Durga P. Dash

Diabetes Downregulates Large-Conductance Ca2+-Activated Potassium β1 Channel Subunit in Retinal Arteriolar Smooth Muscle

Retinal vasoconstriction and reduced retinal blood flow precede the onset of diabetic retinopathy. The pathophysiological mechanisms that underlie increased retinal arteriolar tone during diabetes remain unclear. Normally, local Ca2+ release events (Ca2+-sparks), trigger the activation of large-conductance Ca2+-activated K+(BK)-channels which hyperpolarize and relax vascular smooth muscle cells, thereby causing vasodilatation. In the present study, we examined BK channel function in retinal vascular smooth muscle cells from streptozotocin-induced diabetic rats. The BK channel inhibitor, Penitrem A, constricted nondiabetic retinal arterioles (pressurized to 70mmHg) by 28%. The BK current evoked by caffeine was dramatically reduced in retinal arterioles from diabetic animals even though caffeine-evoked [Ca2+]i release was unaffected. Spontaneous BK currents were smaller in diabetic cells, but the amplitude of Ca2+-sparks was larger. The amplitudes of BK currents elicited by depolarizing voltage steps were similar in control and diabetic arterioles and mRNA expression of the pore-forming BK&agr; subunit was unchanged. The Ca2+-sensitivity of single BK channels from diabetic retinal vascular smooth muscle cells was markedly reduced. The BK&bgr;1 subunit confers Ca2+-sensitivity to BK channel complexes and both transcript and protein levels for BK&bgr;1 were appreciably lower in diabetic retinal arterioles. The mean open times and the sensitivity of BK channels to tamoxifen were decreased in diabetic cells, consistent with a downregulation of BK&bgr;1 subunits. The potency of blockade by Pen A was lower for BK channels from diabetic animals. Thus, changes in the molecular composition of BK channels could account for retinal hypoperfusion in early diabetes, an idea having wider implications for the pathogenesis of diabetic hypertension.

Association of polymorphisms in the hepatocyte growth factor gene promoter with keratoconus

PURPOSE Keratoconus is a progressive disorder of the cornea that can lead to severe visual impairment or blindness. Although several genomic regions have been linked to rare familial forms of keratoconus, no genes have yet been definitively identified for common forms of the disease. METHODS Two genome-wide association scans were undertaken in parallel. The first used pooled DNA from an Australian cohort, followed by typing of top-ranked single-nucleotide polymorphisms (SNPs) in individual DNA samples. The second was conducted in individually genotyped patients, and controls from the USA. Tag SNPs around the hepatocyte growth factor (HGF) gene were typed in three additional replication cohorts. Serum levels of HGF protein in normal individuals were assessed with ELISA and correlated with genotype. RESULTS The only SNP observed to be associated in both the pooled discovery and primary replication cohort was rs1014091, located upstream of the HGF gene. The nearby SNP rs3735520 was found to be associated in the individually typed discovery cohort (P = 6.1 × 10(-7)). Genotyping of tag SNPs around HGF revealed association at rs3735520 and rs17501108/rs1014091 in four of the five cohorts. Meta-analysis of all five datasets together yielded suggestive P values for rs3735520 (P = 9.9 × 10(-7)) and rs17501108 (P = 9.9 × 10(-5)). In addition, SNP rs3735520 was found to be associated with serum HGF level in normal individuals (P = 0.036). CONCLUSIONS Taken together, these results implicate genetic variation at the HGF locus with keratoconus susceptibility.

Mutational screening of VSX1 in keratoconus patients from the European population

PurposeTo perform mutational screening of the visual system homeobox gene 1 (VSX1; MIM#605020) in patients with sporadic and familial keratoconus (MIM#148300) in a European population and, for the first time, report the mutational analysis of the two newly identified VSX1exons.MethodsVSX1sequence variants in patients with keratoconus were evaluated by direct sequencing of the entire coding region, including two novel exons. In familial keratoconus cases, segregation of potentially pathogenic VSX1variants was assessed to determine pathogenicity. Transcript analysis was carried out on splice site and synonymous sequence variants not detected in controls.ResultsA total of 66 unrelated patients with keratoconus from the European population (27 with familial keratoconus; 39 with sporadic keratoconus) were analysed for VSX1mutations. Four sequence variants were not observed in 100 healthy control individuals: c.432C>G (p.D144E), c.479G>A (p.G160D), c.789C>T (p.S263S), and an intronic change c.844-13T>A (numbered with respect to NM_014588). Segregation was not detected for p.D144E and c.844-13T>A. The change in p.G160D was observedin two patients with sporadic keratoconus. Although predicted to alter VSX1splicing, p.S263S had no effect on transcript processing. Four known SNPs were detected and the following polymorphic variants were observed in keratoconus patients and controls: c.711T>A (NM_199425; p.P237P), c.844-5_-6insT (NM_014588), c.*28G>T (DQ854811/DQ854812), and c.*50G>A (DQ854809/DQ854810).ConclusionsVSX1has a minor role in keratoconus pathogenesis. The pathogenicity of p.G160D remains controversial and this change may represent a rare polymorphism or genetic modifier. Further evidence is provided that the previously reported variant, p.D144E, is a polymorphism.

TRPV2 Channels Contribute to Stretch-Activated Cation Currents and Myogenic Constriction in Retinal Arterioles

Purpose Activation of the transient receptor potential channels, TRPC6, TRPM4, and TRPP1 (PKD2), has been shown to contribute to the myogenic constriction of cerebral arteries. In the present study we sought to determine the potential role of various mechanosensitive TRP channels to myogenic signaling in arterioles of the rat retina. Methods Rat retinal arterioles were isolated for RT-PCR, Fura-2 Ca2+ microfluorimetry, patch-clamp electrophysiology, and pressure myography studies. In some experiments, confocal immunolabeling of wholemount preparations was used to examine the localization of specific mechanosensitive TRP channels in retinal vascular smooth muscle cells (VSMCs). Results Reverse transcription-polymerase chain reaction analysis demonstrated mRNA expression for TRPC1, M7, V1, V2, V4, and P1, but not TRPC6 or M4, in isolated retinal arterioles. Immunolabeling revealed plasma membrane, cytosolic and nuclear expression of TRPC1, M7, V1, V2, V4, and P1 in retinal VSMCs. Hypoosmotic stretch-induced Ca2+ influx in retinal VSMCs was reversed by the TRPV2 inhibitor tranilast and the nonselective TRPP1/V2 antagonist amiloride. Inhibitors of TRPC1, M7, V1, and V4 had no effect. Hypoosmotic stretch-activated cation currents were similar in Na+ and Cs+ containing solutions suggesting no contribution by TRPP1 channels. Direct plasma membrane stretch triggered cation current activity that was blocked by tranilast and specific TRPV2 pore-blocking antibodies and mimicked by the TRPV2 activator, Δ9-tetrahydrocannabinol. Preincubation of retinal arterioles with TRPV2 blocking antibodies prevented the development of myogenic tone. Conclusions Our results suggest that retinal VSMCs express a range of mechanosensitive TRP channels, but only TRPV2 appears to contribute to myogenic signaling in this vascular bed.

Pharmacological Profiling of Store-Operated Ca2+ Entry in Retinal Arteriolar Smooth Muscle

Please cite this paper as: McGahon MK, McKee J, Dash DP, Brown E, Simpson DA, Curtis TM, McGeown JG, Scholfield CN. Pharmacological profiling of store‐operated Ca2+ entry in retinal arteriolar smooth muscle. Microcirculation 19: 586–597, 2012.