The role of IP₃ receptors (IP₃R) and/or ryanodine receptors (RyR) in the regulation of microvascular permeability by endothelial cell Ca²⁺ has not yet been defined. Our aim was to determine if activating the IP₃R/RyR was sufficient to increase the permeability of microvessels without extracellular Ca²⁺ influx. The Landis-Michel method (Michel, 1974) was used to measure hydraulic conductivity (L_p) ( X 10^-7 cm s^-1 cmH₂O^-1) in single mesenteric microvessels of frog. Erythrocytes were collected by cardiac puncture from 5 % halothane-anaesthetised rats (killed by cervical dislocation). Frogs were anaesthetised by immersion in 1 mg ml^-1 MS222 in frog Ringer (FR) and maintained by superfusing the mesentery with 0.25 mg ml^-1 MS222. They were killed by cranial destruction. All experiments conformed with the national guidelines for the usage of animals. Caffeine (100 µM), an IP₃R/RyR agonist, was perfused to determine if Ca²⁺ store release affects permeability. Nine vessels were perfused with 1 % BSA in FR and erythrocytes to determine the baseline L_p. Due to the non-normally distributed L_p measurements, actual values are expressed as median ± interquartile range and fold increases normalised as means ± S.E.M. The Wilcoxon signed ranks test was used in all statistical analyses unless otherwise stated. Vessels were then perfused with caffeine for 10 min. L_p transiently increased on average 2.0 ± 0.5-fold from 1.5 ± 0.5 to 2.8 ± 0.5 within 2 min (P < 0.02). To ensure that the effects seen with caffeine were due directly to store release and not Ca²⁺ influx induced by store release the experiments were repeated with the Ca²⁺ influx inhibitor 100 µM SK&F96365 (SK&F). Nine vessels were perfused with 1 % BSA followed by SK&F for 10 min then SK&F with caffeine for another 10 min. SK&F decreased the baseline L_p from 3.2 ± 0.8 to 1.5 ± 0.2 (P < 0.05). When SK&F and caffeine were perfused the L_p transiently increased 11.1 ± 9.2-fold to 6.5 ± 1.0 (P < 0.02 vs. BSA, P < 0.005 vs. SK&F). There was no significant difference between the fold increase in L_p measured with caffeine compared with that with SK&F and caffeine (P > 0.1, Mann-Whitney), indicating that caffeine increases permeability by releasing Ca²⁺ from stores and not by inducing Ca²⁺ influx in vivo.

This work was supported by BHF BB2000003 and SS2000057.