Assessing calcium antagonism on vascular smooth muscle: a comparison of three methods.

Three methods relying on depolarization-induced contraction of rabbit aortic rings were compared. The effects of hyperosmolarity induced by adding KCl to the bath and of a lowered NaCl concentration due to an exchange of NaCl for KCl were assessed. A dihydropyridine derivative (PY 108-068) and verapamil were used as antagonists. For the first method, 50.3 mM KCl was either added to the bath to reach a final concentration of 55 mM, or KCl was exchanged for NaCl to keep the bath isoosmotic. We found a noncompetitive antagonism and the results obtained from cumulative concentration-response curves appeared to be based on the slow tonic contraction. For the second method, the rings were suspended in a calcium-free Krebs-Henseleit (KH) or TRIS bath solution containing also 55 mM of KCl and calcium was added as an agonist. We found a competitive inhibition of the calcium effects. The slope of the Schild Plot was, however, -1.3 for PY 108-068 and -0.5 for verapamil. Very high concentrations of calcium had relaxant effects and thus distorted the results. For the third method, the rings were contracted by KCl and then relaxed by adding the calcium antagonist to the bath. Isoosmotic solution and solution made hyperosmotic with sucrose were also used. Hyperosmolarity by itself contributed to the tension development. In hyperosmolar solution, the calcium antagonists appeared to be less active. Such bath conditions can therefore considerably bias the results. The first and the second method, in combination with experiments using receptor stimulating agents such as noradrenaline, appear to be most suitable for investigating calcium antagonists on blood vessels.