| Abstrakt: |
Regulation of tissue blood flow is an important arteriolarfunction. Membrane potential in vascular smooth muscle regulates vascular tone and resistance, and is modulated by ion channel activity. This study directly tested the roles of K+ and Cl- channels in determining retinal blood flow in vivo, using Hooded Lister (HL) rats (male, 320-450g). Channel blocker specificity was first established in vitro using electrophysiological recordings from isolated arterioles(1). Correolide (10µm), a KV1 inhibitor, blocked an A-type current and penitrem A (100nM) blocked a large conductance (BK) Ca2+-activated K+-current. Inhibition of K+-currents using Cs+-pipette solution and 10mM 4-aminopyridine, revealed a Ca2+-activated Cl- current (IClCa), which was blocked by 1mM disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS). This inhibition showed little voltage dependence, and DIDS had no effect on L-type Ca2+-currents, Kv or BK currents(2). The effects of these inhibitors on retinal blood flow were then investigated in vivo. Rats were anaesthetised with an intraperitoneal injection of ketamine (130 mg/kg) and xylazine (17.5 mg/kg). Volumetric blood flow was assessed in arterioles in the superior retina using a confocal scanning laser ophthalmoscope (Heidelberg Engineering, Germany). Intraperitoneal injection of acridine orange was used to label leukocyte DNA and the velocity of leukocyte transit through retinal arterioles determined from sequential confocal images (8.8 frames/s). Fluorescein angiography was then used to estimate the diameter of these arterioles, allowing velocity to be converted to flow. Drug effects were assessed from recordings 15 min after intravitreal drug injection (10µl). Data was summarised (mean ± SEM; n=number of animals) and analysed using nonparametric ANOVA (Kruskal Wallis) and Dunn's post hoc test. In non-injected eyes, mean flow in single arterioles was 12.3 ± 0.2 nL/s (n=9). This was unchanged following injection of Hanks' solution (12.8 ± 0.3 nL/s, n=9, P>0.05). Injection of DIDS (estimated intraocular concentration=10mM) increased volumetric flow (15.7±0.4 nL/s, n=9, P<0.001), while penitrem A (1µM) and correolide (40µM), reduced flow rates (11.2±0.4 nL/s, n=11, P<0.01; and 11.3±0.3 nL/s, n=10, P<0.05, respectively). Et-1 (104nM) induced vasoconstriction and dramatically reduced flow (4.6±0.2 nL/s, n=8, p<0.001). This response was not affected by K+ channel blockers (p>0.05 vs Et-1 alone), but was reversed by DIDS (14.71±0.272 nL/s, n=7, p<0.001 vs Et-1). These results suggest that K+ and Cl- channels in vascular smooth modulate basal blood flow in the retina and activation of IClCa appears to be an important mechanism underpinning the vasoconstrictor actions of Et-1. [ABSTRACT FROM AUTHOR] |
