Regional blood flow is influenced by the contractile behaviour of the smooth muscle of microvessels and altered [Ca²⁺]_i is an important determinant of this contractile behaviour. [Ca²⁺]_i is determined by the balance between influx, extrusion and redistribution. The extrusion mechanism is well studied in cardiac muscle where the Na⁺/Ca²⁺ exchanger contributes to cell pathology. Vascular smooth muscle also expresses Na⁺/Ca²⁺ exchanger and its inhibition can have a profound effect on renal blood flow as well as influence large artery function. The present study aims to assess the relative contributions of Na⁺/Ca²⁺ exchanger and Ca²⁺-ATPase pump in Ca²⁺ homeostasis of microvascular smooth muscle (arterioles/metarterioles).

Sprague Dawley rats (200-300 g) were humanely killed and microvessels mechanically dispersed from fresh retinae using light titruration with a Pasteur pipette. Microvessels were loaded in 10 ÁM Fura-2/AM for 2 h and global cell Ca²⁺ measured by microflourometry in sections of microvessels comprising 6-20 cells (endothelial cells do not load with fura 2). Solutions and drugs were applied to superfusate local to the microvessel and switched within 1 s. [Ca²⁺]_i redistribution was blocked throughout by inhibiting uptake into the sarcoplasmic reticulum (SR), with cyclopiazionic acid (20 ÁM, CPA). It was shown using CCCP and oligomycin that mitochondrial uptake was not detectable. Ca²⁺ extrusion was measured by a challenge with 10 mM caffeine, which rapidly releases Ca²⁺ into the cytosol. The rate at which [Ca²⁺]_i declines is then a measure of Ca²⁺ extrusion. The Ca²⁺-ATPase extruder was blocked by pre-incubation with the membrane permeant carboxyeosin diacetate (5 ÁM) for 10 min. The Na⁺/Ca²⁺ exchanger was blocked by reducing [Na⁺]_i to 10 mM (equimolar Li⁺-replacement). Carboxyeosin slowed by 48 % Ca²⁺ extrusion (half time for decline, mean ± S.E.M., 7.8 ± 1.2 to 16.1 ± 2.4 s, paired t test, P = 0.0006, n = 14) whereas low Na⁺ alone had no effect (6.8 ± 0.7 to 8.5 ± 0.8 s, n = 21) on the decline in the caffeine induced [Ca²⁺]_i transient nor did they have any effect on resting cytosolic Ca²⁺. These agents together induced a rise in resting [Ca²⁺]_i with no recovery after caffeine application ([Ca²⁺]_i continued to rise). Similarly, the blocker of Na⁺/Ca²⁺ exchange KB-R7943 was only effective when applied with carboxyeosin.

These results show that for intact microvascular smooth muscle cells, cell Ca²⁺ is not highly dependent on the Na⁺/Ca²⁺ exchanger and this and the Ca²⁺-ATPase transporter independently handle cell Ca²⁺. Thus pathologies or pharmacological therapies which block one or other will have little adverse effect on retinal blood flow.

We thank the Wellcome Trust and Dublin Docks Development Authority for financial support.