Distortion of KB estimates of endothelin-1 ETA and ETB receptor antagonists in pulmonary arteries: Possible role of an endothelin-1 clearance mechanism.

Dual endothelin ETA and ETB receptor antagonists are approved therapy for pulmonary artery hypertension (PAH). We hypothesized that ETB receptor-mediated clearance of endothelin-1 at specific vascular sites may compromise this targeted therapy. Concentration-response curves (CRC) to endothelin-1 or the ETB agonist sarafotoxin S6c were constructed, with endothelin receptor antagonists, in various rat and mouse isolated arteries using wire myography or in rat isolated trachea. In rat small mesenteric arteries, bosentan displaced endothelin-1 CRC competitively indicative of ETA receptor antagonism. In rat small pulmonary arteries, bosentan 10 µmol L-1 left-shifted the endothelin-1 CRC, demonstrating potentiation consistent with antagonism of an ETB receptor-mediated endothelin-1 clearance mechanism. Removal of endothelium or L-NAME did not alter the EC50 or Emax of endothelin-1 nor increase the antagonism by BQ788. In the presence of BQ788 and L-NAME, bosentan displayed ETA receptor antagonism. In rat trachea (ETB ), bosentan was a competitive ETB antagonist against endothelin-1 or sarafotoxin S6c. Modeling showed the importance of dual receptor antagonism where the potency ratio of ETA to ETB antagonism is close to unity. In conclusion, the rat pulmonary artery is an example of a special vascular bed where the resistance to antagonism of endothelin-1 constriction by ET dual antagonists, such as bosentan or the ETB antagonist BQ788, is possibly due to the competition of potentiation of endothelin-1 by blockade of ETB -mediated endothelin-1 clearance located on smooth muscle and antagonism of ETA - and ETB -mediated contraction. This conclusion may have direct application for the efficacy of endothelin-1 antagonists for treating PAH.

Functional estimation of endothelin-1 receptor antagonism by bosentan, macitentan and ambrisentan in human pulmonary and radial arteries in vitro.

BACKGROUND: Endothelin receptor antagonists are approved for pulmonary arterial hypertension. Development of selective ETA-receptor antagonists over mixed or dual receptor antagonists has depended on a range of receptor binding assays, second messenger assays and functional blood vessel assays. This study compared the 3 clinically-approved endothelin receptor antagonists in assays of human isolated pulmonary and radial arteries in vitro. METHODS: Human isolated pulmonary (i.d. 5.5mm) and human radial (i.d. 3.23mm) artery ring segments were mounted in organ baths for isometric force measurement. Single concentration-contraction curves to endothelin-1 were constructed in the absence or presence of bosentan (1-10µM), macitentan (0.03-0.3µM) or ambrisentan (0.1-1µM). RESULTS: All 3 endothelin antagonists caused competitive rightward shifts in the endothelin-1 concentration-response curves in both arteries. The Clark plot and analysis gave the following pKB values: bosentan, pulmonary artery 6.28±0.13 and radial artery 6.04±0.10; macitentan, pulmonary artery 8.02±0.13 and radial artery 7.49±0.08; and ambrisentan, pulmonary artery 7.38±0.13 and radial artery 6.96±0.10. CONCLUSIONS: Noting the maximum plasma levels attained from recommended oral doses of each antagonist in volunteers, the pKB findings here show that there would be significant antagonism of endothelin-1 contraction in the pulmonary and radial arteries at therapeutic plasma levels. This functional assay confirms in human tissue that much higher plasma concentrations of endothelin-1 receptor antagonists are required to be effective than those predicted from binding or other biochemical assays.

Vasoconstrictor responses to vasopressor agents in human pulmonary and radial arteries: an in vitro study.

BACKGROUND: Vasopressor drugs, commonly used to treat systemic hypotension and maintain organ perfusion, may also induce regional vasoconstriction in specialized vascular beds such as the lung. An increase in pulmonary vascular tone may adversely affect patients with pulmonary hypertension or right heart failure. While sympathomimetics constrict pulmonary vessels, and vasopressin does not, a direct comparison between these drugs has not been made. This study investigated the effects of clinically used vasopressor agents on human isolated pulmonary and radial arteries. METHODS: Isolated pulmonary and radial artery ring segments, mounted in organ baths, were used to study the contractile responses of each vasopressor agent. Concentration-response curves to norepinephrine, phenylephrine, metaraminol, and vasopressin were constructed. RESULTS: The sympathomimetics norepinephrine, phenylephrine, and metaraminol caused concentration-dependent vasoconstriction in the radial (pEC50: 6.99 ± 0.06, 6.14 ± 0.09, and 5.56 ± 0.07, respectively, n = 4 to 5) and pulmonary arteries (pEC50: 6.86 ± 0.11, 5.94 ± 0.05 and 5.56 ± 0.09, respectively, n = 3 to 4). Vasopressin was a potent vasoconstrictor of the radial artery (pEC50 9.13 ± 0.20, n = 3), whereas in the pulmonary artery, it had no significant effect. CONCLUSIONS: Sympathomimetic-based vasopressor agents constrict both human radial and pulmonary arteries with similar potency in each. In contrast, vasopressin, although a potent vasoconstrictor of radial vessels, had no effect on pulmonary vascular tone. These findings provide some support for the use of vasopressin in patients with pulmonary hypertension.

A pharmacological investigation of the venom extract of the Australian box jellyfish, Chironex fleckeri, in cardiac and vascular tissues.

The pharmacology of Australian box jellyfish, Chironex fleckeri, unpurified (crude) nematocyst venom extract (CVE) was investigated in rat isolated cardiac and vascular tissues and in anaesthetised rats. In small mesenteric arteries CVE (0.01-30 µg/ml) caused contractions (EC(50) 1.15±0.19 µg/ml) that were unaffected by prazosin (0.1 µM), bosentan (10 µM), CGRP(8-37) (1 µM) or tetrodotoxin (1 µM). Box jellyfish antivenom (5-92.6 units/ml) caused rightward shifts of the CVE concentration-response curve with no change in the maximum. In the presence of l-NAME (100 µM) the sensitivity and maximum response to CVE were increased, whilst MgSO(4) (6 mM) decreased both parameters. CVE (1-10 µg/ml) caused inhibition of the contractile response to electrical sympathetic nerve stimulation. Left atrial responses to CVE (0.001-30 µg/ml) were bi-phasic, composed of an initial positive inotropy followed by a marked negative inotropy and atrial standstill. CVE (0.3 µg/ml) elicited a marked decrease in right atrial rate followed by atrial standstill at 3 µg/ml. These responses were unaffected by 1 µM of propranolol, atropine or CGRP(8-37). Antivenom (54 and 73 units/ml) caused rightward shifts of the CVE concentration-response curve and prevented atrial standstill in left and right atria. The effects of CVE do not appear to involve autonomic nerves, post-synaptic α(1)- or ß(1)-adrenoceptors, or muscarinic, endothelin or CGRP receptors, but may occur through direct effects on the cardiac and vascular muscle. Box jellyfish antivenom was effective in attenuating CVE-induced responses in isolated cardiac and vascular tissues.