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.

The BK(Ca) channels deficiency as a possible reason for radiation-induced vascular hypercontractility.

It is likely that large-conductance Ca²âº-activated K⁺ (BK(Ca)) channels channelopathy tightly involved in vascular malfunctions and arterial hypertension development. In the present study, we compared the results of siRNAs-induced α-BK(Ca) gene silencing and vascular abnormalities produced by whole-body ionized irradiation in rats. The experimental design comprised RT-PCR and patch clamp technique, thoracic aorta smooth muscle (SM) contractile recordings and arterial blood pressure (BP) measurements on the 30th day after whole body irradiation (6Gy) and following siRNAs KCNMA1 gene silencing in vivo. The expression profile of BK(Ca) mRNA transcripts in SM was significantly decreased in siRNAs-treated rats in a manner similar to irradiated SM. In contrast, the mRNA levels of K(v) and K(ATP) were significantly increased while L-type calcium channels mRNA transcripts demonstrated tendency to increment. The SMCs obtained from irradiated animals and after KCNMA1 gene silencing showed a significant decrease in total K⁺ current density amplitude. Paxilline (500 nM)-sensitive components of outward current were significantly decreased in both irradiated and gene silencing SMCs. KCNMA1 gene silencing increased SM sensitivity to norepinephrine while Ach-induced relaxation had decreased. The silencing of KCNMA1 had no significant effect on BP while radiation produced sustained arterial hypertension. Therefore, radiation alters the form and function of the BK(Ca) channel and this type of channelopathy may contribute to related vascular abnormalities. Nevertheless, it is unlikely that BK(Ca) can operate as a crucial factor for radiation-induced arterial hypertension.

Electrophysiological and contractile evidence of the ability of human mesenchymal stromal cells to correct vascular malfunction in rats after ionizing irradiation.

The effect of intravenous administration of human mesenchymal stromal stem cells (hMSC) has been evaluated by means of large-conductance calcium-dependent potassium channel (BK(Ca)) activity measurements in thoracic aorta smooth muscle cells (SMC) obtained from non-fatal whole-body irradiated rats, using the patch clamp technique in whole-cell modification, and the standard acetylcholine (ACh) test to evaluate functional endothelium integrity using SM contractile recordings. Myofilament calcium sensitivity was estimated using simultaneous contractile recordings versus [Ca(2+)](i). Arterial blood was measured in intact and irradiated rats before and after hMSC administration. Stimulation of isolated SMC from the control group of animals with depolarizing voltage steps showed that outward K(+) currents sensitive to the BK(Ca) inhibitor paxilline were expressed. Outward currents in SMC obtained from irradiated animals were significantly reduced on the 30th day of post-irradiation. Irradiation led to a significant elevation in arterial blood pressure and reduced ACh-induced relaxation responses in irradiated rats as compared with the control group. Simultaneous measurements of contractile force and [Ca(2+)](i) showed that myofilament Ca(2+) sensitivity had increased following irradiation. Intravenously injected hMSC effectively restored BK(Ca) current and the amplitude of ACh-induced endothelium-dependent vasodilatation in vascular tissues obtained from post-irradiated rats. SMC obtained from irradiated rats treated with hMSC demonstrated a significantly increased paxilline-sensitive component of outward potassium currents, indicating that BK(Ca) activity had been restored. hMSC administration normalized increased blood pressure and myofilament Ca(2+) sensitivity in irradiated animals. When administered to healthy rats, hMSC were without effects on either of these. This study does not provide any immunohistochemical proof of hMSC engraftment in the host rats. PCR analysis showed that hMSCs were negative for hematopoietic cell markers and positive for hMSC markers. There were no clinical signs of graft-versus-host disease throughout the experimental period of 30 days. The data obtained suggest that hMSC demonstrate a clearly expressed ability to normalize vascular function damaged following irradiation, i.e. to reduce an elevated arterial blood pressure and myofilament Ca(2+) sensitivity, and to repair BK(Ca) function and endothelium-dependent relaxation in vascular tissues obtained from irradiated animals. Thus, hMSC seem to be worthwhile therapeutic approach in cases of ionizing irradiation accident or radiation beam therapy.

Quercetin-filled phosphatidylcholine liposomes restore abnormalities in rat thoracic aorta BK(Ca) channel function following ionizing irradiation.

The goal of the present study was to investigate the effects of quercetin-filled phosphatidylcholine liposomes (PCL-Q) on the currents carried by large conductance Ca(2+)-dependent K(+) channels (BK(Ca)) in rat thoracic aorta following non-fatal whole-body ionizing irradiation. Using patch-clamp technique, it is found that the outward K(+) currents of isolated smooth muscle cells (SMCs) stimulated by depolarizing voltage steps were sensitive to BK(Ca) inhibitor, paxilline, and this kind of outward K(+) currents in SMCs from irradiated animals demonstrated a significant decrease in amplitude. Radiation-induced BK(Ca) suppression was evident 9 days post-irradiation and progressively increased over 30 days of experimental period. Thus, the vasorelaxing force of these SMCs may be diminished following irradiation. PCL-Q effectively restored BK(Ca) function in post-irradiated SMCs. It is noteworthy that the constituents of PCL-Q, i.e., free quercetin (Q) and "empty" liposomes (PCL), being taken separately, showed a decreased ability to recover BK(Ca) function as compared with combined composition. These results suggest that PCL-Q is able to regain normal function of BK(Ca) following irradiation. The protective effects of PCL-Q can be explained by its antioxidant and membrane repairing properties as well as its ability to inhibit protein kinase C activity. Thus, the lipid encapsulation of flavonoid, PCL-Q, appears to be a potential medication in the case of ionizing irradiation accident, and for the patients with neoplasm who have to receive external radiotherapy as well.

Functional and molecular consequences of ionizing irradiation on large conductance Ca2+-activated K+ channels in rat aortic smooth muscle cells.

AIMS: The goal of this study was to evaluate the influence of gamma-irradiation on Ca(2+)-activated K(+) channel (BK(Ca)) function and expression in rat thoracic aorta. MAIN METHODS: Aortic cells or tissues were studied by the measurement of force versus [Ca(2+)](i), patch-clamp technique, and RT-PCR. KEY FINDINGS: Stimulation of smooth muscle cells with depolarizing voltage steps showed expression of outward K(+) currents. Paxilline, an inhibitor of BK(Ca) channels, decreased outward K(+) current density. Outward currents in smooth muscle cells obtained from irradiated animals 9 and 30 days following radiation exposure demonstrated a significant decrease in K(+) current density. Paxilline decreased K(+) current in cells obtained 9 days, but was without effect 30 days after irradiation suggesting the absence of BK(Ca) channels. Aortic tissue from irradiated animals showed progressively enhanced contractile responses to phenylephrine in the post-irradiation period of 9 and 30 days. The concomitant Ca(2+) transients were significantly smaller, as compared to tissues from control animals, 9 days following irradiation but were increased above control levels 30 days following irradiation. Irradiation produced a decrease in BK(Ca) alpha- and beta(1)-subunit mRNA levels in aortic smooth muscle cells suggesting that the vasorelaxant effect of these channels may be diminished. SIGNIFICANCE: These results suggest that the enhanced contractility of vascular tissue from animals exposed to radiation may result from an increase in myofilament Ca(2+) sensitivity in the early post-irradiation period and a decrease in BK(Ca) channel expression in the late post-irradiation period.

Ionizing non-fatal whole-body irradiation inhibits Ca2+-dependent K+ channels in endothelial cells of rat coronary artery: possible contribution to depression of endothelium-dependent vascular relaxation.

PURPOSE: The goal of this study was to evaluate the influence of ionizing irradiation on large conductance Ca2+-dependent potassium (BKCa) channels in rat coronary endothelial cells. MATERIALS AND METHODS: Rats were exposed to a 6 Gy dose from a cobalt60 source. Experimental design of this study comprised recording of contractile force using isolated rat aortic rings and whole-cell patch clamp techniques to study whole-cell potassium currents in isolated rat coronary artery endothelial cells. RESULTS: It has been shown that outward potassium currents in endothelial cells 9 days after irradiation appear to be suppressed or even totally abolished. The reversal potential for these currents in irradiated cells was shifted to more positive values. Paxilline (500 nM), an inhibitor of BKCa channels, had no or only a negligible effect on irradiated cells. The experiments using isolated aortic rings demonstrated that both paxilline and irradiation significantly shifted the acetylcholine dependent concentration-relaxation response curve to the right. Irradiated tissues were insensitive to paxilline. CONCLUSION: The results suggest that non-fatal, whole-body gamma-irradiation suppresses large conductance, calcium-activated potassium channels, which control the driving force for Ca2+ entry and therefore Ca2+ dependent nitric oxide (NO) synthesis in endothelial cells. This may contribute, in part, to radiation-induced endothelium dysfunction and an increase in arterial blood pressure.

Ionizing radiation alters myofilament calcium sensitivity in vascular smooth muscle: potential role of protein kinase C.

Radiation exposure increases vascular responsiveness, and this change involves endothelial damage, as well as direct effects on vascular smooth muscle. In this study, we tested the hypothesis that myofilament Ca(2+) sensitivity in vascular smooth muscle is increased from single whole body gamma irradiation (6 Gy). We measured contractile responses from intact and permeabilized rat thoracic aortic rings combined with cytosolic Ca(2+) ([Ca(2+)](i)) measurements. The sensitivity to KCl and phenylephrine increased significantly in tissues from animals on the 9th and 30th days postirradiation compared with control. Irradiation also significantly increased Ca(2+) sensitivity in beta-escin permeabilized smooth muscle on the 9th and 30th days postirradiation. Inhibitors of protein kinase C, chelerythrine, and staurosporine, had no effect on the pCa-tension curves in control permeabilized tissues but significantly decreased Ca(2+) sensitivity in permeabilized tissues on the 9th and 30th days postirradiation. Phorbol dibutyrate (PDBu, 10(-7) M) increased Ca(2+) sensitivity in control skinned smooth muscle but was without effect in irradiated vascular rings. Simultaneous measurement of contractile force and [Ca(2+)](i) showed that myofilament Ca(2+) sensitivity defined as the ratio of force change to [Ca(2+)](i) significantly increased following gamma-irradiation. PDBu (10(-6) M) stimulation of intact aorta produced a sustained contraction, while the increase in [Ca(2+)](i) was transient. In irradiated tissues, PDBu-induced contractions were greater than those seen in control tissues but there was no elevation in [Ca(2+)](i). Taken together, these data strongly support the hypothesis that irradiation increases the sensitivity of vascular smooth muscle myofilaments to Ca(2+) and this effect is dependent on activation of protein kinase C.

Mechanisms of endothelial dysfunction after ionized radiation: selective impairment of the nitric oxide component of endothelium-dependent vasodilation.

(1) Gamma radiation impairs vascular function, leading to the depression of endothelium-dependent vasodilatation. Loss of the nitric oxide (NO) pathway has been implicated, but little is known about radiation effects on other endothelial mediators. (2) This study investigated the mechanisms of endothelial dysfunction in rabbits subjected to whole-body irradiation from a cobalt(60) source. (3) The endothelium-dependent relaxation of rabbit aorta evoked by acetylcholine (ACh) or A23187 was impaired in a dose-dependent manner by irradiation at 2 Gy or above. Inhibition was evident 9 days post-irradiation and persisted over the 30 day experimental period. (4) Endothelium-independent responses to glyceryl trinitrate (GTN), sodium nitroprusside (SNP) and 3-morpholino-sydnonimine (SIN-1) were suppressed over a similar dose range at 7-9 days post-irradiation, but recovered fully by 30 days post-irradiation. (5) In healthy vessels, ACh-induced relaxation was inhibited by L-N(omega)-nitroarginine (L-NA; 3 x 10(-4) M) and charybdotoxin (10(-8) M) plus apamin (10(-6) M) but resistant to indomethacin, indicating the involvement of NO and endothelium-derived hyperpolarizing factor (EDHF). Supporting this, ACh caused smooth muscle hyperpolarization that was reduced by L-NA and charybdotoxin plus apamin. (6) In irradiated vessels, responses to ACh were insensitive to L-NA but abolished by charybdotoxin plus apamin, indicating selective loss of NO-mediated relaxation. (7) In animals treated shortly after irradiation with the antioxidant, alpha-tocopherol acetate, the NO-dependent relaxation was restored without effect on the EDHF-dependent component. (8) The results imply that radiation selectively impairs the NO pathway as a consequence of oxidative stress, while EDHF is able to maintain endothelium-dependent relaxation at a reduced level.