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T. Bek, M. Misfeldt, C. Aalkjær, U. Simonsen; Recruitment of Intracellular Ca2+ Is Independent of Ryanodine Channels in the Sarcoplasmic Reticulum of Retinal Vascular Smooth Muscle Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3172.
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© ARVO (1962-2015); The Authors (2016-present)
Disturbances in the regulation of the retinal blood flow are involved in the pathophysiology of a variety of sight threatening diseases, including diabetic retinopathy. Therapeutic intervention on these diseases on a rational basis requires a detailed knowledge of the mechanisms involved in the regulation of the tone in retinal resistance arterioles. This tone depends on the pattern of recruitment of intracellular calcium in the vascular smooth muscle cells.
Porcine retinal arterioles with a diameter of approximately 150 µm were mounted in a wire myograph (DMT) and placed in a Zeiss LSM 5 Exciter confocal microscope allowing for simultaneous recording of vascular tone and calcium activity. The vessels were loaded with the calcium sensitive fluorophore Oregon Green and were pre-contracted with the prostaglandin analogue U46619. The vascular tone and the concentration of free calcium was studied after application of Nifedipine 10-10M - 10-6M (blocking of L-type Ca++-channels in the plasma membrane), and cyclopiazonic acid 10-10M - 10-6M and ryanodine 10-10M - 10-6M (Ca++-channels in the sarcoplasmic reticulum). In each vessel the fluorescence was recorded 4-5 times per second over a region of interest containing approximately ten vascular smooth muscle cells which allowed the resolution of spontaneous intracellular calcium spikes.
Ryanodine did not affect the rate of spontaneous calcium spikes nor the vessel tone (p=0.8749 and p=0.98 one-way ANOVA n=6, respectively). Cyclopiazonic acid reduced the rate of intracellular calcium spikes significantly; EC50 2.5*10-8 [2.34*10-7;3*10-9] ( p< 0.0022, n = 5 Repeated Measures ANOVA ). However, vessel tone was not affected (p=0.059 n=6 Repeated Measures ANOVA). Preliminary experiments showed that nifedipine induced an increase in the rate of spontaneous intracellular calcium in parallel with a marked decrease in vascular tone.
The recruitment of intracellular calcium in porcine vascular smooth muscle cells does not depend on ryanodine channels in the sarcoplasmic reticulum, which is different from observations in other tissues. These features may explain the unique disturbances in retinal blood flow in systemic diseases such as diabetic retinopathy.
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