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Cardiovascular Research 2005 68(2):327-335; doi:10.1016/j.cardiores.2005.06.005
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Copyright © 2005, European Society of Cardiology

Involvement of BKCa {alpha} subunit tyrosine phosphorylation in vascular hyporesponsiveness of superior mesenteric artery following hemorrhagic shock in rats

Rong Zhou, Liangming Liu* and Deyao Hu

State Key Laboratory of Trauma, Burns and Combined Injury, Department 2, Research Institute of Surgery, Daping Hospital, The Third Military Medical University, Daping, Chongqing 400042, PR China

* Corresponding author. Tel.: +86 23 68757452; fax: +86 23 68813806. Email address: liuliangming2002{at}yahoo.com

Received 12 December 2004; revised 8 June 2005; accepted 10 June 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: Vascular hyporesponsiveness is a major complication following severe trauma and shock. It plays important roles in the development of shock and seriously interferes with the treatment of shock. The mechanism responsible for the occurrence of vascular hyporesponsiveness has not been fully understood. The purpose of this study was to determine whether the {alpha} subunit tyrosine sites of large conductance calcium-activated potassium channel (BKCa) could be phosphorylated and whether the phosphorylation of BKCa was closely associated with the activation of BKCa and the development of vascular hyporesponsiveness following hemorrhagic shock in rats.

Methods: A hemorrhagic shock (30 mm Hg for 0.5, 2, 4 h) model of Wistar rats was established. Phosphorylation of tyrosine residues of the BKCa {alpha} subunit from vascular smooth muscle cells (VSMC) in superior mesenteric arteries (SMA) was detected by immunoprecipitation and Western blotting. BKCa activity was evaluated by cell-attached patch clamping. The vascular responsiveness of SMA to norepinephrine was measured with an isolated organ perfusion system.

Results: The level of BKCa {alpha} subunit tyrosine phosphorylation was increased in a time-dependent manner following hemorrhagic shock, which was mediated by protein tyrosine kinases (PTK) and protein tyrosine phosphatases (PTP). The activation of VSMC BKCa following hemorrhagic shock was inhibited by genistein (2 x 10–5 mol/L), the permeable isoflavone PTK inhibitor, and was potentiated by the PTP inhibitor sodium orthovanadate (Na3VO4, 10–3 mol/L). The decreased vasoresponsiveness following hemorrhagic shock was partly restored by genistein (10–5 mol/L) or by the BKCa-selective inhibitor tetrabutylammonium chloride (0.1 mmol/L), while it was further decreased by Na3VO4 (10–5 mol/L).

Conclusion: The tyrosine residues of BKCa {alpha} subunit of SMA were phosphorylated following hemorrhagic shock, which was regulated by PTK and PTP and appeared to be related to the activation of BKCa and the development of vascular hyporesponsiveness following hemorrhagic shock.

KEYWORDS Shock; Ion channel; Contractile function; Large conductance calcium activated potassium channel (BKCa); Tyrosine protein kinase


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Vascular hyporesponsiveness is one of the most common complications of serious conditions, such as severe trauma and hemorrhagic shock [1]. The decreased vascular reactivity would result in systemic hypotension, poor perfusion to vital organs, and finally lead to the multiple organ dysfunction (MODS). But the mechanisms responsible for the development of vascular hyporesponsiveness after hemorrhagic shock are not fully understood.

Large conductance, calcium-activated potassium channel (BKCa) plays an important role in regulating myogenic tone of vascular smooth muscle (VSM) and vascular reactivity. Our previous study showed that BKCa of vascular smooth muscle cell (VSMC) in superior mesenteric artery (SMA) was over-activated following hemorrhagic shock, which was associated with the decreased vascular reactivity [2]. But the mechanisms involved in the activation of BKCa of VSMC following hemorrhagic shock have not been elucidated. Continued research showed that, besides the membrane potential and intracellular free calcium, BKCa activity was also regulated by a variety of cellular process including protein phosphorylation, such as serine and threonine phosphorylation mediated by protein kinase C (PKC) and protein kinase A (PKA) [3,4]. Recent studies have indicated that tyrosine phosphorylation mediated by protein tyrosine kinase (PTK) also appears to be implicated in the modulation of BKCa activity. It has been demonstrated that in Chinese hamster ovary cells, the nonreceptor tyrosine kinase JAK2 activated endogenous BKCa, which means BKCa is one of the substrates of PTK signal cascade [5]. Further study showed that the tyrosine residues contained in the –COOH region of the pore-forming {alpha} subunit of mammalian BKCa could be phosphorylated directly, which was associated with the enhanced channel gating when active c-src was coexpressed with {alpha} subunit mouse homolog, mslo [6]. It was strongly suggested that the tyrosine phosphorylation of {alpha} subunit was associated with the activation of BKCa. Furthermore, some inflammatory mediators, such as nitric oxide released following hemorrhagic shock or trauma, could upregulate PTK activity such as src and activate BKCa [7,8]. So, it is reasonable to suppose that the tyrosine sites of BKCa {alpha} subunit might be phosphorylated in VSMC and it might be involved in the activation of BKCa and the development of vascular hyporesponsiveness following hemorrhagic shock.

Previous studies demonstrated that the vascular reactivity, no matter the overall vascular reactivity or a single blood vessel's reactivity, such as aorta, pulmonary vessel, renal artery, cerebral vessels, or coronary artery, were all reduced following prolonged hemorrhagic shock. Our previous study showed that although there were some differences of vascular hyporeactivity among select vasculatures following hemorrhagic shock, the total trend of all observed vasculatures was decreased [1]. So, in the present study, we used superior mesenteric artery from hemorrhagic shock rats as representative to observe the changes of BKCa {alpha} subunit tyrosine phosphorylation following hemorrhagic shock and its roles in the activation of BKCa and the development of vascular hyporesponsiveness.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1 Animal model
This study was approved by the Science and Technological Committee and Animal Use and Care Committee of the Third Military Medical University (Chongqing, China), and all procedures were conducted in compliance with the NIH Guidelines. Wistar rats, weighing 250 ± 20 g, were anesthetized by pentobarbital sodium (40 mg/kg, ip, Sigma, St. Louis Mo, USA). The left femoral artery was cannulated and connected to a pressure recorder for the measurement of mean arterial pressure (MAP) and heparinized with sodium heparin (50 U/kg, Wanbang Biochemical Pharmaceutical Co., Shanghai, China). Rats were hemorrhaged and the MAP were maintained at 30 mm Hg for 0.5 h, 2 h or 4 h via femoral artery catheter.

2.2 Experimental protocol
2.2.1 BKCa {alpha} subunit tyrosine phosphorylation of SMA and its modulation following hemorrhagic shock
2.2.1.1 BKCa {alpha} subunit tyrosine phosphorylation
Wistar rats from sham-operated control group (with the same surgical operation but without hemorrhage) and hemorrhagic shock (MAP at 30 mm Hg for 0.5, 2, 4 h) group were sacrificed and SMA were isolated rapidly. After the fat and connective tissue were stripped and the adventitia and the endothelium removed, SMAs were homogenized with RIPA1 and the total lysates were prepared as previously described [6]. For the measurement of phosphotyrosine-containing protein, the lysates were mixed with Laemmli sample buffer and fractioned by 8% SDS-polyacrylamide gel electrophoresis (PAGE) and electrotransferred to PVDF membrane at 4 °C at 35 V overnight. The membranes were incubated with anti-tyrosine phosphorylation antibodies (1:1500, Santa Cruz, California, USA) and anti-mouse secondary antibody (1:2000, Santa Cruz, California, USA). For the measurement of tyrosine phosphorylation of BKCa {alpha} subunit, the lysate (500 µg) was precleared by incubation with 20 µL of protein A/G-agarose (Sigma, St. Louis Mo, USA) on a rotator for 1 h and centrifuged at 10000 g for 5 min and immunoprecipitated with anti-phosphotyrosine monoclonal antibody (5 µg per tube, PY20) for 4 h. Following the addition of 20 µL of protein A/G-agarose per tube, the samples were again rotated at 4 °C for another 1 h and then centrifuged for 2 min at 10000 g. The pelleted materials were washed by gentle resuspension in lysate buffer and then analyzed by 8% SDS-PAGE and Western blotting with anti-BKCa {alpha} subunit monoclonal antibody (1:2000, Santa Cruz, California, USA) and anti-goat secondary antibody (1:3000, Santa Cruz, California, USA). For the measurement of the expression of BKCa {alpha} subunit, the lysates were fractioned by 8% SDS-PAGE and electrotransferred to PVDF membrane. The membranes were incubated with anti-BKCa {alpha} subunit monoclonal antibody and anti-goat secondary antibody. The intensity of the aimed band on the film was measured with image analysis software and quantified by densitometry.

2.2.1.2 Modulation of BKCa {alpha} subunit tyrosine phosphorylation
Single smooth muscle cells of SMA were freshly isolated from normal rats by an enzymatic method as previously described [9]. Briefly, after removing the endothelium, the media intima of small segments (1 cm2) of SMA were incubated in a modified Ca2+-free physiological salt solution (mmol/L: NaCl 127, KCl 5.9, MgCl2 1.2, glucose 12, HEPES 10, pH 7.4) containing 2 mg/mL bovine serum albumin (BSA), 1 mg/mL collagenase (type 1, GIBCO, California, USA) and 0.36 mg/mL soybean trypsin inhibitor (Sigma, St. Louis Mo, USA) at 37 °C for 40 min. Cells were dispersed by trituration and the isolated VSMCs were cultured in Dulbeco's Modified Eagle's medium (DMEM)/F12 containing 20% fetal bovine serum (FBS, HyClone, Logan, Utah, USA) in a CO2 incubator for 5~7 days. The cultured cells were divided into four groups: control, Na3VO4 (10–3 mol/L), Na3VO4 (2 x 10–3 mol/L), Na3VO4 (2 x 10–3 mol/L)+genistein (5 x 10–5 mol/L). Na3VO4 is a widely used PTP inhibitor which could upregulate the tyrosine phosphorylation and genistein, a specific PTK inhibitor, could downregulate the tyrosine phosphorylation (both were purchased from Sigma, St. Louis Mo, USA) [10]. After VSMC exhibited the ‘hill-and-valley’ growth pattern upon reaching confluence, the cells were FBS starved for 24 h in FBS-free DMEM/F12 before Na3VO4 or genistein insults. In Na3VO4 groups, the cells were incubated with Na3VO4 for 30 min, while in Na3VO4 plus genistein group, the cells were preincubated with genistein for 10 min before incubated with Na3VO4. The expression of BKCa {alpha} subunit tyrosine phosphorylation was detected as described above.

2.2.2 Relationship between tyrosine phosphorylation of BKCa and its activation following hemorrhagic shock
The single channel patch clamp was used to measure the activity of BKCa. The freshly isolated SMA smooth muscle cell from sham-operated control and hemorrhagic shock rats (30 mm Hg, 2 h) were plated on cover slips at 4 °C and used within 6–8 h. Single BKCa current was measured in cell-attached and inside-out patch at room temperature. The recording set-up has been described previously [11,12]. Patch pipettes were prepared from capillary glass with a two-stage puller (PP-83, Narishige, Tokyo, Japan). The tip of each pipette was fire-polished. In cell-attached configuration, the patch pipette (5~8 M{Omega}) was filled with a ‘high’ K+ solution containing (mmol/L: CaCl2 1, HEPES 10, KCl 40, KOH 100, L-Asp 100, pH 7.4). The bath solution contained the identical K+ concentration (mmol/L: CaCl2 0.55, EGTA 1, HEPES 10, KCl 40, KOH 100, L-Asp 100, pH 7.4, free calcium concentration 10–7 mol/L). Voltage across the patch was controlled by clamping the cell at 0 mV with the high extracellular K+ concentration solution [13,18]. For inside-out patches, the bathing solution and the pipette solution were as the same as for the cell-attached mode. The desired free calcium concentration of the bathing solution was obtained by adding an appropriate concentration of CaCl2.

VSMC of SMA from hemorrhagic shock or sham-operated rats were randomized into four groups, including control (n = 8), hemorrhagic shock (n = 6), hemorrhagic shock+genistein (n = 6), hemorrhagic shock+genistein+Na3VO4 (n = 6). After a giga seal was made, the cells were incubated with genistein (2 x 10–5 mol/L) for 5 min in hemorrhagic shock+genistein group. Further incubation of the same patch was followed with the treatment of Na3VO4 (10–3 mol/L) for 30 min in hemorrhagic shock+genistein+Na3VO4 group. Single channel currents were filtered at 3 kHz and monitored using an Axopatch amplifier (Axopatch 200A, CEZ2300, Nihon Kohden, Japan). Data were digitized at sampling rates of 50 kHz and were stored on a computer disk via an analog-to-digital interface (DigiData 1200, Axon Instruments, New South Wales, Australia) and analyzed with pClamp6.0. "n" represented the numbers of patch from VSMC of different animals. BKCa activity was quantified using the methods described by Singer and Walsh [13]. The channel current amplitude was fitted by Gauss curve and the open-time and closed-time histogram of BKCa was fitted by a two-exponential curve. The open-state probability of BKCa (Po) was calculated as the total open duration at the first level divided by the total recording duration.

2.2.3 Role of the tyrosine phosphorylation of BKCa in vascular hyporesponsiveness following hemorrhagic shock
The endothelium-denuded SMA rings (3 mm in length) from hemorrhagic shock (30 mm Hg for 2 h) and sham-operated control rats were prepared and randomly divided into seven groups: control (n = 7), hemorrhagic shock (n = 7), hemorrhagic shock+TEA (0.1 mol/L, n = 6), hemorrhagic shock+genistein (10–5 mol/L, n = 7), hemorrhagic shock+Na3VO4 (10–5 mol/L, n = 6), hemorrhagic shock+genistein+TEA (n = 7), and hemorrhagic shock+Na3VO4+ TEA (n = 6). Each vascular ring was mounted in a 10 mL organ perfusion system filled with modified Krebs solution (mmol/L: NaCl 118, KCl 4.7, NaHCO3 25, KH2PO4 1.03, MgSO4.7H2O 0.45, CaCL2 2.5, glucose 11.1, pH 7.4), continuously bubbled with 95% O2 and 5% CO2 and maintained at 37 °C. Each mesenteric arterial ring was stretched to a passive force about 0.6~0.8 g preload and equilibrated for 2 h. The contractile response of each SMA ring to norepinephrine (NE, Shanghai Harvest Pharmaceutical Co., Shanghai, China) was recorded by a Powerlab polygraph (AD instrument, Castle Hill, Australia) through a force transducer. The incubation time of SMA rings with TEA (0.1 mmol/L) was 15 min, with genistein (10–5 mol/L) was 30 min and with Na3VO4 (10–5 mol/L) was 4 h. SMA rings were preincubated with TEA (0.1 mmol/L) for 15 min before insulted with genistein or Na3VO4 in hemorrhagic shock+genistein+TEA group and hemorrhagic shock+Na3VO4+TEA group. NE was given cumulatively from 10–9 to 10–5 mol/L. The contractile force of each SMA ring was calculated as the percentage of the maximal tension of SMA at 10–5 mol/L of NE in normal control. pD2 of NE was calculated as described previously [14]. The cumulative dose–response curve of SMA to NE and the maximal contraction (Emax) and pD2 were used to evaluate the vascular reactivity of SMA.

2.3 Statistical analysis
All data are expressed as mean ± SD of "n" observations. Comparison between each group was performed by one-way analysis of variance (ANOVA) with SPSS 10.0. The p value less than 0.05 was considered to be statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1 BKCa {alpha} subunit tyrosine phosphorylation in SMA following hemorrhagic shock in rats and its modulation
Western blotting of SMA lysates showed a significantly higher expression of 44~66 kDa and 125 kDa tyrosine-phosphoproteins at 0.5 h after hemorrhagic shock as compared with controls. This response persisted for the entire duration of hemorrhagic shock. The 125 kDa tyrosine-phosphorylated protein was increased about 1.4 and 1.8 folds at 2 and 4 h after hemorrhagic shock, respectively (Fig. 1). In order to explore whether the tyrosine site(s) of BKCa {alpha} subunit was phosphorylated following hemorrhagic shock, the lysates were further analyzed by immunoprecipitation (IP) with anti-phosphotyrosine monoclonal antibody (PY20) and Western blotting (WB) with anti-BKCa {alpha} subunit monoclonal antibody. The results indicated that the expression of tyrosine-phosphorylated BKCa {alpha} subunit was significantly increased following hemorrhagic shock, and the results also showed that tyrosine-phosphorylated protein band around 125 kDa in Fig. 1 mainly contained BKCa {alpha} subunit. In order to exclude the influence of {alpha} subunit expression level on BKCa {alpha} subunit phosphorylation, the expression level of BKCa {alpha} subunit was detected before and after hemorrahagic shock. The results showed that there were no significant increase of BKCa {alpha} subunit expression following hemorrhagic shock as compared to the sham-operated group (Fig. 2). VSMC studies showed that Na3VO4 (1~2 x 10–3 mol/L) significantly induced BKCa {alpha} subunit tyrosine phosphorylation in a dose-dependent manner in cultured SMA cells, which could be prevented by genistein (5 x 10–5 mol/L) (Fig. 3).


Figure 1
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  		Fig. 1 The changes of protein tyrosine phosphorylation in total lysate of SMA following hemorrhagic shock in rats. The total lysate of SMA were immunoblotted with anti-phosphotyrosine monoclonal antibody (PY20). Intensities of the band (125 kDa) in A was measured using image analysis software and the results are shown as percentages of the control group in B. The position of molecular mass markers are shown on the right (A). Lane 1: control group; Lane 2: hemorrhagic shock (0.5 h); Lane 3: hemorrhagic shock (2 h); Lane 4: hemorrhagic shock (4 h) . Data are expressed as mean ± SD, n = 5, ** p< 0.01 vs. control group. SMA, superior mesenteric artery.

 

Figure 2
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  		Fig. 2 Identification of tyrosine phosphorylation of BKCa {alpha} subunit by immunoprecipitation and immunoblotting. (A) The total lysates were immunoprecipitated with anti-phosphotyrosine monoclonal antibody (PY20) and the immunoprecipitates were immunoblotted with anti-BKCa {alpha} subunit. (B) Western blot analyses of BKCa {alpha} subunit expression in SMA before and after hemorrhagic shock. Lane 1: control group; Lane 2: hemorrhagic shock (2 h); Lane 3: hemorrhagic shock (4 h). SMA, superior mesenteric artery. BKCa, large conductance Ca2+ activated potassium channel.

 

Figure 3
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  		Fig. 3 Effects of genistein and Na3VO4 on BKCa {alpha} subunit tyrosine phosphorylation in cultured SMA smooth muscle cells. Lane 1: control group; Lane 2: Na3VO4 (1 x 10–3 mol/L); Lane 3: Na3VO4 (2 x 10–3 mol/L); Lane 4: Na3VO4 (2 x 10–3 mol/L) plus genistein (5 x 10–5 mol/L). Data are expressed as mean ± SD, n = 5, *p<0.05 vs. control group, #p<0.05 vs. Na3VO4 (2 x 10–3 mol/L) group. SMA, superior mesenteric artery. BKCa, large conductance Ca2+ activated potassium channel.

 
3.2 The relationship between PTK-dependent BKCa tyrosine phosphorylation and BKCa activation in hemorrhagic shock
In the cell-attached configuration, the K+ channel showed a linear current–voltage relationship between 0 and +60 mV of membrane potential with a slope conductance of 167.7 ± 7.88 pS (r = 0.998, n = 8). Channel activity of smooth muscle cells from normal rats revealed minimal gating events (Po was 0.02 ± 0.011 at +40 mV membrane potential) with the channel current amplitude of 6.33 ± 0.499 pA (Fig. 4A). When it was made into an inside-out configuration, its activity was increased with the increase of free [Ca2+] in the bath solution, and the channel open probability (Po) may increase to 0.05 ± 0.037 when the free [Ca2+] of the bath solution was at 10–6 mol/L. It was suggested that the potassium channel current recorded in our experiment was BKCa current.


Figure 4
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  		Fig. 4 Effects of genistein and Na3VO4 on BKCa activity of SMA smooth muscle cells following hemorrhagic shock in rat. (A) Typical record of single-channel current of BKCa from the cell-attached patch with identical K+ between bath and patch solution. (B) I–V curve. (C) Typical record of the effects of genistein and Na3VO4 on BKCa current. (D) Effects of genistein and Na3VO4 on channel open probability (Po). Open (o) state and close (c) state were showed in the left. The membrane potentials (Vm) in C was +40mV. Data are shown as means ± SD. "n" represents the numbers of cell-patches from different rats. **p<0.01 vs. control group, $p<0.05 vs. shock group, ##p<0.01 vs. shock plus genistein group. SMA, superior mesenteric artery. BKCa, large conductance Ca2+ activated potassium channel.

 
BKCa was significantly activated following severe hemorrhagic shock (MAP at 30 mm Hg for 2 h). In the cell-attached configuration, the open probability (Po) of BKCa was increased nearly 4 folds. The open-time histogram and the closed-time histogram of BKCa were fitted by a two-exponential curve with a mean open time of (3.6 ± 1.2 ms) and a mean close time of (504.6 ± 289.3 ms) in normal animals. Further analysis demonstrated that the increase of BKCa open probability (Po) following hemorrhagic shock was owing to the increase of slow close time constant ({tau}sc) and mean close time (tmc) (Table 1). Treating SMA smooth muscle cells from hemorrhagic shock rats with inhibitors of PTK (genistein) for 5 min, BKCa activity was attenuated. The open probability (Po) of BKCa at the level of +40 mV membrane potential was decreased from 0.07 ± 0.02 to 0.05 ± 0.01 (n = 6, p<0.05 vs. hemorrhagic shock group) without significant changes of single channel current amplitude (p>0.05). Further incubation of the same patch with Na3VO4 for 30 min could reverse the effects of genistein on BKCa activity in SMA smooth muscle cells, and the open probability (Po) of BKCa at the level of +40 mV membrane potential was increased to 0.38 ± 0.24 (n = 6, p<0.01 vs. hemorrhagic shock+genistein group) (Fig. 4B, C, Table 1).


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  		Table 1 The changes of kinetic parameters of VSMC BKCa of SMA

 
3.3 Role of PTK-mediated BKCa tyrosine phosphorylation in vascular hyporesponsiveness following hemorrhagic shock
As compared with the sham-operated group, the reactivity of SMA to NE was significantly decreased following hemorrhagic shock (30 mm Hg, 2 h), and the cumulative concentration–response curve of NE was shifted to the right. TEA (0.1 mmol/L) partly improved the vasoreactivity of SMA with the significant ascending response to NE (Table 2, Fig. 5A), which meant that BKCa played an important role in the occurrence of vascular hyporesponsiveness following hemorrhagic shock. In order to determine whether the tyrosine phosphorylation of BKCa was involved in the development of vascular hyporesponsiveness following hemorrhagic shock, the non-selective PTK inhibitor genistein (10–5 mol/L) was used to downregulate protein tyrosine phosphorylation and Na3VO4 (10–5 mol/L) was used to upregulate protein tyrosine phosphorylation of SMA. The results showed that neither incubation genistein for 30 min nor incubation Na3VO4 for 4 h alone could affect the basal tension of SMA significantly before NE was applied (data are not shown). Genistein (10–5 mol/L) enhanced the contractile response of SMA to NE and made the cumulative dose–response curve of NE shift to the left, while incubation of Na3VO4 (10–5 mol/L) for a much longer time (4 h) slightly but significantly caused a further decrease of vascular responsiveness and the cumulative dose–response curve of NE shift to the right as compared with the hemorrhagic shock group (Table 2, Fig. 5 B). Genistein plus TEA treatment synergistically shifted the cumulative dose–response curve of NE to the left and increased the contractile response of SMA to NE (Table 2, Fig. 5 C). Na3VO4 (10–5 mol/L) induced decreased vascular responsiveness was partly prevented by lower concentration of TEA (0.1 mmol/L) (Table 2, Fig. 5 D).


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  		Table 2 The changes of pD2 and Emax of the contractile response of SMA to NE

 

Figure 5
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  		Fig. 5 Effects of TEA, genistein, and Na3VO4 on vascular responsiveness of SMA to NE following hemorrhagic shock in rats. (A) Effects of TEA (0.1 mmol/L). (B) Effects of genistein and Na3VO4. (C) Effects of genistein plus TEA. (D) Effects of TEA on Na3VO4 induced-decrease of vascular reactivity. Data are expressed as means ± SD. "n" represents the numbers of observations from different rats. **p<0.01 vs. control group; #p<0.05, ##p<0.01 vs. hemorrhagic shock group; @p<0.05 vs. hemorrhagic shock plus Na3VO4 group. SMA, superior mesenteric artery. TEA, tetrabutylammonium chloride. NE, norepinephrine.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
Vascular hyporesponsiveness in peripheral blood vessels is one of the most serious complications of shock, and vascular reactivity to vasoconstrictor (such as NE, angiotensin, endothelin-1, and even the vasoconstriction induced by KCl) and vasodilators is greatly reduced after hemorrhagic shock. Many factors, including desensitized adrenoreceptors, the decreased {alpha} receptor expression, the decreased Ca2+ influx and release, the decrease in the sensitivity of vascular contractile components to intracellular free Ca2+, endogenous mediators such as opioid peptides and inflammatory cytokines (such as IL-1, NO, ET-1, etc.), the dysfunction of the K+ and Ca2+ channel and the hyperpolarization of cell membrane, have been proposed to be the important inducers of the decreased vascular reactivity during shock. Our previous work showed that although there were some differences of vascular hyporeactivity among vasculatures following hemorrhagic shock, the lose of responsiveness was prevalent in celiac, left renal, SMA and left femoral arteries [1]. The further studies in our lab showed that BKCa in SMA smooth muscle cells was over-activated following hemorrhagic shock, which was closely associated with the decrease of vascular reactivity [2]. As the predominant K+ channel subtype that carried outward current in vascular smooth muscle cells, BKCa activation would decrease the membrane reactivity through closing voltage-sensitive calcium channel and induce vascular hyporesponsiveness. But until now, the mechanism involved in the activation of BKCa following hemorrhagic shock remains unclear.

It has been demonstrated that the membrane potential and intracellular free calcium ion ([Ca2+]i) in vascular smooth muscle cells (VSMC) are all important regulators of BKCa opening[15]. Electrophysiological and pharmacological studies showed that BKCa activity is also modulated by phosphorylation/dephosphorylation dependent pathway, including PKC and PKA mediated serine and threonine phosphorylation and PTK mediated tyrosine phosphorylation [3–6]. The role of PTK-mediated protein tyrosine phosphorylation in the regulation of BKCa activity attracted more and more attentions in recent years. Although some reports showed that BKCa activity could be modulated by different tyrosine kinases in an opposite direction, it has been demonstrated that the pore-forming {alpha} subunit of the mammalian BKCa can undergo direct tyrosine phosphorylation in the presence of cSrc tyrosine kinase and this phosphorylation correlated with an enhancement of channel gating [6]. As the BKCa and PTK activity were stimulated following hemorrhagic shock [2,7], it is rational to suppose that BKCa {alpha} subunit tyrosine sites could be directly phosphorylated, which might play important roles in the activation of BKCa and the development of vascular hyporeactivity following hemorrhagic shock.

Our results showed that there was a higher expression of tyrosine-phosphorylated proteins over a wide range of molecular weight following hemorrhagic shock as compared with controls. Immunoprecipitation and Western blotting showed that 125 kDa tyrosine-phosphorylated BKCa {alpha} subunit in SMA was significantly increased at 2 and 4 h following hemorrhagic shock in a time-dependent manner, which was negatively associated with the decrease of vascular hyporeactivity. Tyrosine phosphorylation of BKCa {alpha} subunit following hemorrhagic shock may be associated with prolonged stimulation of high concentrations of catecholamines (CA), angiotensin II and inflammatory mediators (including nitric oxide and tumor necrosis factor-{alpha}) released excessively after hemorrhagic shock [16,17]. In order to confirm whether BKCa {alpha} subunit tyrosine phosphorylation was mediated by PTK and/or PTP, the primary cultured SMA smooth muscle cells were treated with Na3VO4 or Na3VO4 plus genistein. The results indicated that Na3VO4, the PTP inhibitor, could increase the tyrosine phosphorylation of BKCa {alpha} subunit in a concentration-dependent manner, and genistein, the permeable non-selective PTK inhibitor, could prevent Na3VO4 induced BKCa {alpha} subunit tyrosine phosphorylation. All of these suggested that PTK and PTP were involved in the modulation of BKCa {alpha} subunit tyrosine phosphorylation.

To understand the role of BKCa {alpha} subunit tyrosine phosphorylation in activation of BKCa following hemorrhagic shock, a cell-attached patch clamp was adopted and the relationship of protein tyrosine phosphorylation with the opening of BKCa in VSMC was observed. It was showed that BKCa was significantly activated following hemorrhagic shock, which was characterized with the shortening of mean close time (Tmc), the decrease of slow close time constant ({tau}cs) and the increase of channel open probability (Po). The results were consistent with our previous study [2]. Genistein could lengthen the Tmc and {tau}cs and decrease Po while Na3VO4 reversed the effects of genistein, which suggested that the inhibitory effect of genistein on BKCa activity in hemorrhagic shock was associated with a PTK-mediated protein phosphorylation cascade. Although we had no direct data to show which tyrosine site(s) phosphorylation was associated with the regulation of BKCa activity, the report by Ling et al. suggested that phosphorylation of Tyr766 in the C-terminus of BKCa {alpha} subunit might be closely associated with the over-activation of BKCa following hemorrhagic shock, because they found the site-directed mutagenesis at position of Tyr766 in the –COOH region of BKCa {alpha} subunit prevented phosphorylation and channel opening of BKCa in the presence of cSrc [6]. Further experiment on artery ring in vitro indicated that the vascular reactivity of SMA following hemorrhagic shock was significantly decreased. Downregulation of tyrosine phosphorylation by genistein could partly restore vascular hyporesponsiveness of SMA, while Na3VO4 could cause a further decrease of vascular hyporesponsiveness, which was partly inhibited by 0.1 mmol/L TEA (a selective BKCa inhibitor at this concentration [18]). These results suggested that BKCa tyrosine phosphorylation mediated by PTK and/or PTP was partly associated with the development of vascular hyporesponsiveness following hemorrhagic shock.

BKCa and PTK were abundant in VSM tissue which were both related to the contraction and the reactivity of resistant artery [19,20]. Most reports showed that PTK could not only increase the intracellular [Ca2+] through voltage-sensitive calcium channel, but also activate myosin light chain kinase (MLCK) and increase the Ca2+ sensitivity of the intracellular contractile apparatus [21]. Previous studies and current results showed that PTK-dependent signal cascade was also coupled to BKCa through PTK-dependent protein tyrosine phosphorylation. This could be one of the negative feedback mechanisms of agonist-induced vasoconstriction. PTK and/or PTP mediated BKCa {alpha} subunit tyrosine phosphorylation and BKCa activation could lead to the hyperpolarization of VSMC membrane and the close of voltage-sensitive calcium channel, which could facilitate the development of vascular hyporesponsiveness in hemorrhagic shock. Salomonsson and Arendshorst reported that genistein could inhibit the NE-induced vasoconstriction through decreasing intracellular [Ca2+] and the sensitivity of contractile apparatus to Ca2+ at higher concentration (5 x 10–2 mol/L) [22], while genistein induced the restitution of the decreased vascular reactivity in the present study at much lower concentration (10–5 mol/L). It was suggested that genistein could have different dose-dependent effects on NE-induced vasoconstriction and vascular reactivity through different mechanisms. But it is worthy to point out that in the present study there were many limitations and problems that need to be further confirmed, for example, was one or more tyrosine residues phosphorylated following hemorrhagic shock? Can isolated SMA and VSMC completely reflect the in vivo situation? Whether or not the Wistar rats and the hemorrhagic shock model used in the present study can completely reflect the human body and represent the other types of shock and clinical shock situations?

In summary, our results revealed that hemorrhagic shock could enhance BKCa {alpha} subunit tyrosine phosphorylation, which was regulated by PTK and PTP. BKCa {alpha} subunit tyrosine phosphorylation was closely related to the over-activation of BKCa and played an important role in the development of vascular hyporesponsivenesses following hemorrhagic shock. Isoflaves (such as genistein) may have potential clinical utility to restore vascular responsiveness at the proper dosage during hemorrhagic shock.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
This work was supported by the National Natural Science Foundation of China (no. 30271266, 30370563) and the National Basic Research Program of China (2005CB522601). The authors thank Dr. Michael A. Dubick of the US Army Institute of Surgical Research for his editorial assistance in the preparation of this manuscript.


   Notes
 
Time for primary review 32 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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