We have shown previously that cytokines such as ATP and VEGF cause increases in both L_p and [Ca²⁺]_i in individually perfused frog mesenteric microvessels. However, although [Ca²⁺]_i has been measured in vessels in which L_p has previously been determined (Bates et al. 2001), simultaneous measurement of the two parameters has not been possible. We have therefore developed a system to measure L_p and [Ca²⁺]_i simultaneously in capillaries and postcapillary venules of living animals.

Frogs (Rana temporaria) were anaesthetised by immersion in MS222 (1 mg ml^-1 ) solution, laid supine and lightly secured to a supporting tray. The gut was exposed and the exteriorised mesentery was gently stretched over a coverslip and pinned. The tray was placed on an extended microscope stage with the mesentery over a Ω40 Fluotar objective on an inverted epifluorescence microscope (Leica DMIRB). The mesentery was continuously superfused with frog Ringer solution containing MS222 (0.25 g l^-1), to visualise the mesenteric microvessels and to maintain anaesthesia. A straight, free-flowing capillary or post-capillary venule with at least 300 µm between side-branches, 20-35 µm in diameter and free of white cells, was cannulated using a bevelled glass micropipette, bent by approximately 45-60 deg at the shoulder. The vessel was perfused with 10 µM fura-2 AM ester in 1 % bovine serum albumin (BSA, in frog Ringer solution at pH 7.4) at 30 cmH₂O. The vessel was illuminated by a xenon light source (Cairn High Intensity Arc lamp). A rotating (50 Hz) disc containing 340, 360 and 380 nm excitation filters was used to control the excitation wavelength. Loading of endothelial cells was monitored at 10 min intervals by measuring fluorescence intensity (I_f ) within a defined window at 340, 380 and 360 nm wavelengths (F₃₄₀, F₃₈₀ and F₃₆₀) excitation and 510 nm emission and comparing this with a window of mesentery away from the perfused vessel (background). The vessel was checked visually to ensure even loading. In successfully loaded vessels, F₃₈₀ was 4- to 10-fold higher than background. The perfusate was then changed to a 1 % BSA solution containing washed rat erythrocytes, using a syringe refiller (Hillman et al. 2001). Rat blood was obtained by cardiac puncture of 5 % halothane-anaesthetised adult male Wistar rats, killed by cervical dislocation. Baseline L_p was recorded using the Landis-Michel method (Michel et al. 1974). Occlusion was determined by watching the flow of rat erythrocytes on a monitor (Sony SSM125CE). This was connected to a video (Panasonic AG7350) and Video Timer (ForA VTG-33). Incident white light was passed through an infrared (IR) filter (750 nm longpass) to visualise the preparation with an IR camera (Watec WAT-902B). A dichroic filter was placed in front of the photometer to reflect light < 700 nm wavelength to the photomultiplier tube (PMT). Light > 700 nm passed through the dichroic filter to the IR camera. The PMT was controlled by a Cairn spectrophotometer connected to a PowerLab/4SP (AdInstruments) system. Thus L_p was measured off-line from a video recording of vessel occlusions and [Ca²⁺]_i measured using Chart software. Occlusion sites were checked regularly by switching the light emission from the PMT to the eyepiece. At the end of experiments the Ca²⁺-sensitive fura fluorescence was quenched by perfusing with 10 µM ionomycin/5 mM MnCl₂, and this value used to calculate background I_f. Frogs were killed by cranial destruction.

[Ca²⁺]_i was calculated as:

[Ca²⁺]_i = K [(R – 0.85R_min )/(0.85R_max – R),

where R is the normalised ratios of I_f (R = R_exp/R_min) where R_exp = (F₃₄₀ – B₃₄₀ )/(F₃₈₀ – B₃₈₀ ), B₃₄₀ and B₃₈₀ are the background I_f values at excitations of 340 and 380 nm, respectively, and R_min is the in vitro ratio for zero [Ca²⁺ ]. R_max is the in vitro ratio at saturating calcium, and K is the product of the effective dissociation constant for fura-2 and the in vitro I_f ratio of zero to saturating calcium. K was estimated from an in vitro calibration of fura-2.

This work was supported by the BHF (FS98023 and PG2000030).

Bates, D.O., Heald, R.I., Curry, F.E. & Williams, B. (2001). J. Physiol. 533, 263-272. abstract

Hillman, N.J., Whittles, C.E., Pocock, T.M., Williams, B. & Bates, D.O. (2001). J. Vas. Res. 38, 176-186.

Michel, C.C., Mason, J.C., Curry, F.E., Tooke, J.E. & Hunter, P.J. (1974). Q. J. Exp. Physiol. Cogn. Med. Sci. 59, 283-309.