THE BRADYKININ ANALOG RMP-7 INCREASES INTRACELLULAR FREE CALCIUM LEVELS IN RAT-BRAIN MICROVASCULAR ENDOTHELIAL-CELLS

The vasoactive peptide bradykinin is believed to cause increased Vascu lar permeability by the activation of B-2 receptors on the vascular en dothelium. A bradykinin analog, H-Arg-Pro-Hyp-Gly-Thi-Ser-Pro-4-Me-Tyr (psi CH2NH)-Arg-OH (RMP-7), was designed and it was proposed that it m ight increase cerebrovascular permeability by activating B-2 receptors on brain microvasculature. In this report, the effects of RMP-7 and r elated peptides on bradykinin receptor-induced calcium signaling were examined in rat brain microvascular endothelial (RBME) cultures. RBME cells are responsive to bradykinin and exhibit specific [H-3]-bradykin in binding, with Scatchard analysis indicating a major class of sites with a K-d of 3.9 +/- 1.4 nM and a minor class of higher affinity site s (K-d similar to 33 pM). RMP-7 displaces a significant component of s pecific [H-3]-bradykinin binding from RBME cells; RMP-10, an RMP-7 dia stereomer with a D-amino acid substitution in the number 9 position, d oes not. [H-3]-bradykinin binding to RBME cells is not displaced by de sArg(9)-bradykinin, which indicates that neither class of sites is a B -1 bradykinin receptor. RMP-7 induces an increase in intracellular fre e calcium levels in RBME cells. The time course, magnitude and concent ration dependence of RMP-7-induced calcium signaling is similar to tha t induced by bradykinin in RBME and other endothelial cells. Compared with RMP-7, RMP-10 is at least 1000 times less potent. RMP-12, another diastereomer with a D-amino acid substitution in the number 8 positio n, is at least 100 times less potent. However, the level of contaminat ion by RMP-7 in the RMP-12 preparation accounts for its activity, an i ndication that both the RMP-10 and RMP-12 diastereomers are essentiall y inactive. Overall, these results support the hypothesis that RMP-7 i s an agonist for B-2 receptors on brain endothelial cells and that thi s activity is highly dependent on the presence of L-amino acids in the carboxyl-terminal-reduced dipeptide.