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

Life Sciences 84 (2009) 164–171

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Functional and molecular consequences of ionizing irradiation on large conductance Ca2+-activated K+ channels in rat aortic smooth muscle cells Anatoly Soloviev a,⁎, Sergey Tishkin a, Irina Ivanova a, Sergey Zelensky a, Victor Dosenko b, Sergey Kyrychenko a, Robert S. Moreland c a b c

Institute of Pharmacology and Toxicology, Academy of Medical Science, 14 Eugene Pottier Str., 03057, Kiev, Ukraine Bogomoletz Institute of Physiology, National Academy of Sciences, 4 Bogomoletz Str., 01024, Kiev, Ukraine Drexel University College of Medicine, Department of Pharmacology and Physiology, Philadelphia, USA

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Article history: Received 20 May 2008 Accepted 10 November 2008 Keywords: Ionizing irradiation Vascular smooth muscle Ca2+-activated K+ channels Vasoconstriction Arterial hypertension

a b s t r a c t Aims: The goal of this study was to evaluate the influence of γ-irradiation on Ca2+-activated K+ channel (BKCa) function and expression in rat thoracic aorta. Main methods: Aortic cells or tissues were studied by the measurement of force versus [Ca2+]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 BKCa 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 BKCa channels. Aortic tissue from irradiated animals showed progressively enhanced contractile responses to phenylephrine in the postirradiation period of 9 and 30 days. The concomitant Ca2+ 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 BKCa α- and β1-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 Ca2+ sensitivity in the early post-irradiation period and a decrease in BKCa channel expression in the late post-irradiation period. © 2008 Elsevier Inc. All rights reserved.

Introduction The most common health problems in the Ukrainian population exposed to radiation as a result of the Chernobyl catastrophe are diseases of the cardiovascular system, and specifically hypertension (Soloviev et al., 2003). Arterial hypertension is a reasonably common disorder in the normal population, but its occurrence is significantly higher among survivors of the Chernobyl catastrophe. A similar finding was concluded from longitudinal studies of survivors of the Nagasaki and Hiroshima atomic bombs (Yamada et al., 2004). Therefore, exposure to excess levels of radiation, even in non-fatal doses, leads to vascular contractile anomalies. A major contributor to this vascular dysfunction is the loss of endothelium-dependent vasodilatation. Specifically, radiation increases vascular tone due to the selective impairment of the nitric oxide (NO)-dependent component of vasodilatation (Soloviev et al., 2003). The loss of NOdependent vasodilatation may be due to the reduction of large ⁎ Corresponding author. Tel.: +380 44 536 1340; fax: +380 44 536 1341. E-mail address: [email protected] (A. Soloviev). 0024-3205/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2008.11.015

conductance Ca2+-activated K+ channel (BKCa) activity as observed in rat coronary artery endothelial cells (Tishkin et al., 2007). Furthermore rat systolic blood pressure is significantly increased 9 days after exposure to a 6 Gy dose of radiation and remained elevated as compared to control animals for up to six months (Soloviev et al., 2002). Taken together, the findings from longitudinal studies of humans exposed to radiation and those from acute animal studies strongly support the hypothesis that there is a direct and consistent linkage between exposure to ionizing irradiation and vascular abnormalities. Several lines of evidence clearly demonstrate that blood vessels may represent one of the main targets for ionizing irradiation. At the same time there is a lack of information on the direct effect of irradiation on smooth muscle cells and, in particular, of the cellular mechanism(s) leading to smooth muscle abnormalities. This missing information prevents the development of new drugs for therapeutic strategies aimed at reversing radiation-induced vascular disorders and hypercontractility. The primary K+ current-carrying channels in vascular smooth muscle cells are the BKCa channels and the Kv channels (Brakemeier

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decreases endothelial cell BKCa channel activity resulting in a decrease in endothelial-dependent vascular relaxation and an increase in vascular contractility (Tishkin et al., 2007). Furthermore, we also showed that irradiation increases the Ca2+ sensitivity of vascular smooth muscle myofilaments, and that this effect is mediated by an enhanced activation of protein kinase C (Soloviev et al., 2005). Thus, irradiation-induced effects at both endothelial and smooth muscle levels may result in an enhanced vascular contractility. Moreover, in the membrane of the vascular smooth muscle cell changes in ion channel content and activity cannot be ruled out. It is well accepted that K+ channels play an important role in regulation of the membrane potential of smooth muscle cells and therefore of vascular disease (Jackson and Blair, 1998). Four main types of K+ channels have been described in smooth muscle cells: voltage

et al., 2003). It has been suggested that Kv, but not BKCa plays a major role in the regulation of the excitability and contractility in rat aorta (Tammaro et al., 2004). However, these two types of K+ channels operate at different transmembrane potentials; Kv currents appear at potentials ≥−40 mV, while BKCa currents are seen at potentials more positive than −20 mV (Tammaro et al., 2004). It is important to add that BKCa channels have a low Ca2+ sensitivity that renders them silent under resting conditions even in arterioles with substantial resting tone (Lu et al., 2006). BKCa channels are also very sensitive to reactive oxygen species (Brakemeier et al., 2003; Tishkin et al., 2007) and it is well accepted that radiation impact is a form of oxidative stress. In endothelial cells, activation of BKCa channels causes membrane hyperpolarization that, in turn, promotes membrane potential-driven Ca2+ influx and Ca2+dependent synthesis of vasodilator factors such as NO. Irradiation

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activated K+ channels (Kv) which are encoded by the Kv gene family, inward rectifiers (KIR) which are encoded by the Kir2.0 gene, ATPsensitive K+ channels (KATP) which are encoded by Kir6.0 and sulphonylurea receptor genes, and BKCa which are encoded by the Slo gene (Standen and Quayle, 1996). At the molecular level, BKCa channels are composed of pore-forming α-subunits which are coded by the gene Slo 1 (KNCMA1) and regulatory β1-subunits (Standen and Quayle, 1996), the latter being responsible for channel sensitivity to Ca2+ making the BKCa channel an efficient modulator of smooth muscle function in health and disease. Therefore, we attempted to clarify the role of the BKCa channel in the regulation of vascular tone following radiation impact. The goal of this study was to evaluate the influence of non-fatal whole-body γirradiation (6 Gy) on BKCa function and mRNA expression in rat thoracic aortic smooth muscle cells using the combined results from contractile force versus [Ca2+]i studies, patch-clamp technique in the whole-cell configuration, and RT-PCR analysis.

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All animal studies were approved by the Institutional Animal Care and Use Committees of all institutions listed. Isolated smooth muscle cells were dispersed from rat thoracic aorta obtained from six–eight month old male Wistar rats (250–300 g) by papain treatment. Briefly, the rats were anesthetized with ketamine (37.5 mg/kg b.w., IP) and xylazine (5 mg/kg b.w., IP). Segments of the thoracic aorta (1.0–1.5 cmlong) were excised and cleaned of adipose and connective tissue. The aorta was then cut into small pieces (1.5 × 1.5 mm) in a cold low-Ca2+ solution containing (in mM): 140 NaCl; 6 KCl; 3 MgCl2; 10 glucose; 10 HEPES for 15 min. The vascular tissues were transferred to a fresh lowCa2+ solution containing: 0.2 mg/ml papain (11.55 U/mg), 0.3 mg/ml dithiothreitol, and 0.3 mg/ml bovine serum albumin. The tissues were then stored at +5 °C for 18 h and then incubated for 15 min at 37 °C. The tissues were then washed (2–3 min) twice in a fresh low-Ca2+ solution to remove the papain. Cells were dispersed by agitation using a glass pipette, and placed in normal Krebs bicarbonate buffer. Aliquots of the myocytes were stored at +5 °C and remained functional for at least 5 h.

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Segments of thoracic aorta (1.5 cm-long) were obtained as described above, cleaned of both connective and adipose tissue, and cut into 1 to 1.5 mm width rings. All procedures were performed at room temperature in a nominally Ca2+-free physiological salt solution. 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 (Russian Federation) equipped with epifluorescence collection equipment. The aortic rings were mounted isometrically between a stationary stainless steel hook and a force transducer (AE 801, SensoNor A/S, Norten, Norway). Except for during the Fura-2AM loading procedure, the rings were continuously perfused with Krebs solution preheated to 35 °C at a rate of 2.0 ml/min; Ca2+ measurements tended to be more stable at 35° as compared to 37 °C. 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 allowed to equilibrate in normal physiological salt solution for at least 30 min. Following the equilibration period, the tissues were exposed several times to phenylephrine (PE; 0.1 μM), or high KCl (60 mM), until reproducible

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mV Fig. 2. Effects of paxilline (500 nM) on the I–V relationship of BK channel currents recorded in rat thoracic aortic smooth muscle cells obtained before (A), Ca 9 days after (B) and 30 days after (C) irradiation. Currents were elicited by 10 mV step pulses with 300 ms duration between −100 and +70 mV from a holding potential of −60 mV in control and irradiated cells before and after treatment with paxilline (500 nM). Data shown are mean±SEM (n=7–8). ⁎P b 0.05 versus control.

contractile responses were obtained. High KCl solution was prepared by equimolar replacement of NaCl with KCl in order to avoid a change in osmolarity of the solution.

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Fura-2 fluorescence was excited at 340 and 380 nm wavelength (λ) and recorded at 510 nm emission wavelength from a central region (approximately 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 the 510 nm emission fluorescence intensity [I510(λ)] at λ = 340 nm and λ = 380 nm excitation signals: R =I510 (340) /I510(380). At the end of the experiment, the maximum fluorescence ratios were determined. The maximum fluorescence was determined in a phosphate-free, bicarbonate-free, 120 mM KCl, 5 mM CaCl2, salt solution containing 10 μM ionomycin and 50 μM PE. The minimum fluorescence ratio was determined by adding 10 mM ethylene glycol-bis (β-aminoethyl ether)-N, N, N′, N′-tetraacetic acid (EGTA). [Ca2+]i was determined as described by Grynkiewicz et al. (1985) using the formula [Ca2+]i (nM) =Kd × [(R −Rmin) / (Rmax −R)] × (Sf2 /Sb2), where: Kd (224 nM) is the dissociation constant of Fura-2 for Ca2+; R is the ratio of fluorescence of the sample at 340 nm 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; Sf2 /Sb2 is the ratio of fluorescence of Fura-2 at 380 nm in zero Ca2+ to that in saturating Ca2+ respectively. Preliminary experiments indicated that contractions induced by high KCl, PE, and caffeine were not significantly affected by Fura-2AM loading.

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ton, DE, USA). Reverse transcription was performed using a RevertAid™ H Minus First Strand cDNA Synthesis Kit (Fermentas, Lithuania), using 1.2–1.5 µg of total RNA and a random hexamer primer. The single-strand DNA used for PCR was obtained using the following primers:α subunit (GeneBank accession no. U40603): sense — 5′-TAC TTC AAT GAC AAT ATC CTC ACC CT-3′ and antisense — 5′-ACC ATA ACA ACC ACC ATC CCC TAA G-3′; β1 subunit (GeneBank accession no. AF020712): sense — 5′-GTA TCA CAC AGA AGA CAC TCG GGA-3′ and antisense — 5′-AAG AAG GAG AAG AGG AGG ATT TGG G-3′. Primers were synthesized by Metabion (Martinsried, Germany). Amplification mixture contained 5 µl of 5× PCR-buffer with MgSO4, 200 µM mixture of four nucleotide triphosphates, 40 pM of each primer, and 0.5 U of Taq polymerase (AmpliSense, Russian Federation), the volume was brought up to 25 µl with deionized water. Amplification of the fragment of BKCa consisted of 43 cycles: denaturation 94 °С, 45 s, primer annealing 62 °С, 45 s, and elongation 72 °С, 45 s (GeneAmp System 2700, Applied Biosystems, Foster City, CA, USA). To control the quality of RNA isolation and for comparison of intensity of the BKCa gene expression, a fragment of β-actin gene was amplified as an internal control. Amplified fragments were separated on 2% agarose gels containing ethidium bromide. Visualization and estimation of the density of amplified fragments after horizontal electrophoresis (160 V, 30 min) was performed using a transilluminator and Vitran software (Biocom, Russian Federation).

Electrophysiology Whole-body animal γ-irradiation The whole-cell patch clamp method was used to study whole-cell K+ currents (voltage clamp mode). Data acquisition and voltage protocols were performed using an Axopatch 200B Patch-Clamp amplifier and Digidata 1200B interface (Axon Instruments Inc., Foster City, CA, USA) coupled to an IBM-compatible computer equipped with pClamp software (version 6.02, Axon Instruments Inc., Foster City, CA, USA). Membrane currents were filtered at 2 kHz and digitized at a sampling rate of 10 kHz. The reference electrode was a Ag-AgCl plug electrically connected to the bath. At the beginning of each experiment, the junction potential between the pipette solution and bath solution was electronically adjusted to zero. No leakage current subtraction was performed on the original recordings, and all cells with input resistances below 1 GΩ were excluded from further analysis. Macroscopic current values were normalized as pA/pF. The membrane capacitance of each cell was estimated by integrating the capacitive current generated by a 10 mV hyperpolarizing pulse after electronic cancellation of pipette-patch capacitance using Clampfit software (version 6.02, Axon Instruments Inc., Foster City, CA USA). Average cell capacitance in the present study was 12.9 ± 2.5 pF (control), 12.2 ± 1.9 pF (9 days following irradiation) and 12.5 ± 1.8 pF (30 days following irradiation). All electrophysiological experiments were carried out at room temperature (20 °C). Electrophysiology, fluorescence, and PCR data analysis were performed using Origin 7.5 (Micrococal Software, Northampton, MA, USA) software. Patch pipettes were made from borosilicate glass (Clark Electromedical Instruments, Pangbourne Reading, England) and backfilled with intracellular solution (in mM): 140 KCl, 2 MgCl2, 1 Na2ATP, 10 HEPES, 2 EGTA, 1 CaCl2, adjusted to pH 7.2 with KOH, resulting in a free [Ca2+] of approximately 170 nM. Pipettes had resistances of 2.5– 5.0 MΏ. The standard Krebs external solution contained (in mM): 133 NaCl, 5.0 KCl, 16.3 NaHCO3, 1.38 NaH2PO4, 2.5 CaCl2, 1.2 MgCl2, and 7.8 glucose at pH 7.4.

Rats were exposed to a 6 Gy dose of ionizing irradiation for approximately 7.5 min. The dose of 6 Gy was used based on previous studies showing that this dose induced vascular effects while maintaining survival of the animals (Taranenko et al., 1992). The animals were euthanized either 9 or 30 days after the brief exposure to radiation. Whole-body irradiation was performed with gamma rays delivered at a rate of 0.80–0.84 Gy min− 1 from a cobalt60 source (TGT ROCUS M, Russian Federation) positioned 50 cm from the animal. During irradiation, animals were restrained in a plastic box specifically designed for this study and the radiation beam was focused on the animal's chest. There was no change in housing, standard food, or drink following irradiation. All animals survived the 9–30 day experimental period following the exposure to irradiation. Chemicals Fura-2AM was obtained from Molecular Probes Inc. (Eugene, OR, USA). Papain was obtained from Fluka Chemie AG (Buchs, Switzerland). PE, caffeine, paxilline, and all constituents of the Krebs solution were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). Statistics and analysis Data are shown as means ± S.E.M. n indicates the number of cells or preparations tested; each cell or preparation used for a specific experimental protocol was obtained from different animals. Comparisons between two values were performed using the Student's t-test. Multiple comparisons were performed using one way analysis of variance (ANOVA). If any significant difference was found the Tukey's multiple comparison test was applied. Differences were considered to be statistically significant when P was less than 0.05. Results

RNA isolation and semi-quantitative reverse transcriptase (RT)-polymerase chain reaction (PCR) Total RNA was isolated from rat aorta using a Trizol RNA-prep kit (Isogen, Russian Federation) according to the manufacturer's protocol. RNA concentration was determined with the use of a NanoDrop spectrophotometer ND 1000 (NanoDrop Technologies Inc., Wilming-

The first series of experiments were performed to study BKCa channel activity in smooth muscle from control and irradiated animals. To measure BKCa channel activity, we performed whole-cell patch-clamp experiments using freshly isolated thoracic aortic smooth muscle cells. Isolated smooth muscle cells from healthy rats were stimulated with increasing depolarizing voltage steps. Fig. 1A–C

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shows representative current tracings obtained in isolated cells from control animals (Fig. 1A) and cells from animals 9 days (Fig. 1B) and 30 days (Fig. 1C) after irradiation. The current–voltage (I–V) relationship shown in Fig. 1D clearly demonstrates outward rectification with a reversal potential of −40 ± 5 mV. The current density amplitude of cells from control animals was 52 ± 6 pA/pF (n = 8) at +70 mV (Fig. 1D). Outward currents in smooth muscle cells obtained from irradiated animals 9 and 30 days after irradiation demonstrated a significant decrease in K+ current density amplitude from 52 ± 6 pA/pF to 35 ± 4 pA/pF (9 days post-irradiation, P b 0.05, n = 7) and 20 ± 3 pA/pF (30 days post-irradiation, P b 0.001, n = 8) (Fig. 1D). There was no significant shift in reversal potential in cells obtained from irradiated animals as compared to cells from control animals. The I–V relationship in intact cells exhibited near linear behavior at potentials more negative than −40 mV and became non-linear at potentials above − 40 mV (Fig. 1D). This non-linearity becomes pronounced at potentials more positive than +25 mV. Paxilline (500 nM), a selective inhibitor of BKCa channels (Li and Cheung, 1999), added to the external bathing solution sharply decreased outward K+ current density from 52 ± 6 pA/pF to 12 ± 2 pA/pF at +70 mV (P b 0.001, n = 8) in cells from control animals (Fig. 2A). Paxilline was without effect on the reversal potential in cells from control and irradiated animals (Fig. 2A–C). Paxilline significantly altered K+ currents in cells from animals maintained for 9 days after irradiation. Paxilline depressed K+ currents in these cells at potentials more positive than +50 mV (Fig. 2B). Paxilline significantly decreased K+ current density from 35 ± 4 to 20± 5 pA/pF at +70 mV (P b 0.05, n = 7) in cells obtained 9 days after irradiation (Fig. 2B). Paxilline was without effect on cells obtained from animals 30 days following irradiation suggesting the absence of BKCa channel conductance a month after exposure to the radiation (Fig. 2C). It is interesting to note that the value of residual paxilline insensitive current in cells from animals 9 and 30 days following irradiation appears to be increased as compared to cells from control animals. To further investigate the effects of irradiation on aortic muscle cells, we compared basal and stimulated levels of [Ca2+]i in smooth muscle thoracic aortas obtained from control and irradiated animals. Aortic rings were activated with a high K+ solution (KCl), PE, or caffeine to primarily induce Ca2+ entry (KCl), intracellular mobilization (caffeine), or a combination of both (PE). Fig. 3A–C shows typical force/[Ca2+]i tracings and Fig. 3D–F averaged data from experiments performed using PE as the stimulus. The steady state contractile response to PE was significantly increased in tissues from animals maintained for 9 days after irradiation and this increase was again significantly increased in tissues from animals maintained for 30 days following irradiation as compared to vascular tissues from control animals. In contrast, the corresponding Ca2+ transient was significantly smaller in tissues from animals maintained for 9 days following irradiation. Surprisingly, in tissues from animals maintained for 30 days following irradiation, the PE induced Ca2+ transient increased to levels higher than that observed in tissues from control animals. Smooth muscle obtained from irradiated animals demonstrated a greater force-[Ca2+]i relationship as compared to that in tissues from control animals demonstrating an irradiation induced increase in myofilament Ca2+ sensitivity (Fig. 3F). Irradiation had no significant effect on either the contractile responses or concomitant Ca2+ transients in response to caffeine or high-K+ (data not shown), suggesting the effects of irradiation are more specific for G-protein coupled receptor responses. The results presented in Fig. 3G demonstrate that basal [Ca2+]i in aorta from irradiated animals are significantly lower 9 and

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Fig. 4. RT-PCR analysis of BKCa channel α and β1 subunits mRNA in rat aortic smooth muscle cells from control animals and from animals 9 days and 30 days after non-fatal whole body ionizing irradiation (6 Gy). Panel A — agarose electrophoretic gel showing the expression of the α-subunit in freshly isolated smooth muscle. The left arrow indicates the 209 bp fragment representing the α-subunit. The next two panels show expression of the β1 subunit (215 bp, B) and β-actin (300 bp, C) in aortic smooth muscle cells. Left lane in all panels are base-pair ladders in 50 bp increments.

30 days following irradiation as compared to basal Ca2+ levels in aorta from control animals. We used RT-PCR to determine whether the irradiation induced changes in BKCa channel activity correlated with an alteration in levels of α- and/or β1-subunit mRNA. Aortic smooth muscle from control animals expressing functional BKCa channel activity contained both the α- and β1-subunit mRNA (Figs. 4A and B and 5). α-Subunit mRNA levels were not significantly altered 9 days after irradiation (from 0.8 ± 0.05 to 0.7 ± 0.07 relative units; P N 0 05, n = 6) however a trend towards lower values was noted. BKCa channel alpha subunit was significantly decreased 30 days after irradiation (from 0.8 ±0.05 to 0.6 ±0.02 relative units; P b 0.05, n = 10) as compared to levels in aorta from control animals. Similar results were obtained with measurements of β1-subunit mRNA.

Fig. 3. Effect of non-fatal whole-body ionizing irradiation (6 Gy) on phenylephrine (PE, 10− 7 M)-induced increase in contractile force and intracellular Ca2+ concentration ([Ca2+]i) in isolated rings of rat thoracic aortic smooth muscle. Panels A–C are representative tracings of the simultaneous recording of force and [Ca2+]i in aortic rings from control animals (A) and from animals 9 days after (B) and 30 days after (C) irradiation. “a” denotes the force tracing and “b” denotes the [Ca2+]i response. Panel D shows averaged force and panel E shows averaged [Ca2+]i measurements in response to PE in aortic rings from control animals and from animals 9 and 30 days following irradiation. Panel F shows the force generated in N/µM [Ca2+]i in aorta from control animals and from animals 9 and 30 days following irradiation. Panel G shows basal [Ca2+]i in aorta from control animals and from animals 9 and 30 days following irradiation. Data shown are mean ± SEM (n = 9). ⁎P b 0.05 versus control. †P b 0.05 9 days versus 30 days post-irradiation.

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Fig. 5. Comparison of the mean levels± SEM of mRNA corresponding to BKCa channel α- and β1-subunits 9 days and 30 days following irradiation. Aortic RNA was isolated from: 6 control rats, 6 rats 9 days following irradiation and 10 rats 30 days following irradiation and then reverse transcripted to obtain gene-specific cDNAs. Bands corresponding to BKCa channel α- and β1 subunits and β-actin were excised from gels and aliquots were quantified by scintillation counting. Bars show the BKCa channel α- and β1 transcript level following irradiation versus normal condition. Single and double asterisks indicate the statistical significance as compared to control (P b 0.05 and P b 0.01, respectively).

There were no changes in β1-subunit mRNA 9 days after irradiation (from 0.8 ± 0.1 to 0.6 ± 0.08 relative units; P N 0.05, n =6) although a trend toward lower levels was again noted. A significant decrease in β1-subunit mRNA was shown 30 days after irradiation (from 0.8 ± 0.1 to 0.4 ±0.02 relative units, P b 0.01, n =10). These results indicate that irradiation produces a decrease in BKCa channel subunit mRNA levels in aortic smooth muscle cells and this may contribute to the increased contractility that occurs following exposure to radiation. Discussion The main finding of this study is the significant suppression of outward K+ current through BKCa channels in aortic smooth muscle cells obtained from rats after exposure to whole-body ionizing irradiation. Inhibition of the outward current was time-dependent and appeared as early as 9 days after irradiation, and persisted over the 30 day experimental period. Radiation-induced suppression of outward K+ currents was not accompanied by a change in the reversal potential of whole-cell current, thus suggesting that irradiation did not affect the resting membrane potential of the aortic smooth muscle cells. This is in contrast with the known effects of irradiation on the resting membrane potential of endothelial cells (Tishkin et al., 2007). This difference in response to irradiation suggests that BKCa channels are not important in the regulation of the resting membrane potential in rat aortic smooth muscle cells (Tammaro et al., 2004). Our results suggest that irradiation-induced suppression of BKCa channel activity is important in the increase in contractility that occurs during the late post-irradiation period. Sensitivity to paxilline was used as the primary criteria for determining the contribution of BKCa channels to the total K+ outward current. Irradiation produced a progressive decrease in BKCa channel activity (Fig. 2). In addition, irradiation increased a paxillineinsensitive outward K+ current likely resulting from a compensatory response to the decrease in BKCa channel activity. There are two pathways by which smooth muscle cells increase cellular Ca2+ to initiate a contraction (Somlyo and Himpens, 1989). One pathway is the influx of extracellular Ca2+ and the other is release of Ca2+ from intracellular stores. These two pathways of Ca2+ mobilization often overlap during different stimulation conditions. In our studies, we used a

sub-maximal concentration of PE, which mobilizes primarily, but not exclusively, intracellular Ca2+ to stimulate the aortic strips. Our results showed that tissues from irradiated animals produced greater levels of force in response to PE while the magnitude of the concomitant Ca2+ transient was smaller 9 days following irradiation as compared to corresponding parameters in tissues from control animals. Caffeine which stimulates Ca2+ release from the sarcoplasmic reticulum and high K+ solutions that depolarize the plasma membrane and activate voltagegated Ca2+ channels, did not show any differences in force or [Ca2+]i in the aortic tissues from irradiated animals as compared to those from controls. Therefore, a direct effect on either Ca2+ influx or intracellular release alone does not seem to underlie the irradiation induced decrease in magnitude of the Ca2+ transient. Rather, signaling activated in response to alpha-adrenergic stimulation but not by caffeine or high K+ might be involved. At the same time it is important to note that Ca2+ transients in tissues from animals 30 days following irradiation were significantly increased as compared to tissues from animals 9 days following irradiation. Whether this is the result of an increase in Ca2+ influx or release from intracellular stores is not known. As suggested above, a signaling step that is activated by PE but not caffeine or high K+ may be involved in the irradiation induced changes in Ca2+ sensitivity of force. A possible explanation for the increased levels of smooth muscle contraction during states of lower [Ca2+] involves protein kinase C. The potential involvement of protein kinase C in the mechanism(s) responsible for enhanced myofilament Ca2+ sensitivity is well established (Nishimura et al., 1992; Horowitz et al., 1996; Eto et al., 2001). We have shown that smooth muscle Ca2+ sensitivity is increased following irradiation and the increase is the result of changes in protein kinase C activity (Soloviev et al., 2005). As neither caffeine nor high K+ would be expected to activate protein kinase C while it is known that PE stimulation does, this provides an attractive hypothesis to justify the enhanced force development observed in irradiated tissues. The radiation-induced hypercontractility seen during the early stage of irradiation, therefore, may be the result of an increase in myofilament Ca2+ sensitivity. Our results demonstrate that myofilament Ca2+ sensitivity is increased 9 days following irradiation which was the earliest time point measured. Although the magnitude of the PE induced Ca2+ transient increases 30 days following irradiation as compared to that at 9 days, the enhancement of myofilament Ca2+ sensitivity is retained. BKCa channels are composed of a pore-forming α-subunit and an accessory β-subunit (Kaczorowski et al., 1996). There are four family members of the β-subunit that have been identified in different tissues. The β1-subunit is expressed predominantly in arterial smooth muscle and is believed to be responsible for BKCa channel Ca2+ sensitivity (Cox and Aldrich, 2000). The RT-PCR experiments of this study clearly demonstrated that BKCa channel α- and β1-subunit mRNA levels in aortic smooth muscle were significantly lower in tissues from irradiated animals as compared to those from control animals. This suggests that the ability of these channels to increase K+ outward current and therefore relax a contraction may be diminished in blood vessels exposed to irradiation. It is possible, therefore, that the β1-subunit is sensitive to irradiation and plays a predominant role in the irradiationinduced increase in blood pressure. On a larger scale, it is known that animals without the BKCa channel β1-subunit have increased mean arterial pressure (Brenner et al., 2000) and down-regulation of the BKCa channel β1-subunits occurs in rats made hypertensive by chronic exposure to angiotensin II (Amberg et al., 2003). These observations (Brenner et al., 2000; Amberg et al., 2003) support the hypothesis that alterations in β1-subunit expression, function, or both could also contribute to the development of non-radiation-induced hypertension. Conclusion In conclusion, the results of our study suggest that non-fatal, whole-body γ-irradiation suppresses both expression and function of BKCa channels and this, in turn, may be responsible for the enhanced

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