| Part One:The electrotonic transduction along the rat retinal vasculaturePresent study was to investigate the electrotonic coupling of retinal microvasculature and understand its way of modulating blood supply by contraction and relaxation. Freshly isolated retinal microvessels were harvested from adult Long-Evans rats after sacrifice. Trypan blue exclusion test was used to assess viability of cells on isolated vessels. Perforated patch-clamp was employed to show the current-voltage relations of different vessles through single-recording from abluminal cells. Electrotonic transmission along each segment of retinal vessels was documentd by double recording and its mode of transmission was speculated.Results:fresh retinal microvasculature could be readily harvested with cell viability higher than 90%. Retinal microvessels could be subdivided into three parts (secondary arterioles, tertiary arterioles and capillaries) according to the lumen of the vessels, the shape and density of abluminal cells. Abluminal cells from these vessels were called smooth muscle cells from seconday arterioles, myocytes from tertiary arterioles and pericytes from capillaries, respectively. They were easily identified under phase contrast microscope and thus were appropriate for patch-clamping. Single recordings from pipette sealed onto abluminal cells showed that resting membrane potential of pericytes, myocytes and smooth muscle cells were-38±1 mV (n=27),-42±1 mV (n=29) and-45±1 mV (n=10), respectively, with no significant difference from each other (one-way ANOVA, P=0.27). With membrane potential held at-50mV, the membrane resistance of pericytes, myocytes and smooth muscle cells were 175±19 M?(n=27),151±9 MQ(n=29) and 127±15M?(n= 10), with no significant difference from each other (one-way ANOVA, P=0.58). Dual recording from pairs of ablummal cells showed that electrotonic transmission was most efficient along capillaries, with efficacy of 0.56±0.15 (n=27), and a decay rate of 2±2% per 100?m (n=27). Along tertiary arterioles, the efficacy of transmission was 0.27±0.09 (n=22), and the decay was 6±8%(n=22) per 100?m. Along secondary arterioles, the efficacy of transmission was 0.25±0.19 (n= 10), and the decay was 6±8%(n=10) per 100?m. There was no difference in the decay rate within these vessels.In summary, the electrotonic transmission pathway is the abluminal cells to endothelial cells and then to abluminal cells. The retinal microvasculature is not simply a well-coupled synctium since we detected significant voltage dissipation with radial abluminal cell-to-endothelium transmission and also at branch points between a capillary and its tertiary arteriole and between tertiary and secondary arterioles.Part Two:The role of angiotensin II on the electrotonic transmission along the rat retinal vasculatureTo further study if electrotonic architecture was modulated by vasoactive signals such as angiotensin II, retinal microvessels were isolated from adult Long-Evans rats. Immunohistochemistry was performed to study expression of angiotensin II type I receptors (AT1R) on the microvascular complexes. Electrophysiological responses to 500nM angiotensin II were shown with single recording of perforated patch-clamp. A micromanipulator-controlled micropipette was used to transect isolated microvessels at the capillary/tertiary arteriole junction or tertiary/secondary arteriole junction. Calcium-imaging was used to monitor intracellular calcium in cells loaded with fura-2. Effect of Angiotensin II on electrotonic transmission was investigated via dual perforated patch clamp recording.Results:500nM Angiotensin II activated a larger non-specific cation conductance in pericytes of capillaries. At each tested voltage form-38 mV to-108 mV, the inward current was significantly (P<0.0001) larger during exposure to angiotensin; at 12 mV and 22 mV, the outward current during angiotensin exposure was significantly (P < 0.05) larger. In addition, Angiotensin II evoked an increase in pericyte calcium of 325±20 nM (n=81), which was markedly greater than the 55±3nM (n=149, P <0.0001) increase detected in myocytes of tertiary arterioles, and the 10±2 nM (n= 130, P<0.0001) change in smooth muscle cells on secondary arterioles. Angiotensin ?caused the efficacy of axial transmission to decrease significantly (P<0.0001) from 0.98±0.03/100?m to 0.50±0.05/100?m (n=6) in the capillaries, from 0.94±0.08/100?m to 0.52±0.07/100?m (n=10) in the tertiary arterioles, and from 0.94±0.08/100?m to 0.55±0.11/100?m (n=8) in the secondary arterioles. In contrast, because the extrapolated?Vresponder/?Vstimulator ratios at 0?m were not altered significantly by angiotensin, it appears that radial transmission in capillaries, tertiary arterioles and secondary arterioles was unaffected by this vasoactive molecule.This study established that angiotensin?selectively and profoundly inhibits axial transmission within capillaries, tertiary arterioles and secondary arterioles. As a consequence, the angiotensin-induced depolarization generated in the capillaries remains localized to the capillary network and fails to spread to the calcium channel-rich proximal portions of the microvasculature. Of general physiological importance, angiotensin's inhibition of axial transmission establishes the operational concept that the electrotonic architecture of the retinal microvasculature is not static, but rather, is dynamically modulated by vasoactive signals. |
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