Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, C55
Ca2+-sparks Promote Development of Myogenic Tone in Rat Retinal Arterioles
J. Kur1, P. Bankhead1, N. Scholfield1, T. Curtis1, J. G. McGeown1
1. CVVS, Queen's University Belfast, Belfast, United Kingdom.
High speed confocal Ca2+-imaging reveals brief localised Ca2+-sparks and global waves/oscillations in vascular smooth muscle. The original observations of Ca2+-sparks in arteries demonstrated they could result in vascular relaxation by activating Ca2+-activated K+-currents(1). This model, which emphasises the inhibitory role of sparks, is now widely accepted. We have previously reported that sparks can summate to generate Ca2+-oscillations in retinal arterioles, however, leading to constriction(2). In the current study we tested the functional significance of Ca2+-sparks in arterioles under conditions of myogenic tone. Male Sprague-Dawley rats were humanely killed using intraperitoneal sodium pentobarbital and the eyes removed. Retinal arterioles (25-40µm external diameter) were mechanically isolated from the retina and cannulated for pressure myography. Intracellular [Ca2+] was imaged in the arteriolar smooth muscle using Fluo4 and confocal laser microscopy. Summary data is presented as means±SEM and statistical tests were carried out on raw rather than normalised data. Pressurization to 70mmHg led to an initial passive increase to 121.7±2.8% of the diameter at 0mmHg but, as myogenic tone developed, this decreased to 114.2±2.7% within 5-10min (P<0.01, n=10 arterioles, repeated measures ANOVA). Removal of extracellular Ca2+ led to a mean dilatation of 5.9±1.6% relative to the steady-state diameter at 70mmHg (P<0.01, n=6). Myogenic tone development was associated with an increase in the frequency of sparks from 0.435±0.051/cell/sec at 0mmHg to 1.168±0.113/cell/sec at 70mmHg (P<0.001, n=6, Mann-Whitney U-test). Oscillation frequency also increased from 0.011±0.003/cell/sec at 0mmHg to 0.118±0.013/cell/sec at 70mmHg (P<0.001, Mann-Whitney U-test). This reflected both a doubling in the number of cells generating Ca2+-oscillations and an increase in oscillation frequency within active cells. Vasomotion was observed in 40% of arterioles and was associated with increased synchronization of Ca2+-oscillations, with a rise in the cross-correlation coefficient from 0.26±0.05 in the absence of vasomotion to 0.57±0.08 when it was present (P<0.02, n=6 arterioles, unpaired t-test). When pressurized arterioles were superfused with drugs previously shown to inhibit Ca2+-sparks in these vessels(2), dilatation was observed. Mean increases in diameter, normalised to the steady state diameter at 70mmHg, were as follows: ryanodine (100µM) 4.8±1.0% (P<0.05, n=7, ANOVA), tetracaine (100µM) 2.7±0.7% (P<0.05, n=6), cyclopiazonic acid (20µM) 4.6±1.5% (P<0.01, n=10), and nimodipine (1µM) 7.9±1.8% (P<0.01, n=6). These results suggest that Ca2+-sparks play a constrictor role in arterioles, in contrast with the dilator effect more usually ascribed to sparks in vascular myocytes from larger vessels.
Where applicable, experiments conform with Society ethical requirements