Cytosolic Ca2+ concentration and rate of increase of the cytosolic Ca2+ concentration in the regulation of vascular permeability in Rana in vivo

Abstract

Vascular permeability is assumed to be regulated by the cytosolic Ca(2+) concentration ([Ca(2+)](c)) of the endothelial cells. When permeability is increased, however, the maximum [Ca(2+)](c) appears to occur after the maximum permeability increase, suggesting that Ca(2+)-dependent mechanisms other than the absolute Ca(2+) concentration may regulate permeability. Here we investigate whether the rate of increase of the [Ca(2+)](c) (d[Ca(2+)](c)/dt) may more closely approximate the time course of the permeability increase. Hydraulic conductivity (L(p)) and endothelial [Ca(2+)](c) were measured in single perfused frog mesenteric microvessels in vivo. The relationships between the time courses of the increased L(p), [Ca(2+)](c) and d[Ca(2+)](c)/dt were examined. L(p) peaked significantly earlier than [Ca(2+)](c) in all drug treatments examined (Ca(2+) store release, store-mediated Ca(2+) influx, and store-independent Ca(2+) influx). When L(p) was increased in a store-dependent manner the time taken for L(p) to peak (3.6 +/- 0.9 min during store release, 1.2 +/- 0.3 min during store-mediated Ca(2+) influx) was significantly less than the time taken for [Ca(2+)](c) to peak (9.2 +/- 2.8 min during store release, 2.1 +/- 0.7 min during store-mediated influx), but very similar to that for the peak d[Ca(2+)](c)/dt to occur (4.3 +/- 2.0 min during store release, 1.1 +/- 0.5 min during Ca(2+) influx). Additionally, when the increase was independent of intracellular Ca(2+) stores, L(p) (0.38 +/- 0.03 min) and d[Ca(2+)](c)/dt (0.30 +/- 0.1 min) both peaked significantly before the [Ca(2+)](c) (1.05 +/- 0.31 min). These data suggest that the regulation of vascular permeability by endothelial cell Ca(2+) may be regulated by the rate of change of the [Ca(2+)](c) rather than the global [Ca(2+)].

Figure 1. Total intracellular store release increases [Ca²⁺]_c

Fluorescence measurements from a single microvessel. A, I_f340 (upper trace) and I_f380 (lower trace) during basal conditions (bovine serum albumin (BSA) solution), during Ca²⁺ influx inhibition (SKF 96365; SKF) and during total store release without Ca²⁺ influx (SKF/TG/IM). B, R_norm, the ratio of I_f340 to I_f380, normalized to the minimum ratio for zero calcium (R_min), for the same experiment. Time of drug applications are indicated by the bars. Inhibition of Ca²⁺ influx resulted in a small increase in the baseline R_norm (point A). Application of thapsigargin (TG) and ionomycin (IM) (point B) resulted in a significant transient increase in R_norm that returned to below baseline within 10 min (point C).

Figure 2. Total store release increases [Ca²⁺]_c

A, mean ± s.e.m.R_norm under basal conditions (BSA), during Ca²⁺ influx inhibition (SKF) and during total store release without Ca²⁺ influx (SKF/TG/IM). * Significant difference versus SKF. B, [Ca²⁺]_c during Ca²⁺ influx inhibition (SKF) significantly correlated with [Ca²⁺]_c during total store release (peak SKF/TG/IM).

Figure 3. The time for L_p to reach a maximum is similar to the time taken for the maximum rate of change of [Ca²⁺]_c to occur during total store release

A, the top graph shows an individual microvessel where L_p was measured during total store release without Ca²⁺ influx (SKF/TG/IM added at time point zero). The bottom graph shows R_norm in a separate microvessel treated with the same protocol as the top graph. The rate of change of R_norm is shown. The time frame before the dashed arrow indicates when the L_p will be maximum. These two vessels gave the fastest permeability and calcium responses. The highest L_p value was reached before the highest fluorescence ratio was reached. B, mean ± s.e.m. time to peak L_p, time to peak [Ca²⁺]_c and time to peak rate of change of [Ca²⁺]_c during total store release in the absence of Ca²⁺ influx (SKF/TG/IM). * Significant difference versus time to peak L_p. In general, the permeability peaks before the calcium, but at a similar time to the rate of change of calcium.

Figure 4. The time taken for the maximum rate of change of [Ca²⁺]_c to occur is very similar to the time for ATP-induced L_p increases to reach a maximum

Mean ± s.e.m. time to peak L_p, time to peak [Ca²⁺]_c and time to peak rate of change of [Ca²⁺]_c with ATP. * Significant difference versus time to peak L_p.

Figure 5. The time taken for VEGF-induced L_p increases to reach a maximum is less than the time for the maximum [Ca²⁺]_c to occur, but similar to that taken for the maximum rate of change of [Ca²⁺]_c to occur

A, measurement of calcium and rate of change of calcium in vessel exposed to 1 nm VEGF. The [Ca²⁺]_c reaches a maximum at around 90 s (point i), by which time all the permeability responses measured have returned to control. The maximum rate of change, however, occurs within 30 s (point ii), similar to the peak permeability measurement. B, mean ± s.e.m. time to peak L_p, time to peak [Ca²⁺]_c and time to peak rate of change of [Ca²⁺]_c with VEGF. * Significant difference versus time to peak L_p.

Figure 6. Simultaneous measurement of L_p and [Ca²⁺]_c in two individual microvessels during store-dependent Ca²⁺ influx

ATP (30 μm) was added at time zero to induce store-dependent Ca²⁺ influx. Each filled square is a L_p measurement. The thin lines show the change in L_p and the thick interrupted line indicates [Ca²⁺]_c as represented by R_norm. Note that L_p reaches a maximum before R_norm.

Figure 7. The time to the peak [Ca²⁺]_c is positively correlated to the time to the peak d[Ca²⁺]_c/dt

The time taken for the [Ca²⁺]_c to peak was highly significantly correlated to the time taken for the maximum rate of increase of the [Ca²⁺]_c to occur when the [Ca²⁺]_c was increased by 1 nm VEGF (•), ATP (Δ) and thapsigargin (□).