Plasmonic gold nanoparticles possess the ability to open potassium channels in rat thoracic aorta smooth muscles in a remote control manner.

Colloidal gold nanoparticles (AuNPs) of ~5nm core size and Zeta-potential of -35mV, having absorption maximum and plasmon resonance in the range of 510-570nm, were studied as a potential K(+)-channel opener in vascular smooth muscle (SM) cells. Experimental design of the study comprised SM contractile recordings. When externally applied to the organ bath, AuNPs (10(-6)-3×10(-4)M) led to decrease in amplitude of norepinephrine-induced contractions in a concentration-dependent and endothelium-independent manner in SM thoracic aorta, with mean value of pD2 (-log EC50) 4.2±0.03, Emax=55±4%. Being added to the bath solution in concentration of 10(-4)M, AuNPs significantly increased whole cell peak outward current at +70mV from 32±2pA/pF to 59±5pA/pF (n=14, P<0.05). External irradiation using a 5mW/532nm green laser, to facilitate plasmon resonance, led to an increment in the AuNPs-induced macroscopic outward potassium current (IK) from 59±5pA/pF to 74±1pA/pF (n=10, P<0.05). Paxilline (500nM), when added to the external bathing solution, significantly decreased AuNPs-induced increment of IK in SM cells. Single channel recordings provided a direct confirmation of BKCa activation by AuNPs at the single-channel level. Application of AuNPs to the bath potentiated BKCa activity with a delay of 1-2min, as was seen initially by more frequent channel openings followed by the progressive appearance of additional open levels corresponding to multiple openings of channels with identical single-channel amplitudes. Eventually, after 10-15min in the presence of AuNPs and especially when combined with the green laser illumination, there was a massive increase in channel activity with >10 channels evident. When irradiated by laser, AuNPs significantly increased the amplitude of maximal AuNPs-induced relaxation from 55±5% to 85±5% (n=10, P<0.05) while the sensitivity of SM to AuNPs was without changes. In summary, plasmonic AuNPs possess the ability to activate BKCa channel opening in vascular SM. Laser irradiation facilitates this effect due to local plasmon resonance that, in turn, further increases BKCa channel activity causing SM relaxation.

PKC-δ isozyme gene silencing restores vascular function in diabetic rat.

Abstract Background: Endothelium and K+ channel functionality in smooth muscle cells (SMCs) regulates vascular function and is exposed to damage in diabetes. The regulatory enzyme protein kinase C (PKC) is known to play a key role in vascular tone regulation in health and disease. In this study, we evaluated the effect of PKC-δ gene silencing using small interfering RNAs (siRNAs) on endothelial dysfunction and acquired potassium channelopathy in vascular SMCs in diabetes. Methods: The experimental design comprised diabetes induction by streptozotocin (65 mg/kg) in rats, RNA interference, isolated aortic ring contractile recordings, whole-cell patch-clamp technique, measurements of reactive oxygen species (ROS), and real-time polymerase chain reaction technique. Animals were killed by cervical dislocation following ketamine (45 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) anesthesia administration on the third month of diabetes and on the seventh day after intravenous injection of siRNAs. Results: The aortas of diabetic rats demonstrated depressed endothelium-dependent relaxation and integral SMCs outward K+ currents as compared with those of controls. On the seventh day, PKC-δ gene silencing effectively restored K+ currents and increased the amplitude of vascular relaxation up to control levels. An increased level of PKC-δ mRNA in diabetic aortas appeared to be reduced after targeted PKC-δ gene silencing. Similarly, the level of ROS production that was increased in diabetes came back to control values after siRNAs administration. Conclusions: The silencing of PKC-δ gene expression using siRNAs led to restoration of vasodilator potential in rats with diabetes mellitus. It is likely that the siRNA technique can be a good therapeutic tool to normalize vascular function in diabetes.

Correction of vascular hypercontractility in spontaneously hypertensive rats using shRNAs-induced delta protein kinase C gene silencing.

Potassium conductance in vascular smooth muscle (VSM) is known to be altered in arterial hypertension. High level of protein kinase C (PKC) activity is a common feature for hypertension of different genesis. The main goal of this study was to investigate the efficacy of the RNA interference (RNAi) technique targeting PKC delta-isoform gene as a possible pharmacological tool to restore vasodilator potential in spontaneously hypertensive rats (SHR). Experimental design of the study comprised RNAi and patch-clamp techniques, RT-PCR analysis and standard acetylcholine test. Total outward currents and acetylcholine-induced endothelium-dependent relaxant responses were blunted in SHR. BKCa alpha subunit mRNA expression in SHR was unchanged whereas KV and KATP mRNA expression appeared significantly increased. PKC inhibitor, chelerythrine (100 nM), restored potassium channels activity in SHR. PKC-delta-isoform protein expression and PKC-delta-isoform mRNA expression are 2.5-4 fold increased in VSM from SHR. PKC gene silencing with the short hairpin RNAs (shRNAs)-plasmid delivery system administered intravenously led to an increment in maximal amplitude of acetylcholine-relaxation, restored outward K(+) currents and PKC-delta-isoform mRNA and protein expression. Arterial blood pressure in SHR was normalized following shRNAs administration. We conclude that BKCa channels are likely to be the most PKC-dependent member of K(+) channels family responsible for vascular hypercontractility in SHR while Kv and KATP channels may constitute a reserve mechanism for the maintenance of vasodilator potential under BKCa channelopathy. It is likely that RNAi technique is a good therapeutic approach to inactivate PKC gene and to normalize vascular functions and high arterial blood pressure in SHR.