Log EC50 Research Articles

Functional characterization of adenosine receptors and coupling to ATP-sensitive K+ channels in Guinea pig urinary bladder smooth muscle.

Although multiple adenosine receptors have been identified, the subtype and underlying mechanisms involved in the relaxation response to adenosine in the urinary bladder remain unclear. The present study investigates changes in the membrane potential, as assessed by fluorescence-based techniques, of bladder smooth muscle cells by adenosine receptor agonists acting via ATP-sensitive potassium (K(ATP)) channels. Membrane hyperpolarization evoked by adenosine and various adenosine receptor subtype-selective agonists was attenuated or reversed by the K(ATP) channel blocker glyburide. Comparison of adenosine receptor agonist potencies eliciting membrane potential effects showed a rank order of potency 5'-N-ethyl-carboxamido adenosine (NECA; -log EC50 = 7.97) approximately 2-p-(2-carboxethyl)phenethyl-amino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS-21680; 7.65) > 2-chloro adenosine (5.90) approximately 2-chloro-N6-cyclopentyladenosine (CCPA; 5.51) approximately N6-cyclopentyladenosine approximately N6-(R)-phenylisopropyladenosine > 2-chloro- N6-(3-iodobenzyl)-adenosine-5'-N-methyl-carboxamide (2Cl-IBMECA; 4.78). Membrane potential responses were mimicked by forskolin, a known activator of adenylate cyclase, and papaverine, a phosphodiesterase inhibitor. The A(2A)-selective antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino] ethyl)phenol (ZM-241385), and the adenylate cyclase inhibitor N-(cis-2-phenyl-cyclopentyl) azacyclotridecan-2-imine-hydrochloride (MDL-12330A) inhibited the observed change in membrane potential evoked by adenosine and adenosine-receptor agonists. The rank order potency for relaxation of K+-stimulated guinea pig bladder strips, NECA (-log EC50 = 6.41) approximately CGS-21680 (6.38) > 2-chloro adenosine (5.90) >> CCPA approximately 2Cl-IBMECA (>4.0) was comparable to that obtained from membrane potential measurements. Collectively, these studies demonstrate that adenosine-evoked membrane hyperpolarization and relaxation of bladder smooth muscle is mediated by A(2A) receptor-mediated activation of K(ATP) channels via adenylate cyclase and elevation of cAMP.

Attenuation of cardiac fibrosis by pirfenidone and amiloride in DOCA-salt hypertensive rats.

1. This study has administered pirfenidone (5-methyl-1-phenyl-2-[1H]-pyridone) or amiloride to attenuate the remodelling and associated functional changes, especially an increased cardiac stiffness, in DOCA-salt hypertensive rats. 2. In control rats, the elimination half-life of pirfenidone following a single intravenous dose of 200 mg kg(-1) was 37 min while oral bioavailability at this dose was 25.7%. Plasma pirfenidone concentrations in control rats averaged 1.9 +/- 0.1 microg ml(-1) over 24 h after 14 days' administration as a 0.4% mixture in food. 3. Pirfenidone (approximately 250-300 mg kg(-1) day(-1) as 0.4% in food) and amiloride (1 mg kg(-1) day(-1) sc) were administered for 2 weeks starting 2 weeks post-surgery. Pirfenidone but not amiloride attenuated ventricular hypertrophy (2.69 +/- 0.09, UNX 2.01 +/- 0.05. DOCA-salt 3.11 +/- 0.09 mg kg(-1) body wt) without lowering systolic blood pressure. 4. Collagen deposition was significantly increased in the interstitium after 2 weeks and further increased with scarring of the left ventricle after 4 weeks; pirfenidone and amiloride reversed the increases and prevented further increases. This accumulation of collagen was accompanied by an increase in diastolic stiffness constant; both amiloride and pirfenidone reversed this increase. 5. Noradrenaline potency (positive chronotropy) was decreased in right atria (neg log EC50: control 6.92 +/- 0.06; DOCA-salt 6.64 +/- 0.08); pirfenidone but not amiloride reversed this change. Noradrenaline was a more potent vasoconstrictor in thoracic aortic rings (neg log EC50: control 6.91 +/- 0.10; DOCA-salt 7.90 +/- 0.07); pirfenidone treatment did not change noradrenaline potency. 6. Thus, pirfenidone and amiloride reverse and prevent cardiac remodelling and the increased cardiac stiffness without reversing the increased vascular responses to noradrenaline.

Altered Vascular Function in Young Women with Polycystic Ovary Syndrome

Polycystic ovary syndrome (PCOS) is characterized by hyperinsulinemic insulin resistance, a metabolic disorder that in other circumstances is associated with increased cardiovascular risk. We compared macrovascular and microvascular function in 19 women with PCOS with 12 control subjects matched as a group for body mass index. Macrovascular function was assessed by recording pulse wave velocity (PWV) across the aorta and brachial artery. Microvascular function was studied by wire myography, by measuring the concentration response curve to norepinephrine (NE) before and after incubation with insulin (100 and 1,000 pM). PWV at the level of the brachial artery was found to be significantly elevated in the PCOS group [9.08 (range, 8.34-11.15) m/sec(-1) vs. 8.27 (range, 7.5-9.01) m/sec(-1); P = 0.03]. In contrast, PWV measured in the aorta did not differ between the two groups [7.49 +/- 1.21 vs. 7.84 +/- 1.44 m/sec(-1); P = 0.8]. In vessels from control subjects, insulin reduced the contraction response to NE. At an insulin concentration of 100 pM, NE negative log EC50 (pD(2)) was 6.2 +/- 0.24 vs. 6.7 +/- 0.15 (P = 0.02). At a concentration of 1,000 pM, NE pD(2) was 6.4 +/- 0.14 vs. 6.9 +/- 0.19 (P = 0.0006). Both concentrations also caused attenuation in maximal tension developed in response to NE (insulin 100 pM, 12 +/- 3%, P = 0.002; insulin 1,000 pM, 17 +/- 5%, P = 0.009). In contrast, there was no change in the PCOS group with insulin at 100 pM for either pD(2) (6.7 +/- 0.24 vs. 6.8 +/- 0.27; P = 0.3) or maximum contraction (-0.4 +/- 2%; P = 0.8). At 1,000 pM, there was a change in pD(2) (6.4 +/- 0.2 vs. 6.8 +/- 0.2; P = 0.003) but not maximum contraction (4 +/- 3%; P = 0.2). In conclusion, this study is the first to demonstrate increased vascular stiffness and a functional defect in the vascular action of insulin ex vivo in patients with PCOS. We suggest that these findings are indicative of insulin resistance at a vascular level in women without overt cardiovascular disease.

The Acute Toxicity of α-Branched Phenylsulfonyl Acetates in Photobacterium phosphoreum Test

The acute toxicity (15-min EC50) of 20 α-substituted phenylsulfonyl acetates was measured using the Microtox test with Photobacterium phosphoreum. On the basis of TLSER, quantitative structure–activity relationships (QSARs) were calculated for the inhibition of bioluminescence. With the parameters molecular volume (Vmc), dipolarity/polarizability (π*), and the covalent contribution to Lewis basicity (εb), a squared correlation coefficient (r2adj) of 0.868 and standard error of 0.094 for log EC50 were obtained. By introducing a structural indicator variable (I), the regressive quality was improved (correlation coefficient of 0.948 and standard error of 0.059); the descriptors Vmc, π*, and the electrostatic basicity contribution (q−) were significant. –NO2– and –SO2– groups are active centers of these compounds. The strong hydrogen bonding effects between the compound active centers and FMNH2 play an important role in the greater toxicity of these chemicals.

Effects of methylenedioxymethamphetamine on noradrenaline-evoked contractions of rat right ventricle and small mesenteric artery

We have compared the effects of methylenedioxymethamphetamine (MDMA) and cocaine on contractions to noradrenaline in 1 Hz paced rat right ventricular strips, and in rat small mesenteric artery and aorta. Noradrenaline increased the force of contraction of 1 Hz paced ventricular strips with a pD 2 (−log EC 50) of 5.64±0.07. Both cocaine (10 μM) and MDMA (10 μM) significantly increased the potency of noradrenaline to 6.31±0.11 and 6.42±0.13, respectively. However, in the presence of cocaine (10 μM) which increased the potency of noradrenaline to 6.78±0.15, MDMA (10 μM) no longer increased the potency of noradrenaline (pD 2 of 6.78±0.32). Likewise, following chemical sympathectomy, MDMA failed to increase the potency of noradrenaline. The potency of the agonist isoprenaline, which is not a substrate for the noradrenaline transporter, was not increased by either cocaine or MDMA. In rat small mesenteric artery, but not aorta, MDMA and cocaine significantly increased the potency of noradrenaline, but in the presence of cocaine, MDMA had no further effect. Hence, MDMA shares with cocaine an ability to potentiate the actions of noradrenaline, an action in the case of MDMA which may involve competitive blockade of the noradrenaline transporter, rather than simply displacement of noradrenaline. Since cocaine is linked to an increased incidence of myocardial infarction, these results may have implications in terms of cardiac morbidity of MDMA.

Beta-3 versus beta-2 adrenergic agonists and preterm labour: in vitro uterine relaxation effects

Objective 1. To investigate the effects of the selective beta-3 adrenoreceptor agonist, BRL 37344, on human pregnant myometrial contractility in vitro. 2. to compare these effects with those of the beta-2 adrenoreceptor agonist, ritodrine. Methods Isometric tension recording was performed under physiological conditions in isolated myometrial strips from biopsies obtained at elective caesarean section. Following pre-incubation with oxytocin (10 -9 M), the effects of cumulative additions of BRL 37344 or ritodrine (10 -8–10 -3.5 M) on myometrial contractility were investigated. Results were expressed as -log EC 50 (pD 2) and mean maximal inhibition achieved for both drug compounds. Results BRL 37344 exerted a concentration dependant relaxant effect on myometrial contractions in all strips exposed [pD 2, 7.26 (0.48) (SEM); mean maximal inhibition 61.98 (4.89%); n= 6]. Similarly, ritodrine exerted a concentration dependant inhibition of myometrial contractility in all strips exposed [pD 2 = 7.40 (0.28); mean maximal inhibition 59.49 (3.97%); n = 6]. There was no significant difference between calculated pD 2 values ( P = 0.65) or mean maximal inhibition achieved ( P = 0.79). Conclusions The beta-3 adrenoreceptor agonist BRL 37344 induced relaxation of human myometrial contractions with similar potency to that of the most commonly used tocolytic agent ritodrine. This raises the possibility that the novel beta-3 adrenoreceptor agonists may have potential as therapeutic agents for human preterm labour. In view of their reported reduced cardiovascular side effects their potential clinical use requires further evaluation.

Maturation depresses cGMP-mediated decreases in [Ca2+]i and Ca2+ sensitivity in ovine cranial arteries.

Because cerebrovascular cGMP levels vary significantly during maturation, we examined the hypothesis that the ability of cGMP to relax cerebral arteries also changes during maturation. In concentration-response experiments, potassium-induced tone in basilar arteries was significantly more sensitive to a nonmetabolizable cell-permeant cGMP analogue 8-(p-chlorophenylthio)-cGMP (8-pCPT-cGMP) in term fetal [-log one-half maximal concentration (EC(50)) = 4.4 +/- 0.1 M] than in adult (-log EC(50) = 4.0 +/- 0.1 M) ovine basilar arteries. Serotonin-induced tone also revealed significantly greater sensitivity to the cGMP analogue in fetal (-log EC(50) = 4.9 +/- 0.1 M) than in adult (-log EC(50) = 4.7 +/- 0.1 M) basilars. In fura 2-loaded preparations, 8-pCPT-cGMP had no significant effect on cytosolic calcium concentrations in potassium-contracted arteries but at 6 microM significantly reduced calcium only in fetal basilars (Delta = 33 +/- 8%). Higher 8-pCPT-cGMP concentrations reduced cytosolic calcium in both fetal and adult basilars. Similarly, in both potassium- and 5-hydroxytryptamine (5-HT)-contracted preparations, low concentrations of 8-pCPT-cGMP reduced myofilament calcium sensitivity only in fetal basilars (Delta = 29 +/- 6 and Delta = 42 +/- 10%, respectively), whereas higher concentrations reduced calcium sensitivity in both fetal and adult arteries. In beta-escin-permeabilized arteries, equivalent reductions in basal and agonist-enhanced myofilament calcium sensitivity were produced by much lower 8-pCPT-cGMP concentrations in fetal (172 and 61 microM, respectively) than in adult (410 and 231 microM, respectively) basilars. The mechanisms mediating cGMP-induced vasorelaxation appear similar in fetal and adult arteries, with the exception that they are much more sensitive to cGMP in fetal than adult arteries. These age-related differences in the sensitivity of cytosolic calcium concentration, basal, and agonist-enhanced myofilament calcium sensitivity to cGMP can easily explain why both potassium- and 5-HT-induced tone are more sensitive to cGMP in fetal than adult cerebral arteries.

Relationship of Airway Responsiveness with Airway Morphometry in Normal and Immunized Rabbits

Airway responses to chemical stimuli occur over a wide range of concentrations, with overlap between severe, moderate and mild asthmatic groups and with normal healthy individuals. Mathematical modelling has suggested that relative thickness of the airway wall may account for this range of responsiveness. We have investigated whether in vivo airway responsiveness varies as a function of airway wall thickness in terms of airway smooth muscle area in normal and immunized New Zealand White (NZW) rabbits. Airway responsiveness to inhaled methacholine (MCh) was determined in vivo under neuroleptanalgesia. Subsequently, ex vivo responsiveness to MCh (pD2=−log EC50) of isolated bronchi from the same animal was established. Smooth muscle area per mm basement membrane (SM/mmBM) was also measured morphometrically in the tested bronchi and the findings related to in vivo and ex vivo responsiveness. We found no relationship between airway responsiveness in vivo and pD2values in either immunized or control rabbits. In both control and immunized rabbits, no correlation was found between SM/mmBM and in vivo airway responsiveness. Only in immunized animals with a PCA titre >0, was there a significant correlation (=−0.5986, P<0.05) between SM/mmBM and pD2. We conclude that airway smooth muscle area per se is not the sole contributor of airway responsiveness in vivo in normal rabbits.

Reversal of cardiovascular remodelling with candesartan.

Cardiovascular remodelling, defined as ventricular and vascular hypertrophy together with fibrosis, characterises hypertension following inhibition of the production of the endogenous vasodilator, nitric oxide (NO). This study has determined whether the cardiovascular remodelling following chronic NO synthase inhibition can be reversed by administration of the selective angiotensin II AT1-receptor antagonist, candesartan. Male Wistar rats were treated with L-nitroarginine methyl ester (L-NAME, 400 mg/l in drinking water) for eight weeks and with candesartan cilexetil (2 mg/kg/day by oral gavage) for the last four weeks. L-NAME-treated rats became hypertensive with systolic blood pressure increasing from 110±4 mmHg (control) to 170±10 mmHg. Rats developed left ventricular hypertrophy (control 1.70±0.06; L-NAME 2.10±0.04 mg/kg body wt) with markedly increased deposition of perivascular and interstitial collagen. Candesartan returned blood pressure, left ventricular weights and collagen deposition to control values. Echocardiographic assessment showed concentric hypertrophy with an increased fractional shortening; this was reversed by candesartan treatment. Heart failure was not evident. In the isolated Langendorff heart, diastolic stiffness increased in L-NAME-treated rats while the rate of increase in pressure (+dP/dt) increased after eight weeks only; candesartan reduced collagen deposition and normalised +dP/dt. In isolated left ventricular papillary muscles, the potency (negative log EC50) of noradrenaline as a positive inotropic compound was unchanged, (control 6.56±0.14); maximal increase in force before ectopic beats was reduced from 5.0±0.4 mN to 2.0±0.2 mN. Noradrenaline potency as a vasoconstrictor in thoracic aortic rings was unchanged, but maximal contraction was markedly reduced from 25.2±2.0 mN to 3.0±0.3 mN; this was partially reversed by candesartan treatment. Thus, chronic inhibition of NO production with L-NAME induces hypertension, hypertrophy and fibrosis with increased toxicity and significant decreases in vascular responses to noradrenaline. These changes were at least partially reversible by treatment with candesartan, implying a significant role of AT1-receptors in L-NAME-induced cardiovascular changes.

Limited signal transduction repertoire of human Y 5 neuropeptide Y receptors expressed in HEC-1B cells

In HEC-1B cells transfected with human Y 5 neuropeptide Y (NPY) receptors (but not in non-transfected cells) NPY inhibited forskolin-stimulated cAMP accumulation in a pertussis toxin-sensitive manner (−log EC 50 8.88 ± 0.25). Elevations of intracellular Ca 2+ were largely restricted to very high NPY concentrations and similar in transfected and nontransfected cells. NPY did not increase inositol phosphate accumulation and did not activate a variety of isoforms of protein kinase C or mitogen-activated protein kinases. We conclude that at least upon expression in HEC-1B cells the signal transduction of Y 5 NPY receptors is limited to inhibition of cAMP accumulation.

CYTOCHROME P450 INDUCTION BY NITRATED POLYCYCLIC AROMATIC HYDROCARBONS, AZAARENES, AND BINARY MIXTURES IN FISH HEPATOMA CELL LINE PLHC-1

Nitrated polycyclic aromatic hydrocarbons (NP AH) and N-heterocyclic aromatic hydrocarbons (azaarenes) are as ubiquitous in the environment as their parent PAH compounds, although occurring at lower concentrations. The toxicological importance of NPAHs and azaarenes is based on their mutagenic and carcinogenic potential. Azaarenes possess a higher solubility and mobility in the environment than PAHs. However, very little is known about the toxicity and cytochrome P450 (CYP)1A induction potencies of NPAHs and azaarenes in fish. Here we report on the cytotoxicities and relative CYP1A induction potencies of 12 NPAHs, 12 azaarenes, and 11 PAHs, determined as neutral red uptake and ethoxyresorufin-O-deethylase (EROD) activity, respectively, in fish hepatoma PLHC-1 cells. Additionally, CYP1A enzyme protein was determined by ELISA for two NPAHs, azaarenes, PAHs, and binary mixtures. Compared with the structurally analogous PAHs, 2-nitronaphthalene, 3-nitrofluoranthene, 2-aza- and 7-azafluor-anthene, 1,6-dinitropyrene, benzo[a]acridine and benzo[h]quinoline revealed higher induction potencies, whereas the other compounds showed similar or less activity. The induction potency was highly dependent on the compounds structural properties, reflected by significant correlations between the half-maximal EROD induction (-log EC50) and the molecular descriptors lipophilicity (log Kow) and maximal molecular length (Lmax). Binary mixtures of 6-nitrochrysene + benzo[a]anthracene, 6-nitrochrysene + ben-zo[a]acridine, and benzo[a]acridine + benzo[a]anthracene showed an additive interaction. The CYP1A induction potencies of NPAHs and azaarenes, demonstrated here for the first time in fish hepatoma cells, suggest that their contribution to the overall CYP1A induction potencies in PAH-contaminated environmental samples have to be taken into account.

Cytochrome P450 induction by nitrated polycyclic aromatic hydrocarbons, azaarenes, and binary mixtures in fish hepatoma cell line PLHC‐1

Nitrated polycyclic aromatic hydrocarbons (NPAHs) and N-heterocyclic aromatic hydrocarbons (azaarenes) are as ubiquitous in the environment as their parent PAH compounds, although occurring at lower concentrations. The toxicological importance of NPAHs and azaarenes is based on their mutagenic and carcinogenic potential. Azaarenes possess a higher solubility and mobility in the environment than PAHs. However, very little is known about the toxicity and cytochrome P450 (CYP)1A induction potencies of NPAHs and azaarenes in fish. Here we report on the cytotoxicities and relative CYP1A induction potencies of 12 NPAHs, 12 azaarenes, and 11 PAHs, determined as neutral red uptake and ethoxyresorufin-O-deethylase (EROD) activity, respectively, in fish hepatoma PLHC-1 cells. Additionally, CYP1A enzyme protein was determined by ELISA for two NPAHs, azaarenes, PAHs, and binary mixtures. Compared with the structurally analogous PAHs, 2-nitronaphthalene, 3-nitrofluoranthene, 2-aza- and 7-azafluoranthene, 1,6-dinitropyrene, benzo[a]acridine and benzo[h]quinoline revealed higher induction potencies, whereas the other compounds showed similar or less activity. The induction potency was highly dependent on the compounds structural properties, reflected by significant correlations between the half-maximal EROD induction (-log EC50) and the molecular descriptors lipophilicity (log Kow) and maximal molecular length (Lmax). Binary mixtures of 6-nitrochrysene + benzo[a]anthracene, 6-nitrochrysene + benzo[a]acridine, and benzo[a]acridine + benzo[a]anthracene showed an additive interaction. The CYP1A induction potencies of NPAHs and azaarenes, demonstrated here for the first time in fish hepatoma cells, suggest that their contribution to the overall CYP1A induction potencies in PAH-contaminated environmental samples have to be taken into account.

Autoradiographic Localisation and Contractile Properties of Prostatic Endothelin Receptors in Patients with Bladder Outlet Obstruction

Objectives: Previous studies have used endothelin (ET) receptor agonists and antagonists to localise ET receptor subtypes in prostatic tissue. We have utilised high affinity ET_A ([¹²⁵I]PD151242) and ET_B ([¹²⁵I]BQ3020) receptor–specific radioligands to determine the density and distribution of ET receptor subtypes in prostatic tissues obtained from patients with symptomatic benign prostatic hyperplasia (BPH). The contractile properties of the ET receptor subtypes as well as the effect of ET–1 on α₁–adrenergic receptor–mediated prostatic smooth muscle contraction were assessed. Patients and Methods: Saturation binding and quantitative autoradiographic studies were performed using specific radioligands for ET_A and ET_B receptors on prostate sections obtained from patients with bladder outflow obstruction secondary to BPH. In vitro isometric tension studies were carried out to characterise the ET receptor subtypes in prostatic smooth muscle strips from the same group of patients. In addition, the effect of ET–1 on α₁–adrenergic receptor–induced prostatic smooth muscle contraction was also investigated. Results: There were dense ET_A and ET_B receptor–binding sites in the prostatic stroma. ET_A receptor–binding sites were also prominent on the prostatic epithelium. ET–1 and sarafotoxin 6 c (ET_B receptor agonist) elicited prostatic smooth muscle contraction (–log EC₅₀ 8.31±0.15 and 8.22±0.22 M, respectively). Both BQ123 (ET_A antagonist) and BQ788 (ET_B antagonist) significantly inhibited ET–1– and S6c–mediated prostatic smooth muscle contractile responses, respectively. ET–1 at sub–threshold concentrations significantly enhanced α₁–adrenergic receptor–mediated prostatic smooth muscle contractile responses. Conclusions: ET_A receptor–binding sites are prominent in both prostatic stroma and epithelium, whereas ET_B receptor–binding sites were predominantly seen in the prostatic stroma in symptomatic BPH. Both ET_A and ET_B receptors mediate prostatic smooth muscle contraction. ET–1 enhances α₁–adrenergic receptor–mediated contractile responses, suggesting that ET may play a pathophysiological role in bladder outlet obstruction associated with BPH.

Both beta(2)- and beta(1)-adrenergic receptors mediate hastened relaxation and phosphorylation of phospholamban and troponin I in ventricular myocardium of Fallot infants, consistent with selective coupling of beta(2)-adrenergic receptors to G(s)-protein.

In adult human heart, both beta(1)- and beta(2)-adrenergic receptors mediate hastening of relaxation; however, it is unknown whether this also occurs in infant heart. We compared the effects of stimulation of beta(1)- and beta(2)-adrenergic receptors on relaxation and phosphorylation of phospholamban and troponin I in ventricle obtained from infants with tetralogy of Fallot. Myocardium dissected from the right ventricular outflow tract of 27 infants (age range 21/2 to 35 months) with tetralogy of Fallot was set up to contract 60 times per minute. Selective stimulation of beta(1)-adrenergic receptors with (-)-norepinephrine (NE) and beta(2)-adrenergic receptors with (-)-epinephrine (EPI) evoked phosphorylation of phospholamban (at serine-16 and threonine-17) and troponin I and caused concentration-dependent increases in contractile force (-log EC(50) [mol/L] NE 5.5+/-0.1, n=12; EPI 5.6+/-0.1, n=13 patients), hastening of the time to reach peak force (-log EC(50) [mol/L] NE 5.8+/-0.2; EPI 5.8+/-0.2) and 50% relaxation (-log EC(50) [mol/L] NE 5.7+/-0.2; EPI 5.8+/-0.1). Ventricular membranes from Fallot infants, labeled with (-)-[(125)I]-cyanopindolol, revealed a greater percentage of beta(1)- (71%) than beta(2)-adrenergic receptors (29%). Binding of (-)-epinephrine to beta(2)-receptors underwent greater GTP shifts than binding of (-)-norepinephrine to beta(1)-receptors. Despite their low density, beta(2)-adrenergic receptors are nearly as effective as beta(1)-adrenergic receptors of infant Fallot ventricle in enhancing contraction, relaxation, and phosphorylation of phospholamban and troponin I, consistent with selective coupling to G(s)-protein.

2-Phenylmelatonin: A Partial Agonist at Enteric Melatonin Receptors

The effect of the melatonin receptor ligand, 2-phenylmelatonin, has been assessed in isolated strips of the guinea-pig proximal colon. 2-Phenylmelatonin (0.01 nM-1 microM) caused a concentration-dependent contractile response. The potency value (-log EC50) was 9.3 +/- 1.0. The maximum effect was 25 +/- 4%, of that elicited by the maximally effective concentration (0.3 microM) of 5-HT and 43 +/- 3%, of that by the maximally effective concentration (10 microM) of melatonin. When used as an antagonist, 2-phenylmelatonin (0.01 nM and 0.1 nM) concentration-dependently inhibited melatonin-induced contractions with depression of the maximum response by 25% and 54%, respectively. Higher (1 nM) 2-phenylmelatonin concentrations failed to antagonize melatonin-induced response. Prazosin (0.3 microM), a selective antagonist of melatonin MT3 sites, antagonized melatonin-induced contractions to an extent similar to that induced by 0.01 nM 2-phenylmelatonin (with 30% reduction of the maximum effect to melatonin). The combination of 0.3 microM prazosin and 0.01 nM 2-phenylmelatonin caused antagonism similar in extent to that caused by each individual antagonist. 2-Phenylmelatonin at subnanomolar concentrations behaves as an antagonist of melatonin-induced contractile responses while at nanomolar/micromolar concentrations it behaves as a weak contractile agonist.

Heightened Vascular Reactivity Following Sympathetic Denervation Correlates with Increased Expression of C-Kit and Connexin43

P71 Short-term denervation produces supersensitivity to α-adrenergic agonists that resembles the heightened vascular reactivity observed in hypertensive subjects. Gap junctions, transmembrane channels responsible for cell-to-cell communication, may play a role in maintaining increased vascular reactivity. We examined the effects of denervation with intra-peritoneal reserpine 3 mg/kg/day (N=5) or topical 5% phenol-glycerol (N=7) on agonist-induced oscillatory activity. Wistar-Kyoto (WKY) rat tail arteries were exposed to step-wise addition of norepinephrine (NE) 10 -9 M-10 -5 M. The concentration for half-maximal response for NE-induced contraction was lower in reserpine- and phenol-treated vessels than controls (log EC 50 [mol]/L control -6.89 vs reserpine -7.16, p&lt;0.05); control -6.86 vs phenol -7.32, p&lt;0.0001). Incubation in K + -free solution produced an 8-fold increase in isometric tension in control vs denervated vessels (138.4±23.2 vs 17.2±4.5 % of contraction to phenylephrine 10 -6 M, p&lt;0.001). These findings are consistent with adrenergic denervation. Agonist-induced oscillatory activity was observed in 5/5 reserpine- and 4/7 phenol-treated vessels vs 0/12 controls (Fischer’s exact test p&lt;0.05). The highly selective gap junction inhibitor, Gap27 (10 mM), nearly abolished NE-induced vascular smooth muscle oscillations. Reverse transcriptase polymerase chain reaction was carried out for connexin43 (Cx43) and c-Kit, a marker for non-neural interstitial cells that serve as pacemaker cells, using specific oligonucleotide primers. Following denervation, mRNA levels of Cx43 remained unchanged while levels of c-Kit increased markedly (0.16±0.03 vs 0.62±0.13 a.u., p&lt;0.05). Western blot analysis revealed near doubling of Cx43 protein after denervation (80.0±13.7 vs 41.4±5.7 a.u., p&lt;0.05). In conclusion, sympathetic denervation produced increased agonist-induced oscillatory contractile activity in vascular smooth muscle. The heightened vascular reactivity may result from upregulation of pacemaker cells and increased Cx43 expression.

Heightened Vascular Reactivity Following Sympathetic Denervation Correlates with Increased Expression of C-Kit and Connexin43

P71 Short-term denervation produces supersensitivity to α-adrenergic agonists that resembles the heightened vascular reactivity observed in hypertensive subjects. Gap junctions, transmembrane channels responsible for cell-to-cell communication, may play a role in maintaining increased vascular reactivity. We examined the effects of denervation with intra-peritoneal reserpine 3 mg/kg/day (N=5) or topical 5% phenol-glycerol (N=7) on agonist-induced oscillatory activity. Wistar-Kyoto (WKY) rat tail arteries were exposed to step-wise addition of norepinephrine (NE) 10 -9 M-10 -5 M. The concentration for half-maximal response for NE-induced contraction was lower in reserpine- and phenol-treated vessels than controls (log EC 50 [mol]/L control -6.89 vs reserpine -7.16, p + -free solution produced an 8-fold increase in isometric tension in control vs denervated vessels (138.4±23.2 vs 17.2±4.5 % of contraction to phenylephrine 10 -6 M, p

Gender Difference in the Contribution of an Indomethacin-Sensitive Constrictor Factor to Phenylephrine Responsiveness in Rat Mesenteric Arteries

P22 The mechanism for gender differences in cardiovascular disease may be due, in part, to differences in vascular function. Based on previous studies from our laboratory, we hypothesized that there is enhanced production of a vasoconstrictor cyclooxygenase product in arteries from males when compared to those from females. We studied three month-old male and female Sprague-Dawley rats (n=6 in each group). Small mesenteric arteries (∼400 μm) were isolated, pressurized, and the outer diameter was measured. Arteries from each rat were paired: one had intact endothelium (ENDO +) and the other was mechanically denuded (ENDO -). Dose-response curves to the α 1 -adrenergic agonist, phenylephrine (PE), were performed in the absence and presence of the cyclooxygenase inhibitor, indomethacin (INDO; 10 μM). Negative log EC 50 values were calculated as an index of PE sensitivity and compared between groups using ANOVA and lsd. There were no differences in the sensitivity to PE in ENDO + arteries from males (6.07±0.07) and females (6.12±0.10, p=0.93). In ENDO + arteries from females, the PE sensitivity was unchanged in the presence of INDO (6.11±0.13, p=0.94 when compared to control). However, INDO significantly decreased PE sensitivity in ENDO + arteries from males (5.70±0.08, p=0.01 when compared to control). Endothelium denudation had no significant effect on PE sensitivity in males or females. In denuded arteries from females, INDO produced no change in PE sensitivity (denuded female: 6.08±0.09, denuded + INDO: 5.88±0.13, p=0.23). In contrast, INDO produced a significant decrease in sensitivity in denuded arteries from males (denuded male: 6.33±0.14, denuded + INDO (5.94±0.09, p=0.03). The magnitude of the shift in PE sensitivity produced by INDO (Δ EC 50 )in males was equivalent in ENDO + (0.37±0.07) and ENDO - (0.39±0.09, p=0.9, Student’s t-test) arteries. In conclusion, arteries from male rats produce more of an indomethacin-sensitive vasoconstrictor factor than arteries from female rats. The site of enhanced production of this cyclooxygenase product in males is not the endothelium, but may be vascular smooth muscle.

Source of calcium for contractile responses of large and small human intramyometrial arteries.

The role of calcium (Ca(2+)) released from intracellular stores and the entry of extracellular Ca(2+) for vasopressin (AVP)-induced responses in large and small, human, intramyometrial arteries was investigated. There was no statistical difference as revealed by pD(2) values (-log EC(50)), in the sensitivity of large and small vessels to AVP. Nimodipine caused an inhibition of contractions induced by low concentrations (10(-10) mol/l) of AVP in both types of vessels but, at higher concentration (>10(-10) mol/l), whereas responses in small arteries were diminished, in large arteries they remained unchanged. In Ca(2+)-free solution, responses of large and small arteries to potassium and to 10(-10) mol/l AVP were abolished. With 10(-6) mol/l AVP, response in small arteries was completely inhibited, whereas in large arteries it was reduced by approximately 50%. Additional experiments were done on large arteries. Thapsigargin (TSG), which causes depletion of internal Ca(2+) stores, caused a significant reduction in responses. Following treatment with TSG, responses to AVP in Ca(2+)-free solution were almost completely inhibited but arteries responded again when incubated in normal physiological salt solution. The results indicate that in contrast to large arteries, small arteries are highly dependent on extracellular Ca(2+). Response of large arteries showed considerable dependence on Ca(2+) stored internally particularly, for maximum activation.

Modulation of cholinergic airway reactivity and nitric oxide production by endogenous arginase activity.

Cholinergic airway constriction is functionally antagonized by agonist-induced constitutive nitric oxide synthase (cNOS)-derived nitric oxide (NO). Since cNOS and arginase, which hydrolyzes L-arginine to L-ornithine and urea, use L-arginine as a common substrate, competition between both enzymes for the substrate could be involved in the regulation of cholinergic airway reactivity. Using a perfused guinea-pig tracheal tube preparation, we investigated the modulation of methacholine-induced airway constriction by the recently developed, potent and specific arginase inhibitor N(Omega)-hydroxy-nor-L-arginine (nor-NOHA). Intraluminal (IL) administration of nor-NOHA caused a concentration-dependent inhibition of the maximal effect (E(max)) in response to IL methacholine, which was maximal in the presence of 5 microM nor-NOHA (E(max)=31.2+/-1.6% of extraluminal (EL) 40 mM KCl-induced constriction versus 51.6+/-2.1% in controls, P<0.001). In addition, the pEC(50) (-log(10) EC(50)) was slightly but significantly reduced in the presence of 5 microM nor-NOHA. The inhibition of E(max) by 5 microM nor-NOHA was concentration-dependently reversed by the NOS inhibitor N(Omega)-nitro-L-arginine methyl ester (L-NAME), reaching an E(max) of 89.4+/-7.7% in the presence of 0.5 mM L-NAME (P<0.01). A similar E(max) in the presence of 0.5 mM L-NAME was obtained in control preparations (85.2+/-9.7%, n.s.). In the presence of excess of exogenously applied L-arginine (5 mM), 5 microM nor-NOHA was ineffective (E(max)=33.1+/-5.8 versus 31.1+/-7.5% in controls, n.s.). The results indicate that endogenous arginase activity potentiates methacholine-induced airway constriction by inhibition of NO production, presumably by competition with cNOS for the common substrate, L-arginine. This finding may represent an important novel regulation mechanism of airway reactivity.