Abstract
Purpose::
Mammalian transient receptor potential (TRP) channels form a novel cation channel family consisting of nearly 30 members. Recent studies have suggested that these channels may play a pivotal role in controlling vascular contractility and permeability. In the present study we sought to identify systematically the TRP channel subtypes expressed in rat retinal arterioles.
Methods::
Adult male Sprague-Dawley rats were anaesthetized with CO2 and killed by cervical dislocation. Retinas were removed and intact arteriole segments isolated. TRP channel mRNA expression was evaluated by RT-PCR, and rat brain and kidney samples were used as positive controls. Immunofluorescence staining with commercially available polyclonal antibodies was also used to test for cell-specific expression of TRP channels in retinal arterioles embedded within retinal flatmount preparations. Retinas were counter-stained with propidium iodide nuclear stain. The specificity of the antibodies was investigated by parallel control experiments in the absence of primary antibody.
Results::
With RT-PCR, mRNA of 13 TRP channel subtypes was detected in retinal arterioles: TRPC1, TRPC3, TRPC4, TRPC7, TRPV1, TRPV2, TRPV4, TRPM1, TRPM2, TRPM3, TRPM7, TRPML1 and TRPML3. Lack of expression of other TRP channels could not be attributed to RT-PCR conditions since all of the other TRP channel family members could be detected in brain or kidney. Immunofluorescence labelling revealed a punctuate distribution of TRPV2 throughout the vascular smooth muscle cell layer of retinal arterioles, while TRPV4 was specifically localised to the plasma membrane of endothelial cells. Consistent with the RT-PCR results, TRPC5 protein could not be detected in retinal arterioles. This was not due to a lack of antibody reactivity because intense staining was observed in the ganglion cell layer of the retina.
Conclusions::
Our results indicate that multiple TRP channels are expressed in retinal arterioles. These data may serve as molecular basis for future work exploring the physiological significance of non-selective cation channels in the retinal vasculature.
Keywords: vascular cells • ion channels • calcium