Fold Rightward Shift Research Articles

Synergistic interaction between the agonism of cebranopadol at nociceptin/orphanin FQ and classical opioid receptors in the rat spinal nerve ligation model.

Cebranopadol (trans‐6′‐fluoro‐4′,9′‐dihydro‐N,N‐dimethyl‐4‐phenyl‐spiro[cyclohexane‐1,1′(3′H)‐pyrano[3,4‐b]indol]‐4‐amine) is a novel analgesic nociceptin/orphanin FQ opioid peptide (NOP) and classical opioid receptor (MOP, DOP, and KOP) agonist with highly efficacious and potent activity in a broad range of rodent models of nociceptive, inflammatory, and neuropathic pain as well as limited opioid‐type side effects such as respiratory depression. This study was designed to explore contribution and interaction of NOP and classical opioid receptor agonist components to cebranopadol analgesia in the rat spinal nerve ligation (SNL) model. Assessing antihypersensitive activity in SNL rats intraperitoneal (IP) administration of cebranopadol resulted in ED 50 values of 3.3 and 3.58 μg/kg in two independent experiments. Pretreatment (IP) with J‐113397 (4.64 mg/kg) a selective antagonist for the NOP receptor or naloxone (1 mg/kg), naltrindole (10 mg/kg), or nor‐BNI (10 mg/kg), selective antagonists for MOP, DOP, and KOP receptors, yielded ED 50 values of 14.1, 16.9, 17.3, and 15 μg/kg, respectively. This 4‐5 fold rightward shift of the dose‐response curves suggested agonistic contribution of all four receptors to the analgesic activity of cebranopadol. Combined pretreatment with a mixture of the antagonists for the three classical opioid receptors resulted in an 18‐fold potency shift with an ED 50 of 65.5 μg/kg. The concept of dose equivalence was used to calculate the expected additive effects of the parent compound for NOP and opioid receptor contribution and to compare them with the observed effects, respectively. This analysis revealed a statistically significant difference between the expected additive and the observed effects suggesting intrinsic synergistic analgesic interaction of the NOP and the classical opioid receptor components of cebranopadol. Together with the observation of limited respiratory depression in rats and humans the synergistic interaction of NOP and classical opioid receptor components in analgesia described in the current study may contribute to the favorable therapeutic index of cebranopadol observed in clinical trials.

Dynorphin A (1-17) was Selective toµ-Opioid Receptor in Agonist-Stimulated [35S] GTPγS Binding in Cortical and Thalamic Membranes of Monkey

Background: Dynorphin A (1-17) is conceived as an endogenous opioid peptide with a high degree of selectivity for -opioid receptor even though it has been reported to sometimes act like a - opioid agonist. The aim of this study was to investigate [S] GTPS binding stimulated activation by dynorphin A (1-17) in the cerebral and thalamic membranes of a rhesus monkey. Methods: The rhesus monkey (Macaca mulatta, male, n = 1) was euthanized for the preparation of the cerebral and thalamic membranes. Protein concentrations were determined by the Bradford method. In the dynorphin A (1-17)-stimulated [S] GTPS binding dose-response curve, (effective concentration 50 nM) and maximum stimulation (% over basal) were determined in the absence or presence of the -and -opioid receptor antagonists naloxone (20 nM) and norbinaltorphimine (nor-BNI, 3 nM), respectively. E2078-stimulated [S] GTPS binding was also determined in the absence or presence of -and -opioid receptor antagonists in the cortical membrane and compared with dynorphin A (1-17). Results: Values of and maximum stimulation of dynorphin A (1-17)-stimulated [S] GTPS binding were as follows: cortex (474 nM/32.0%) and thalamus (423 nM/45.3%). Nor-BNI (3 nM) did not antagonize dynorphin A (1 17)-stimulated [S] GTPS binding at all in cortical or thalamic membrane, but naloxone (20 nM) produced a 12.2 fold rightward shift of the dynorphin A (1-17)-stimulated [S] GTPS binding dose-response curve in the thalamic membrane. The and the maximum stimulation of E2078-stimulated [S] GTPS binding were 65.6 nM and 22.7%, respectively. In E2078-stimulated [S] GTPgammaS binding, the dose-response curve was antagonized not by nor-BNI but by naloxone but in the cortical membrane (a 14.2 times rightward shift). Conclusions: Dynorphin A (1-17) is selective form-opioid receptor in agonist-stimulated [S] GTPS binding in the cortical and thalamic membranes of rhesus monkey.

Anandamide-induced relaxation of sheep coronary arteries: the role of the vascular endothelium, arachidonic acid metabolites and potassium channels.

1. The effects of the endocannabinoid, anandamide, and its metabolically stable analogue, methanandamide, on induced tone were examined in sheep coronary artery rings in vitro. 2. In endothelium-intact rings precontracted to the thromboxane A(2) mimetic, U46619, anandamide (0.01 - 30 microM) induced slowly developing concentration-dependent relaxations (pEC(50) [negative log of EC(50)]=6.1+/-0.1; R(max) [maximum response]=81+/-4%). Endothelium denudation caused a 10 fold rightward shift of the anandamide concentration-relaxation curve without modifying R(max). Methanandamide was without effect on U46619-induced tone. 3. The anandamide-induced relaxation was unaffected by the cannabinoid receptor antagonist, SR 141716A (3 microM), the vanilloid receptor antagonist, capsazepine (3 and 10 microM) or the nitric oxide synthase inhibitor, L-NAME (100 microM). 4. The cyclo-oxygenase inhibitor, indomethacin (3 and 10 microM) and the anandamide amidohydrolase inhibitor, PMSF (70 and 200 microM), markedly attenuated the anandamide response. The anandamide transport inhibitor, AM 404 (10 and 30 microM), shifted the anandamide concentration-response curve to the right. 5. Precontraction of endothelium-intact rings with 25 mM KCl attenuated the anandamide-induced relaxations (R(max)=7+/-7%), as did K(+) channel blockade with tetraethylammonium (TEA; 3 microM) or iberiotoxin (100 nM). Blockade of small conductance, Ca(2+)-activated K(+) channels, delayed rectifier K(+) channels, K(ATP) channels or inward rectifier K(+) channels was without effect. 6. These data suggest that the relaxant effects of anandamide in sheep coronary arteries are mediated in part via the endothelium and result from the cellular uptake and conversion of anandamide to a vasodilatory prostanoid. This, in turn, causes vasorelaxation, in part, by opening potassium channels.

Structure-activity relationship studies of (+/-)-terbutaline and (+/-)-fenoterol on beta3-adrenoceptors in the guinea pig gastric fundus.

(+/-)-Terbutaline and (+/-)-fenoterol are both arylethanolamine analogs that have tertbutyl and aryliso-propyl substituents respectively at the a position on the nitrogen of the ethanolamine side chain. In the present study, we have investigated the structure-activity relationships of (+/-)-terbutaline and (+/-)-fenoterol as beta3-adrenoceptor agonists in the guinea pig gastric fundus. (+/-)-Terbutaline and (+/-)-fenoterol induced concentration-dependent relaxation of the precontracted gastric fundus with pD2 values of 4.45+/-0.10 and 5.90+/-0.09, and intrinsic activities of 1.00+/-0.03 and 0.99+/-0.01 respectively. The combination of the selective beta1-adrenoceptor antagonist (+/-)-atenolol (100 microM), and the selective beta2-adrenoceptor antagonist (+/-)-butoxamine (100 microM), produced a 2 and 6 fold rightward shift of the concentration-response curves for (+/-)-terbutaline and (+/-)-fenoterol respectively, without depressing the maximal responses. The order of potency of these agonists was (pD2 value): (+/-)-fenoterol (5.09+/-0.10) > (+/-)-terbutaline (4.13+/-0.08). In the presence of (+/-)-atenolol and (+/-)-butoxamine, however, the non-selective beta1, beta2- and beta3-adrenoceptor antagonist (+/-)-bupranolol caused a concentration-dependent rightward shift of the concentration-response curves for (+/-)-terbutaline and (+/-)-fenoterol. Schild plot analyses of the effects of (+/-)-bupranolol against these agonists gave pA2 values of 6.21+/-0.07 ((+/-)-terbutaline) and 6.37+/-0.06 ((+/-)-fenoterol) respectively, and the slopes of the Schild plot were not significantly different from unity (p>0.05). These results suggest that the relaxant responses to (+/-)-terbutaline and (+/-)-fenoterol are mainly mediated through beta3-adrenoceptors in the guinea pig gastric fundus. The beta3-adrenoceptor agonist potencies of arylethanolamine analogs depend on the size of the end of the alkylamine side chain.

Differential A(1)-adenosine receptor reserve for inhibition of cyclic AMP accumulation and G-protein activation in DDT(1) MF-2 cells.

1. The A(1)-adenosine receptor (A(1)AdoR) reserve for N(6)-cyclopentyladenosine (CPA) mediated inhibition of (-)isoprenaline stimulated cyclic AMP accumulation and stimulation of [(35)S]-guanosine-5'-O-(thiotriphosphate) (GTPgammaS) binding, a measure of guanine nucleotide binding protein (G-protein) activation, was determined in DDT(1) MF-2 cells. 2. Inactivation of the A(1)AdoRs with the chemoreactive ligand 8-cyclopentyl-3-[3-[[4-(fluorosulphonyl)benzoyl]oxy]propyl]-1-p ropylx anthine (FSCPX) caused a progressive rightward shift of the concentration-response curves for CPA to inhibit cyclic AMP accumulation, with a maximum of 10 fold increase in the EC(50) value. In contrast, inactivation of A(1)AdoR's caused only a 1.7 fold rightward shift in the CPA concentration-response for stimulation of [(35)S]-GTPgammaS binding. 3. The A(1)AdoR occupancy-response relationship for CPA inhibition of cyclic AMP accumulation was hyperbolic with 43% receptor occupancy required to elicit the maximal response, i.e. a 57% A(1)AdoR reserve. In contrast, the A(1)AdoR occupancy-response relationship for CPA mediated stimulation of [(35)S]-GTPgammaS binding was linear indicating little or no receptor reserve for G-protein activation. The relationship between CPA stimulation of [(35)S]-GTPgammaS binding and cyclic AMP inhibition was also hyperbolic with 44% G-protein activation sufficient to cause maximal inhibition. 4. The data suggest that the A(1)AdoR reserve for CPA mediated inhibition of cyclic AMP accumulation occurs at the level of G-protein interaction with adenylyl cyclase. However, each A(1)AdoR appears to activate a constant fraction of the total G-protein population suggesting signal amplification at the receptor-G-protein level which may also contribute to the receptor reserve for CPA.

Role of L-arginine in the vascular actions and development of tolerance to nitroglycerin.

The goal of this work was to test the role of nitric oxide synthase (NOS) and its substrate L-arginine in development of tolerance to nitroglycerin's (GTN) vasodilator actions. GTN's effects on NOS activity and NO formation were tested in cultured bovine aortic endothelial cells (BAECs). The arginine to citrulline conversion assay showed that GTN stimulated NOS basal activity in BAECs by approximately 40%, comparable with acetylcholine (ACh)-treated controls. Both effects were blocked by L-NMMA. Photometric assays showed that both GTN and ACh-stimulated NO formation. Both effects were potentiated by L-arginine and inhibited by L-NAME. L-NAME inhibited ACh responses approximately 80% compared with approximately 40% for GTN responses. The aortic ring assay showed that 2 h pretreatment with GTN caused substantial tolerance to GTN's vasodilating effects as evidenced by a 38 fold rightward shift of the concentration-relaxation curve. In contrast to D-arginine, addition of L-arginine substantially inhibited this effect, reducing the rightward shift to 4.4 fold of control values. GTN tolerance was associated with a 40% reduction in L-arginine tissue levels. GTN had a biphasic effect on BAEC uptake of L-arginine, stimulating uptake at 5 and 15 min, and suppressing uptake after 1 and 4 h In summary, acute GTN treatment stimulates endothelial NOS activity in producing NO and increases cellular uptake of L-arginine. Prolonged GTN exposure reduces GTN's vasodilator actions, decreases L-arginine tissue levels and depresses BAECs uptake of L-arginine. Supplementation of L-arginine reduces development of GTN tolerance. These data indicate that GTN tolerance depends in part on activation of the NOS pathway.

Cannabinoid agonists and antagonists discriminated by receptor binding in rat cerebellum.

1. The effect of allosteric regulators on the binding affinity of a number of cannabinoid receptor ligands of varying efficacy in the rat cerebellum was investigated. 2. Radioligand ([3H]-SR141716A) competition curves were constructed in the presence or absence of sodium ions, magnesium ions and guanine nucleotides. 3. It was found that the presence of these allosteric regulators did not affect the affinity of the two antagonists used but did cause a significant decrease in the affinity of full and partial agonists. 4. This reduction in affinity ranged from a 3.67 fold rightward shift of the displacement curve of a mixed agonist/antagonist (3-(6-cyano-2-hexynyl)-delta-8-tetrahydrocannabinol-O-823) to a 38 fold rightward shift for 3-(1, 1-dimethyl-6-dimethylcarboxamide)-delta-8-tetrahydrocannabinol (O-1125), a full agonist. 5. In summary, the results of this study suggest a simple method for the inference of functional data using the classical radioligand binding assay.

The cyclo-oxygenase-dependent regulation of rabbit vein contraction: evidence for a prostaglandin E2-mediated relaxation.

1. Arachidonic acid (0.01-1 microM) induced relaxation of precontracted rings of rabbit saphenous vein, which was counteracted by contraction at concentrations higher than 1 microM. Concentrations higher than 1 microM were required to induce dose-dependent contraction of vena cava and thoracic aorta from the same animals. 2. Pretreatment with a TP receptor antagonist (GR32191B or SQ29548, 3 microM) potentiated the relaxant effect in the saphenous vein, revealed a vasorelaxant component in the vena cava response and did not affect the response of the aorta. 3. Removal of the endothelium from the venous rings, caused a 10 fold rightward shift in the concentration-relaxation curves to arachidonic acid. Whether or not the endothelium was present, the arachidonic acid-induced relaxations were prevented by indomethacin (10 microM) pretreatment. 4. In the saphenous vein, PGE2 was respectively a 50 and 100 fold more potent relaxant prostaglandin than PGI2 and PGD2. Pretreatment with the EP4 receptor antagonist, AH23848B, shifted the concentration-relaxation curves of this tissue to arachidonic acid in a dose-dependent manner. 5. In the presence of 1 microM arachidonic acid, venous rings produced 8-10 fold more PGE2 than did aorta whereas 6keto-PGF1alpha and TXB2 productions remained comparable. 6. Intact rings of saphenous vein relaxed in response to A23187. Pretreatment with L-NAME (100 microM) or indomethacin (10 microM) reduced this response by 50% whereas concomitant pretreatment totally suppressed it. After endothelium removal, the remaining relaxing response to A23187 was prevented by indomethacin but not affected by L-NAME. 7. We conclude that stimulation of the cyclo-oxygenase pathway by arachidonic acid induced endothelium-dependent, PGE2/EP4 mediated relaxation of the rabbit saphenous vein. This process might participate in the A23187-induced relaxation of the saphenous vein and account for a relaxing component in the response of the vena cava to arachidonic acid. It was not observed in thoracic aorta because of the lack of a vasodilatory receptor and/or the poorer ability of this tissue than veins to produce PGE2.

Modulation of AT1 receptor-mediated contraction of rat uterine artery by AT2 receptors.

The aim of this study was to characterize the angiotensin II receptors in isolated uterine arteries from non pregnant and pregnant rats, since it has been reported from binding studies that ovine uterine arteries contain AT2 receptors. Uterine arterial segments were obtained from virgin, non-pregnant and late pregnant (18-21 days) Sprague-Dawley rats and mounted in small vessel myographs. Concentration-response curves were constructed to angiotensin II (1 nM-10 microM) in the absence and presence of various angiotensin II receptor subtype selective compounds. These included losartan (AT1 antagonist; 1, 10 and 100 nM), PD 123319 (AT2 antagonist; 1 microM) and CGP 42112 (AT2 agonist; 1 microM). Responses to angiotensin II were measured as increases in force (mN) and expressed as a per cent of the response to a K+ depolarizing solution. Losartan (1, 10 and 100 nM) caused significant concentration-dependent rightward shifts of the angiotensin II concentration-response curve in uterine arteries from non-pregnant and pregnant rats. The pA2 values calculated from these data were 9.8 and 9.2, respectively, although the slope of the Schild plot in the non-pregnant group was less than unity. PD 123319 (1 microM) caused significant 6- and 3 fold leftward shifts of the angiotensin II concentration-response curve in uterine arteries from non-pregnant and pregnant rats, respectively. In vessels from pregnant rats, PD 123319 also significantly increased the maximum response to angiotensin II. CGP 42112 (1 microM) attenuated the response to angiotensin II of uterine arteries from non-pregnant rats. This was reflected by a 14 fold rightward shift of the angiotensin II concentration-response curve and a decrease in the maximum response. In uterine arteries from pregnant rats, CGP 42112 (1 microM) caused a 3 fold rightward shift of the angiotensin II concentration-response curve, but had no effect on the maximum response. PD 123319 (1 microM) and CGP 42112 (1 microM) had no effect on the concentration-response curves to phenylephrine (PE) of uterine arteries from non-pregnant or pregnant rats. In addition, CGP 42112 (1 nM-1 mM) had no vasodilator effect on tissues precontracted with phenylephrine. These results suggest that the contractile responses of the rat uterine artery are mediated by the AT1 receptor. Furthermore, in this vascular preparation, the AT2 receptor appears to inhibit the response mediated by the AT1 receptor, although, this is not uniform between the non-pregnant and pregnant states.

The actions of the cannabinoid receptor antagonist, SR 141716A, in the rat isolated mesenteric artery.

1. The actions of the cannabinoid receptor antagonist, SR 141716A, were examined in rat isolated mesenteric arteries. At concentrations greater than 3 microM, it caused concentration-dependent, but endothelium-independent, relaxations of both methoxamine- and 60 mM KCl-precontracted vessels. 2. SR 141716A (at 10 microM, but not at 1 microM) inhibited contractions to Ca2+ in methoxamine-stimulated mesenteric arteries previously depleted of intracellular Ca2+ stores. Neither concentration affected the phasic contractions induced by methoxamine in the absence of extracellular Ca2+ 3. SR 141716A (10 microM) caused a 130 fold rightward shift in the concentration-response curve to levcromakalim, a K+ channel activator, but had no effect at I microM. 4. SR 141716A (10 microM) attenuated relaxations to NS 1619 (which activates large conductance. Ca2+-activated K+ channels; BK(Ca)). The inhibitory effect of SR 141716A on NS 1619 was not significantly different f'rom, and was not additive with, that caused by a selective BKc,, inhibitor, iberiotoxin (100 nM). SR 141716A (1 microM) did not effect NS 1619 relaxation. 5. SR 141716A (10 microM) had no effect on relaxations to the nitric oxide donor S-nitroso-N-acetylpenicillamine, or relaxations to carbachol in the presence of 25 mM KCl. 6. The results show that, at concentrations of 10 microM and above. SR 141716A causes endothelium-independent vasorelaxation by inhibition of Ca2+ entry. It also inhibits relaxations mediated by K+ channel activation. This suggests that such concentrations of SR 141716A are not appropriate for investigation of cannabinoid receptor-dependent processes.

Characterization of NS 2028 as a specific inhibitor of soluble guanylyl cyclase.

1 The haeme-containing soluble guanylyl cyclase (alpha1beta1-heterodimer) is a major intracellular receptor and effector for nitric oxide (NO) and carbon monoxide (CO) and mediates many of their biological actions by increasing cyclic GMP. We have synthesized new oxadiazolo-benz-oxazins and have assessed their inhibitory actions on guanylyl cyclase activity in vitro, on the formation of cyclic GMP in cultured cells and on the NO-dependent relaxation of vascular and non-vascular smooth muscle. 2 Soluble guanylyl cyclase, purified to homogeneity from bovine lung, was inhibited by 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one (NS 2028) in a concentration-dependent and irreversible manner (IC50 30 nM for basal and 200 nM for NO-stimulated enzyme activity). Evaluation of the inhibition kinetics according to Kitz & Wilson yielded a value of 8 nM for Ki, the equilibrium constant describing the initial reversible reaction between inhibitor and enzyme, and 0.2 min(-1) for the rate constant k3 of the subsequent irreversible inhibition. Inhibition was accompanied by a shift in the soret absorption maximum of the enzyme's haem cofactor from 430 to 390 nm. 3 S-nitroso-glutathione-enhanced soluble guanylyl cyclase activity in homogenates of mouse cerebellum was inhibited by NS 2028 (IC50 17 nM) and by 17 structural analogues in a similar manner, albeit with different potency, depending on the type of substitution at positions 1, 7 and 8 of the benzoxazin structure. Small electronegative ligands such as Br and Cl at position 7 or 8 increased and substitution of the oxygen at position 1 by -S-,- NH- or -CH2- decreased the inhibition. 4 In tissue slices prepared from mouse cerebellum, neuronal NO synthase-dependent activation of soluble guanylyl cyclase by the glutamate receptor agonist N-methyl-D-aspartate was inhibited by NS 2028 (IC50 20 nM) and by two of its analogues. Similarly, 3-morpholino-sydnonimine (SIN-1)-elicited formation of cyclic GMP in human cultured umbilical vein endothelial cells was inhibited by NS 2028 (IC50 30 nM). 5 In prostaglandin F2alpha-constricted, endothelium-intact porcine coronary arteries NS 2028 elicited a concentration-dependent increase (65%) in contractile tone (EC50 170 nM), which was abolished by removal of the endothelium. NS 2028 (1 microM) suppressed the relaxant response to nitroglycerin from 88.3+/-2.1 to 26.8+/-6.4% and induced a 9 fold rightward shift (EC50 15 microM) of the concentration-relaxation response curve to nitroglycerin. It abolished the relaxation to sodium nitroprusside (1 microM), but did not affect the vasorelaxation to the KATP channel opener cromakalim. Approximately 50% of the relaxant response to sodium nitroprusside was recovered after 2 h washout of NS 2028. 6 In phenylephrine-preconstricted, endothelium-denuded aorta of the rabbit NS 2028 (1 microM) did not affect relaxant responses to atrial natriuretic factor, an activator of particulate guanylyl cyclase, or forskolin, an activator of adenylyl cyclase. 7 NO-dependent relaxant responses in non-vascular smooth muscle were also inhibited by NS 2028. The nitroglycerin-induced relaxation of guinea-pig trachea preconstricted by histamine was fully inhibited by NS 2028 (1 microM), whereas the relaxations to terbutaline, theophylline and vasoactive intestinal polypeptide (VIP) were not affected. The relaxant responses to electrical field stimulation of non-adrenergic, non-cholinergic nerves in the same tissue were attenuated by 50% in the presence of NS 2028 (1 microM). 8 NS 2028 and its analogues, one of which is the previously characterized 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ), appear to be potent and specific inhibitors of soluble guanylyl cyclase present in various cell types. Oxidation and/or a change in the coordination of the haeme-iron of guanylyl cyclase is a likely inhibitory mechanism.

Analysis of relaxation and repolarization mechanisms of nicorandil in rat mesenteric artery.

1. The mechanisms by which nicorandil causes relaxation of rat isolated small mesenteric arteries mounted on a Mulvany myograph was investigated by use of a combination of putatively mechanism-specific antagonists. 2. In arteries precontracted by the thromboxane-mimetic, U46619, the EC50 for cromakalim and levcromakalim-induced relaxation curves were raised by 36 and 17 fold by glibenclamide (3 microM) while the EC50 for nicorandil-induced relaxation was unaffected by either glibenclamide or methylene blue (10 microM). A combination of these antagonists raised the EC50 for nicorandil by 8 fold. 3. In U46619-contracted arteries, nifedipine (100 nM) did not affect the cromakalim relaxation curve but it raised the EC50 for nicorandil by 5 fold. The combination of methylene blue, glibenclamide and nifedipine further inhibited the maximum relaxation to nicorandil. 4. In separate experiments, membrane potential (Em) and force responses were measured simultaneously. In arteries depolarized and contracted by U46619 both nicorandil and cromakalim repolarized (delta Em, 35 mV) and relaxed (100%) the vessels. Glibenclamide (3 microM) did not alter the relaxation-concentration curve to nicorandil but reduced the maximum repolarization to delta 10.8 mV. In contrast to Em and relaxation-response curves to cromakalim were shifted to the right by glibenclamide by 30-100 fold. 5. In unstimulated arteries, nicorandil (but not cromakalim) -induced hyperpolarization was significantly antagonized by methylene blue (10 microM) (6.6 fold rightward shift) or nifedipine (100 nM) (2.6 fold). In depolarized arteries (U46619), nifedipine but not methylene blue inhibited the nicorandil-induced hyperpolarization. 6. In arteries precontracted to 50% tissue maximum by either KCl or U46619, nifedipine (100 nM) relaxed the artery but failed to repolarize the Em. Presumably voltage-operated calcium channels (VOCC) were blocked preventing contraction but the artery remained depolarized, presumably through non VOCC mechanisms. 7. These data suggest that nicorandil may relax small arteries through 3 parallel pathways, (i) NO-donor mediated stimulation of guanylate cyclase and increase in cyclic GMP, (ii) K+ATP channel opening, and (iii) nifedipine-sensitive VOCC inhibition. Em data suggest that nicorandil-induced repolarization is caused principally through opening K+ATP channels. Blockade of this hyperpolarization by glibenclamide is not sufficient to alter the relaxation, indicating dissociation of nicorandil-induced changes in membrane potential and relaxation. 8. These results highlight the 'chameleon' actions of nicorandil where there is no apparent association of Em repolarization with relaxation, in contrast to the parallel responses for cromakalim.

Changes in sensitivity of 5-HT receptor mediated functional responses in the rat oesophagus, fundus and jejunum following chronic infusion with 5-hydroxytryptamine.

The effects of chronic infusion with 5-hydroxytryptamine (5-HT, 75 micrograms/kg per hour) for 5 or 10 days in vivo on the responses of rat oesophagus, fundus and jejunum to 5-HT and partial 5-HT receptor agonists in vitro were investigated. In the rat oesophagus, chronic treatment produced rightward shifts of the 5-HT4 receptor-mediated concentration-effect curves to 5-HT (dose-ratio = 3.8, day 5 and 2.8, day 10) and SC 53116 (1-S,8-S)-4-amino-5-chloro-N-[(hexahydro-1H-pyrrolizin-1-yl) methyl]-2-methoxy-benzamide hydrochloride, dose-ratio = 7.1, day 5 and 8.9, day 10) as compared to control tissues. The maximum effect of 5-HT and SC 53116 in rat oesophagus was reduced following the 10 day treatment. The 5-HT2B receptor-mediated contractile effect of 5-HT on rat fundus from treated animals responded with a significantly reduced potency (dose-ratio = 4.1, 5-day; 4.2, 10-day) compared to control tissues. The maximum response to 5-HT was reduced in tissues from animals treated for 10 days. The concentration-effect curve to the partial agonist 1-(3-chlorophenyl)-piperazine dihydrochloride (mCPP) was shifted to the right in fundic tissue from treated animals (dose-ratio = 2.5, 5-day; 2.8, 10-day) compared to control tissues. The maximum response to mCPP was also reduced in tissues from 5-HT treated animals. It has recently been shown in a preliminary characterisation that in rat jejunum, 5-HT produces a biphasic concentration-effect curve which is mediated by a putative 5-ht7 (first phase) and 5-HT3 (second phase) receptor mechanism. In the present study 5-HT produced a control biphasic concentration-effect curve in rat jejunum with a pEC50-1 value of 8.5 +/- 3.5 and a pEC50-2 value of 5.9 +/- 0.3 and maximum response values of 43.8 (32.5-55.0)% (Emax1) and 65.3 (41.7-88.9)% (Emax 2) of the response to acetylcholine. In jejunal tissues from treated animals, 5-HT produced its contractile effect in vitro with a 3.7 and 2.8 fold (5-day) and a 1.3 and 1.4 fold (10-day) rightward shift of the first and second phase respectively of the control concentration-effect curve to 5-HT. The maximum response produced by 5-HT in the first phase in jejunal tissues from animals treated for 10 days was significantly reduced by 49.1%. These findings represent the first report that chronic infusion of 5-HT produces a residual desensitisation of the 5-HT4, 5-HT2B, and putative 5-ht7/5-HT3 receptor-mediated responses in rat oesophagus, fundus and jejunum respectively when measured in vitro.

Time course of changes in ETB receptor density and function in tracheal airway smooth muscle during respiratory tract viral infection in mice

1. In the current study, the density and function of ETA and ETB receptors in mouse tracheal airway smooth muscle were determined over the time course of respiratory tract infection with influenza A/PR-8/34 virus. 2. Quantitative autoradiographic studies using [125I]-endothelin-1 revealed that the tracheal airway smooth muscle from control mice contained ETA and ETB sites in the ratio of 49%:51% (+/- 2%, n = 29 mice). Respiratory tract viral infection was associated with increases in the density of ETA sites and decreases in the density of ETB sites at days 1, 2 and 4 post-inoculation which were reversible by day 19. For example, at day 4 post-inoculation, a time when the manifestations of viral infection were at or near their peak, the ratio of ETA:ETB sites was 72%:28% (+/- 4%, n = 6 mice, P < 0.05). In contrast, at day 19 post-inoculation, by which time viral infection had essentially resolved, the ratio of ETA:ETB sites was similar to control (51%:49% (+/- 3%), n = 6 mice). 3. Endothelin-1 was a potent spasmogen in isolated tracheal airway smooth muscle preparations from control mice (ED70 = concentration producing 70% of contraction induced by 10 microM carbachol = 6.3 nM (95% confidence limits, 4.0-10; n = 6 mice)). Neither the ETA receptor-selective antagonist, BQ-123 (3 microM), nor the ETB receptor-selective antagonist, BQ-788 (1 microM) alone had any significant inhibitory effect on endothelin-1-induced contractions of mouse isolated tracheal smooth muscle. However, simultaneous treatment with BQ-123 (3 microM) and BQ-788 (1 microM) resulted in a 10 fold rightward shift in the concentration-effect curve to endothelin-1 (ED70 = 60 nM, (44-90; n = 6 mice, P < 0.05)), indicating that contraction was mediated via both ETA and ETB receptors. 4. Endothelin-1 evoked similar concentration-dependent contractions of tracheal smooth muscle isolated from control and virus-inoculated mice. In the presence of the ETB receptor-selective-antagonist, BQ-788 (1 microM), the potency and maximum response to endothelin-1 were similar in preparations from control and virus-inoculated mice at all time points investigated. However, unlike control responses, endothelin-1-induced contractions in preparations from virus-infected mice were significantly inhibited by the ETA receptor-selective antagonist, BQ-123. For example, at day 4 post-inoculation, the contractile response to 30 nM endothelin-1, in the presence of BQ-123 (3 microM), was only 20 +/- 12% (n = 6 mice, P < 0.05) of that produced in control preparations under similar conditions. However, at day 19 post-inoculation, contraction evoked by 30 nM endothelin-1 in the presence of BQ-123 (3 microM), was similar to that in preparations from control mice. 5. In summary, during the early stages (days 1-8 post-inoculation) of respiratory tract infection with influenza A/PR-8/34 virus, we observed decreases in the density of tracheal airway smooth muscle ETB receptors which were reflected in decreases in ETB receptor-mediated airway smooth muscle contraction. In addition, during the same period of viral infection we observed increases in the density of tracheal airway smooth muscle ETA receptors which were not associated with increased function of the ETA receptor-effector system linked to contraction. Virus-associated modulation of ETA and ETB receptor density and function was reversible with recovery from infection.

ETB but not ETA receptor-mediated contractions to endothelin-1 attenuated by respiratory tract viral infection in mouse airways.

1. The current study investigated the effects of respiratory tract viral infection on the density of ETA and ETB receptors in murine tracheal smooth muscle and on the contractile response to endothelin-1 mediated by these receptors. 2. Quantitative autoradiographic studies using [125I]-endothelin-1 revealed that tracheal smooth muscle from control mice contained ETA and ETB receptors in the ratio of 42%:58% (+/- 4%, n = 10 mice), respectively. In contrast, tracheal smooth muscle obtained from mice 2 days post-inoculation with Influenza A/PR-8/34 virus contained 23 +/- 2% fewer receptors for [125I]-endothelin-1 (n = 10, P < 0.01). This reflected a selective reduction in ETB receptor density and a change in the ratio of ETA and ETB receptors to 77%:23% (+/- 5%, n = 10 mice), respectively. 3. The ETB receptor-selective agonist, sarafotoxin S6c, was a potent spasmogen of murine isolated tracheal smooth muscle and the EC50 for contraction was similar in preparations from control (3.6 nM [95% confidence limits, 2.7-4.8 nM], n = 16 preparations from 8 mice) and virus-inoculated mice (3.0 nM [2.4-3.7 nM], n = 16 preparations from 8 mice). However, the maximum contractions induced by sarafotoxin S6c (100 nM) in the preparations from virus-inoculated mice (37 +/- 5% Cmax, where 100% Cmax was the response to 10 microM carbachol) were significantly smaller than those from control mice (85 +/- 4% Cmax, P < 0.01). 4. Contractions induced by endothelin-1 in tracheal smooth muscle preparations obtained from virus inoculated mice (EC50 for contraction, 6.5 nM [95% confidence limits, 2.7-16 nM]; maximum contraction,112 +/- 5% Cm.; n = 4) were similar to those induced by endothelin-1 in control preparations (ECm9.3 nM (4.2-21); maximum contraction, 110 +/- 3% Cmax; n = 4). Endothelin-1-induced contractions in control preparations were only marginally inhibited by the ETA receptor-selective antagonist BQ-123 (in the presence of 3 micro M BQ-123; EC50 for contraction, 5.9 nM [4.1-8.5]; maximum contraction, 82 +/- 4%Cmax; n = 4). In contrast, 3 microM BQ-123 caused a 50 fold rightward shift (17-160, n =4) of the concentration-effect curve to endothlin-1 in preparations obtained from virus-inoculated mice (measured at the 30% Cmax level of contraction).5. Tracheal smooth muscle preparations exposed to 100 nM sarafotoxin S6c for 30 min (followed by a 30 min washout period) did not contract to subsequently administered sarafotoxin S6c (1-100 nM;n = 8), but contracted normally in response to endothelin-1 (EC50 6.5 nM (2.3-18); maximum contraction,109 +/- 2% Cmax; n = 4). Endothelin-l-induced contractions in these ETB receptor desensitized preparations were markedly inhibited by 3 microM BQ-123, irrespective of whether the preparations were obtained from control (63 fold shift (10-400) at the 30% Cma. level of contraction, n = 4) or virus inoculated mice (46 fold shift (18-120), n = 4).6. In summary, tracheal smooth muscle obtained from mice infected with a respiratory tract virus,Influenza A/PR-8/34 had a reduced density of ETB receptors and an attenuated ETB receptor-mediated contractile response to sarafotoxin S6c and endothelin-1. Virus-inoculation was also associated with a modest increase in tracheal smooth muscle ETA receptor density, although no significant change in ETA receptor-mediated contractile activity was seen. Thus, virus infection in murine airways produced profound alterations in endothelin receptor density, some of which were associated with changes in receptor-mediated contractile activity.

Dependence of endotoxin-induced vascular hyporeactivity on extracellular L-arginine.

1. The dependence on extracellular L-arginine of vascular hyporeactivity induced by bacterial lipopolysaccharide (LPS) was studied in vivo in rats infused with LPS and in vitro in endothelium-denuded rat thoracic aortic rings exposed to LPS. 2. Infusion of LPS during 50 min at a dose of 10 mg kg-1 h-1 produced a significant impairment of the pressor effect of noradrenaline, while in tissues collected 60 min after the start of LPS infusion, no significant alteration in either plasma arginine concentration or aortic arginine content was found compared to saline-infused controls (where plasma arginine was 78.5 +/- 7 microM and aortic arginine 394 +/- 124 nmol g-1 tissue). 3. Incubation of isolated, endothelium-denuded aortic rings with LPS (10 micrograms ml-1) in the absence of L-arginine for 4 h at 37 degrees C produced a 6 fold (P < 0.01) rightward shift in the noradrenaline concentration-effect curve compared to polymyxin B (1 micrograms ml-1, a LPS neutralizing agent) and reduced by 15% the maximum observed tension. 4. The presence of L-arginine (100 microM) during the incubation with LPS and throughout the following contraction experiments caused a 15 fold (P < 0.01) increase in the EC50 of noradrenaline and greater depression (45%) of the maximum observed tension compared to polymyxin B-treated controls. Responses in control, non LPS-treated rings were unaffected by the presence of L-arginine. 5. The addition of L-arginine to rings incubated with LPS in the absence of L-arginine and maximally precontracted with noradrenaline (10 microM) induced a dose-dependent relaxation. The EC50 of L-arginine was 8.0+/-0.3mu.6. The reactivity of LPS-treated rings to noradrenaline both in the absence and presence of L-arginine was restored to control levels by N0-nitro-L-arginine methyl ester (L-NAME, 300 mu), an inhibitor of NO production and by methylene blue (3 JAM), an inhibitor of guanylate cyclase.7. Incubation of isolated aortae in the absence of L-arginine did not significantly decrease the tissue arginine content, whether LPS (10 fg ml-') was present or not. Similarly, the presence of L-arginine(100 mu) in the incubation medium did not modify the tissue arginine content.8. These results show that the LPS-induced impairment of vasoconstriction elicited by noradrenaline is dependent on extracellular L-arginine, although the tissue arginine content is not depleted after LPS pretreatment, and that circulating L-arginine is sufficient to activate maximally the vascular L-arginine/NO pathway in endotoxaemic rats.

Impaired endothelium-dependent relaxation of dog coronary arteries after myocardial ischaemia and reperfusion: prevention by amlodipine, propranolol and allopurinol.

1. Anaesthetized, open-chest dogs were subjected to 60 min of left circumflex coronary artery occlusion followed by 90 min of reperfusion. Endothelium-dependent and -independent relaxant responses of the isolated coronary arterial rings were then investigated. 2. The endothelium-dependent, acetylcholine-induced relaxation of ischaemic/reperfused arterial rings was significantly attenuated in comparison to control rings (1.9 fold rightward shift, ischaemic/reperfused maximum relaxation = 57 +/- 13% of control maximum relaxation; P less than 0.05). In contrast, glyceryl trinitrate produced similar relaxant responses in control and ischaemic rings. 3. Pretreatment of dogs with either amlodipine (3 micrograms kg-1 min-1, i.v.) or propranolol (1 mg kg-1, i.v.) completely prevented the postischaemic impairment of endothelium-dependent relaxant responses (100 +/- 3% and 90 +/- 5% of control maximum relaxation, respectively). 4. Allopurinol pretreatment (25 mg kg-1, p.o. 24 h previously, plus 50 mg kg-1 i.v. 5 min before arterial occlusion) partially protected against endothelial dysfunction by preventing the ischaemia-induced rightward shift of the acetylcholine relaxation curve and increasing the maximum relaxation response (83 +/- 7% of control rings). 5. These results confirm that endothelium-dependent coronary vascular relaxation is impaired by ischaemia and reperfusion, and that the ischaemia-induced impairment is reduced by pretreatment with amlodipine, propranolol or allopurinol.

Characterization of catecholamine‐mediated relaxations in rat isolated gastric fundus: evidence for an atypical β‐adrenoceptor

1. Experiments were carried out in order to characterize the receptors mediating relaxant responses to catecholamines in the rat gastric fundus. The effects of noradrenaline, isoprenaline and the 'atypical' or beta 3-adrenoceptor agonist, BRL 37344, on methacholine-induced tone were measured. Prazosin, propranolol and cyanopindolol were used as antagonists. 2. Relaxant responses to noradrenaline, in the presence of propranolol (1 microM) were antagonized in a concentration-dependent manner by prazosin (0.01 to 1 microM), although this antagonism was weak and non-competitive in nature. Relaxant responses to isoprenaline, in the presence of prazosin (0.1 microM), were antagonized only by the highest concentration of propranolol (1 microM) giving a pKB of 6.3 BRL 37344 also relaxed the rat gastric fundus in the presence of prazosin (0.1 microM), and the responses to BRL 37344 were unaffected by propranolol (1 microM). 3. Tachyphylaxis to BRL 37344 was observed, a second concentration-response curve being significantly shifted to the right. Exposure of tissues to BRL 37344 (1 microM) between concentration-response curves also caused an 11 fold rightward shift in the response to isoprenaline. 4. In the presence of prazosin (0.1 microM) and propranolol (1 microM), the rank order of potency of the agonists was: (-)-isoprenaline (1.0) greater than (-)-noradrenaline (0.39) greater than BRL 37344 (0.10). 5. Responses to BRL 37344 in the presence of prazosin (0.1 microM) and propranolol (1 microM) were antagonized by (+/-)-cyanopindolol (1 microM), with a pKB of 6.56. Responses to isoprenaline, under the same conditions, were antagonized in a competitive manner by (+/-)-cyanopindolol (0.1-1 microM), with the slope of a Schild plot close to unity and a pA2 value of 7.44. 6. The resistance to blockade by prazosin and propranolol and the antagonism by cyanopindolol of the responses mediated by isoprenaline and BRL 37344 suggest that atypical beta-adrenoceptors similar to 'atypical',beta-adrenoceptors in rat adipocytes and other tissues are present in the rat gastric fundus.

Effects of calcium entry blockers on contractions evoked by endothelin‐1, [Ala3,11]endothelin‐1 and [Ala1,15]endothelin‐1 in rat isolated aorta

1. The aim of the present study was to examine the contractile responses evoked by the recently characterized vasoactive peptide endothelin-1 (ET-1) and by two of its structural analogues, [Ala3,11]ET-1 and [Ala1,15]ET-1 in endothelium-denuded rat isolated aorta, and also to assess the extent of dependence of these responses on extracellular calcium entry. 2. ET-1 (0.3 to 10 nM), [Ala3,11]ET-1 (2.25 to 225 nM) and [Ala1,15]ET-1 (0.04 to 1.36 microM) evoked concentration-dependent contractions in normal, calcium-containing medium with the order of potency: ET-1 greater than [Ala3,11]ET-1 greater than [Ala1,15]ET-1. 3. Preincubation of tissues for 60 min with diltiazem (1 microM) induced a significant 3 fold rightward shift of concentration-effect curves to ET-1 without affecting maximal responses elicited by 10 nM of this peptide, whereas the same treatment failed to modify concentration-effect curves to [Ala3,11]ET-1. 4. Preincubation of tissues for 60 min with nifedipine (0.1 or 1 microM) markedly inhibited contractions elicited by either ET-1 (10 nM) or [Ala1,15]ET-1 (0.41 microM). Furthermore, when added cumulatively to tissues maximally contracted by ET-1 (10 nM), nifedipine (3 nM to 1 microM) induced concentration-dependent relaxations with an IC50 value of 21.8 +/- 5.9 nM. Maximal relaxation to nifedipine, 1 microM, amounted to 56.9 +/- 11.5%. 5. Submaximal concentrations of ET-1 (3 nM), [Ala3,11]ET-1 (75 nM) and [Ala1,15]ET-1 (0.41 microM), gave about 85% of maximal contractions elicited by noradrenaline (1 microM) in normal, calcium-containing medium. These contractile responses were all reduced by about 70% in calcium-free medium. These contractile responses were all reduced by about 70% in calcium-free medium. Pretreatment of tissues with diltiazem (1 microM) or with nifedipine (0.1 or 1 microM) did not affect these residual contractions. 6. Upon readdition of calcium (10 microM to 10mM) to tissues in calcium-free medium, preincubated with submaximal concentrations of one or the other of the peptides, concentration-dependent contractions were elicited with EC50 values of 1.21 + 1.1 mm (ET-1-exposed rings), 74.6 + 9.1 microM ([Ala31 ']ET-1-exposed rings) and 102 + 27 microM ([Ala' 15]ET-1-exposed rings). Calcium-induced responses were significantly inhibited by diltiazem (1 microM) in both ET-1- and [Ala3"1 ']ET-1-exposed tissues and by nifedipine (0.1 and 1 microM) in ET-1-exposed tissues. However, nifedipine did not significantly affect calcium induced responses in [Ala"'15]ET-1-exposed rings. 7. Readdition of calcium to a calcium-free medium containing 40mm K+, evoked concentrationdependent responses that were unaffected by the presence of ET-1 (3 nM). 8. Overall, these results indicate that contractions to ET-1 and its analogues in rat aorta are to some extent dependent on extracellular calcium. However, the mode of antagonism by diltiazem and nifedipine of responses to the peptides in normal medium, as well as of contractions induced by readdition of calcium in peptide-exposed tissues in calcium-free medium, argue against a direct activation by endothelin or its analogues of L-type calcium channels. Finally, removal of one or the other of the disulphide bonds of ET-1 reduces the potency of the peptide.

Antagonism of PGD2 vasodepressor responses in the rat in vivo by the novel, selective antagonist, BW A868C.

1. Bolus intravenous injection of prostaglandin D2 (PGD2, 1-160 micrograms kg-1), the hydantoin prostanoid BW245C (0.25-160 micrograms kg-1) or prostacyclin (PGI2, 0.05-0.5 microgram kg-1) caused a dose-dependent fall in systemic arterial blood pressure (BP) in the anaesthetized rat, lasting 2-4 min. 2. Intravenous infusion of the novel 3-benzyl substituted hydantoin, BW A868C (1-10 micrograms kg-1 min-1), in doses that had no direct effect on BP, dose-dependently reduced the vasodepressor action of PGD2. 3. Bolus injection of BW A868C (30 and 100 micrograms kg-1, i.v.) likewise dose-dependently antagonized the vasodepressor responses to PGD2, causing a 3.4 and 13.2 fold rightward shift of the dose-response curve. 4. The thromboxane-receptor antagonist, BM 13.177 (2.5 mg kg-1 i.v.) had little effect on the PGD2 vasodepressor responses, suggesting minimal contribution of a PGD2 interaction at thromboxane receptor-sites in the systemic vasculature of this species. 5. BW A868C (10 micrograms kg-1 min-1 i.v.) caused a rightward shift (59 fold) of the dose-response relationship for BW245C, the putative PGD2-receptor agonist. This antagonism lasted for at least 1h after termination of the BW A868C infusion. Higher doses of BW A868C (20-100 micrograms kg-1 min-1) caused no further antagonism of the vasodepressor responses to BW245C, suggesting that this prostanoid also acts at vascular receptors other than of the DP-type. 6. BW A868C (10 micrograms kg-1 min-1, i.v.) failed to alter the vasodepressor actions of prostacyclin. 7. These findings in the rat in vivo support the characterization of BW A868C as a potent and selective antagonist of the cardiovascular actions of PGD2 at the DP-receptor.