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Am J Physiol Regul Integr Comp Physiol 289: R755-R762, 2005. First published May 12, 2005; doi:10.1152/ajpregu.00748.2004
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NEUROHUMORAL CONTROL OF CARDIOVASCULAR FUNCTION

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

Anatoly I. Soloviev,1 Sergey M. Tishkin,1 Sergey N. Zelensky,1 Irina V. Ivanova,1 Igor V. Kizub,1 Alexandra A. Pavlova,1 and Robert S. Moreland2

1Institute of Pharmacology and Toxicology, Academy of Medical Sciences, Kiev, Ukraine; and 2Drexel University College of Medicine, Department of Pharmacology and Physiology, Philadelphia, Pennsylvania

Submitted 3 November 2004 ; accepted in final form 6 May 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 Ca2+ 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 Ca2+ ([Ca2+]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 Ca2+ 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 Ca2+ sensitivity in permeabilized tissues on the 9th and 30th days postirradiation. Phorbol dibutyrate (PDBu, 10–7 M) increased Ca2+ sensitivity in control skinned smooth muscle but was without effect in irradiated vascular rings. Simultaneous measurement of contractile force and [Ca2+]i showed that myofilament Ca2+ sensitivity defined as the ratio of force change to [Ca2+]i significantly increased following {gamma}-irradiation. PDBu (10–6 M) stimulation of intact aorta produced a sustained contraction, while the increase in [Ca2+]i was transient. In irradiated tissues, PDBu-induced contractions were greater than those seen in control tissues but there was no elevation in [Ca2+]i. Taken together, these data strongly support the hypothesis that irradiation increases the sensitivity of vascular smooth muscle myofilaments to Ca2+ and this effect is dependent on activation of protein kinase C.

ionized irradiation; chelerythrine; fura-2; staurosporine; phorbol dibutyrate

LONGITUDINAL STUDIES OF ATOMIC BOMB survivors from Hiroshima and Nagasaki, Japan, have clearly shown that the second leading cause of radiation-dependent deaths is associated with cardiovascular events (26, 30). Moreover, the relative risk of death due to a cardiovascular event increases sharply as the survivors age compared with a population of similarly aged subjects not exposed to radiation (26). The cause of these cardiovascular deaths is due to both myocardial infarct and hypertension (46) and therefore involves the heart as well as the vasculature.

Similar results have been published from studies on the general population near the Chernobyl, Ukraine Atomic Energy Station accident and the workers assigned to clean the facility. Second only to childhood thyroid disease were health problems associated with the cardiovascular system, with hypertension being the most prominent pathophysiological state (15). Additionally, the workers, also termed "liquidators," have been shown to have statistically increased incidences of carotid arterial stenosis (17), atherosclerosis (19), and decreased cerebral blood flow (29, 42).

If exposure to excess levels of radiation leads to vascular anomalies, then it would be reasonable to propose that the mechanism for these anomalies lies either in a decrease in endothelium-dependent relaxation of the vasculature or in an increase in the contractility within the vascular smooth muscle cell. We have previously shown that direct irradiation of rats produced a significant increase in systolic blood pressure (34). We postulated that irradiation impaired vascular function by depression of the endothelium-dependent vasodilatation. This is accomplished by selective impairment of the nitric oxide (NO)-dependent component of vasodilatation (35). Our results and postulates are consistent with previously published work examining the effect of irradiation on the role of the endothelium on vascular function (2, 13, 21, 27, 40, 41). Interestingly, one study demonstrated an increase in NO generation after irradiation, but this was performed in cultured vascular smooth muscle and therefore the cells may or may not have been in the contractile phenotype (47).

In contrast to the numerous studies demonstrating that irradiation depresses endothelium-dependent vasorelaxation, other investigations have shown that irradiation enhances vascular contractility in an endothelium-independent manner (20, 39). This information suggests that, in addition to the indirect effect through a decrease in endothelial-dependent relaxation, a direct effect on vascular contractility may be important in the vascular anomalies induced by irradiation.

Vascular smooth muscle is known to contract in response to an increase in cellular free calcium ([Ca2+]i). The increase in [Ca2+]i activates the calmodulin-dependent myosin light chain (MLC) kinase, which catalyzes the phosphorylation of the 20,000 Da MLC. MLC phosphorylation, in turn, activates the myosin molecule, which can then interact with actin and initiate contraction (14). In addition to this primary regulatory pathway, several modulatory pathways exist in smooth muscle that can alter the magnitude of force that is developed for any given level of cellular Ca2+ (37). Alterations in myofilament Ca2+ sensitivity can be either positive or negative depending on the pathways stimulated. For increases in myofilament Ca2+ sensitivity, two primary hypotheses have been proposed: G-protein-dependent activation of Rho kinase and protein kinase-C-dependent phosphorylation of a cellular protein called CPI-17 (37). Both proposals have a common end point, that being inhibition of the MLC phosphatase resulting in a greater level of MLC phosphorylation for any given level of Ca2+ and activity of the MLC kinase.

Hypertension has been clearly shown to be associated with an increase in vascular reactivity. Recently, the enhanced reactivity during hypertension has been shown to be due in part to an increase in myofilament Ca2+ sensitivity (28). Hypertension has also been shown to be associated with blunted endothelium-dependent responses due to a decreased sensitivity to NO (31). Both an increase in myofilament Ca2+ sensitivity and a decrease in sensitivity to NO may result from a change in protein kinase C (PKC) activity (7, 25, 32, 33). Taken together, this suggests the possibility that alterations in the activity or regulation of PKC may be a unifying hypothesis in the irradiation-induced hypertensive state and alterations in vascular function.

Therefore, the goal of this study was to test the hypothesis that the increase in vascular responsiveness that results following whole body irradiation is due to a change in the PKC-dependent increase in myofilament Ca2+ sensitivity in the vascular smooth muscle cell.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All experiments involving the use of animals were approved by the Institutional Animal Care and Use Committees of the Institute of Pharmacology and Toxicology, Academy of Medical Sciences, Kiev, Ukraine, and Drexel University College of Medicine, Philadelphia, PA. Experiments were performed on intact and chemically skinned thoracic aortic rings obtained from adult Wistar-Kyoto rats weighing from 200 to 400 g. Single whole body irradiation was performed with gamma rays delivered at a rate of 0.9 Gy/min from a cobalt-60 source (TGT ROCUS M) positioned 50 cm from the animal. During irradiation, the animals were restrained inside individual plastic boxes specifically designed for this study, which were placed such that the axis of the radiation beam, with a square of 20 x 20 cm, was centered on the animal’s chest. There was no change in housing, standard food, or water after irradiation. Irradiation increased systolic blood pressure which remained elevated for up to 6 mo postirradiation.

Preparation of vascular smooth muscle and contractile recordings. Control and irradiated animals were anesthetized using diethyl ether and euthanized by cervical dislocation. After euthanasia, a 2-cm-long segment of the thoracic aorta was dissected and cleaned of both connective and adipose tissues. This segment was cut into 1- to 1.5-mm-wide rings and turned inside-out when used for [Ca2+]i measurements. When appropriate, the rings were denuded of endothelium by gently rolling the luminal surface over filter paper. All procedures were performed at room temperature in nominally Ca2+-free solution. Aortic rings were mounted isometrically under a resting tension of 10 mN in a flowing tissue bath, between a stationary stainless steel hook and an isometric force transducer (AE 801, SensoNor A/S, Norten, Norway) coupled to a chart recorder (model 202, Cole-Palmer Instrument, Vernon Hills, IL). Vessels were maintained at 37°C in an organ bath containing 0.5 ml of modified Krebs-bicarbonate buffer. Rings were allowed to equilibrate for 1 h under resting tension before experiments commenced. After the equilibration period, they were exposed several times to high KCl (60 mM) solution until reproducible contractile responses were obtained. High KCl solution was prepared by replacement of NaCl with equimolar KCl to avoid a change in ionic strength or tonicity of the solution.

Permeabilization. Aortic rings were stretched to a resting tension of 10 mN, a tension that produces maximal active force, and equilibrated for at least 30 min in a HEPES-buffered solution of the following composition (in mM): 135.5 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgCl2, 11.6 N-2-hydroxyethylpiperazine-N-2 ethanesulfonic acid (HEPES), and 11.5 glucose. Aortic rings were stimulated with a high-KCl solution to verify viability. Rings not responding to stimulation were discarded. Permeabilization procedures were performed at room temperature (21°C). The aortic rings were permeabilized using 0.05% (wt/vol) {beta}-escin in a solution of pCa 6.0 for 25–30 min. This resulted in a gradual increase in force from the basal level to a plateau. The increase in force to stable levels in response to 1 µM Ca2+ [the 50% effective concentration (EC50) for Ca2+-dependent force development] suggested efficient and complete permeabilization.

Measurement of [Ca2+]i. Experiments for the simultaneous measurement of [Ca2+]i and contractile force were carried out in a 500 µl tissue chamber mounted on the stage of a fluorescence microscope LUMAM-2 equipped with epifluorescence collection. The aortic rings were mounted isometrically between a stationary stainless steel hook and a force transducer (AE 801, SensoNor A/S, Norten). Except for during the fura-2 AM loading procedure, the rings were continuously perfused with preheated Krebs solution at 35°C at a rate of 2.0 ml/min. The rings were loaded with 10 µM fura 2 AM in a physiological solution of the following composition (mM): 122 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgCl2, 11.6 HEPES, 11.5 glucose, and a pH of 7.3–7.4. The loading solution also contained 2.5% DMSO and 5 mg/ml Pluronic F-127. Loading continued for 2 h at room temperature. The tissues were then mounted in a tissue chamber for measurement of force and [Ca2+]i and equilibrated in normal physiological salt solution for at least 30 min.

Fura-2 fluorescence was excited at 340- and 380-nm wavelengths ({lambda}) and recorded at 510-nm emission wavelength from a central region (~0.5 mm in diameter) on the blood surface of the aortic ring. The fluorescence emitted from the tissue was collected by a photomultiplier through a 510-nm filter. The results of [Ca2+]i measurements are presented as the ratio (R) of a 510-nm emission fluorescence intensity [I510({lambda})] at {lambda} = 340 nm and {lambda} = 380 nm excitation signals: R = I510340/I510380. [Ca2+]i was determined, as described by Grynkiewicz et al. (9), using the formula [Ca2+]i (nM) = Kd x [(R – Rmin)/(Rmax – R)] x (Sf2/Sb2), where Kd (224 nM at 37°C) is the dissociation constant of fura-2 for Ca2+; R is the ratio of fluorescence of the sample at 340 to that at 380 nm; Rmin and Rmax represent the ratios of fluorescence at the same wavelengths in the presence of zero and saturating Ca2+, respectively; and Sf2/Sb2 is the ratio of fluorescence of fura-2 at 380 nm in zero Ca2+ to that in saturating Ca2+, respectively. Before initiating experiments, it was determined that contractions induced by high KCl were not affected by fura-2 loading.

Solutions. All tissues were stored in a physiological salt solution of the following composition (in mM): 122 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgCl2, 15.5 NaHCO3, 1.2 KH2PO4, and 11.5 glucose, pH 7.3–7.4. Nominally Ca2+-free solutions were prepared by simply omitting CaCl2. Ca2+ free/EGTA solutions used for removal of cellular calcium were prepared by omitting CaCl2 and including 1 mM EGTA. Depolarizing KCl solutions were prepared by the equimolar exchange of KCl for NaCl. Solutions for experiments performed using permeabilized muscle contained (in mM): 3.2 MgATP, 2 Mg2+, 12 phosphocreatine, 0.5 sodium azide, 30 TES, pH 6.9 at 21°C. Unless otherwise stated, creatine phosphokinase (15 U/ml) and calmodulin (0.5 µM) were added to all solutions. Desired free Ca2+ levels (expressed as pCa) were obtained by mixing stock solutions containing K2EGTA and K2CaEGTA. The total EGTA concentration was 4 mM. Ionic strength was adjusted to 150 mM with potassium propionate.

Chemicals. Fura-2 AM was obtained from Molecular Probes, (Eugene, OR). All other compounds were obtained from Sigma Chemical (St. Louis, MO). DMSO (0.1% final concentration) was used as a solvent for phorbol dibutyrate (PDBu). This concentration of DMSO had no effect on any parameter of contraction.

Statistics. The data are shown as means ± SE; n indicates the number of vascular preparations tested. Curves were fitted to the Hill equation. Half-maximally effective concentration (EC50) values were expressed as pD2 (Log EC50). Comparison of variables obtained by various treatments with basal values were made by one-way ANOVA with a repeated measurements design, and if any significant difference was found, the Scheffé’s multiple-comparison test was applied. Differences were considered to be statistically significant when P was <0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Intact vascular strips from control and irradiated animals. The first series of experiments performed were designed to determine the effects of whole body irradiation on vascular contractile sensitivity. Figure 1A shows the results of concentration-response curves from the cumulative addition of KCl. The addition of KCl (8–120 mM) produced concentration-dependent increases in force development in the thoracic aorta from both control and irradiated animals. The experiments were performed on the 9th and 30th days postirradiation. Thoracic aortic rings obtained from irradiated animals on the 9th day postirradiation were significantly more sensitive to the addition of KCl compared with aortic rings from control animals. Maximum force development was similar in aortic rings from the two animal groups (control: 7.6 ± 0.8 mN; irradiated: 8.6 ± 1.2 mN; P > 0.05, n = 12). Similar results were obtained on the 30th day postirradiation (maximal force development was 10.0 ± 1.1 mN, P > 0.05, n = 12 compared with control).



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  		Fig. 1. Effect of whole body irradiation (6 Gy) on KCl- and phenylephrine-induced contractions of rat thoracic aorta. A: cumulative concentration-response curves for KCl were obtained with aortic rings from control ({circ}) and irradiated rats on the 9th ({bullet}) and 30th ({blacksquare}) days postirradiation. Data shown are means ± SE. Curve fits of mean values were performed using the following: F = Fmax/[1 + exp(log EC50 – log [agonist+])·h], where F and Fmax are actual and maximal force, h is the Hill coefficient, EC50 is 50% effective concentration. Mean values of pD2 (–log EC50) are 1.70 ± 0.03 control, and 1.90 ± 0.05 (P < 0.01, n = 12) and 1.90 ± 0.03 (P < 0.01, n = 12), irradiated (9th and 30th days, respectively). B: cumulative concentration-response curves to phenylephrine obtained with aortic rings from control ({circ}) and irradiated rats on the 9th ({bullet}) and 30th ({blacksquare}) days postirradiation. Mean values of pD2: control, 6.30 ± 0.07; and irradiated: 6.92 ± 0.05 (P < 0.01, n = 12) and 6.89 ± 0.05 (P < 0.01, n = 12), 9th and 30th days, respectively. Contractions are expressed as a percentage of the tension evoked by maximal KCl (120 mM) or phenylephrine (10–5 M) concentration.

 
Similar experiments using the cumulative addition of phenylephrine showed increased sensitivity as evidenced by a shift to the left in the stimulation response curve in vascular rings from animals subjected to whole body irradiation compared with those from control animals (Fig. 1B). On the basis of these results, whole body {gamma}-irradiation produces a general increase in contractile sensitivity in response to all forms of stimulation, whether in response to membrane depolarization or agonist activation.

Experiments on chemically permeabilized vascular strips from control and irradiated animals. Although many mechanisms exist by which irradiation could increase vascular sensitivity, the two most probable mechanisms are alterations in calcium metabolism in response to stimulation or a direct effect on the contractile proteins. One of the best models to use to differentiate between these two possibilities is the chemically permeabilized strip. This preparation allows one to examine the contractile response under conditions of constant intracellular [Ca2+]i. The data shown in Figs. 2 and 3 were obtained from experiments performed on {beta}-escin-permeabilized aortic strips. All forces shown were normalized to the maximal Ca2+-dependent force attained in each vascular preparation. The results shown in these figures clearly demonstrate that on the 9th and 30th day of the postirradiation period, a significant shift to the left in the calcium-tension relationship was seen denoting an increase in myofilament calcium sensitivity. On the 9th day after a single whole-body {gamma}-irradiation, an increase in myofilament Ca2+ sensitivity of 0.38 ± 0.09 pCa units was observed in the series of experiments performed to generate Figs. 2 and 3.



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  		Fig. 2. Force development in {beta}-escin-permeabilized rat aortic rings as a function of calcium concentration in the presence and absence of chelerythrine. Aortic rings from control ({circ}) and irradiated ({bullet}) animals were subjected to the cumulative addition of calcium. Irradiated animals were studied on the 9th (A) and 30th (B) days postirradiation period following exposure {gamma}-radiation (6 Gy). In addition, the calcium response of irradiated tissues was examined in the presence of 1 µM chelerythrine ({blacksquare}). The values shown are means ± SE. Data are normalized to the force developed at pCa 5.0. Curves shown were fit to the Hill equation; see Fig. 1 for details. Mean values of pD2 (or pCa50 as generally accepted for permeabilized smooth muscle) for the 9th day postirradiation were 5.60 ± 0.09, control; 5.98 ± 0.06, irradiated (P < 0.01, n = 12), and 5.33 ± 0.15 irradiated+chelerythrine (P < 0.05, n = 8, comparison with irradiated). The data for the 30th day postirradiation were 5.60 ± 0.09 for control, 6.48 ± 0.15 for irradiated (P < 0.01, n = 12), and 5.99 ± 0.09 for irradiated+chelerythrine (P < 0.01, n = 8, comparison with irradiated).

 


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  		Fig. 3. Force development in {beta}-escin permeabilized rat aortic rings as a function of calcium concentration in the presence and absence of staurosporine. Aortic rings from control ({circ}) and irradiated ({bullet}) animals were subjected to the cumulative addition of calcium. Irradiated animals were studied on the 9th (A) and 30th (B) days postirradiation period after exposure {gamma}-radiation (6 Gy). In addition, the calcium response of irradiated tissues was examined in the presence of 0.1 µM of staurosporine ({blacksquare}). The values shown are means ± SE. Data are normalized to force developed at pCa 5.0. Curves shown were fit to the Hill equation; see Fig. 1 for details. Mean values of pD2 (or pCa50 as generally accepted for permeabilized smooth muscle) for the 9th day were 5.60 ± 0.09, control; 5.98 ± 0.06, irradiated (P < 0.01, n = 12), and 5.30 ± 0.10, irradiated+staurosporine (P < 0.05, n = 6, comparison with irradiated). The data for the 30th day postirradiation were 5.60 ± 0.09, control; 6.48 ± 0.15, irradiated (P < 0.01, n = 9), and 5.53 ± 0.08, irradiated+staurosporine (P < 0.05, n = 7, comparison with irradiated).

 
In an attempt to uncover potential mechanism(s) responsible for the {gamma}-irradiation-induced increase in myofilament Ca2+ sensitivity, we performed experiments using inhibitors of PKC. PKC has been shown to be involved in agonist induced increases in myofilament Ca2+-sensitivity (45) and, in fact, has been shown to produce Ca2+-independent contractions of vascular smooth muscle (7, 10). Figure 2 shows the results of experiments using chelerythrine (10–6 M) and Fig. 3 shows the results of experiments using staurosporine (10–7 M). Both inhibitors reversed the increase in myofilament Ca2+ sensitivity induced by single whole body {gamma}-irradiation on the 9th day of the postirradiation period. On the 9th day postirradiation, chelerythrine shifted the Ca2+ concentration response curve 0.65 ± 0.10 µM to the right, whereas staurosporine shifted the curve 0.68 ± 0.08 µM to the right. On the 30th day of the postirradiation period, chelerythrine partially reversed the increase in Ca2+ sensitivity (Fig. 2B), whereas staurosporine completely reversed the change in sensitivity (Fig. 3B). Of particular interest was the total lack of effect of the PKC inhibitors on the calcium responsiveness of permeabilized aortic tissues from control animals. This is an especially important finding given the potential for nonspecific inhibition of other kinases by these inhibitors, such as the MLC kinase.

PDBu (10–7 M), a potent PKC activator, shifted the pCa-tension response relationship for skinned smooth muscles to the left in control permeabilized tissues but had no effect in permeabilized thoracic aorta rings from irradiated animals (Fig. 4).



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  		Fig. 4. Effect of phorbol dibutyrate (PDBu, 10–7 M) on force development in control and irradiated {beta}-escin permeabilized rat aortic rings as a function of calcium concentration. {circ}, control; {bullet}, PDBu-treated control tissues; {blacksquare}, PDBu-treated irradiated tissues. Mean values of pD2 (or pCa50 as generally accepted for permeabilized smooth muscle) for the 9th day postirradiation were 5.30 ± 0.03 for control, 5.63 ± 0.11 for PDBu-treated control (P < 0.05, n = 6), and 5.81 ± 0.04 for irradiated+PDBu (P < 0.01, n = 6, comparison with irradiated).

 
Simultaneous measurements of contractile force and [Ca2+]i. The next series of experiments were designed to simultaneously measure stimulation-induced changes in intracellular calcium concentration and force in the intact aortic tissue from control and irradiated animals. It is important to note that in these experiments the endothelial cells were mechanically removed from the area in which fura-2 calcium fluorescence was measured. Therefore, fluorescent changes were derived specifically from the vascular smooth muscle cells and not influenced by any potential changes in the endothelium. The goal of these experiments was to determine whether the irradiation-induced change in myofilament calcium sensitivity demonstrated in the permeabilized preparation could also be demonstrated in an intact tissue where calcium is mobilized by normal physiological pathways.

Intact aortic preparations were stimulated with 60 mM KCl and the increase in force and preceding increase in [Ca2+]i measured simultaneously. Representative tracings from such experiments are shown in Fig. 5. Membrane depolarization by 60 mM KCl produced a slightly smaller increase in [Ca2+]i in aorta from irradiated compared with that from control animals. In contrast, aorta from irradiated animals produced significantly more force than tissues from control animals.



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  		Fig. 5. Representative recordings of the effect of irradiation (6 Gy) on KCl-induced increase in intracellular free Ca2+ concentration [ratio of F340 to F380 (R340/380)] and tension in intact rings of rat thoracic aorta. KCl-mediated muscle contraction (bottom) and increment in (R340/380) (top) were measured simultaneously in the same aortic ring. Aortic rings from both animal groups produced similar increases in cellular free calcium ([Ca2+]i) in response to 60 mM of KCl. Aortic rings from irradiated animals produced significantly more force in response to 60 mM KCl compared with rings from control animals.

 
The compilation of several simultaneous force and calcium measurements is shown in Fig. 6. As expected, a positive correlation exists between the force developed at different KCl concentrations in the organ bath and the [Ca2+]i. Similar to the results obtained from the experiments performed on permeabilized arterial preparations, greater levels of force are developed at any given [Ca2+]i in tissues from irradiated animals compared with those from control animals. Aortic rings obtained from irradiated animals produced 0.068 mN force/nM Ca2+, whereas arteries from control animals produced 0.026 mN force/nM Ca2+.



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  		Fig. 6. Effect of irradiation (6 Gy) on the KCl-mediated [Ca2+]i-tension relationship in the thoracic aorta. Several experiments of the type shown in Fig. 4 were used to develop the [Ca2+]i-tension relationship shown in this figure. Rings loaded with fura-2 were subjected to the cumulative addition of KCl and levels of force and the simultaneous measurement of [Ca2+]i recorded. The results are shown as ({square} control) and ({bullet} irradiated). The slope (mN tension/nM [Ca2+]i) was significantly increased in irradiated tissues compared with control, indicating that the Ca2+ sensitivity of the myofilaments had increased.

 
Stimulation of intact aortic smooth muscle from control animals with PDBu (10–6 M) produced a slow but tonic contraction that required 26–28 min for maximal tension to be developed. In contrast, the PDBu stimulation induced change in [Ca2+]i was only transiently increased (Fig. 7A). Stimulation of intact aortic smooth muscle from irradiated animals with PDBu also produced the slow development of a tonic contraction; however, the maximal levels attained were greater than that produced in aortas from control animals (Fig. 7B). Interestingly, although levels of developed force were greater in tissues from irradiated compared with control animals, there was no change from basal in cellular [Ca2+]i in response to PDBu stimulation in the aortas from irradiated animals.



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  		Fig. 7. Typical recordings of the effect of PDBu, (10–6 M) on intracellular calcium concentration (R340/380; upper tracings) and contractile force development (lower tracings) in both control (left) and irradiated (right) thoracic aorta tissues. Force and R340/380 were measured in the same aortic ring. PDBu produced greater levels of force in rings from irradiated animals. Conversely, PDBu produced a transient increase in [Ca2+]i in rings from control animals but no visible increase in [Ca2+]i in rings from irradiated animals.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Nuclear events, whether accidental such as that which occurred in Chernobyl or during wartime as occurred in Hiroshima and Nagasaki, are fortunately uncommon, and we can only hope will never happen again. However, in our changing world, the ever-present danger of the so-called "dirty atomic bomb" exists. Moreover, radiation therapy is commonly employed as a primary and adjuvant therapeutic modality in patients with neoplasm, and despite many advances in technology, normal tissues, including the vasculature are often affected. The effects of radiation therapy in the arterial system manifest as a blunting in vasorelaxation and hypercontractility of the vascular smooth muscle (1). The current challenge in radiation medicine therefore is to determine how to prevent radiation-induced vascular abnormalities that complicate the treatment of patients or to minimize the consequences of nuclear accidents. However, before effective treatments can be devised, the mechanisms underlying the abnormalities must be known.

Work from our group, as well as others (1, 13, 27, 35, 40), has suggested that a major site of radiation-induced vascular injury is the endothelium. Endothelial damage in response to radiation exposure specifically depresses NO-dependent vascular relaxation resulting in an increase in systolic blood pressure and hypercontractility of the vasculature (35). In addition to effects on the endothelial lining of blood vessels, it has been shown that radiation may directly impact vascular smooth muscle contractility (2, 20, 21, 41).

The results in the present study now clearly demonstrate that single whole body {gamma}-irradiation significantly increases the myofilament calcium sensitivity of the vascular smooth muscle cell independent of any action on the endothelium. Moreover, our results strongly suggest that the mechanism by which irradiation increases myofilament calcium sensitivity is through a PKC-dependent pathway.

Alterations in myofilament calcium sensitivity have been shown to underlie, at least in part, the changes in smooth muscle function that occur in response to hypertension, asthma, benign prostatic hyperplasia (28, 38, 44), and now based on our results, in response to {gamma}-irradiation. The question then becomes, what are the mechanisms underlying the increase in myofilament calcium sensitivity that we observed after radiation exposure? Myofilament calcium sensitivity may be altered by influencing the enzymes that initiate or modulate actin and myosin interactions or by direct actions on the contractile proteins. Although theoretically possible, the second suggestion is not supported by experimental evidence. The first possibility is supported by a vast amount of literature (11, 37). As discussed in the introduction, two pathways have been proposed for the enhancement of myofilament calcium sensitivity in smooth muscle, one involving G-proteins and the other CPI-17. We know that functional G-proteins are retained in a {beta}-escin permeabilized smooth muscle preparation, and Bonnevier and Arner (2) have shown that CPI-17 is retained and can be phosphorylated in a {beta}-escin permeabilized smooth muscle. Therefore, both pathways are most likely functional in our permeabilized muscles.

Our results would suggest that exposure to {gamma}-irradiation increases myofilament calcium sensitivity by augmenting the PKC pathway. This is based on the fact that the increase in calcium sensitivity in vessels from irradiated animals was reversed by either staurosporine or chelerythrine and the activator of protein kinase C, PDBu, had no effect on pCa-force relationship in irradiated tissue, while shifting the pCa-force relationship in control aortic rings to the left. It is important to point out again that neither antagonist had any effect on the calcium sensitivity of vessels from control animals. Thus nonspecific actions of these PKC inhibitors were apparently not an issue in this study. In addition, because {beta}-escin disrupts endothelial cell function, our experiments are examining the vascular smooth muscle cells directly without modulatory effects from the endothelium. Moreover, the calcium concentration curves in the permeabilized tissues were performed in the absence of GTP, and activation of the intact tissues was in response to membrane depolarization. Our results using the permeabilized tissues aid in alleviating any concerns that KCl may activate RhoA in the intact tissues as has been previously suggested in human myometrial smooth muscle (18). Moreover, because similar findings were obtained with norepinephrine and KCl, this also supports the similar effect of irradiation in both activation regimens. Importantly, PKC has been shown to be activated in KCl stimulated intact vascular smooth muscle (31), and PKC is retained and can be activated in permeabilized vascular tissues (5).

Simultaneous measurement of [Ca2+]i and force also supports this point of view. PDBu-induced contractions of control intact tissues showed a concomitant rise in force and [Ca2+]i. In stark contrast, PDBu-induced contractions of irradiated intact tissues were not accompanied by any increase in [Ca2+]i, even though the amplitude of PDBu-induced contractions were greater on average than that in intact preparations of control tissues. The enhanced force in tissues from irradiated animals in the absence of a change in cellular calcium concentration in response to PDBu suggests the irradiation-induced increase in myofilament calcium sensitivity was increased primarily by a PKC-dependent pathway.

If, as we speculate, PKC activity is enhanced after irradiation, then are there potential mechanisms that could produce this end result? It is well known that after irradiation, the content of oxygen-derived free radicals, including superoxide anion, hydroxyl anions, and others, increases significantly in living cells and biological liquids. These reactive oxygen species are associated with radiation-induced cytotoxicity and are responsible for radiation-induced lipid peroxidation and related vascular lesions. It is interesting to speculate that irradiation induces a state of oxidative injury on vascular tissue. This possible but speculative role for oxidative stress in radiation-induced vascular dysfunction is supported by the protective effect of the antioxidant, {alpha}-tocopherol in rabbit thoracic aorta (35). {alpha}-Tocopherol was shown to prevent the loss of endothelium-dependent relaxation when administered as a single dose shortly after radiation exposure. Nevertheless, this problem remains yet unsolved, and hopefully, more convincing evidence will be forthcoming.

Radiation has been shown to have a direct effect on altering the expression of PKC. Radiation therapy dose-dependently increased PKC in mouse lymphocytes (43). Interestingly, differential responses were found in the radiation-induced expression of {alpha}, {delta}, {zeta} isoforms of PKC. In addition, it is known that radiation induces the translocation of PKC from the cytosol to the membrane as a result of reactive oxygen formation producing membrane lipid peroxidation (24). These findings are relevant to our present study as the {alpha} isoform of PKC has been implicated in calcium-dependent PKC-induced contractions of smooth muscle and the novel PKC isoform {epsilon}, of which {zeta} is also a member, has been implicated in calcium-independent PKC-induced contractions (10, 19).

It is important in any discussion of irradiation-induced vascular dysfunction to bear in mind that the underlying mechanism producing the alterations is multifaceted. In addition to any role for PKC or Rho kinase in irradiation-induced changes in vascular contractility, a role for alterations in NO metabolism has been clearly shown. It is known that phorbol esters can inhibit NO release (6), and thus an elevation of PKC activity might interfere with NO synthesis. In turn, it is likely that NO induces rat aorta dilation via inhibition of Rho-kinase (4). Recently, it has been shown that NO can relax smooth muscle via activation of protein phosphatases (36). Additionally, NO-generating agents can decrease the phosphotransferase activity of purified PKC with an IC50 7.5x10–5 M, and this effect is blocked by the NO scavenger oxyhemoglobin or reversed by dithiothreitol (8). In a situation such as whole body irradiation, when the endothelial cell and NO-dependent component of relaxation and NO effects on PKC are abolished, PKC activity in the vascular wall could be elevated, producing an increase in myofilament calcium sensitivity and the resultant increase in contractile force. At the same time, the experiments using permeabilized smooth muscle clearly demonstrate that irradiation has direct effects on PKC-mediated pathways, not dependent on NO, including an increase in myofilament calcium sensitivity.

Therefore, we propose the hypothesis that whole body {gamma}-irradiation causes the production of reactive oxygen species, which alters vascular function by an effect on both the endothelial layer and the smooth muscle cell. In the smooth muscle cell, specific isoforms of PKC are upregulated and activated, which produce an apparent increase in myofilament calcium sensitivity. The combination of decreased NO release from damaged endothelial cells and hypercontractility of the vascular smooth muscle cells acts in concert to produce increases in arterial blood pressure and vasospasm in response to normally occurring levels of endogenous agents.


   ACKNOWLEDGMENTS
 
This study was supported, in part, by grant UB1–2452-KV-02 from the U.S. Civilian Research and Development Foundation to A. I. Soloviev and R. S. Moreland and HL 37956 from the National Institutes of Health to R. S. Moreland.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. I. Soloviev, Institute of Pharmacology and Toxicology, Academy of Medical Sciences, 14 Eugene Pottier St., Kiev, 03057, Ukraine (e-mail: s.a.pharm{at}naverex.kiev.ua)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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