It remains incompletely understood how homocysteine impairs endothelial function. Whether mechanisms such as calcium-activated potassium (KCa) channels are involved is uncertain and the significance of endoplasmic reticulum (ER) stress in KCa channel-dependent endothelial function in hyperhomocysteinemia remains unexplored. We investigated the effect of homocysteine on endothelial KCa channels in coronary vasculature with further exploration of the role of ER stress. Vasorelaxation mediated by intermediate- and small-conductance KCa (IKCa and SKCa) channels was studied in porcine coronary arteries in a myograph. IKCa and SKCa channel currents were recorded by whole-cell patch-clamp in coronary endothelial cells. Protein levels of endothelial IKCa and SKCa channels were determined for both whole-cell and surface expressions. Homocysteine impaired bradykinin-induced IKCa and SKCa-dependent EDHF-type relaxation and attenuated the vasorelaxant response to the channel activator. IKCa and SKCa currents were suppressed by homocysteine. Inhibition of ER stress during homocysteine exposure enhanced IKCa and SKCa currents, associated with improved EDHF-type response and channel activator-induced relaxation. Homocysteine did not alter whole-cell protein levels of IKCa and SKCa whereas lowered surface expressions of these channels, which were restored by ER stress inhibition. Homocysteine induces endothelial dysfunction through a mechanism involving ER stress-mediated suppression of IKCa and SKCa channels. Inhibition of cell surface expression of these channels by ER stress is, at least partially, responsible for the suppressive effect of homocysteine on the channel function. This study provides new mechanistic insights into homocysteine-induced endothelial dysfunction and advances our knowledge of the significance of ER stress in vascular disorders.

While the role of hyperhomocysteinemia in cardiovascular pathogenesis continuously draws attention, deficiency of hydrogen sulfide (H₂S) has been growingly implicated in cardiovascular diseases. Generation of H₂S is closely associated with the metabolism of homocysteine via key enzymes such as cystathionineβ-synthase (CBS) and cystathionineγ-lyase (CSE). The level of homocysteine and H₂S is regulated by each other. Metabolic switch in the activity of CBS and CSE may occur with a resultant operating preference change of these enzymes in homocysteine and H₂S metabolism. This paper presented an overview regarding (1) linkage between the metabolism of homocysteine and H₂S, (2) mutual regulation of homocysteine and H₂S, (3) imbalance of homocysteine and H₂S in cardiovascular disorders, (4) mechanisms underlying the protective effect of H₂S against homocysteine-induced vascular injury, and (5) the current status of homocysteine-lowering and H₂S-based therapies for cardiovascular disease. The metabolic imbalance of homocysteine and H₂S renders H₂S/homocysteine ratio a potentially reliable biomarker for cardiovascular disease and development of drugs or interventions targeting the interplay between homocysteine and H₂S to maintain the endogenous balance of these two molecules may hold an even bigger promise for management of vascular disorders than targeting homocysteine or H₂S alone.

Endothelial dysfunction is caused by reduced nitric oxide (NO) bioavailability and/or over-produced reactive oxygen species (ROS). The present study investigated a vascular benefit of AVE3085, an endothelial nitric oxide synthase (eNOS) enhancer, in preserving endothelial function in diabetic mice and the mechanisms involved. Male db/db and db/m⁺ mice were orally administered AVE3085 for 7 days (10 mg kg⁻¹ day⁻¹). Vascular reactivity of arteries was studied via myography under both isometric and isobaric conditions. ROS levels in aortas were determined using dihydroethidium fluorescence dye and electron paramagnetic resonance spin trapping. Chronic treatment with AVE3085 reduced blood pressure, enhanced endothelium-dependent relaxations (EDR) to acetylcholine in aortas, mesenteric, and renal arteries, lowered oxidative stress, and augmented the attenuated flow-dependent dilatation in mesenteric resistance arteries from db/db mice. Incubation of aortas from C57BL/6J mice in high glucose (30 mmol L⁻¹) culture medium for 48 h impaired EDR and elevated ROS generation, and these effects were reversed by co-treatment with AVE3085 (1 µmol L⁻¹). Benefits of AVE3085 were abolished by the transcription inhibitor actinomycin D, the NOS inhibitor N^G-nitro-l-arginine methyl ester, and in eNOS^−/− mice. NO production in primary endothelial cells from mouse aortas was detected with a NO-sensitive fluorescence dye. Protein expression was assayed by western blotting. Treatment with AVE3085 enhanced NO production in endothelial cells and eNOS expression in aortas. AVE3085 ameliorates endothelial dysfunction in db/db mice through increased NO bioavailability, which reduces oxidative stress in the vascular wall. Targeting eNOS and NO production may be a promising approach to combat diabetic vasculopathy.

Intracellular Ca²⁺ ([Ca²⁺]_i) regulation in endothelial cells depends on transient receptor potential channels (TRPs), and the role of canonical TRPs (TRPCs) during hypoxia–reoxygenation (H–R) is unclear. We hypothesized that TRPC3 contributes to endothelial nitric oxide (NO) release and that H–R may reduce TRPC3 activity and the associated endothelial function, including NO release. Measurements of [Ca²⁺]_i and patch-clamp study in primary cultured porcine coronary endothelial cells, measurements of NO and endothelium-dependent relaxation in porcine coronary arteries, and RT-PCR and western blot were conducted. Pre-treatment with SKF96365 (an inhibitor of TRPCs) or the selective TRPC3 inhibitor Pyr3 significantly decreased bradykinin-induced vasorelaxation. One hour of hypoxia followed by reoxygenation significantly reduced the vasorelaxation (70.3 ± 6.4 vs. 88.9 ± 3.5%) and NO concentration (24.0 ± 1.3 vs. 45.2 ± 2.8 nmol/L), and they were restored by pre-incubation with the TRPC3/6/7 activator 1-oleoyl-2-acetyl-sn-glycerol (96.4 ± 1.8% and 41.1 ± 4.7 nmol/L, respectively). In porcine coronary endothelial cells, H–R inhibited bradykinin-activated membrane current (8.6 ± 0.4 vs. 14.0 ± 1.5 pA/pF) and Pyr3-sensitive TRPC3 current (3.8 ± 0.3 vs. 6.3 ± 0.6 pA/pF; P< 0.01). H–R also inhibited bradykinin-induced Ca²⁺ influx and the Ca²⁺ influx via TRPC3. Cell surface expression of TRPC3 was decreased after H–R. We have, for the first time, demonstrated that Ca²⁺ entry via endothelial TRPC3 contributes to NO release and have revealed that H–R is associated with inhibition of TRPC3 activity. Inhibition of channel trafficking to the cell surface is involved in the underlying mechanism of the decrease of TRPC3 current and the reduction in Ca²⁺ entry through TRPC3 during H–R. This study suggests that TRPC3 may have the potential to be a new target for endothelial protection during H–R.