Simple and specific assessment of Ca-entry-blocking activities of drugs by measurement of Ca reversal.

A new, simple, and specific screening method for Ca-entry blocker is reported. In the presence of various blockers of Ca-entry (Ca-channel blockers), Ca2+ (1 x 10(-4) M) causes Ca reversal, i.e., it causes relaxation of rat uterine smooth muscle that has been tonically contracted with oxytocin in Ca-free medium (Ca-free contraction) after prolonged preincubation with 3 mM EGTA. The potencies of various drugs to cause Ca reversal, assessed on an all-or-none basis, were found to reflect their activities of blocking Ca entry. The potencies of drugs for blocking Ca entry via voltage-operated Ca-channels could be determined in 125.6 mM K+ Ca-free medium. Drugs that inhibited Ca-free contraction directly have some other antispasmodic activity. Conventionally, activity for blocking Ca entry is screened by measuring inhibition of Ca-induced contraction, but true Ca-entry blocking activity cannot be differentiated clearly from other antispasmodic activity when a drug has both activities. However, by measuring Ca reversal, activity for blocking Ca entry only can easily be found, when done in solution with high K+ content for blocking voltage-operated channels and when done in solution with normal K+ content for blocking voltage-independent channels.