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.

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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 AChinduced 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 paxillinesensitive 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.

The abilities of such therapeutic nitrovasodilators as sodium nitroprusside (SNP) and glyceryl trinitrate (GTN) to dilate vascular smooth muscles (VSM) and affect intracellular calcium concentration level ([Ca2+]i) in a rat tail artery were tested under different types of preactivation. To shed light on mechanisms underlying possible differences in the action of these two nitric oxide (NO) donors, simultaneous measurements of [Ca2+]i and contractile force were done. All vascular rings were precontracted either using a high-K+-Krebs solution or phenylephrine (PE). It was shown that the effect of both NO donors strongly depended on a type of VSM preactivation. The EC50 for GTN under K+ stimulation of VSM comprised (2.48 +/- 1.6) x 10(-5) M, whereas the mean EC50 under PE stimulation was (3.05 +/- 2.3) x 10(-4) M (p < 0.05, n = 9). The EC50 for SNP under K+ stimulation of VSM comprised (1.09 +/- 0.47) x 10(-7) M, whereas the EC(50) under PE stimulation was (8.01 +/- 2.4) x 10(-6) M (p < 0.05, n = 9). GTN demonstrated a significant discrepancy in the magnitude of changes in [Ca2+]i and related VSM relaxant responses, indicating the ability of GTN to relax VSM in the absence of a proportional decrease in [Ca2+]i. The main peculiarity of SNP action under K+ stimulation as compared to PE stimulation was the transient decrease in [Ca2+]i while relaxation was sustained. Therefore, both NO donors demonstrated their ability to produce vasorelaxation as a result of an alteration in myofilament calcium sensitivity. These data clearly indicate that the sensitivity of VSM to NO donors is higher under K+ depolarization than that seen under PE stimulation, indicating that Ca2+ entry through voltage-operated calcium channels is more sensitive to NO as compared to calcium mobilization by means of Ca2+ entry through receptor- operated calcium channels or intracellular Ca2+ release, or both.