The role of thromboxane A2 (TxA2) in allergic cutaneous reactions and the effect of (E)-3-[p-(1H-imidazol-1-ylmethyl) phenyl]-2-propenoic acid hydrochloride (OKY-046), a TxA2 synthetase inhibitor

To study the role of thromboxane A2 (TxA2) in cutaneous allergic reactions, the effect of (E)-3-[p-(1H-Imidazol-1-ylmethyl)phenyl]-2-propenoic acid hydrochloride (OKY-046), a selective TxA2 synthetase inhibitor, on cutaneous reactions in rats and mice was studied. Simultaneously, the effect of 9,11-methanoepoxy-prostaglandin H2 (U-46619), a stable analogue of TxA2, on capillary permeability in mouse and rat skin was investigated. Passive cutaneous anaphylaxis (PCA) in mouse ear was clearly inhibited by OKY-046 but not by indomethacin. The inhibitory action of OKY-046 was not influenced by pretreatment with indomethacin. Moreover, prostaglandin I2, which accumulated as a result of the inhibition of TxA2 synthetase, did not affect the PCA. But, the dye leakages caused by histamine, serotonin and leukotriene C4 in mouse ear were clearly inhibited by OKY-046. In addition, OKY-046 inhibited rat reversed cutaneous anaphylaxis, but its inhibitory action was not affected by pretreatment with indomethacin. Contrary to the above results, rat footpad passive Arthus reaction and mouse footpad tuberculin delayed hypersensitivity reaction were not affected by OKY-046. Additionally, U-46619 did not cause an increase of capillary permeability in either mouse and rat skin. These results suggest a slight role of TxA2 in cutaneous allergic reactions in mice and rats and the efficacy of OKY-046 on Type I and II reactions regardless of the inhibition of TxA2 synthetase activity.     

Effects of platelet-activating factor and thromboxane A2 on isolated perfused guinea pig liver

Lipid mediators, thromboxane A2 (TxA2) and platelet-activating factor (PAF), are potent vasoconstrictors, and have been implicated as mediators of liver diseases, such as ischemic-reperfusion injury. We determined the effects of a TxA2 analogue (U-46619) and PAF on the vascular resistance distribution and liver weight (wt) in isolated guinea pig livers perfused with blood via the portal vein. The sinusoidal pressure was measured by the double occlusion pressure (P(do)), and was used to determine the pre- (R(pre)) and post-sinusoidal (R(post)) resistances. U-46619 and PAF concentration-dependently increased the hepatic total vascular resistance (R(t)). The minimum concentration at which significant vasoconstriction occurs was 0.001 microM for PAF and 0.1 microM for U-46619. Moreover, the concentration of U-46619 required to increase R(t) to the same magnitude is 100 times higher than PAF. Thus, the responsiveness to PAF was greater than that to U-46619. Both agents increased predominantly R(pre) over R(post). U-46619 caused a sustained liver weight loss. In contrast, PAF also caused liver weight loss at lower concentrations, but it produced liver weight gain at higher concentrations (2.5 +/- 0.3 per 10g liver weight at 1 microM PAF), which was caused by substantial post-sinusoidal constriction and increased P(do). In conclusion, both TxA2 and PAF contract predominantly the pre-sinusoidal veins. TxA2 causes liver weight loss, while PAF at high concentrations increases liver weight due to substantial post-sinusoidal constriction in isolated guinea pig livers.     

A thromboxane mimetic, U-46619, produces plasma exudation in airways of the guinea pig.

Thromboxane A2 (TxA2) has been implicated in airway responses to allergen and in the bronchial hyperresponsiveness observed in asthma. Furthermore a TxA2 receptor antagonist and a TxA2 synthase inhibitor inhibit plasma exudation in airways induced by inhaled platelet-activating factor. To evaluate whether TxA2 has any direct effect on plasma exudation in the airways, we studied the effect of a stable TxA2 mimetic (U-46619; 2, 20, and 200 nmol/kg iv) on lung resistance (RL) and Evans blue dye extravasation (marker of plasma albumin; 20 mg/kg iv) at the airway levels of trachea, main bronchi, and proximal and distal intrapulmonary airways in anesthetized, tracheostomized, and mechanically ventilated guinea pigs. Injection of U-46619 produced an immediate and marked dose-dependent increase in RL, which peaked at approximately 30 s. At the highest dose of U-46619, we also observed a later increase in RL, starting at approximately 3 min and reaching a second peak at approximately 8 min. Mean systemic blood pressure increased in a dose-dependent manner [maximum 82 +/- 8 (SE) mmHg]. U-46619 also produces dose-dependent plasma exudation, measured as Evans blue dye extravasation, at all airway levels as well as into the tracheal lumen. Airway responses to U-46619 (200 nmol/kg iv) were abolished in animals pretreated with the TxA2 receptor antagonist ICI-192605 (0.5 mg/kg iv). We conclude that U-46619, despite being a vasoconstrictor, is potent in inducing plasma exudation in airways and that this effect is mediated via a TxA2 receptor.     

Role of serotonin in thromboxane A2-induced coronary chemoreflex

We reported previously that the thromboxane A(2) (TxA(2)) mimetic U-46619 stimulates cardiac vagal afferent nerves, eliciting a reflex decrease in heart rate (HR) and arterial blood pressure (ABP). The present experiments were designed to test the hypothesis that TxA(2) evokes these changes via the release of serotonin [5-hydroxytryptamine (5-HT)] and activation of the 5-HT(3) receptor. Injections of the 5-HT(3) antagonist tropisetron (1 mg of 3-tropanyl-indole-3-carboxylate or ICS-205-930) attenuated the decreases in HR and ABP induced by left atrial injections of U-46619 (20 microg). Tropisetron administration also eliminated the U-46619-induced increase in impulse frequency in a majority of cardiac, vagal afferent units tested. Measurement of serum 5-HT levels revealed an elevation in serum 5-HT levels after U-46619 injection in those rabbits that displayed a significant HR change following injection of U-46619. These results indicate that although other factors may also contribute to these reflex responses, the release of 5-HT and stimulation of the 5-HT(3) receptor plays a significant role in coronary reflexes induced by TxA(2).     

Myocardial ischemia-mediated excitatory reflexes: a new function for thromboxane A2?

Clinical and experimental evidence has shown that myocardial ischemia activates cardiac spinal afferents that mediate sympathoexcitatory reflex responses. During myocardial ischemia, thromboxane A2 (TxA2) is released in large quantities by activated platelets in the coronary circulation of patients with coronary artery disease. We hypothesized that endogenous TxA2 contributes to sympathoexcitatory reflexes during myocardial ischemia through stimulation of TxA2/prostaglandin endoperoxide (TP) receptors. Regional myocardial ischemia was induced by occlusion of a diagonal branch of left anterior descending coronary artery of anesthetized cats. Hemodynamic parameters and renal sympathetic nerve activity were recorded after sinoaortic denervation and bilateral vagotomy. Regional myocardial ischemia evoked significant increases in mean blood pressure (122+/-10 vs. 139+/-12 mmHg, before vs. ischemia), aortic flow (153+/-18 vs. 167+/-20 ml/min), first derivative of left ventricular pressure at 40-mmHg developed pressure (2,736+/-252 vs. 2,926+/-281 mmHg/s), systemic vascular resistance (0.6+/-0.1 vs. 0.9+/-0.12 peripheral resistance units), and renal sympathetic nerve activity (by 22%). The reflex nature of the excitatory responses was confirmed by observing its disappearance after blockade of cardiac nerve transmission with intrapericardial 2% procaine treatment. Moreover, application of U-46619 (2.5-10 microg), a TxA2 mimetic, on the heart caused graded increases in mean arterial pressure and renal nerve activity, responses that were abolished 3 min after local blockade of cardiac neural transmission with intrapericardial procaine. BM 13,177 (30 mg/kg iv), a selective TP receptor antagonist, eliminated the reflex responses to U-46619 and significantly attenuated the excitatory responses during brief (5 min) regional myocardial ischemia. The sympathoexcitatory reflex responses to U-46619 were unchanged by blockade of histamine H1 receptors with pyrilamine and serotonin 5-HT3 receptors with tropisetron, indicating specificity of this TP receptor agonist. These data indicate that endogenous TxA2 participates in myocardial ischemia-mediated sympathoexcitatory reflex responses through a TP receptor mechanism.     

Nongenomic vasodilator action of progesterone on primate coronary arteries.

In the present investigation, we test the hypothesis that progesterone can rapidly relax, via a nongenomic mechanism, persistent flow occluding, agonist-activated coronary artery (CA) vasospasm, and hyperreactive vascular muscle cell (VMC) Ca(2+) responses in ovariectomized rhesus monkeys. CA vasospasm, induced by injection of 100 microM serotonin and 1 microM U-46619 (5-HT+U; 1 ml/30 s), resulted in a decrease in CA diameter (phi) from 1.8 +/- 0.2 to 0.3 +/- 0.1 mm at the site of focal constriction. Injection of 100 ng progesterone into the CA significantly relieved the severe vasoconstriction (1.3 +/- 0.2 mm) and reestablished distal flow in 3 min; the preconstriction phi was completely restored in 8.2 +/- 2.6 min (n = 6). Similarly, cell impermeant albumin-conjugated progesterone, but not albumin-conjugated 17 beta-estradiol, decreased 5-HT+U stimulated VMC Ca(2+) responses (250 +/- 34% of basal 30 min after stimulation) back to the prestimulation level (113 +/- 17% of basal) in 25 min (half time = 7 min). The presence of a rapid vasodilator action of progesterone in the primate CA and isolated VMC suggests its benefits in hormone replacement therapy may also include nongenomic vascular relaxant actions.     

Characterization of thromboxane receptor blocking effects of SQ 29548 in the feline pulmonary vascular bed.

The effects of SQ 29548, a thromboxane (Tx) A2 receptor blocking agent, on responses to the TxA2 mimic U46619 were investigated in the pulmonary vascular bed of the intact-chest cat under constant-flow conditions. The administration of SQ 29548 in doses of 0.25-1 mg/kg iv reduced vasoconstrictor responses to U-46619; however, responses to prostaglandins (PG) F2 alpha and D2 and to serotonin were also decreased. After administration of SQ 29548 in doses of 0.05-0.1 mg/kg iv, responses to U-46619 and U-44069 were reduced significantly, and the dose-response curves for these TxA2 mimics were shifted to the right in a parallel manner at a time when responses to PGF2 alpha and PGD2 were not altered. The low doses of the TxA2 receptor blocking agent significantly reduced responses to the PG and TxA2 precursor arachidonic acid but were without significant effect on vasoconstrictor responses to serotonin; histamine; norepinephrine; angiotensin II; the major PGD2 metabolite 9 alpha,11 beta-PGF2; BAY K 8644, an agent that enhances calcium entry; and endothelin-1. The present data show that at low doses SQ 29548 selectively blocks TxA2 receptor-mediated responses in a competitive and reversible manner in the pulmonary vascular bed. These data suggest that responses to arachidonic acid are mediated in large part by the formation of TxA2 and provide evidence in support of the hypothesis that a discrete TxA2 receptor unrelated to PGF2 alpha or PGD2 receptors is present in undefined resistance vessel elements in the feline pulmonary vascular bed.     

Influence of SQ 30741 on thromboxane receptor-mediated responses in the feline pulmonary vascular bed.

The effects of SQ 30741, a thromboxane A2 (TxA2) receptor blocking agent, on responses to the TxA2 mimic, U-46619, were investigated in the pulmonary vascular bed of the intact-chest cat under constant-flow conditions. The administration of SQ 30741 in doses of 1-2 mg/kg iv markedly reduced vasoconstrictor responses to U-46619 without altering responses to prostaglandin (PG) F2 alpha or PGD2 and serotonin. SQ 30741 had no significant effect on mean vascular pressures in the cat, and the dose-response curve for U-46619 was shifted to the right in a parallel manner with a similar apparent maximal response. In addition to not altering responses to PGF2 alpha, PGD2 alpha, or serotonin, SQ 30741 (2 mg/kg iv) was without significant effect on pulmonary vasoconstrictor responses to the PGD2 metabolite 9 alpha, 11 beta-PGF2, norepinephrine, angiotensin II, BAY K 8644, endothelin 1, or endothelin 2. Although responses to vasoconstrictor agents, which act through a variety of mechanisms, were not altered, responses to the PG and TxA2 precursor, arachidonic acid, were reduced significantly. The duration of the TxA2 receptor blockade was approximately 30 and 75 min at the 1- and 2-mg/kg iv doses of the antagonist, respectively. The present data show that SQ 30741 selectively blocks TxA2 receptor-mediated responses in a competitive and reversible manner in the pulmonary vascular bed. These data suggest that responses to arachidonic acid are due in large part to the formation of TxA2 and that discrete TxA2 receptors unrelated to receptors activated by PGD2 or PGF2 alpha are most likely located in resistance vessel elements in the feline pulmonary vascular bed.     

Daltroban blocks thromboxane responses in the pulmonary vascular bed of the cat.

The influence of daltroban (BM13.505; SK&F 96148), a thromboxane (Tx) A2-receptor-blocking agent, on responses to the TxA2 mimics U-46619 and U-44069 was investigated in the pulmonary vascular bed of the intact-chest cat under constant-flow conditions. Daltroban (5 mg/kg iv) had no significant effect on mean baseline vascular pressures but significantly decreased responses to the TxA2 mimics without altering responses to prostaglandin (PG) F2 alpha or PGD2 or the PGD2 metabolite 9 alpha, 11 beta-PGF2. Dose-response curves for U-46619 and U-44069 were shifted to the right in a parallel manner, and daltroban had no significant effect on responses to norepinephrine, serotonin, angiotensin II, BAY K 8644, endothelin-(ET) 1, ET-2, or platelet-activating factor (PAF). After administration of daltroban, responses to U-46619 returned to 50% of control in 90 min and responses to the PG and TxA2 precursor arachidonic acid were decreased significantly. These results suggest that daltroban selectively antagonizes TxA2-receptor-mediated responses in a competitive and reversible manner. These data provide support for the hypothesis that discrete TxA2 receptors unrelated to receptors stimulated by PGF2 alpha, PGD2, or 9 alpha, 11 beta-PGF2 are present in the pulmonary vascular bed of the cat. The present data suggest that pulmonary vasoconstrictor responses to PAF and ET peptides are not dependent on activation of TxA2 receptors in the cat.     

Pregnancy reduces RhoA/Rho kinase and protein kinase C signaling pathways downstream of thromboxane receptor activation in the rat uterine artery

During pregnancy, reduced vascular responses to constrictors contribute to decreased uterine and total vascular resistance. Thromboxane A(2) (TxA(2)) is a potent vasoconstrictor that exerts its actions via diverse signaling pathways, and its biosynthesis increases in preeclampsia. In this study, we hypothesized that maternal vascular responses to TxA(2) will be attenuated via Rho kinase, PKC, p38 MAPK, and ERK1/2 signaling pathways. Isolated ring segments of uterine and small mesenteric arteries from late pregnant (19-21 days) and virgin rats were suspended in a myograph, and isometric force was measured. Pregnancy did not affect uterine and mesenteric artery responses to the TxA(2) analog U-46619 (10(-9)-10(-5) M), but transduction signals associated with these contractions were different between pregnant and nonpregnant rats. Inhibition of Rho kinase (10(-6) M Y-27632) reduced sensitivity to U-46619 in virgin uterine vessels but did not inhibit these contractions in pregnant uterine arteries and had no effect on mesenteric vessels. Treatment of arterial segments with a PKC inhibitor (10(-6) M bisindolylmaleimide I) reduced U-46619-induced contractions in virgin uterine and mesenteric arteries and in pregnant mesenteric arteries. Pregnant uterine arteries, however, were unresponsive to PKC inhibition. Inhibition of ERK1/2 (10(-5) M PD-98059) and p38 MAPK (10(-5) M SB-203580) reduced U46619-induced contractions in nonpregnant vessels and in pregnant uterine and mesenteric vessels. These data suggest that normal pregnancy does not affect uterine and mesenteric contractile responses to TxA(2) but reduces the contribution of Rho kinase and PKC signaling pathways to these contractions in the uterine vasculature. In contrast, the role of ERK1/2 and p38 MAPK in U-46619-induced uterine contractions remains unchanged with pregnancy. TxA(2)-associated transduction signals and its regulators might present potential targets for the development of new treatments for preeclampsia and other pregnancy-associated vascular diseases.     

Effects of platelet-activating factor, thromboxane A2 and leukotriene D4 on isolated perfused rat liver

Vasoconstrictive lipid mediators, thromboxane A(2) (TxA(2)), platelet-activating factor (PAF) and leukotriene D(4) (LTD(4)) have been implicated as mediators of liver diseases. There are species differences in the primary site of hepatic vasoconstriction in response to these mediators. We determined the effects of a TxA(2) analogue (U-46619), PAF and LTD(4) on the vascular resistance distribution, weight and oxygen consumption of isolated rat livers portally perfused with blood. The sinusoidal pressure was measured by the double occlusion pressure (P(do)), and was used to determine the pre- (R(pre)) and post-sinusoidal (R(post)) resistances. All these three mediators increased the hepatic total vascular resistance (R(t)). The responsiveness to PAF was 100 times greater than that to U-46619 or LTD(4). Both of PAF and U-46619 predominantly increased R(pre) over R(post). At the comparable increased R(t) levels, U-46619 more preferentially increased R(pre) than PAF. In contrast, LTD(4) increased both the R(pre) and R(post) to similar extent. U-46619 caused liver weight loss, while high concentrations of either LTD(4) or PAF produced liver weight gain, which was caused by substantial post-sinusoidal constriction and increased P(do). PAF and U-46619 decreased hepatic oxygen consumption while LTD(4) induced biphasic change of an initial transient decrease followed by an increase. In conclusion, PAF is the most potent vasoconstrictor of rat hepatic vessels among these three mediators. Both TxA(2) and PAF constrict the pre-sinusoidal veins predominantly. TxA(2) more preferentially constricts the pre-sinusoids than PAF, resulting in liver weight loss. However LTD(4) constricts both the pre- and post-sinusoidal veins similarly. High concentrations of LTD(4) and PAF cause liver weight gain by substantial post-sinusoidal constriction. PAF and TxA(2) decrease hepatic oxygen consumption, whereas LTD(4) causes a biphasic change of it.     

Prostaglandins potentiate U-46619-induced pulmonary microvascular dysfunction.

The induction of cyclooxygenase is an important event in the pathophysiology of acute lung injury. The purpose of this study was to examine the synergistic effects of various cyclooxygenase products (PGE(2), PGI(2), PGF(2alpha)) on thromboxane A(2) (TxA(2))-mediated pulmonary microvascular dysfunction. The lungs of Sprague-Dawley rats were perfused ex vivo with Krebs-Henseleit buffer containing indomethacin and PGE(2) (5 x 10(-8) to 1 x 10(-7) M), PGF(2alpha) (7 x 10(-9) to 5 x 10(-6) M), or PGI(2) (5 x 10(-8) to 2 x 10(-5) M). The TxA(2)-receptor agonist U-46619 (7 x 10(-8) M) was then added to the perfusate, and then the capillary filtration coefficient (K(f)), pulmonary arterial pressure (Ppa), and total pulmonary vascular resistance (RT) were determined. The K(f) of lungs perfused with U-46619 was twice that of lungs perfused with buffer alone (P = 0.05). The presence of PGE(2), PGF(2alpha), and PGI(2) within the perfusate of lungs exposed to U-46619 caused 118, 65, and 68% increases in K(f), respectively, over that of lungs perfused with U-46619 alone (P < 0.03). The RT of lungs perfused with PGE(2) + U-46619 was approximately 30% greater than that of lungs exposed to either U-46619 (P < 0.02) or PGE(2) (P < 0.01) alone. When paired measurements of RT taken before and then 15 min after the addition of U-46619 were compared, PGI(2) was found to attenuate U-46619-induced increases in RT (P < 0.01). These data suggest that PGE(2), PGI(2), and PGF(2alpha) potentiate the effects of TxA(2)-receptor activation on pulmonary microvascular permeability.     

Hepatocyte heterogeneity in response to icosanoids. The perivenous scavenger cell hypothesis

1. The metabolic and hemodynamic effects of prostaglandin F2 alpha, leukotriene C4 and the thromboxane A2 analogue U-46619 were studied during physiologically antegrade (portal to hepatic vein) and retrograde (hepatic to portal vein) perfusion and in a system of two rat livers perfused in sequence. 2. The stimulatory effects of prostaglandin F2 alpha (3 microM) on hepatic glucose release, perfusion pressure and net Ca2+ release were diminished by 77%, 95% and 64%, respectively, during retrograde perfusion when compared to the antegrade direction, whereas the stimulation of 14CO2 production from [1-14C]glutamate by prostaglandin F2 alpha (which largely reflects the metabolism of perivenous hepatocytes) was lowered by only 20%. Ca2+ mobilization and glucose release from the liver comparable to that seen during antegrade perfusion could also be observed in retrograde perfusions; however, higher concentrations of the prostaglandin were required. 3. The glucose, Ca2+ and pressure response to leukotriene C4 (20 nM) or the thromboxane A2 analogue U-46619 (200 nM) of livers perfused in the antegrade direction were diminished by about 90% during retrograde perfusion. Sodium nitroprusside (20 microM) decreased the pressure response to leukotriene C4 (20 nM) and U-46619 (200 nM) by about 40% and 20% in antegrade perfusions, respectively, but did not affect the maximal increase of glucose output. 4. When two livers were perfused antegradely in series, such that the perfusate leaving the first liver (liver I) entered a second liver (liver II), infusion of U-46619 at concentrations below 200 nM to the influent perfusate of liver I increased the portal pressure of liver I, but not of liver II. At higher concentrations of U-46619 there was also an increase of the portal pressure of liver II and with concentrations above 800 nM the pressure responses of both livers were near-maximal [19.6 +/- 0.8 (n = 7) cm H2O and 16.5 +/- 1.1 (n = 8) cm H2O for livers I and II, respectively]. There was a similar behaviour of glucose release from livers I and II in response to U-46619 infusion. When liver I was perfused in the retrograde direction, a significant pressure or glucose response of liver II (antegrade perfusion) could not be observed even with U-46619 concentrations up to 1000 nM. 5. Similarly, the perfusion pressure increase and glucose release induced by leukotriene C4 (10 nM) observed with liver II was only about 20% of that seen with liver I.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in serotonin2A and GABA(A) receptors in schizophrenia: studies on the human dorsolateral prefrontal cortex

Having shown a decrease in serotonin2A receptors in the dorsolateral prefrontal cortex (DLPFC) from schizophrenic subjects, we have now determined if this change was reflective of widespread changes in neurochemical markers in DLPFC in schizophrenia. In Brodmann's area (BA) 9 from 19 schizophrenic and 19 control subjects, we confirmed a decrease in the density of [3H]ketanserin binding to serotonin2A receptors in tissue from the schizophrenic subjects [39 +/- 3.3 vs. 60 +/- 3.6 fmol/mg estimated tissue equivalents (ETE); p < 0.005]. In addition, the density of [3H]muscimol binding to GABA(A) receptors was increased in the schizophrenic subjects (526 +/- 19 vs. 444 +/- 28 fmol/mg ETE; p < 0.02). [3H]YM-09151-2, N-[1-(2-thienyl)cyclohexyl]-3,4-[3H]piperidine, [3H]SCH 23390, [3H]mazindol, and N(G)-nitro-L-[3H]arginine binding to BA 9 did not differ between groups, and there was no specific binding of [3H]raclopride or 7-hydroxy-2-(di-n-[3H]propylamino)tetralin to BA 9 from either cohort of subjects. This suggests the density of dopamine D1-like and NMDA receptors, the dopamine transporter, and nitric oxide synthase activity are not altered in BA 9 from schizophrenic subjects. The selective nature of the changes in serotonin2A and GABA(A) receptors in DLPFC could indicate that these changes are involved in the pathology of schizophrenia.     

Involvement of KCNQ2 subunits in [3H]dopamine release triggered by depolarization and pre-synaptic muscarinic receptor activation from rat striatal synaptosomes

KCNQ2 and KCNQ3 subunits encode for the muscarinic-regulated current (I(KM)), a sub-threshold voltage-dependent K+ current regulating neuronal excitability. In this study, we have investigated the involvement of I(KM) in dopamine (DA) release from rat striatal synaptosomes evoked by elevated extracellular K+ concentrations ([K+]e) and by muscarinic receptor activation. [3H]dopamine ([3H]DA) release triggered by 9 mmol/L [K+]e was inhibited by the I(KM) activator retigabine (0.01-30 micromol/L; Emax = 54.80 +/- 3.85%; IC50 = 0.50 +/- 0.36 micromol/L). The I(KM) blockers tetraethylammonium (0.1-3 mmol/L) and XE-991 (0.1-30 micromol/L) enhanced K+-evoked [3H]DA release and prevented retigabine-induced inhibition of depolarization-evoked [3H]DA release. Retigabine-induced inhibition of K+-evoked [3H]DA release was also abolished by synaptosomal entrapment of blocking anti-KCNQ2 polyclonal antibodies, an effect prevented by antibody pre-absorption with the KCNQ2 immunizing peptide. Furthermore, the cholinergic agonist oxotremorine (OXO) (1-300 micromol/L) potentiated 9 mmol/L [K+]e-evoked [3H]DA release (Emax = 155 +/- 9.50%; EC50 = 25 +/- 1.80 micromol/L). OXO (100 micromol/L)-induced [3H]DA release enhancement was competitively inhibited by pirenzepine (1-10 nmol/L) and abolished by the M3-preferring antagonist 4-diphenylacetoxy N-methylpiperidine methiodide (1 micromol/L), but was unaffected by the M1-selective antagonist MT-7 (10-100 nmol/L) or by Pertussis toxin (1.5-3 microg/mL), which uncouples M2- and M4-mediated responses. Finally, OXO-induced potentiation of depolarization-induced [3H]DA release was not additive to that produced by XE-991 (10 micromol/L), was unaffected by retigabine (10 micromol/L), and was abolished by synaptosomal entrapment of anti-KCNQ2 antibodies. Collectively, these findings indicate that, in rat striatal nerve endings, I(KM) channels containing KCNQ2 subunits regulate depolarization-induced DA release and that I(KM) suppression is involved in the reinforcement of depolarization-induced DA release triggered by the activation of pre-synaptic muscarinic heteroreceptors.     

Effects of the thromboxane receptor antagonist SK&F 88046 in the canine, monkey and human coronary vasculature

U-46619, a stable "functional" thromboxane/endoperoxide receptor agonist, produced potent contractile responses in isolated canine, rhesus monkey and human left circumflex coronary arteries (EC50 = 9.11 x 10(-9)M, 1.98 x 10(-8)M and 3.50 x 10(-9)M, respectively). Canine intrapulmonary veins were also contracted potently by U-46619 (EC50 = 1.22 x 10(-9)M). SK&F 88046, a thromboxane A2 (TxA2) end-organ receptor antagonist, blocked the vasoconstrictor effects of U-46619 in the canine circumflex artery (KB = 1.33 x 10(-8)M), canine intrapulmonary vein (KB = 1.46 x 10(-9)M), monkey circumflex artery (KB = 8.47 x 10(-8)M), and human circumflex artery (KB = 8.49 x 10(-7)M). SK&F 88046 was 10-60 times more potent in the canine and rhesus monkey coronary vasculature than in the human coronary preparations. Intracoronary administration of U-46619 to anesthetized, open chest dogs produced a dose-related decrease in left circumflex coronary artery blood flow which resulted in decreases in left ventricular developed pressure, left ventricular positive and negative dP/dt, ascending aortic blood flow, and an increase in left ventricular end-diastolic pressure. The decrease in coronary blood flow and the hemodynamic changes were either attenuated or completely inhibited by i.v. administration of SK&F 88046 (2.5 mg/kg + 0.05 mg/kg/min or 5.0 mg/kg + 0.1 mg/kg/min). SK&F 88046 was compared to two other TxA2 receptor antagonists in canine isolated intrapulmonary veins. SQ 29,548 was approximately 2-times more potent than SK&F 88046 as an antagonist of U-44619 mediated contractions (KB = 7.0 x 10(-10)M). In contrast, BM 13.177 was 150-fold less potent (KB = 2.19 x 10(-7)M) than SK&F 88046. Thus, the present study demonstrates species variability in response to TxA2 agonists and antagonists and reconfirms the relative importance of species selection in studying these agents.     

Metabolite ligands of estrogen receptor-beta reduce primate coronary hyperreactivity

Previous reports showed that 17beta-estradiol implants attenuate in vivo coronary hyperreactivity (CH), characterized by long-duration vasoconstrictions (in coronary angiographic experiments), in menopausal rhesus monkeys. Prolonged Ca2+ contraction signals that correspond with CH in coronary vascular muscle cells (VMC) to the same dual-constrictor stimulus, serotonin + the thromboxane analog U-46619, in estrogen-deprived VMC were suppressed by >72 h in 17beta-estradiol. The purpose of this study was to test whether an endogenous estrogen metabolite with estrogen receptor-beta (ER-beta) binding activity, estriol (E3), suppresses in vivo and in vitro CH. E3 treatment in vivo for 4 wk significantly attenuated the angiographically evaluated vasoconstrictor response to intracoronary serotonin + U-46619 challenge. In vitro treatment of rhesus coronary VMC for >72 h with nanomolar E3 attenuated late Ca2+ signals. This reduction of late Ca2+ signals also appeared after >72 h of treatment with subnanomolar 5alpha-androstane-3beta,17beta-diol (3beta-Adiol), an endogenous dihydrotestosterone metabolite with ER-beta binding activity. R,R-tetrahydrochrysene, a selective ER-beta antagonist, significantly blocked the E3- and 3beta-Adiol-mediated attenuation of late Ca2+ signal increases. ER-beta and thromboxane-prostanoid receptor (TPR) were coexpressed in coronary arteries and aorta. In vivo E3 treatment attenuated aortic TPR expression. Furthermore, in vitro treatment with E3 or 3beta-Adiol downregulated TPR expression in VMC, which was blocked for both agonists by pretreatment with R,R-tetrahydrochrysene. E3- and 3beta-Adiol-mediated reduction in persistent Ca2+ signals is associated with ER-beta-mediated attenuation of TPR expression and may partly explain estrogen benefits in coronary vascular muscle.     

Stereoselective inhibition of thromboxane-induced coronary vasoconstriction by 1,4-dihydropyridine calcium channel antagonists

The biological activity of the (+)-S- and (-)-R-enantiomers of niguldipine, of the (-)-S- and (+)-R-enantiomers of felodipine and nitrendipine, and of rac-nisoldipine and rac-nimodipine was investigated in vitro and in vivo. Inhibition of coronary vasoconstriction due to the thromboxane A2 (TxA2)-mimetic U-46619 in guinea pig Langendorff hearts, displacement of (+)-[3H]isradipine from calcium channel binding sites of guinea pig skeletal muscle T-tubule membranes, and blood pressure reduction in spontaneously hypertensive rats were determined. The enantiomers were obtained by stereoselective synthesis. Cross-contamination was less than 0.5% for both S- and R-enantiomers of niguldipine and nitrendipine and less than 1% for those of felodipine. From the doses necessary for a 50% inhibition of coronary vasoconstriction, stereoselectivity ratios for (+)-(S)-/(-)-(R)-niguldipine, (-)-(S)-/(+)-(R)-felodipine, and (-)-(S)-/(+)-(R)-nitrendipine of 28, 13, and 7, respectively, were calculated. The potency ratio rac-nisoldipine/rac-nimodipine was 3.5. Ratios obtained from binding experiments and antihypertensive activity were (+)-(S)-/(-)-(R)-niguldipine = 45 and 35, (-)-(S)-/(+)-(R)-felodipine = 12 and 13, (-)-(S)-/(+)-(R)-nitrendipine = 8 and 8, and rac-nisoldipine/rac-nimodipine = 8 and 7, respectively. Highly significant correlations were found between the in vitro potency of the substances to prevent U-46619-induced coronary vasoconstriction and their affinity for calcium channel binding sites as well as their antihypertensive activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

2[125I]iodomelatonin binding sites in spleens of guinea pigs.

2-[125I]Iodomelatonin was found to bind specifically to the membrane preparations of the spleens of guinea pigs with high affinity. The binding was rapid, stable, saturable and reversible. Scatchard analysis of the binding assays revealed an equilibrium dissociation constant (Kd) of 49.8 +/- 4.12 pmol/l and binding site density (Bmax) of 0.69 +/- 0.082 fmol/mg protein at mid-light (n = 10). There was no significant change in the Kd (41.8 +/- 3.16 pmol/l) or the Bmax (0.58 +/- 0.070 fmol/mg protein) at mid-dark (n = 10). Kinetic analysis showed a Kd of 23.13 +/- 4.81 pmol/l (mean +/- SE, n = 4), in agreement to that derived from the saturation studies. The 2-[125I]iodomelatonin binding sites have the following order of potency: 2-iodomelatonin greater than melatonin greater than 6-chloromelatonin much greater than N-acetylserotonin, 6-hydroxymelatonin greater than 5-methoxytryptamine, 5 methoxytryptophol greater than serotonin, 5-methoxyindole-3-acetic acid greater than 5-hydroxytryptophol, 3-acetylindole, 1-acetylindole-3-carboxyaldehyde, L-tryptophan greater than tryptamine, 5-hydroxyindole-3-acetic acid. Differential centrifugation studies showed that the binding sites are localized mainly in the nuclear fraction (65.5%), the rest are distributed in the microsomal fraction (17.4%), mitochondrial fraction (14.7%) and cytosolic fraction (0.3%). The demonstration of 2-[125I]iodomelatonin binding sites in the spleen suggests the presence of melatonin receptors and a direct mechanism of action of melatonin on the immune system.     

In vitro binding properties and autoradiographic imaging of 3-iodobenzamide ([125I]-IBZM): a potential imaging ligand for D-2 dopamine receptors in SPECT

The in vitro binding properties of the [125I] labeled benzamide (S(-)-N-[(1-ethyl-2-pyrrolidinyl)-methyl]-2-hydroxy-3-iodo-6-methoxy- benzamide, IBZM) were determined in bovine and mouse caudate membrane homogenates and by autoradiography of mouse brain slices. [125I]-IBZM binding is saturable and reversible with a Bmax of 373 +/- 51 fmol/mg protein and a Kd of 3.1 +/- 0.62 nM (mean +/- SD, Scatchard analyses) and 0.56 nM as calculated by association and dissociation time constants. In competition experiments, Ki values for the D-2 antagonists YM-09151-2 and spiperone are 4 orders of magnitude lower than the Ki value for the D-1 antagonist SCH-23390 and S(-)-IBZM is ten-fold more potent than R(+)-IBZM. [125I]-IBZM has a low affinity for serotonin S-2 and for alpha receptors. Therefore, it is a highly selective ligand for dopamine D-2 receptors. Autoradiographic images of brain sections incubated with [125I]-IBZM show the dopamine D-2 receptors of the striatum, nucleus accumbens and olfactory tubercle with a high ratio of specific to nonspecific binding. Thus, S(-)-IBZM, when labeled with [123I], may be useful for in vivo imaging of dopamine D-2 receptors by single photon emission computerized tomography (SPECT).