relaxation in both vessel types. There were no differences between responses to hαCGRP for vessels pretreated with ET-1 or vehicle. CONCLUSIONS AND CLINICAL RELEVANCE: Both hαCGRP and capsaicin induced digital vasodilation unaffected by a functional endothelium. This suggested that endogenous CGRP likely emanates from sensory-motor nerves and may contribute to digital vasodilation.

Arachidonic acid epoxygenase and 12(S)-lipoxygenase: evidence of their concerted involvement in ductus arteriosus constriction to oxygen.

Oxygen promotes closure of the ductus arteriosus at birth. We have previously presented a scheme for oxygen action with a cytochrome P450 (CYP450) hemoprotein and endothelin-1 (ET-1) being, respectively, sensor and effector, and a hypothetical monooxygenase product serving as a coupling link. We have also found in the vessel arachidonic acid (AA) 12(S)-lipoxygenase (12-lipoxygenase) undergoing upregulation at birth. Here, we examined the feasibility of a sensor-to-effector messenger originating from AA monooxygenase and 12-lipoxygenase pathways. The epoxygenase inhibitor, N-methylsulfonyl-6-(2-)hexanamide, suppressed the tonic contraction of ductus to oxygen. A similar effect was obtained with 12-lipoxygenase inhibitors baicalein and PD 146176. By contrast, none of the inhibitors modified the endothelin-1 contraction. Furthermore, an AA ω-hydroxylation product, 20-hydroxyeicosatetraenoic acid (20-HETE), reportedly responsible for oxygen contraction in the systemic microvasculature, had no such effect on the ductus. We conclude that AA epoxygenase and 12-lipoxygenase jointly produce a hitherto uncharacterized compound acting as oxygen messenger in the ductus.

Evaluation of the serotonin receptor blockers ketanserin and methiothepin on the pulmonary hypertensive responses of broilers to intravenously infused serotonin.

The pathogenesis of pulmonary hypertension remains incompletely understood. Many factors have been implicated; however, there has been great interest in the potent pulmonary vasoconstrictor serotonin (5-HT) due to episodes of primary pulmonary hypertension in humans triggered by serotoninergic appetite-suppressant drugs. Pulmonary hypertensive patients have elevated blood 5-HT levels and pulmonary vasoconstriction induced by 5-HT is believed to be mediated through 5-HT1B/1D and 5-HT2A receptors that are expressed by pulmonary smooth muscle cells. The vascular remodeling associated with pulmonary hypertension also appears to require the serotonin transporter. We investigated the roles of 5-HT receptor blockers on the development of pulmonary hypertension induced by infusing 5-HT i.v. in broilers. For this purpose, we treated broilers with the selective 5-HT2A receptor antagonist ketanserin (5 mg/ kg of BW) or with the nonselective 5-HT1/2 receptor antagonist methiothepin (3 mg/kg of BW). Receptor blockade was followed by infusion of 5-HT while recording pulmonary arterial pressure and pulmonary arterial blood flow. The results demonstrate that methiothepin, but not ketanserin, eliminated the 5-HT-induced pulmonary hypertensive responses in broilers. The 5-HT2A receptor does not, therefore, appear to play a role in the 5-HT-induced pulmonary hypertensive responses in broilers. Methiothepin did not inhibit pulmonary vascular contractility per se, because the pulmonary hypertensive response to the thromboxane A2 mimetic U44069 remained intact in methiothepin-treated broilers. Methiothepin will be a useful tool for evaluating the role of 5-HT in the pathogenesis of pulmonary hypertension syndrome (ascites) as well as the onset of pulmonary hypertension triggered by inflammatory stimuli such as bacterial lipolysaccharide.

Glibenclamide inhibits agonist-induced vasoconstriction of placental chorionic plate arteries.

BACKGROUND: Preliminary data suggest that K(ATP) channels may be expressed in placental arteries and veins [Wareing M, Turner C, Greenwood SL, Baker PN, Fyfe GK. Expression of mRNA encoding K+ channels in chorionic plate arteries and veins. J Soc Gynecol Investig 2004;11:353A]. However, no data exist on glibenclamide's effects in placental chorionic plate arteries. AIM: To assess the effect of glibenclamide on placental chorionic plate arterial vasoconstriction. METHODS: Arteries were dissected from placental chorionic plate biopsies obtained at term from uncomplicated pregnancies (N=63). Arteries were mounted onto a wire myograph in HCO3- -buffered physiological salt solution (PSS) at 37 degrees C (5% O2/5% CO2 bubbling) and normalised at 0.9 of L5.1 kPa. Constriction viability was assessed with 120 mmol l(-1) potassium solution (KPSS). Dose-response curves were produced with the thromboxane-mimetics U46619 and U44069 (10(-10)-2 x 10(-6)M), arginine vasopressin (10(-10)-5 x 10(-8)M) and endothelin-1 (10(-11)-3 x 10(-7)M) in the presence or absence of 50 micromol l(-1) glibenclamide. The effect of glibenclamide on arginine vasopressin- and U46619-induced constriction was also assessed in the presence of the cyclo-oxygenase inhibitor indomethacin (10 micromol l(-1)). RESULTS: Pre-incubation with 50 micromol l(-1) glibenclamide significantly right-shifted dose-response curves to all vasoconstrictive agonists tested (repeated measures ANOVA). Indomethacin did not modify the inhibitory effect of glibenclamide. CONCLUSION: Glibenclamide's effects on agonist-induced constrictions are unlikely to be via an inhibition of ATP-sensitive K+ channels, and with U46619- and U44069-induced constrictions, glibenclamide may be acting as a competitive antagonist of thromboxane receptors.

Glibenclamide inhibits thromboxane-mediated vasoconstriction by thromboxane receptor blockade.

Because sulfonylureas, such as glibenclamide, are used to treat Type 2 diabetes and because this disease is associated with various cardiovascular complications that may be mediated by thromboxane (TX), this study was designed to characterize the role of glibenclamide on TX-mediated contractions in isolated ring segments of bovine coronary arteries and rabbit aortas. A series of TXA(2) analogs [9,11 Dideoxy-9alpha, 11alpha-methanoepoxy prostaglandin F(2alpha) (U46619), [1S-(1alpha, 2beta(5Z),3alpha(1E, 3R*),4alpha)]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo [2.2.1]heptan-2-yl]-5-heptenoic acid (I-BOP), carbocyclic TXA(2) (CTA(2)) and 9,11-dideoxy-9alpha,11alpha-epoxymethano prostaglandin F(2alpha) (U44069)], endothelin and phenylephrine contracted both types of blood vessels. Glibenclamide (10 microM) inhibited the contraction to each of the TX agonists but had no effect on endothelin- or phenylephrine-induced contractions. We hypothesized that this effect was due to a direct effect to block the vascular smooth muscle cell TX receptor. Receptor binding studies were performed in rabbit vascular smooth muscle cells and indicated that glibenclamide (10 microM) inhibited (125)I-BOP binding by more than 80%. The inhibition constants or K(i) for glibenclamide was 0.53 microM. These studies provide the first evidence that the ability of glibenclamide to inhibit TX-mediated contractions occurs independent of the vascular K(ATP) channel and is, instead, mediated by the blockade of the vascular TX receptor.

Crucial role of the peroxyketal function for antimalarial activity in the G-factor series.

New endoperoxides, related to the natural phytohormones known as G factors (G1, G2, G3), were modified on the side chain and the ketalic position. An unexpected rearrangement, specific to one diastereoisomer was observed in the deprotection step of O-silylated compounds and attributed to a hexacoordinated fluorosilicon intermediate. The reduction potential of these new peroxides was determined. They exhibited good to moderate antimalarial activity, greatly related to the presence of peroxyketal function.

Intravenous endotoxin triggers pulmonary vasoconstriction and pulmonary hypertension in broiler chickens.

Bacterial endotoxins stimulate endothelin-mediated, thromboxane-dependent increases in pulmonary vascular resistance in mammals, and thromboxane has been shown to cause an immediate but transient pulmonary vasoconstriction in broiler chickens. In the present study, i.v. injections of 1 mg endotoxin into anesthetized male broilers caused a pulmonary vasoconstrictive response that was delayed in onset by 15 min and that elevated the pulmonary arterial pressure by 10 mm Hg within 25 min postinjection. Thereafter, pulmonary hemodynamic variables gradually (> or = 15 min) returned toward pre-injection levels, and supplemental injections of 4 mg endotoxin during this recovery period failed to reinitiate pulmonary hypertension. In contrast, injecting the thromboxane A2 mimetic U44069 during the endotoxin recovery period triggered pulmonary vasoconstriction and pulmonary hypertension similar in magnitude to the responses triggered by U44069 before endotoxin had been administered. The time course and magnitude of the pulmonary hemodynamic responses to endotoxin were highly variable among individual broilers, whereas the individual responses to U44069 were more consistent. Unanesthetized broilers resembled anesthetized broilers in the time course, magnitude, and variability of their pulmonary hemodynamic responses to endotoxin. Overall, these observations are consistent with the hypothesis that endotoxin initiates a biochemical cascade, culminating in the delayed onset of pulmonary vasoconstriction and pulmonary hypertension within 20 min postinjection. Subsequently, the pulmonary vasculature remains responsive to large bolus injections of exogenous thromboxane mimetic; however depletion of endogenous vasoconstrictive components of the endotoxin-mediated cascade, a compensatory increase in endogenous vasodilators, or the induction of a transient cellular tolerance to endotoxin prevented fourfold higher doses of endotoxin from reversing the return toward a normal pulmonary vascular tone. Individual differences among broilers in their susceptibility to pulmonary hypertension syndrome (ascites) may be related to innate or acquired variability in their pulmonary vascular responsiveness to vasoactive mediators.

Spontaneous contractions in elasmobranch vessels in vitro.

Isolated vessels from four elasmobranchs, yellow stingray (Urolophus jamaicensis), clearnose skate (Raja eglanteria), ghost shark (Hydrolagus novaezelandiae), and spiny dogfish (Squalus acanthias), were examined for the presence of spontaneous contractions (SC). SC were observed in otherwise unstimulated dorsal aortas (DA) from stingray and ghost shark, but not in skate DA. Unstimulated ventral aortas (VA) did not exhibit SC. After treatment of VA with a contractile agonist, SC appeared in stingray and skate but not ghost shark or dogfish. SC in stingray VA were subsequently inhibited by either epinephrine (10(-5) M) or indomethacin (10(-4) M). Agonist contraction also elicited strong SC in ductus Cuvier from stingray, but not from ghost shark or dogfish. SC in dogfish hepatic portal veins (HPV) produced a rhythmical oscillation in tension. The frequency of HPV SC was highest (approximately 1 min(-1)) in intact veins and lower (approximately 3 min(-1)) in vein segments, indicative of a dominant pacemaker in the intact vessel. SC in HPV were depressed during the first 30 min of hypoxia, but there was substantial recovery over an additional 30 min of hypoxia and complete recovery upon return to normoxia. Addition of 80 mM KCl completely inhibited HPV SC and lowered resting tone. These results show that SC are a common feature of elasmobranch vessels and there appears to be a correlation between swimming behavior and the propensity for SC. KCl inhibition of SC and tonus in HPV is highly unusual for vascular smooth muscle.

Endothelin alters the reactivity of vasa vasorum: mechanisms and implications for conduit vessel physiology and pathophysiology.

1 The walls of certain large blood vessels are nourished by the vasa vasorum, a network of microvessels that penetrate the adventitia and media of the vessel wall. The purpose of this study was to characterize endothelin-1 (ET-1)-mediated contraction of vasa and to investigate whether threshold concentrations of ET-1 alters the sensitivity to constrictors. Arterial vasa were dissected from the walls of porcine thoracic aorta and mounted in a tension myograph. 2 ET-1 and ETB-selective agonist, sarafotoxin 6c (S6c), produced concentration-dependent contraction. ETA receptor antagonist, BQ123 (10 microM), caused a biphasic rightward shift of ET-1 response curves. ETB receptor antagonist, BQ788 (1 microM), produced a rightward shift of response curves to ET-1 and S6c of 5- and 80 fold respectively. 3 ET-1 responses were abolished in Ca2+-free PSS but unaffected by selective depletion of intracellular Ca2+ stores. Nifedipine (10 microM), an L-type Ca2+ channel blocker, attenuated ET-1 responses by 44%. Inhibition of receptor-operated Ca2+ channels or non-selective cation entry using SKF 96365 (30 microM) and Ni2+ (1 mM) respectively, attenuated ET-1 contractions by 60%. 4 ET-1 (1-3 nM) enhanced responses to noradrenaline (NA) (4 fold) but not to thromboxane A2-mimetic, whilst K+ (10-20 mM) sensitized vasa to both types of constrictor. 5 Therefore, ET-1-induced contraction of isolated vasa is mediated by ETA and ETB receptors and involves Ca2+ influx through L-type and non-L-type Ca2+ channels. Furthermore elevation of basal tone of vasa vasorum alters the profile of contractile reactivity. These results suggest that ET-1 may be an important regulator of vasa vasorum reactivity.

Nonadrenergic noncholinergic vasodilation of guinea pig pulmonary arteries is mediated by nitric oxide.

Nonadrenergic noncholinergic (NANC) mediated vasodilation may contribute to the maintenance of low pulmonary vascular tone. The NANC neurotransmitters, nitric oxide (NO) and the sensory neuropeptides, substance P and calcitonin gene related peptide (CGRP), were investigated as possible mediators of NANC vasodilation in guinea pig pulmonary arteries. Fresh guinea pig pulmonary artery rings, with and without an intact endothelium, were mounted in organ baths containing Krebs solution and precontracted with the prostaglandin F2alpha analogue U44069. In both endothelium-intact and denuded vessels, electrical field stimulation (1-12 Hz) in the presence of guanethidine and atropine resulted in a frequency-dependent vasodilation. The peptide fragment hCGRP8-37, a competitive antagonist of the CGRP receptors, the peptide fragment NK1 antagonist SP4-11, and the nonpeptide NK1 antagonist RP67580 had no effect on NANC vasodilation. In both endothelium-intact and denuded vessels, N(G)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthesis, inhibited NANC vasodilation, an effect that was reversible with L-arginine. We conclude that NANC vasodilation in guinea pig pulmonary arteries is mediated predominantly through NO activity.

On the regulation of tone in vasa vasorum.

OBJECTIVE: The vasa vasorum form a network of microvessels in and around the walls of large blood vessels and are thought to be necessary to delivery oxygenated blood to the outer parts of the vessel wall that are inadequately nourished by diffusion from luminal blood. This study was undertaken to investigate directly the mechanisms which control tone in the vasa vasorum. METHODS: Arterial vasa vasorum were dissected from the walls of porcine or bovine thoracic aorta and mounted in a tension myograph. Concentration-response curves were constructed to vasoconstrictors; endothelin-1(ET-1), noradrenaline (NA) angiotensin II (Ang II) and thromboxane A2-mimetics (U44069 or U46619) or vasodilators; substance P (SP) bradykinin (BK), calcitonin gene-related peptide (CGRP) or isoprenaline. Strips of porcine aorta were mounted in 25 ml organ baths. RESULTS: Potent concentration-dependent contraction of vasa vasorum was produced by ET-1. NA was a weak constrictor, Ang II had no effect or produced contraction that underwent tachyphylaxis and thromboxane A2-mimetics had no effect. In contrast NA, Ang II, U-44069 and ET-1 all produced potent concentration-dependent contraction of aortic strips. SP and BK produced endothelium-dependent relaxation while CGRP produced endothelium-independent relaxation of ET-1-precontracted vasa vasorum. Isoprenaline had no relaxant effect. CONCLUSIONS: We have demonstrated functional responses of arterial vasa vasorum to vasodilators and vasoconstrictors. Additionally these microvessels appear to respond to constrictors differently from the large host vessel.

Dextran-70 inhibits equine platelet aggregation induced by PAF but not by other agonists.

Dextrans of mean molecular weight 70 kDa (dextran-70) have had clinical use as anti-thrombotics in man. A major part of the anti-thrombotic action is mediated via inhibition of platelet function. Greatorex (1975, 1977) treated thromboembolic colic in horses with infusions of dextran-70 and reported a 90% recovery rate, but this treatment is nonetheless rarely used. We have used an in vitro method to examine the effect of dextran-70 on equine platelet suspensions, in the hope that understanding the mechanism of action of dextran-70 might lead to the development of alternative therapeutic agents. The effects of dextran-70 on equine platelets occurred immediately in vitro with an initial activation and shape change. Subsequent assessment of aggregation revealed a dose-dependent specific inhibition of platelet-activating factor (PAF)-induced aggregation, significant in rate of aggregation at dextran-70 concentrations >40 g/l (P<0.05) and in extent of aggregation at dextran-70 concentrations >50 g/l (P<0.05). Pre-incubation with 60 g/l dextran-70 significantly inhibited the rate and extent of aggregation in response to PAF (1 nmol/l) (P<0.001 and P = 0.003, respectively) but this was not dependent on the duration of pre-incubation (from 0 to 150 min). No effects were seen when the agonist was adenosine 5'-diphosphate (200 nmol/l), collagen (10 mg/l), 5-hydroxytryptamine (100 micromol/l) or U44069 (600 nmol/l) (all P>0.1). Analysis of PAF concentration-aggregation curves after pre-incubation with 60 g/l dextran-70 indicated significant noncompetitive inhibition by dextran-70 (P<0.001 for rate and extent of aggregation). The ability of dextran-70 to inhibit responses of equine platelets to PAF is probably an important component of its beneficial effect as an anti-thrombotic in colic cases.

5-Hydroxytryptamine-induced contraction of the marmoset aorta is mediated by a 5-HT1-like receptor.

1. 5-Hydroxytryptamine (5-HT) exerts both contractile and relaxant effects in the marmoset isolated aorta, actions that are unaffected by the 5-HT2 antagonist ketanserin. The aim of the present study was to define the receptors mediating the contractile activity of 5-HT in the marmoset aorta. 2. Contractile responses were elicited in aortic rings that were either: (i) precontracted submaximally with the thromboxane A2 agonist U44069 in order to amplify the responses; or (ii) exposed to N(omega)-nitro-L-arginine (100 micromol/L) plus LY 53857 (0.1 micromol/L; a 5-HT2 receptor antagonist shown previously to inhibit relaxation). The effect of 5-HT on adenosine 3',5'-cyclic monophosphate (cAMP) formation was also investigated. 3. The effects of agonists and antagonists comprised: (i) agonist potencies in the order 5-carboxamidotryptamine > 5-HT > sumatriptan > 8-hydroxy-2-(di-n-propylamino)tetralin; (ii) inhibition of contractile action of 5-HT by the 5-HT1D antagonist GR 127935; (iii) a contractile response to methysergide; (iv) a lack of effect of tropisetron, an antagonist of 5-HT3 and 5-HT4 receptors; and (v) inhibition of forskolin-stimulated cAMP formation by 5-HT (in the presence of LY 53857), indicative of negative coupling to adenylate cyclase. 4. The above effects fulfill the criteria for a 5-HT1-like receptor. In view of the previous finding that this contractile response is insensitive to ketanserin, it is concluded that the contractile effects of 5-HT in the marmoset aorta are mediated exclusively by a 5-HT1-like receptor.

Plasma 5-hydroxytryptamine constricts equine digital blood vessels in vitro: implications for pathogenesis of acute laminitis.

Cumulative concentration response curves to 5-hydroxytryptamine (5-HT; 10(-10)-10(-4) mol/l) were constructed using isolated rings of equine digital, facial, tail and coronary arteries (endothelium intact). 5-HT was 17.7 and 41 times more potent as a vasoconstrictor of digital arteries than facial and tail arteries respectively. Removal of the endothelium increased the vasoconstrictor potency of 5-HT in the facial artery by 3.7-fold (P<0.05) but did not alter the sensitivity of digital arteries to 5-HT. Coronary arteries failed to contract to 5-HT. Coronary arteries pre-contracted with U44069 showed concentration dependent relaxation to 5-HT, a response which was partially dependent on the presence of the endothelium. No vasorelaxant effects were found in the digital or facial arteries. The concentration of 5-HT in platelet poor and platelet rich equine plasma was found to be 6.70+/-1.1 x 10(-8) mol/l and 1.77+/-0.36 x 10(-6) mol/l (mean +/-s.e.) respectively by high performance liquid chromatography (HPLC). Plasma which contained no detectable platelets had a 5-HT concentration of 1.12+/-0.48 x 10(-8) mol/l. Isolated digital arteries constricted when exposed to dilutions of platelet poor and platelet depleted equine plasma. These plasma induced contractions were almost completely inhibited by 5-HT receptor antagonists, ketanserin and methiothepin. The change in isometric tension in rings of equine digital artery in vitro was therefore used as a bioassay for plasma 5-HT and the results obtained by this method showed an excellent correlation (r2 = 97.2%, P<0.001) with the concentration estimated by HPLC. Circulating free concentrations of 5-HT in normal horses may be sufficient to constrict digital blood vessels partially in vivo but are well below the threshold for contraction of other peripheral blood vessels examined.

The direct vasomotor effect of thyroid hormones on rat skeletal muscle resistance arteries.

UNLABELLED: The present study examines the hypothesis that the hormones have direct vasodilatory effects and attempts to determine whether the effects are endothelium-dependent. Rat skeletal muscle resistance arteries of approximately 100 microns were dissected, and vessel diameter changes were monitored using a videodetection system. After equilibration at 37 degrees C, each vessel was preconstricted with the thromboxane analog U46619 1 microM, and the percentage of dilation was measured after exposure to increasing concentrations of triiodothyronine (T3) or levothyroxine (T4) (10(-10) to 10(-7) M). Dilation in response to T3 was also measured after endothelial denudation and pretreatment with the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine (L-NNA) 10 microM, the cyclooxygenase inhibitor indomethacin 10 microM, the adenosine triphosphate-sensitive K+ channel blocker glibenclamide 1 microM, or the beta-adrenergic antagonist propranolol 1 microM. Both T3 and T4 demonstrated concentration-dependent dilation of the U46619-preconstricted vessels (P < 0.001 each), with T3 having a greater effect than T4 (P < 0.05) (36% +/- 9% [mean +/- SD] dilation at 10(-7) M T3 vs 24% +/- 6% dilation at 10(-7) M T4). In comparison, isoproterenol 10(-7) M produced 56% +/- 6% dilation. T3-mediated vasodilation was attenuated but not abolished by endothelial denudation (18% +/- 3% dilation at 10(-7) M T3) (P < 0.01), L-NNA (15% +/- 7% dilation at 10(-7) M T3) (P < 0.01), indomethacin (20% +/- 9% dilation at 10(-7) M T3) (P < 0.05), and glibenclamide (22% +/- 7% dilation at 10(-7) M T3) (P < 0.01), but it was not affected by propranolol (37% +/- 20% dilation at 10(-7) M T3) (P = 0.99). We conclude that thyroid hormones possess direct vasodilatory effects with both endothelium-independent and endothelium-dependent components. IMPLICATIONS: Thyroid hormones may have modest direct vasodilatory effects. This may partially account for the cardiovascular actions of the hormones in hyperthyroidism or when administered pharmacologically in cardiac surgery.

Isoprostane 8-epi-prostaglandin F2 alpha is a potent contractor of human peripheral lymphatics.

Isoprostanes are products of free radical-catalyzed peroxidation and 8-epi-prostaglandin (PG) F2 alpha is the most important vasomodulator of this group of compounds. In human lower leg lymphatics isolated from 5 different patients without a smoking history or hyperlipidemia, 8-epi-PGF2 alpha stimulated in vitro contraction more strongly than the thromboxane receptor agonist U46619. Other isoprostanes (8-epi-PGE1, 8-epi-PGE2) had only limited lymphatic contractile potency. These data suggest a potentially relevant role for epi-8-PGF2 alpha in facilitating lymph transport especially in conditions of inflammation.

Enhanced constrictor responses of skeletal muscle arterioles during chronic myocardial infarction.

The goal of this study was to test the hypothesis that chronic myocardial infarction potentiates agonist-induced constrictor responses of rat skeletal muscle arterioles in vivo. Eight weeks after we performed coronary artery ligation or sham (control) surgery, the spinotrapezius muscle was prepared for direct visualization of the microcirculation. Diameter of third-order arterioles (40.7 +/- 0.5 microns) to topical suffusion of angiotensin II (ANG II; 0.1-10 nM), arginine vasopressin (AVP; 0.1-10 nM), endothelin-1 (ET-1; 1.0-100 pM), and the thromboxane analog U-46619 (1.0-100 nM) was measured in both groups. Myocardial-infarcted rats exhibited enhanced arteriolar constrictor responses to ANG II and AVP compared with the responses in controls. In contrast, ET-1- and U-46619-induced constrictor responses were similar in control and myocardial-infarcted rats. Additional experiments explored the impact of NG-monomethyl-L-arginine (L-NMMA; 0.1 mM) on arteriolar reactivity. In control animals, L-NMMA potentiated ANG II- and AVP-induced vasoconstriction, achieving values similar to those observed in myocardial-infarcted rats. L-NMMA did not alter vasoconstrictor responses in rats with chronic myocardial infarction. These observations suggest that enhanced agonist-induced vasoconstriction during heart failure may reflect a loss of nitric oxide-mediated modulation of arteriolar tone.

Vascular effects of PTHrP (1-34) and PTH (1-34) in the human fetal-placental circulation.

The aim of this study was to examine the vasodilatory effects of parathyroid hormone-related protein (PTHrP) (1-34) and parathyroid hormone (PTH) (1-34) on the human fetal-placental circulation utilising an in vitro placental perfusion model. In all experiments, the vasculature of an isolated human placental cotyledon was pre-constricted with the thromboxane A2 mimetic U46619. A simple dose of PTHrP (1-34) or PTH (1-34) (1.7-300 nM) was then infused into the fetal-placental circulation of the cotyledon. In other experiments, cotyledons were repeatedly infused with PTHrP (1-34) or PTH (1-34) (51.3 nM). Vasodilatory responses were significantly reduced in response to repeated exposure to PTHrP (1-34) (P < 0.001), indicating that this peptide desensitizes the fetal-placental vasculature. PTHrP (1-34) and PTH (1-34) equipotently stimulated a significant vasodilation of the fetal-placental circulation (P < 0.0001). The PTHrP receptor antagonist [Asn10, Leu 11]PTHrP (7-34) (102 nM) was infused in U46619-constricted placentae in the presence and absence of PTHrP (1-34) (10.2 nM). The PTHrP antagonist alone had no significant effect in the fetal-placental circulation. The antagonist significantly attenuated the response to PTHrP (1-34) (P < 0.015). Based on the data obtained in this study it is suggested that locally produced PTHrP (1-34) may be involved in the regulation of normal human fetal-placental vascular tone in autocrine and/or paracrine fashion.

Regulatory role of prostaglandin E2 in induction of cyclo-oxygenase-2 by a thromboxane A2 analogue (U46619) and basic fibroblast growth factor in porcine aortic smooth-muscle cells.

U46619, a thromboxane A2 analogue, and basic fibroblast growth factor (FGF-2) both induced the expression of the inducible cyclo-oxygenase (Cox)-2 in porcine aortic smooth-muscle cells. This induction was dose-dependent (submaximal at 300 nM for U46619 and 1 ng/ml for FGF-2) and time-dependent, with similar intensity and maximal expression at 2 h. Under these conditions, both inducers stimulated rapid activation of extracellular signal-regulated kinase (ERK2) at 5-10 min, a transient and lower intensity being induced by U46619 whereas that induced by FGF-2 was sustained (>1 h). PD98059, an inhibitor of the ERK pathway, inhibited the expression of Cox-2. In contrast, activation of Jun-N-terminal kinase (JNK1) was sustained with U46619 but poorly induced by FGF-2. Cox-2 expression induced by U46619 or FGF-2 was similarly reduced by prostaglandin (PGE2), forskolin or dibutyryl-cAMP, suggesting a regulatory effect of adenylate cyclase on Cox-2 expression. However, activation of ERK2 by FGF-2 was not affected by PGE2 whereas that of JNK1 by U46619 was inhibited, suggesting that inhibition of COX-2 expression by cAMP may be downstream of ERK2. The effects of PGE2 and forskolin on Cox-2 and phosphorylation of JNK1 were reversed with the protein kinase A inhibitor H89. In addition, endogenous PGE2 down-regulated the expression of Cox-2 by the two inducers, as stimulation of the cells in the presence of different Cox inhibitors increased the expression of the protein. Overall, these results suggest that exogenous and endogenous PGE2 exert negative inhibitory effects on Cox-2 expression mediated by stimulation of protein kinase A.

Characterization of the prostanoid receptor(s) on human blood monocytes at which prostaglandin E2 inhibits lipopolysaccharide-induced tumour necrosis factor-alpha generation.

1. The prostanoid receptor(s) that mediates inhibition of bacterial lipopolysaccharide (LPS)-induced tumour necrosis factor-alpha (TNF-alpha) generation from human peripheral blood monocytes was classified by use of naturally occurring and synthetic prostanoid agonists and antagonists. 2. In human monocytes that were adherent to plastic, neither prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), prostaglandin F(2 alpha) (PGF(2 alpha)) nor the stable prostacyclin and thromboxane mimetics, cicaprost and U-46619, respectively, promoted the elaboration of TNF alpha-like immunoreactivity, as assessed with a specific ELISA, indicating the absence of excitatory prostanoid receptors on these cells. 3. Exposure of human monocytes to LPS (3 ng ml-1, approximately EC84) resulted in a time-dependent elaboration to TNF alpha which was suppressed in cells pretreated with prostaglandin E1 (PGe1), PGE2 and cicaprost. This effect was concentration-dependent with mean pIC50 values of 7.14, 7.34 and 8.00 for PGE1, PGE2 and cicaprost, respectively. PGD2, PGF(2 alpha) and U-46619 failed to inhibit the generation of TNF alpha at concentrations up to 10 microM. 4. With respect to PGE2, the EP-receptor agonists, 16,16-dimethyl PGE2 (non-selective), misoprostol (EP2/EP3-selective), 11-deoxy PGE1 (EP2-selective) and butaprost (EP2-selective) were essentially full agonists as inhibitors of LPS-induced TNF alpha generation with mean pIC50 values of 6.21, 6.02, 5.67 and 5.59, respectively. In contrast to the results obtained with butaprost and 11-deoxy PGE1, another EP2-selective agonist, AH 13205, inhibited TNF alpha generation by only 21% at the highest concentration (10 microM) examined. EP-receptor agonists which have selectively for the EP1- (17-phenyl-omega-trinor PGE2) and EP3-receptor (MB 28,767, sulprostone) were inactive or only weakly active as inhibitors of TNF alpha generation. 5. Pretreatment of human monocytes with the TP/EP4-receptor antagonist, AH 23848B, at 10, 30 and 100 microM suppressed LPS-induced TNF alpha generation by 10%, 28% and 77%, respectively, but failed to shift significantly the location of the PGE2 concentration-response curves. 6. Given that AH 13205 was a poor inhibitor of TNF alpha generation, studies were performed to determine if it was a partial agonist and whether it could antagonize the inhibitory effect of PGE2. Pretreatment of human monocytes with 10 and 30 microM AH 13205 inhibited the generation of TNF alpha by 31% and 53%, respectively, but failed to shift significantly the location of the PGE2 concentration-response curves at either concentration examined. 7. Since PGD2 and 17-phenyl-omega-trinor PGE2 (EP1-agonist) did not suppress TNF alpha generation, the EP1/EP2/DP-receptor antagonist, AH 6809, was employed to assess if EP2-receptors mediated the inhibitory effect of PGE2. Pretreatment of human monocytes with 10 microM AH 6809 did not affect LPS-induced TNF alpha generation but produced a parallel 3.5 fold rightwards shift of the PGE2 concentration-response curve. 8. Collectively, these data suggest that human peripheral blood monocytes express at least two distinct populations of inhibitory prostanoid receptors that mediate inhibition of LPS-induced TNF alpha generation. One of these probably represents i.p. receptors based upon the selectivity of cicaprost for this subtype. The other population has the pharmacology of EP-receptors, but the rank of potency for a range of synthetic EP-receptor agonists was inconsistent with an interaction with any of the currently defined subtypes. Given the pharmacological behaviour of butaprost, AH 6809 and AH 23848B in these cells, we propose that multiple (EP2- and/or EP-4- and/or i.p.) or novel EP-receptors mediate the inhibitory effect of PGE2 on TNF alpha generation.