Quantitation of bradykinin-induced microvascular leakage of FITC-dextran in rat cremaster muscle

Abstract

We studied the in vivo effects of bradykinin (BK) on transvascular movement of macromolecules in the rat cremaster muscle. The muscle was fashioned as a single layer, placed in a transparent chamber, and superfused with a bicarbonate buffer solution (pH 7.4, 35 degrees). FITC-Dextran 150 (MW 150,000) was injected iv as a macromolecular tracer. The fluorescent tracer remained in the vascular compartment during control observations. BK (8 X 10(-8) to 8 X 10(-7) M) was topically applied for periods of 5 min. Depending on the dose, leakage of FITC-Dextran 150 started 2-3 min after the application of BK. Discrete leakage sites were detected for a short period at the beginning of the response only. Subsequently, the entire interstitial space became fluorescent as the leakage lasted for several minutes. The preparation returned to control appearance after 40-60 min of continuous superfusion with bicarbonate buffer. Macromolecular leakage was observed in postcapillary venules ranging from 15 to 50 microns in diameter. Neither arterioles nor true capillaries showed leakage of the tracer. To better quantitate the effect of BK on macromolecular transport, the superfusate output was collected at 5-min intervals and analyzed by fluorometry to determine FITC-Dextran 150 concentration. After BK, fluorochrome concentration in the superfusate rose, peaked by 15-20 min, and returned to control levels following 60 min of washout. A direct dose-response relationship was found between BK concentration and clearance of FITC-Dextran 150. These data demonstrate the feasibility of quantitatively studying blood-tissue transport of macromolecules in vivo in skeletal muscle.