Contraction Of Guinea Pig Trachea Research Articles

Nitric oxide pathway-mediated relaxant effect of PPAR (Peracisome proliferator activated receptor)-Gamma agonist in the guinea-pig isolated trachea.

Objectives: This study focuses on the role of the mediator of smooth muscles i.e. nitric oxide in the effect of rosiglitazone on the relaxation of contractions in guinea pig trachea induced by histamine. Study Design: Experimental Study. Setting: Department of Pharmacology, Army Medical College, Rawalpindi. Period: 1st December 2018 till 14th December 2018. Material & Methods: The activity of the trachealis mucle of the guinea pigs was noted by the use of oxygenated solution of Krebs-Henseleit. The contractions were noted with rosiglitazone alone as well as in presence of an ester of L-nitro-arginine-methyl, which can antagonize the activity of NO. The activity was measured by the use of an isometric force displacement transducer and was then recorded on an oscillograph with four channels. Results: The ester L-nitro-arginine-methyl was able to reduce the relaxant effect of rosiglitazone on the contractions induced by histamine. Conclusion: It can be suggested that the relaxation induced by rosiglitazone is mediated in part through NO.

Pharmacological Evaluation of Gut Modulatory and Bronchodilator Activities of Achyranthes aspera Linn.

Achyranthes aspera L. is traditionally used to relieve constipation, diarrhea, and asthma. Its crude extract (Aa.Cr) was evaluated through in vivo and ex vivo experiments to rationalize these medicinal uses of A. aspera and to provide their scientific basis. Aa.Cr, at 3 and 10mg/kg, increased fecal output, similar to castor oil, whereas at 30, 100, 300, and 700mg/kg, it protected against castor oil-induced diarrhea in mice when administered orally. Aa.Cr caused spasmogenic effect on rabbit jejunum and guinea pig ileum preparations, which was partially inhibited by atropine while completely blocked by cyproheptadine preincubation. Aa.Cr also relaxed high K+ (80mM)-induced contraction in rabbit jejunum. Aa.Cr inhibited CCh (100μg/kg)-induced bronchospasm in rats, similar to aminophylline. Like dicyclomine, Aa.Cr relaxed high K+ and CCh (1μM)-induced contractions in guinea pig trachea and caused rightwards parallel shift of CCh concentration-response curves at the lower concentrations followed by non-parallel shift at the higher concentrations. On activity-directed fractionation, spasmogenic and spasmolytic activities of Aa.Cr were concentrated in aqueous and organic fraction, respectively. This study suggests the presence of dose-specific laxative and antidiarrheal effects in A. aspera, possibly mediated through cyproheptadine-sensitive receptors and dual cholinergic and calcium channel blockade, respectively. The latter combination is also a suggested mechanism underlying its bronchodilator effect. Copyright © 2017 John Wiley & Sons, Ltd.

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Is a Second Messenger in Muscarinic Receptor-induced Contraction of Guinea Pig Trachea

Nicotinic acid adenine dinucleotide phosphate (NAADP) is increasingly being demonstrated to be involved in calcium signaling in many cell types and species. Although it has been shown to play a role in smooth muscle cell contraction in several tissues, nothing is known about its possible role in tracheal smooth muscle, a muscle type that is clinically relevant to asthma. To determine whether NAADP functions as a second messenger in tracheal smooth muscle contraction, we used the criteria set out by Sutherland for a molecule to be designated a second messenger. We report that NAADP satisfies all five criteria as follows. First, the NAADP antagonist Ned-19 inhibited contractions in tracheal rings and calcium increases in isolated smooth muscle cells induced by the muscarinic agonist carbachol. Second, NAADP increased cytosolic calcium in isolated cells when microinjected and was blocked by Ned-19. Third, tracheal homogenates could synthesize NAADP by base exchange from exogenous NADP and nicotinic acid and metabolize exogenous NAADP to nicotinic acid adenine dinucleotide by a 2'-phosphatase. Fourth, carbachol induced a rapid and transient increase in endogenous NAADP levels. Fifth, tracheal homogenates contained NAADP-binding sites of high affinity. Taken together, these data demonstrate that NAADP functions as a second messenger in tracheal smooth muscle, and therefore, steps in the NAADP signaling pathway might provide possible new drug targets.

Selectivity profiling of the novel EP2 receptor antagonist, PF‐04418948, in functional bioassay systems: atypical affinity at the guinea pig EP2 receptor

Understanding the role of the EP(2) receptor has been hampered by the lack of a selective antagonist. Recently, a selective EP(2) receptor antagonist, PF-04418948, has been discovered. The aim of this study was to demonstrate the selectivity profile of PF-04418948 for the EP(2) receptor over other EP receptors using a range of isolated tissue systems. PF-04418948 was profiled on a range of isolated tissues to assess its EP receptor potency and selectivity: ONO-DI-004-induced contraction of guinea pig trachea (EP(1)); ONO-AE1-259 and PGE(2)- induced relaxation of mouse and guinea pig trachea (EP(2)); PGE(2)-induced depolarization of guinea pig isolated vagus (EP(3)); PGE(2)-induced relaxation of human and rat trachea (EP(4)). PF-04418948 was also profiled in functional murine TP, IP, DP and FP receptor assays. In bioassay systems, where assessment of potency/selectivity is made against the 'native' receptor, PF-04418948 only acted as an antagonist of EP(2) receptor-mediated events. PF-04418948 competitively inhibited relaxations of murine and guinea pig trachea induced by ONO-AE1-259 and PGE(2) respectively. However, the affinity of PF-04418948 was not equal in the two preparations. Using a wide range of bioassay systems, we have demonstrated that PF-04418948 is a selective EP(2)-receptor antagonist. Interestingly, an atypically low affinity was found on the guinea pig trachea, questioning its utility as an EP(2) receptor assay system. Nevertheless, this compound should be an invaluable tool for investigating the biological activity of PGE(2) and the role of EP(2) receptors in health and disease.

The bitter taste receptor (TAS2R) agonists denatonium and chloroquine display distinct patterns of relaxation of the guinea pig trachea

Activation of taste receptors (TAS2Rs) by bitter taste agonists has been reported to cause bronchodilation. The aim of this study was to extend the information on the effects of bitter taste agonists on responses induced by different contractile mediators in a standard airway physiology preparation. Isometric responses were assessed in guinea pig trachea (GPT). TAS2R agonists were administered either to segments precontracted with different agonists for contraction or given before challenge with the different contractile stimuli, including antigen in tissues from ovalbumin-sensitized animals. TAS2R mRNA expression on GPT epithelium and smooth muscle was measured with real-time PCR. Denatonium, chloroquine, thiamine, and noscapine induced concentration-dependent relaxations (R(max): 98.3 ± 1.6, 100.0 ± 0.0, 100.0 ± 0.0, and 52.3 ± 1.1% of maximum, respectively, in the presence of indomethacin) in segments precontracted with carbachol. The receptors for denatonium (TAS2R4, TAS2R10) and chloroquine (TAS2R3, TAS2R10) were expressed in GPT. Whereas denatonium selectively inhibited contractions induced by carbachol, chloroquine uniformly inhibited contractions evoked by prostaglandin E(2), the thromboxane receptor agonist U-46619, leukotriene D(4), histamine, and antigen. The effects of denatonium, but not those of chloroquine, were partly inhibited by blockers of the large Ca(2+)-activated K(+) channels and decreased by an increase of the level of precontraction. In conclusion, TAS2R agonists mediated strong relaxations and substantial inhibition of contractions in GPT. Chloroquine and denatonium had distinct patterns of activity, indicating different signaling mechanisms. The findings reinforce the hypothesis that TAS2Rs are potential targets for the development of a new class of more efficacious agonists for bronchodilation.

Intestinal, Airway, and Cardiovascular Relaxant Activities of Thymoquinone

Thymoquinone (TQ) is a bioactive component found in many medicinal herbs. In this study, we report the smooth and cardiac muscle relaxant activities of this compound. TQ concentration dependently suppressed spontaneously contracting rabbit jejunum while also relaxed high K+-(80 mM) induced contractions in jejunum and guinea-pig ileum, indicating activity at voltage-operated Ca++ channels (VOCC). Further, TQ displaced Ca++ concentration-response curves, obtained in a Ca++-free environment, to the right, showing blockade of VOCC. Similar activity was observed with verapamil, a standard VOCC blocker. TQ also exhibited nonadrenergic relaxation of agonist-induced contractions in guinea-pig trachea. When tested in fluo-4-loaded mouse lung slices, TQ inhibited ACh-induced airway narrowing and Ca++ signalling in airway smooth muscle cells. In endothelium-intact and endothelium-denuded rat aorta, TQ inhibited high K+-induced contractions at significantly lower concentrations than phenylephrine-(PE-) (1 microM) induced contractions. Relaxation of PE-induced contractions was resistant to blockade by L-NAME and atropine. In guinea-pig atria, TQ showed noncholinergic relaxation of atrial force and rate of contractions. These data suggest smooth and cardiac muscle relaxant activity of TQ possibly mediated, in part, via blockade of VOCC. The results also justify the use of TQ containing plants in related health disorders like colic, diarrhoea, cough, and asthma.

Anticholinergic activity of the dopamine receptor agonist, TL-68 (N,N-dipropyl-2-aminotetralin).

The anticholinergic properties of a dopamine receptor agonist, a non-hydroxylated derivative of N,N-dipropylaminotetralin (TL-68), were evaluated using the guinea-pig isolated tracheal strip and rat phrenic nerve-diaphragm preparations. TL-68 competitively antagonized carbachol-induced contractions in guinea-pig trachea with a pA2 value of 5.88 +/- 0.05. In the rat phrenic nerve-diaphragm preparation, TL-68 was found to be inactive in blocking nicotinic cholinergic receptors.

(+/-)-Govadine and (+/-)-THP, two tetrahydroprotoberberine alkaloids, as selective alpha 1-adrenoceptor antagonists in vascular smooth muscle cells.

(+/-)-Govadine and (+/-)-THP ((+/-)-2,3,10,11-tetrahydroxytetrahydroprotoberberine HBr) have been shown to inhibit noradrenaline-induced contraction of rat thoracic aortae. The pharmacological activity of the compounds was determined in thoracic aortae and cardiac tissue isolated from the rat and in trachea isolated from the guinea-pig to determine the selectivity of the compounds towards different types of receptor. (+/-)-Govadine and (+/-)-THP were found to be alpha 1-adrenoceptor blocking agents in rat thoracic aorta as revealed by their competitive antagonism of vasoconstriction induced by noradrenaline (pA2 = 6.57 +/- 0.07 and 5.93 +/- 0.06, respectively) or phenylephrine (pA2 = 6.74 +/- 0.08 and 6.06 +/- 0.10, respectively). Removal of endothelium did not affect the antagonistic potencies of (+/-)-govadine (pA2 = 6.83 +/- 0.09) and (+/-)-THP (pA2 = 6.25 +/- 0.06) on phenylephrine-induced vasoconstriction. They were more potent than yohimbine (pA2 = 6.05 +/- 0.05), but less so than phentolamine (pA2 = 7.54 +/- 0.11) and prazosin (pA2 = 9.27 +/- 0.12). (+/-)-Govadine and (+/-)-THP, furthermore, inhibited [3H]inositol monophosphate formation caused by noradrenaline (3 microM) in rat thoracic aorta. (+/-)-Govadine and (+/-)-THP were also alpha 2-adrenoceptor blocking agents with pA2 values 5.50 +/- 0.13 and 5.41 +/- 0.11, respectively. A high concentration of (+/-)-govadine (30 microM) or (+/-)-THP (30 microM) did not, however, affect the contraction induced by the thromboxane receptor agonist U46619, prostaglandin F2 alpha (PGF2 alpha), 5-hydroxytryptamine (5-HT), angiotensin II, endothelin or high K+ in rat aorta denuded of endothelium. Neither the cyclic AMP nor cyclic GMP content of rat thoracic aorta was, furthermore, changed by (+/-)-govadine or (+/-)-THP. Contraction of guinea-pig trachea caused by carbachol, histamine, leukotriene C4 or neurokinin A was not affected by (+/-)-govadine or (+/-)-THP. (+/-)-Govadine or (+/-)-THP also did not block beta 1- or beta 2-adrenoceptor-mediated responses induced by isoprenaline in rat right atria and guinea-pig trachea. It is concluded that (+/-)-govadine and (+/-)-THP are selective alpha 1-adrenoceptor antagonists in vascular smooth muscle.

Propofol Preferentially Relaxes Neurokinin Receptor-2-induced Airway Smooth Muscle Contraction in Guinea Pig Trachea

Propofol is the anesthetic of choice for patients with reactive airway disease and is thought to reduce intubation- or irritant-induced bronchoconstriction by decreasing the cholinergic component of vagal nerve activation. However, additional neurotransmitters, including neurokinins, play a role in irritant-induced bronchoconstriction. We questioned the mechanistic assumption that the clinically recognized protective effect of propofol against irritant-induced bronchoconstriction during intubation was due to attenuation of airway cholinergic reflexes. Muscle force was continuously recorded from isolated guinea pig tracheal rings in organ baths. Rings were subjected to exogenous contractile agonists (acetylcholine, histamine, endothelin-1, substance P, acetyl-substance P, and neurokinin A) or to electrical field stimulation (EFS) to differentiate cholinergic or nonadrenergic, noncholinergic nerve-mediated contraction with or without cumulatively increasing concentrations of propofol, thiopental, etomidate, or ketamine. Propofol did not attenuate the cholinergic component of EFS-induced contraction at clinically relevant concentrations. In contrast, propofol relaxed nonadrenergic, noncholinergic-mediated EFS contraction at concentrations within the clinical range (20-100 mum, n = 9; P < 0.05), and propofol was more potent against an exogenous selective neurokinin-2 receptor versus neurokinin-1 receptor agonist contraction (n = 6, P < 0.001). Propofol, at clinically relevant concentrations, relaxes airway smooth muscle contracted by nonadrenergic, noncholinergic-mediated EFS and exogenous neurokinins but not contractions elicited by the cholinergic component of EFS. These findings suggest that the mechanism of protective effects of propofol against irritant-induced bronchoconstriction involves attenuation of tachykinins released from nonadrenergic, noncholinergic nerves acting at neurokinin-2 receptors on airway smooth muscle.

Effect of an alpha-blocker on the contraction of guinea pig trachea with various bronchoconstrictors (author's transl)

Effect of an alpha-blocker on the contraction of guinea pig trachea with various bronchoconstrictors (author's transl)

Y‐27632 relaxation of carbachol‐induced smooth muscle contraction in guinea pig trachea

Smooth muscle (SM) contractions are caused by increases in cytoplasmic Ca2+ ([Ca2+]i). SM contractility may also depend on factors that regulate the sensitivity of intracellular processes to [Ca2+]i. Increasing evidence supports a role for Rho Kinase pathway in regulating smooth muscle contractility, and modulation of Rho Kinase activity may have therapeutic use for diseases such as asthma. Pharmacological inhibition of Rho Kinase using Y‐27632 causes relaxation of airway smooth muscle preparations. However, little is known about how Rho Kinase inhibition affects the sensitivity of airway smooth muscle to a contractile agonist. In this study, the muscarinic agonist carbachol (CCh) was applied to guinea pig trachea rings in a cumulative fashion. Following an initial CCh dose‐response curve, a second CCh dose‐response curve was constructed in the presence of varying concentrations of Y‐27632 (0.01 to 100 µM). EC50 values for CCh‐induced contractions in the presence of Y‐27632 were (in µM) 0.52, 0.4, 0.15, 0.2, 0.24, 0.29, 2, 0.51, 2.2, 0.46 in presence of 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 Y‐27632, respectively. The maximum contractile response to CCh was decreased by 20‐25% in the presence of Y‐27632. Inhibition of Rho Kinase using Y‐27632 appears to decrease the maximum contractility in response to muscarinic stimulation without affecting the sensitivity of the airway SM to the muscarinic agonist.

Chemical Knockdown of Protein-tyrosine Phosphatase 1B by 1,2-Naphthoquinone through Covalent Modification Causes Persistent Transactivation of Epidermal Growth Factor Receptor

1,2-Naphthoquinone (1,2-NQ), an atmospheric contaminant, causes the contraction of guinea pig trachea through the activation of epidermal growth factor receptor (EGFR) by inhibiting protein-tyrosine phosphatases (PTPs). Phosphorylation of EGFR is negatively regulated by PTPs, but details of the mechanism by which 1,2-NQ inhibits PTPs have not been elucidated. Results described in this report demonstrate that 1,2-NQ forms covalent bonds with PTP1B after exposure to human epithelial A431 cells. In this study, a concentration-dependent phosphorylation of EGFR was found to be coupled to the reduction of PTP activity in the cells. The reduction in PTP activity was due to the irreversible modification of PTP1B, and when PTP1B was overexpressed by the cells, the 1,2-NQ-mediated EGFR phosphorylation was suppressed. Studies with purified PTP1B and 1,2-NQ showed that the reduction in enzyme activity was due to a nucleophilic attack by the quinone on the enzyme, to form covalent bonds. Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry analysis and mutation experiments revealed that PTP1B inactivation was primarily due to covalent attachment of the quinone to Cys-121 of the enzyme, with binding to His-25 and Cys-215 as well. Collectively, the results show that covalent attachment of 1,2-NQ to PTP1B is at least partially responsible for the reduction of PTP activity, which leads to prolonged transactivation of EGFR in the cells.

1,2-Naphthoquinone activates vanilloid receptor 1 through increased protein tyrosine phosphorylation, leading to contraction of guinea pig trachea

1,2-Naphthoquinone (1,2-NQ) has recently been identified as an environmental quinone in diesel exhaust particles (DEP) and atmospheric PM2.5. We have found that this quinone is capable of causing a concentration-dependent contraction of tracheal smooth muscle in guinea pigs with EC50 value of 18.7 microM. The contraction required extracellular calcium and was suppressed by L-type calcium channel blockers nifedipine and diltiazem. It was found that 1,2-NQ activated phospholipase A2 (PLA2)/lipoxygenase (LO)/vanilloid receptor (VR1) signaling. Additionally, 1,2-NQ was capable of transactivating protein tyrosine kinases (PTKs) such as epidermal growth factor receptor (EGFR) in guinea pig trachea, suggesting that phosphorylation of PTKs contributes to 1,2-NQ-induced tracheal contraction. Consistent with this notion, this action was blocked by the PTKs inhibitor genistein and the EGFR antagonist PD153035, indicating that contraction was, at least in part, attributable to PTKs phosphorylation that activates VR1, resulting in increased intracellular calcium content in the smooth muscle cells.

ChTX induces oscillatory contraction in guinea pig trachea: role of cyclooxygenase-2 and PGE2.

We examined the possible role of cyclooxygenase (COX) in charybdotoxin (ChTX)-induced oscillatory contraction in guinea pig trachea. Involvement of prostaglandin E(2) (PGE(2)) in ChTX-induced oscillatory contraction was also investigated. ChTX (100 nM) induced oscillatory contraction in guinea pig trachea. The mean oscillatory frequency induced by ChTX was 10.7 +/- 0.8 counts/h. Maximum and minimum tensions within ChTX-induced oscillatory contractions were 68.4 +/- 1.8 and 14.3 +/- 1.7% compared with K(+) (72.7 mM) contractions. ChTX-induced oscillatory contraction was completely inhibited by indomethacin, a nonselective COX inhibitor. Valeryl salicylate, a selective COX-1 inhibitor, did not significantly inhibit this contraction, whereas N-(2-cyclohexyloxy-4-nitro-phenyl)-methanesulfonamide, a selective COX-2 inhibitor, abolished this contraction. Exogenously applied arachidonic acid enhanced ChTX-induced oscillatory contraction. SC-51322, a selective PGE receptor subtype EP(1) antagonist, significantly inhibited ChTX-induced oscillatory contraction. Exogenously applied PGE(2) induced only a slight phasic contraction in guinea pig trachea, but PGE(2) induced strong oscillatory contraction after pretreatment with indomethacin and ChTX. Moreover, ChTX time-dependently stimulated PGE(2) generation. These results suggest that ChTX specifically activates COX-2 and stimulates PGE(2) production and that ChTX-induced oscillatory contraction in guinea pig trachea is mediated by activation of EP(1) receptor.

Pharmacological profiles of R-96544, the active form of a novel 5-HT 2A receptor antagonist R-102444

We examined the pharmacology of (2 R,4 R)-4-hydroxy-2-[2-[2-[2-(3-methoxy)phenyl]ethyl]phenoxy]ethyl-1-methylpyrrolidine hydrochloride (R-96544), the active form of a novel 5-HT 2A receptor antagonist, (2 R,4 R)-4-lauroyloxy-2-[2-[2-[2-(3-methoxy)phenyl]ethyl]phenoxy]ethyl-1-methylpyrrolidine hydrochloride (R-102444). R-96544 produced a concentration-dependent inhibition of platelet aggregation induced by serotonin (5-hydroxytryptamine, 5-HT) alone or in combination with ADP in platelets from humans, monkeys, cats, rabbits, rats and mice. An intravenous administration of R-96544 to rabbits significantly inhibited ex vivo platelet aggregation induced by 5-hydroxytryptamine (5-HT) combined with epinephrine. An oral administration of R-102444 to rats also resulted in significant inhibition of ex vivo platelet aggregation, whereas R-102444 was ineffective in an in vitro platelet aggregation assay. These antiplatelet effects of R-96544 and R-102444 were more potent than those of two other 5-HT 2A receptor antagonists, sarpogrelate and its active metabolite (±)-1-[2-[2-(3-methoxyphenyl)ethyl]phenoxy]-3-(dimethylamino)-2-propanol hydrochloride (M-1). A binding study using cat platelet membranes showed that R-96544 has high affinity for 5-HT 2A receptors but no effect on non-serotonergic [ 3H]ketanserin-binding sites. R-96544 caused a parallel shift to the right of concentration–response curves for 5-HT in rat caudal artery contraction mediated by 5-HT 2A receptors. Schild plot analysis gave a pA 2 value of 10.4 with a slope near unity (1.04). R-96544 also inhibited 5-HT 2A receptor-mediated contraction of guinea pig trachea but not 5-HT 3 receptor-mediated contraction of guinea pig ileum and 5-HT 2B receptor-mediated contraction of rat fundus preparation. R-96544 (i.v.) attenuated the pressor responses evoked by 5-HT (15 μg/kg, i.v.) but not by phenylephrine (5 μg/kg, i.v.) and angiotensin II (0.1 μg/kg, i.v.), after ganglionic blockade in anesthetized spontaneously hypertensive rats. These results show that R-96544, the active form of R-102444, is a novel 5-HT receptor antagonist with potent, competitive, and 5-HT 2A-selective activity.

Characterization of the vanilloid receptor 1 antagonist iodo-resiniferatoxin on the afferent and efferent function of vagal sensory C-fibers.

The effect of iodo-resiniferatoxin (I-RTX) on efferent function (tachykinergic contractions of airway smooth muscle) and afferent function (action potential discharge) of vagal C-fibers mediated by vanilloid receptor 1 (VR1) activation was studied in an isolated guinea pig airway preparation. I-RTX (1 microM) had no VR1 agonist activity in either the afferent or efferent assays. I-RTX (30 nM-1 microM) shifted the resiniferatoxin and capsaicin concentration-response curves for neurokinin-mediated contractions rightward but did not inhibit the maximum response. The pK(B) value calculated from 0.3 microM I-RTX against resiniferatoxin and capsaicin was 7.3 +/- 0.2 and 6.8 +/- 0.2, respectively, showing 10 to 30 times higher potency compared with capsazepine. The slope of Schild plot from the resiniferatoxin efferent studies deviated from unity (~0.6), suggesting complex interactions at VR1 binding site(s). This notion was further supported by lack of additional inhibitory effect of 1 microM I-RTX on capsaicin-evoked contractions compared with 0.3 microM I-RTX. Concentrations of I-RTX up to 1 microM had no effect on trypsin-induced neurokinin-mediated contractions, nor neurokinin A-induced contractions of guinea pig trachea. However, nonselective effects on airway smooth muscle contractions were noted with 10 microM I-RTX. In both afferent and efferent studies I-RTX (30 nM-1 microM) caused a substantial delay of the response to capsaicin. This led to an apparent increase in potency in experiments where the agonist was applied transiently, with insufficient time to reach equilibrium. I-RTX inhibited contractions induced by anandamide and action potential discharge induced by low pH, showing that the I-RTX-antagonism of VR1 does not strictly depend on the vanilloid nature of the agonist.

Anti-Emetic Activity of the Novel Nonpeptide Tachykinin NK1 Receptor Antagonist Ezlopitant (CJ-11,974) against Acute and Delayed Cisplatin-Induced Emesis in the Ferret

The anti-emetic effects of a novel tachykinin NK₁ receptor antagonist, ezlopitant ((2S,3S-cis)-2-diphenylmethyl)- N--1-azabicyclo- [2.2.2]octan-3-amine), were investigated in ferrets. Ezlopitant inhibited [³H]substance P ([³H]SP) binding to the human, guinea pig, ferret and gerbil NK₁ receptors (K_i = 0.2, 0.9. 0.6 and 0.5 nmol/l, respectively), but had no affinity to NK₂ and NK₃ receptors up to 1 µmol/l. Ezlopitant also inhibited SP-induced contraction of guinea pig trachea with a pA₂ value of 7.8, but had no effects on the baseline tension and maximum contractile response. In ferrets, ezlopitant, either orally (0.03–3 mg/kg) or subcutaneously (0.3–3 mg/kg), prevented acute retching and vomiting responses induced by intraperitoneal injection of cisplatin (10 mg/kg). In addition, repeated subcutaneous injection of ezlopitant significantly inhibited delayed retching and vomiting responses that occurred in ferrets treated with the lower dose of cisplatin (5 mg/kg, i.p.). Ezlopitant (0.1–1 mg/kg, s.c.) also produced a dose-dependent inhibition of hindpaw tapping induced by intracerebroventricular injection of [Sar⁹,Met(O₂)¹¹]SP in gerbils, which is known to be mediated by NK₁ receptors in the brain. These findings indicate that ezlopitant is a potent and selective NK₁ receptor antagonist, and that it inhibits both acute and delayed emetic reactions induced by cisplatin in ferrets via acting on NK₁ receptors in the central nervous system.

Mechanisms underlying the contraction induced by bradykinin in the guinea pig epithelium-denuded trachea.

This study investigates some of the mechanisms by which bradykinin (BK) triggers contraction of epithelium-denuded strips of guinea pig trachea (GPT). Cumulative or single additions of BK, T-BK, L-BK, or ML-BK in the presence of captopril (30 microM) produced graded GPT contractions with the following rank order of potency (EC50 level): T-BK (31.3 nM) > BK (40.0 nM) > L-BK (56.0 nM) > ML-BK (77.0 nM). BK-induced contraction (100 nM) in GPT was completely inhibited by either HOE 140 or NPC 17731 with mean IC50 values of 17 and 217 nM, respectively. Addition of BK (100 nM) at 30 min intervals, induced progressive tachyphylaxis, which was complete after 4 h. The tachyphylaxis induced by BK was unaffected by L-NOARG (nitric oxide synthase inhibitor, 100 microM) or valeryl salicylate (a cyclooxygenase-1 (COX-1) inhibitor, 30 microM), but was prevented by a low concentration of indomethacin, diclofenac (non-selective COX inhibitors, 3 nM each) or by NS 398 (a COX-2 inhibitor, 10 nM). Furthermore, higher concentrations of indomethacin, diclofenac, phenidone (a lypooxygenase (LOX) and COX inhibitor), or NS 398, caused graded inhibition of BK-induced contraction, with mean IC50 values of 0.28, 0.08, 46.37, and 0.15 microM, respectively. Together, these results suggest that BK-induced contraction in GPT involves activation of B2 receptors and release of prostanoids from COX-2 pathway. Furthermore, the tachyphylaxis induced by BK was insensitive to the nitric oxide and COX-1 inhibitors, but was prevented by non-selective and selective COX-2 inhibitors, indicating a mediation via COX-2-derived arachidonic acid metabolites.

Ca 2+-activated K + channel blockers induce PKC modulated oscillatory contractions in guinea pig trachea

Mechanisms underlying the Ca 2+-activated K + channel (K Ca) blockers-induced oscillatory contractions were investigated in guinea pig tracheal smooth muscle. The mean oscillatory frequencies induced by charybdotoxin (ChTX; 100 nM) and iberiotoxin (IbTX; 100 nM) were 9.8±0.8 (counts/h) and 8.0±1.3 (counts/h), respectively. Apamin (1 μM), a blocker of SK Ca, induced no contraction in guinea pig trachea and did not affect ChTX-induced oscillatory contractions. In Ca 2+ free solution, no ChTX-induced contraction was observed. Nifedipine (100 nM), a blocker of voltage-dependent Ca 2+ channels, and SK&F 96365 (10 μM), a blocker of capacitative Ca 2+ entry, completely abolished ChTX-induced oscillatory contractions. Ryanodine (1 μM) decreased the amplitude, but increased the frequency of the oscillatory contractions. Thapsigargin (1 μM) changed contractions from the oscillatory type to the sustained type. Moreover, the protein kinase C (PKC) inhibitor, bisindolylamaleimide I (1 μM), decreased the amplitude and frequency, but PKC activator, phorbol 12-myristate 13-acetate (1 μM), increased the frequency of oscillatory contractions. These results suggest that K Ca inhibitors-induced oscillatory contractions are initiated by Ca 2+ influx through L-type voltage-dependent Ca 2+ channels. The ryanodine-sensitive calcium release channels in the sarcoplasmic reticulum may play an important role in maintaining the oscillatory contractions. Moreover, PKC activity modulates these oscillatory contractions.

Cysteinyl Leukotriene Receptor Assays

The cysteinyl leukotrienes (CysLTs), LTC4, LTD4 and LTE4, lipid products derived from arachidonic acid metabolism, have been implicated in the pathophysiology of several inflammatory diseases, in particular, asthma. This unit describes techniques and applications for the measurement of contractile responses to the CysLTs in isolated smooth muscle preparations. The contractions are assessed by standard methods for the isometric measurement of responses (contractile or relaxant) of isolated tissues to exogenous agonists, and a detailed description of the methods employed to assess CysLT-induced contractions in guinea-pig trachea is outlined. However, the same general methodology (other than parameters such as dissection for non-airway tissues) are appropriate for measuring CysLT-induced contractions in airway preparations from other animals, and in non-airways tissues (e.g., the gastrointestinal tract) from different species, and also in exploring the relaxant responses to the CysLTs that have been demonstrated in some tissues (e.g., pulmonary vein or artery).