The effect of altering the external calcium concentration and a calcium channel blocker, verapamil, on microvascular leaky sites and dextran clearance in the hamster cheek pouch

Abstract

The role of calcium in contractile mechanisms has been well documented. Since the formation of intraendothelial gaps may be due to a contractile process, these studies were initiated to describe the effects of altering the external calcium concentration and a calcium blocker, verapamil, on the development of leaky sites and clearance of dextran in the hamster cheek pouch under normal and histamine-stimulated conditions. Adult Syrian hamsters were anesthetized and tracheostomized, and the femoral vein was cannulated for injecting the FITC-Dextran-70K. In all hamsters, a removable plastic chamber was placed in the cheek pouch to observe and collect suffusate from the microvasculature. In one series of experiments, suffusion was begun with either “normal” calcium (1.5 m M) or a calcium-free, Ringer's bicarbonate (pH 7.4, 36°) buffer. After a 30-min period, fluorescein-labeled dextran (FITC-Dextran-70K) was given intravenously and the number of leaky sites and dextran clearance determined for 30–45 min. Two 5-min periods of stimulation with histamine (10 −7 and 10 −6 M) followed by a 30-min equilibration were completed while the above measurements were repeated. Then, the suffusate was switched to the opposite calcium buffer and the same sequence repeated. A similar protocol was followed using either the “normal” buffer or a high (4.5 m M)-calcium buffer. In another series of experiments, similar measurements were completed with and without verapamil (10 −4 M) using a “normal” calcium buffer and histamine-stimulated conditions. In the control, nonstimulated state, there was a significant increase in the leaky sites while suffusing with low-calcium buffer; however, there were no changes in leaky sites while suffusing with the high-calcium buffer. In the histamine-stimulated state, the increase in the number of leaky sites and dextran clearance was attenuated with the low-calcium buffer and potentiated with the high-calcium buffer. While suffusing with the verapamil buffer, there was an increase in the control number of leaky sites and in the histamine-stimulated state there was an attenuation of the histamine-stimulated response. These results indicate that external calcium influences and modulates microvascular leaky sites in normal and stimulated states and that the formation of leaky sites and dextran clearance after histamine stimulation require the transmembrane flux of calcium. Also, the formation of leaky sites was coupled directly to the clearance of dextran.