ABSTRACT
This study characterized the in vitro pharmacological properties of a newly developed endothelin receptor antagonist, N-butanesulfonyl-[N-(3, 5-dimethylbenzoyl)-N-methyl-3-[4-(5-isoxazolyl)-phenyl]-(D)- alanyl]-( L)-valineamide sodium salt (IRL 3630A), and its in vivo effects on respiratory mechanics were determined. IRL 3630A showed highly balanced affinities to human endothelin ET(A) and ET(B) receptors, giving apparent K(i) values of 1.5 and 1.2 nM, respectively. This compound also potently antagonized the endothelin-1-induced intracellular Ca(2+) increases in both embryonic bovine tracheal (EBTr) cells expressing endothelin ET(A) receptors and human Girardi heart (hGH) cells expressing endothelin ET(B) receptors. In guinea pig isolated tracheas having both endothelin ET(A) and ET(B) receptors, IRL 3630A greatly inhibited endothelin-1-induced contraction (pA(2)=7.1), which was partially or scarcely suppressed by the endothelin ET(A) receptor antagonist cyclo[-(D)-Trp-(D)-Asp-(L)-Pro-(D)-Val-(L)-Leu-] (BQ-123) or the endothelin ET(B) receptor antagonist N-(3, 5-dimethylbenzoyl)-N-methyl-3-(4-phenyl)-(D)-phenylalanyl-(L)-t ryptop han (IRL 2500), respectively. Bolus i.v. injections of IRL 3630A administered into anaesthetized guinea pigs at 10 and 30 microg/kg inhibited endothelin-1 (1.3 microg/kg)-induced changes in respiratory resistance and compliance in a dose dependent manner, whereas both sodium 2-benzo[1, 3]dioxol-5-yl-4-(4-methoxy-phenyl)-4-oxo-3-(3,4, 5-trimethoxy-benzyl)-but-2-enoate (an endothelin ET(A) receptor antagonist: PD 156707) and IRL 2500 at doses of up to 30 microg/kg did not affect endothelin-1-induced changes in respiratory mechanics, reflecting the in vitro results. IRL 3630A is thus an effective bifunctional endothelin receptor antagonist, and will be useful in clarifying the role of endothelin in pulmonary diseases such as bronchial asthma.
Subject(s)
Dipeptides/pharmacology , Endothelin Receptor Antagonists , Respiratory Mechanics/drug effects , Airway Resistance/drug effects , Animals , Binding, Competitive , Biphenyl Compounds/pharmacology , CHO Cells , Calcium/metabolism , Cell Line , Cricetinae , Dioxoles/pharmacology , Dipeptides/metabolism , Dose-Response Relationship, Drug , Endothelin-1/pharmacology , Guinea Pigs , Humans , In Vitro Techniques , Lung Compliance/drug effects , Male , Muscle Contraction/drug effects , Receptor, Endothelin A , Receptor, Endothelin B , Receptors, Endothelin/metabolism , Respiratory Mechanics/physiology , Trachea/drug effects , Trachea/physiologyABSTRACT
1. Signalling events responsible for endothelin(A) (ET(A)) and ET(B) receptor-induced contraction were examined in epithelium-denuded guinea-pig tracheal smooth muscle strips. Selective stimulation of each subtype was achieved by a combination of ET-1 (100 nM) and ET(A) and ET(B) receptor-selective antagonists, BQ-123 (10 microM) and BQ-788 (3 microM), respectively. 2. Both ET(A) and ET(B) receptors induced long-lasting contraction that was totally dependent on Ca2+ influx. Stimulation of ET(A) receptor induced both transient and sustained (Ca2+)i increases whereas that of ET(B) receptor induced only a sustained increase. Suppression of the transient (Ca2+)i increase by U73122 (3 microM) did not affect the ET(A)-induced sustained (Ca2+)i increase and tension development. Stimulation of ET(A) receptor, but not ET(B), induced phosphoinositide breakdown and protein kinase C (PKC). The activated PKC contributed to the contraction by increasing the Ca2+ sensitivity of the contractile apparatus. 3. Thus, ET(A) receptor is coupled both with phospholipase C/Ca2+/PKC signalling and Ca2+ influx pathways whereas ET(B) receptor was coupled only with the latter. 4. Stimulation of ET(B) receptor, but not ET(A), caused membrane depolarization measured with a fluorescent indicator, bis-(1,3 dibutylbarbituric acid)-trimethine oxonol. Both nifedipine (1 microM) and verapamil (10 microM) abolished ET(B)-induced Ca2+ influx and contraction, while they barely affected ET(A)-induced responses. 5. Therefore, the Ca2+ influx pathways activated by each subtype appeared to be completely different; ET(A) and ET(B) receptors opens voltage-independent Ca2+ channels and L-type voltage-dependent Ca2+ channels, respectively.
Subject(s)
Muscle, Smooth/physiology , Receptors, Endothelin/physiology , Trachea/physiology , Animals , Calcium/metabolism , Endothelin-1/pharmacology , Guinea Pigs , In Vitro Techniques , Male , Muscle Contraction/drug effects , Protein Kinase C/physiology , Receptor, Endothelin A , Receptor, Endothelin B , Type C Phospholipases/physiologyABSTRACT
In the isolated rabbit trachea, endothelin (ET)-1, ET-3 and the selective ETB receptor agonists, IRL 1620 and sarafotoxin S6c (STXc), induced contraction with EC50 of 2-9 nM. An ETA1 receptor antagonist, BQ-123, was ineffective whereas desensitization of the ETB receptor strongly antagonized the effect of ET-3, IRL 1620 and STXc. An ETB1 receptor antagonist, RES-701-1, antagonized the effects of ET-3 and IRL 1620 whereas the effect of STXc was antagonized by an ETB2 receptor antagonist, BQ-788. In the ETB-desensitized trachea, only ET-1 induced large contraction that was partially antagonized by BQ-123. These results suggest that ET induces tracheal contraction by activating multiple ET receptors: the ET-1-selective ETA (BQ-123-sensitive ETA1 and insensitive ETA2 subtypes) and the isopeptide-nonselective ETB receptors (RES-701-1-sensitive ETB1 and insensitive ETB2 subtypes).
