The roles of intracellular calcium concentration ([Ca2+]i) and Ca2+ sensitization in lipopolysaccharide (LPS)-induced vascular smooth muscle (VSM) hyporesponsiveness are incompletely understood. To investigate these roles, contraction responses to endothelin-1 (ET-1) and 80 mM KCl; relaxation responses to nifedipine; the expression levels of mRNAs of ET-1 and its receptors (ETA or ETB); the expression levels of protein kinase C (PKC) and phosphorylation of Rho kinase (ROKα), CPI-17, and myosin phosphatase target subunit-1 (MYPT1); and changes in aortic VSM cell [Ca2+]i were measured in LPS-treated aortic rings from male Wistar rats (250–300 g). LPS (10 μg/ml, 20 h) decreased contraction induced by ET-1 (0.3–100 nM) or 80 mM KCl. LPS-induced hypocontractility was not observed in the absence of external Ca2+, but LPS-treated aorta remained hypocontractile on subsequent stepwise restoration of extracellular Ca2+ (0.01–10 mM). Vascular relaxation to nifedipine; mRNA expression levels of ET-1, ETA, or ETB; protein expression levels of PKC; and phosphorylation levels of ROKα, CPI-17, and MYPT1 were not affected by LPS. In isolated aortic VSM cells, ET-1 caused a transient initial increase in [Ca2+]i, followed by a maintained tonic increase in [Ca2+]i, which was decreased by LPS pretreatment and was dependent on external Ca2+. Subsequent restoration of extracellular Ca2+ increased [Ca2+]i, but this increase was lower in the LPS-treated group. This difference in response to extracellular Ca2+ addition was not affected by diltiazem, but was abolished by SKF-96365. Therefore, LPS induces hyporeactivity to ET-1 in rat aorta that depends on external Ca2+ influx through non-voltage-operated Ca2+ channels, but not on ET-1 receptor expression or Ca2+ sensitization.
- calcium sensitization
- vascular smooth muscle
septic shock is a complex pathological state characterized by systemic vasodilation and hyporesponsiveness to vasoconstrictor agents (12, 32, 38, 39). Numerous experimental studies of sepsis have clearly documented the presence of both in vivo and in vitro vascular hyporesponsiveness to different vasoconstrictors, including α-adrenergic agonists (7, 12), angiotensin II (38), KCl (38, 39), endothelin-1 (ET-1) (4, 17), and thromboxane A2 (2).
Vascular hyporesponsiveness could be mediated either by an increase in vasodilating or a decrease in vasoconstricting mechanisms. Excessive production of vasodilator molecules, such as nitric oxide or prostacyclin, has been shown to contribute to the hypotension and vasoplegia in septic shock (23, 24, 36); however, inhibition of their production yields conflicting results. We previously showed that lipopolysaccharide (LPS) causes a selective hypocontractility of rat aorta to ET-1 via soluble guanylyl cyclase activation that was nitric oxide, endothelium, and COX independent (6).
Receptor-mediated vasoconstriction, which may also be affected in sepsis, could involve changes in receptor expression, calcium (Ca2+) entry pathways, intracellular calcium concentration ([Ca2+]i), and Ca2+ sensitization. An increase in [Ca2+]i, either through the release of Ca2+ from internal stores or the influx of Ca2+ through voltage-operated Ca2+ channels (VOCCs), receptor-operated Ca2+ channels, and store-operated Ca2+ channels, leads to phosphorylation of the 20-kDa regulatory myosin light chain (MLC20) by MLC kinase (16, 33). MLC20 is dephosphorylated by MLC phosphatase (MLCP) (34). MLCP activity is inhibited by phosphorylation of myosin phosphatase-targeting subunit (MYPT1) (37) or the smooth muscle-specific MLCP inhibitory protein CPI-17 (19). MYPT1 phosphorylation by Rho kinase (Rho-activated kinase/ROKα/ROCKII) (8), or CPI-17 by protein kinase C (PKC) (22) and ROCKII (21), therefore, controls the Ca2+-sensitization pathway.
Only a limited number of studies have examined the role of Ca2+ entry pathways in sepsis-induced vascular hyporeactivity, without investigating Ca2+ sensitization or changes in receptor expression in the same model. For example, mesenteric hyporeactivity to the α-adrenergic agonist methoxamine in rat (7) or hyperreactivity to ET-1 in pig (18) were not associated with changes in [Ca2+]i, while aortic vascular hyporeactivity to KCl was associated with enhanced Ca2+ uptake (39) or with no change in [Ca2+]i release (1).
In the present study, we investigated the mechanisms of LPS-induced hyporesponsiveness to ET-1 in rat aorta using an organ culture method by measuring changes in isometric force generation, expression levels of mRNA of ET-1, ETA, and ETB; expression levels of PKC; phosphorylation of ROKα, CPI-17, and MYPT1; and changes in [Ca2+]i. We find that LPS induces hyporeactivity to ET-1 in rat aorta that depends on external Ca2+ influx through non-VOCCs, but not on ET-1 receptor expression or Ca2+ sensitization.
MATERIALS AND METHODS
Dulbecco's modified Eagle medium, penicillin-streptomycin solution, fetal bovine serum, fura 2-AM, and pluronic F-127 were purchased from Invitrogen (Paisley, UK); ET-1 from American Peptide (Sunnyvale, CA); SKF-96365 from Tocris Bioscience (Avonmouth, UK); collagenase-P from Roche Diagnostics (Mannheim, Germany); and Nonidet P-40, LPS (E. coli O55:B5), protease inhibitor cocktail, nifedipine, diltiazem, papain, bovine serum albumin, soybean trypsin inhibitor, and dithiothreitol from Sigma-Aldrich Chemical (Gillingham, Dorset, UK).
The investigation conforms with the APS' Guiding Principles in the Care and Use of Animals. Male Wistar rats (University of Bath) weighing 250–300 g were maintained and humanely killed by cervical dislocation, in accordance with UK Home Office legislation and the University of Bath Code of ethical practice for research/experimentation involving the use of animals (approved by the University of Bath Ethics Committee, May 8, 2007). The descending thoracic aorta was separated and placed in a cold physiological salt solution (PSS) of the following composition (in mM): 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 25 NaHCO3, and 11.1 glucose, and bubbled with a mixture of 95% O2 and 5% CO2. The dissected thoracic aortas were cut into rings (2–4 mm in length) and placed into 48-well plates under sterile tissue culture conditions. Each well contained one ring in 1-ml Dulbecco's modified Eagle medium supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml), and 10% fetal bovine serum. After 1-h stabilization, vascular rings were transferred to a fresh medium of the same composition (control group) or supplemented with 10 μg/ml LPS (LPS-treated group) and incubated for 20 h at 37°C in humidified atmosphere of 5% CO2 in air. This high-LPS concentration was utilized to maximize the response window for investigation.
In vitro vascular reactivity studies.
Following 20 h of incubation, aortic rings from both the control and the LPS-treated groups were mounted in an organ bath filled with 18 ml of PSS at 37°C, bubbled with a mixture of 95% O2 and 5% CO2
Before each experiment, aortas were preconstricted by several additions of 80 mM KCl until consistent responses were obtained. To measure tissue contractility to ET-1, cumulative concentration (0.3–100 nM)-response curves were constructed in the presence or absence (Ca2+ omitted and 1 mM EGTA added) of external Ca2+. To measure vasorelaxation to the VOCC inhibitor nifedipine, cumulative concentration-response curves (1 nM–30 μM) were constructed in rings preconstricted with 30 nM ET-1. The effect of subsequent increases in extracellular Ca2+ (0.01–10 mM) on vessels preconstricted with 100 nM ET-1 in the presence of 10 μM nifedipine was also measured.
Aortic rings were separated and cultured as before (control and LPS) for 2, 4, 8, and 20 h, flash frozen in liquid nitrogen, and stored at −80°C. RNA was isolated using mechanical homogenization and TRIzol reagent (Invitrogen, Paisley, UK) and then purified and subjected to on-column DNase treatment using a High Pure RNA Tissue Kit (Roche Applied Science, Mannheim, Germany). RNA samples (1 μg each) were reverse-transcriped using Omniscript RT kit (Quiagen, Hilden, Germany) with anchored oligo(dT) primers (ABgene, Surrey, UK) in the presence of RNase inhibitor (RNasin Plus, Promega, Madison, WI), according to the manufacturer's instructions. The produced cDNA was used as the template for quantitative PCR using LightCycler FastStart DNA Master SYBR Green I in a LightCycler 1.5 thermal cycler (Roche Applied Science, Mannheim, Germany). A master mix composed of 0.4 μM primers, 3.5 mM MgCl2, 2 μl SYBR Green Master Mix, and PCR-grade water (to 18 μl) was added to prechilled capillaries, before the addition of 2 μl of 10−1 diluted cDNA. Each template was analyzed in duplicate within the same run. Following 40 amplification cycles, melt-curve analyses were performed to verify specific amplification. PCR efficiency of both the target and reference genes was calculated from the derived slopes of standard curves by LightCycler software (version 4.0). These PCR efficiency values were used to calculate the relative quantification values for calibrator-normalized target gene expression, and transcript levels were normalized to β-actin. The gene-specific primers used in this study, with their annealing temperatures and amplicon sizes, are represented in Table 1.
After 20 h of incubation with either control medium or LPS, aortic rings were mounted in organ bath, as previously mentioned, and then collected at the plateau of maximal ET contraction, rapidly frozen in liquid nitrogen, and stored at −80°C. Immunoblotting was carried out as previously described in detail (6). Briefly, tissue was homogenized in ice-cold lysis buffer containing the following (in mM): 150 NaCl, 1 EDTA, 50 Tris·HCl (pH 7.5), 1 sodium orthovanadate, 10 NaF, and 1 PMSF with 1% Nonidet P-40, 10% glycerol, and 1% protease inhibitor cocktail. Protein samples (40 μg/lane) were then subjected to SDS-PAGE on a 7.5% (for MLC20 and CPI-17) or 15% (for ROKα, PKC, and MYPT1) gels, transferred to nitrocellulose membrane (Whatman) by semidry transfer blot (Transblot SD cell, Bio-Rad), and blocked for 1 h with Tris-buffered saline-Nonidet P-40 containing 5% nonfat milk. Blots were subsequently incubated overnight at 4°C with gentle shaking with one of the following rabbit polyclonal antibodies: anti-phospho-MLC20 (Ser19) antibody (1:1,000 dilution, Cell Signaling Technologies), anti-phospho-ROKα (The396) antibody (2 μg/ml dilution, Abcam, Cambridge, UK), anti-PKC antibody (1:200 dilution, Santa Cruz Biotechnology), anti-phospho-MYPT1 (Thr850) antibody (1 μg/ml dilution, Upstate), or anti-phospho-CPI-17 (Thr38) antibody (2 μg/ml dilution, Upstate). Blots were then incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies (1:10,000 dilution in Tris-buffered saline-Nonidet P-40 with 2% milk, Dako Cytomation, Glostrup, Denmark), and immunoreactive protein bands were detected using ECL Advance kit (Amersham Biosciences) and visualized on an X-ray film (Fujifilm, Tokyo, Japan). The intensity of the specific bands was quantified by densitometric analysis using Labimage software (Kapelan Bio-imaging Solutions, Halle, Germany). For ROKα and PKC, equal loading of proteins was confirmed by reprobing the membranes with rabbit polyclonal anti-β-actin antibody (1:1,000 dilution, Cell Signaling Technologies). For phospho-MLC20, phospho-MYPT1, and phospho-CPI-17 antibodies, membranes were stripped using Re-Blot Plus solution (Chemicon) and then reprobed with anti-MLC20 antibody (1:1,000 dilution, Cell Signaling Technologies), anti-MYPT1 antibody (1:200 dilution, Santa Cruz Biotechnology), or anti-CPI-17 antibody (0.2 μg/ml dilution, Upstate), respectively.
Isolation of vascular smooth muscle cells.
Aortic rings (4–6 mm in length) were separated and incubated as before (control and LPS) for 20 h. After incubation, the endothelium was removed mechanically by gentle rubbing, and aortic rings were washed with Ca2+-free HEPES-buffered PSS composed of the following (in mM): 118 NaCl, 4.7 KCl, 1.2 MgCl2, 10 HEPES, and 11.1 glucose for 15 min at 37°C with continuous shaking. Rings were enzymatically digested in a 2 ml Ca2+-free PSS containing the following (in mg/ml): 1 papain, 2 collagenase-P, 0.5 soybean trypsin inhibitor, 1 dithiothrietol, and 1 bovine serum albumin, for 45 min at 37°C with continuous shaking. The digested rings were washed three times in fresh Ca2+-free PSS (each 5 min), placed in fresh Ca2+-free PSS, and gently triturated by a fire-polished Pasteur pipette to release vascular smooth muscle cells (VSMCs). Undispersed pieces of tissue were removed by nylon mesh (95 μm) filtration, and PSS Ca2+ concentration was gradually increased to a final concentration of 0.5 mM. The viability of the cells (>85%) was tested with trypan blue exclusion. Suspended VSMCs were then placed on poly-l-lysine-coated glass coverslips in a six-well plate for 1 h at room temperature.
Measurement of [Ca2+]i.
Coverslip-adherent VSMCs were loaded with 5 μM membrane-permeant fura 2-AM in normal PSS (with Ca2+ 2.5 mM) containing 0.02% Pluronic F-127 for 1 h at room temperature in the dark. Coverslips were placed in a 0.25-ml chamber on the stage of a Zeiss Axiovert S 100 inverted microscope and examined with a ×40/1.30 Fluar oil immersion objective. Cells were excited alternately at 340- and 380-nm wavelengths of ultraviolet light at a frequency of 0.75 Hz by a Spectramaster I variable excitation wavelength generator (EG&G Wallac LSR, Cambridge, UK). Emitted fluorescence was collected through a 510- to 580-nm bandpass emission filter and acquired using an Ultrapix (PDCI) CCD camera (1024 × 1024 maximum pixels). Cell images were analyzed using Ultraview software version 4 (Perkin-Elmer, Fremont, CA). The background fluorescence signal was subtracted by measuring emitted fluorescence from an area on the coverslip containing no cells.
Cells were perfused with either normal PSS (2.5 mM CaCl2) or Ca2+-free PSS (CaCl2 omitted and 1 mM EGTA added) at 2 ml/min at 37°C for 10 min. [Ca2+]i baseline was recorded for 30 s before the perfusion of 100 nM ET-1 (27, 30). At the end of the experiment, 2 μM ionomycin with 10 mM Ca2+ or 2 mM EGTA were added to obtain the maximal and minimal fluorescence intensity, respectively. [Ca2+]i was determined from the Grynkiewicz equilibrium equation (11), using a dissociation constant (KD) of 224 nM for fura 2 at 37°C.
Data are expressed as means ± SE, where n equals the number of animals, except in qRT-PCR (n = number of independent experiments, each using duplicate PCR reactions from samples prepared from pooled tissue lysates of 2 animals) and in [Ca2+]i experiments (n = number of VSMCs from at least 3 animals). The [Ca2+]i during the sustained phase was expressed as a percentage of the transient [Ca2+]i phase. Vascular relaxation was calculated as percentage of maximal steady-state contraction induced by 30 nM ET-1. The highest response obtained was considered as the maximum response (Emax). pEC50 (= negative log concentration producing 50% of maximal response) was determined from nonlinear regression analysis (4-parameter curve fit) carried out using Graphpad Prism software (Graphpad Software, San Diego, CA). Significant differences between groups were determined with paired Student's t-test, two-way ANOVA, and Bonferroni post tests for dependent data sets, or one-way ANOVA with Student-Newman-Keuls multiple comparisons post hoc test as appropriate.
Effect of LPS treatment on vascular contractility.
Incubation of the aorta with LPS significantly decreased tissue contractility to ET-1 compared with the control group (Fig. 1A). This hyporeactivity to ET-1 was manifested both by a 29.4 ± 4.6% decrease in Emax (from 9.0 ± 0.6 to 6.1 ± 0.3 mN) and an increase in EC50 from 14.0 ± 0.3 to 24.6 ± 1 nM in the control and LPS-treated groups (P < 0.01, n = 9), respectively. Similarly, a 20.0 ± 2.0% decrease in the maximum contraction to 80 mM KCl was also observed (P < 0.001, n = 18) (Fig. 1B).
Removal of external Ca2+ abolished the difference in the ET-1-induced contractions between the control and LPS-treated preparations (Fig. 2A). To determine the role of Ca2+ influx, extracellular Ca2+ (0.01–10 mM) was added to aortic rings preconstricted with 100 nM ET-1, leading to a stepwise increase in vessel contraction. The Emax values of the contractile response to Ca2+ were reduced by 55.7 ± 7.8% after LPS pretreatment (P < 0.01, n = 5) (Fig. 2C). Addition of nifedipine (10 μM), either before or after reintroducing external Ca2+, had no significant effect. Furthermore, no significant changes were found in the relaxation responses to nifedipine (0.1–30 μM) between the control and LPS-treated aortic rings preconstricted with 30 nM ET-1 (Fig. 3), suggesting that changes in VOCCs are not responsible for LPS-induced aortic hyporeactivity to ET-1.
Effect of LPS treatment on gene expression of ET-1, ETA, and ETB.
LPS treatment of the aortas for 4, 8, or 20 h had no significant effect on mRNA expression levels of ET-1 or its receptors (ETA and ETB) compared with nontreated time controls (Fig. 4). This evidence argues against the involvement of changes in gene expression of ET-1 and its receptors in the observed LPS-induced aortic hyporeactivity to ET-1.
Effect of LPS treatment on different proteins involved in Ca2+ sensitization.
LPS treatment decreased ET-1-stimulated phosphorylation of MLC20 in the aorta (Fig. 5, A and B), thus correlating with the observed aortic hypocontractility to ET-1 (Fig. 1A). This decrease, however, was not associated with significant changes in the levels of protein expression of PKC (Fig. 5C) or phosphorylation of ROKα (Fig. 5D), CPI-17 (Fig. 5E), or MYPT1 (Fig. 5F) in LPS-treated aortic rings compared with controls. These results suggest that sensitization to Ca2+ is not altered in LPS-treated aorta.
Effect of LPS on [Ca2+]i.
The resting level of [Ca2+]i was significantly higher in LPS-treated VSMCs (106 ± 5 nM) compared with control (94 ± 5 nM) (P < 0.05, n = 15), but this difference was abolished when external Ca2+ was removed, where the baselines were 111 ± 4 and 108 ± 3 nM for LPS and control groups, respectively. Infusion of 100 nM ET-1 caused an initial increase in [Ca2+]i (transient phase), followed by a plateau elevation in [Ca2+]i (sustained phase) (Fig. 6, A and B). There were no significant differences in the transient [Ca2+]i increase induced by 100 nM ET-1 between control (111 ± 10 nM) and LPS-treated (109 ± 13 nM) aortic VSMCs; however, the sustained phase [Ca2+]i level of control (26 ± 3% of transient phase in each cell) was significantly reduced to 15 ± 2% in the LPS-treated group (P < 0.01, n = 12) (Fig. 6B). Removal of external Ca2+ had no significant effect on the transient phase [Ca2+]i elevation in either the control (103 ± 10 nM) or the LPS-treated (104 ± 11 nM) group, but the sustained phase [Ca2+]i was decreased in both control (11 ± 1%) and LPS-treated (11 ± 2%) groups, which abolished the difference between control and LPS-treated groups (Fig. 6B). Conversely, addition of 2.5 mM Ca2+ during the sustained phase produced a continuous elevation in [Ca2+]i, which was significantly higher in control (210 ± 17%) compared with LPS-treated (155 ± 12%) groups (P < 0.05, n = 12) (Fig. 6, C and D). This continuous elevation in [Ca2+]i was not significantly affected by incubation with 20 μM diltiazem for 5 min before the addition of the 2.5 mM Ca2+ (Fig. 6D). However, incubation with 10 μM SKF-96365 significantly decreased the continuous elevation in [Ca2+]i in both groups and abolished the difference between the control and LPS-treated groups (Fig. 6D).
The evidence in this study indicates that LPS-induced hypocontractility to ET-1 in rat aorta is primarily dependent on Ca2+ influx through non-VOCCs, but not on changes in ET-1 receptor expression or Ca2+ sensitization.
In this study, LPS treatment impaired contractile responses of rat aorta to both ET-1 and 80 mM KCl, indicating that LPS impairs contractile responses to both receptor-dependent and -independent vasoconstrictors. Although changed expression of ET-1-specific receptors ETA and ETB has been suggested as a mechanism of hypocontractility, no systematic study has evaluated this possibility in intact arteries. Previous studies have shown that LPS treatment decreased ETA mRNA in rat heart (13, 17), aortic VSMC line A7r5 (3), and rat pulmonary vascular smooth muscle (5), but increased it in septic pig heart (9). Conversely, increased ETB mRNA was observed in these previous studies, but ETB mRNA was decreased in rat pulmonary endothelial cells (5). Few studies examined ET-1 gene expression in intact arteries, where ET-1 mRNA is increased by in vivo LPS treatment in mouse aorta (31) and in rat aorta and pulmonary artery (4), but was unchanged in porcine aorta (25). Since LPS had no effect on the ET-1, ETA, or ETB receptor mRNA levels, changes in the gene expression of the ET-1 system are unlikely to be involved in LPS-induced hyporeactivity in our model.
To the best of our knowledge, no previous studies are available in which the role of different proteins involved in Ca2+ sensitization was assessed in vascular hyporeactivity in animal models of sepsis. Therefore, our results demonstrating the absence of significant changes between control and LPS-treated aortic rings in the protein expression levels of PKC or the phosphorylation levels of ROKα, CPI-17, or MYPT1 indicate that Ca2+ sensitization is not the primary mechanism responsible for LPS-induced hyporeactivity to ET-1. These observations suggest that changes in Ca2+ mobilization or Ca2+ entry are more important.
Previous studies mainly examined LPS-induced changes in depolarizing KCl (40–100 mM)-stimulated Ca2+ entry via VOCCs. Increasing extracellular Ca2+ up to 30 mM reverses the diminished vascular reactivity to KCl in the aorta from septic rats (1, 39), an effect attributed to the impairment of either Ca2+ sensitization (39) or Ca2+ influx (1), although specific mechanisms were not identified. Our results using the VOCC blockers nifedipine or diltiazem suggest that VOCC-dependent Ca2+ entry is not responsible for LPS-induced hyporeactivity to ET-1. Furthermore, the difference in the sensitivity to external Ca2+ in the aorta preconstricted with ET-1 in the presence of nifedipine suggests that LPS impairs non-VOCC Ca2+ entry. Similarly, rat aorta from endotoxic rats displays reduced sensitivity to Ca2+ in high K+-depolarizing medium (10, 14), and nitrendipine is not able to inhibit vascular hyporeactivity to phenylephrine in septic rat aorta (1). Conversely, preadministration of verapamil and nifedipine (32), or nifedipine (41), is able to prevent hypotension in vivo in endotoxin-shocked rats. Interestingly, amlodipine prevents LPS-induced hypotension in vivo, not in vitro (29), suggesting that results obtained with VOCC modulators in vivo may be affected by other factors inside the body, such as metabolism, interaction with other mediators/cells, and the presence of neuronal factors.
To study LPS-induced changes in Ca2+ influx further, we examined changes in [Ca2+]i levels in isolated aortic VSMCs. The basal [Ca2+]i levels were slightly higher in LPS-treated aortic VSMCs compared with control, a result consistent with previous studies using rat aorta (35) and rat mesenteric arteries (26). Since removal of external Ca2+ abolishes this difference in basal [Ca2+]i levels, it is unlikely to result from an impairment of [Ca2+]i storage (7, 15), but probably from enhanced Ca2+ influx through VOCCs (40) or other Ca2+ channels.
The ET-1-induced biphasic increase in Ca2+ levels, comprising transient and sustained components, has been previously shown (18, 42). The transient phase of [Ca2+]i increase was not affected by external Ca2+ removal, suggesting that it is mainly due to intracellular release of Ca2+, as previously demonstrated (20, 42). Since LPS treatment had no effect on the transient phase increase in [Ca2+]i, it is unlikely that Ca2+ mobilization from intracellular stores was affected under our experimental conditions. However, the ET-1-induced sustained phase [Ca2+]i increase was significantly reduced in LPS-treated aortas. This difference was abolished by removal of external Ca2+, suggesting that it is mediated by voltage-independent Ca2+ influx. Similar differences were observed in response to the addition of external Ca2+ in the presence of ET-1, and the lack of effect of nifedipine and diltiazem confirms this conclusion. The complete inhibition of the difference in the sustained level of [Ca2+]i, by SKF-96365, an inhibitor of both receptor-operated Ca2+ channels and store-operated Ca2+ channels (28, 42), supports the hypothesis that LPS impairs Ca2+ influx through voltage-independent Ca2+ channels.
To summarize, we demonstrated that LPS treatment in vitro induces hyporeactivity to ET-1 in rat aorta, and the mechanism of this hyporeactivity depends on external Ca2+ influx through non-VOCCs, but not on ET-1 receptor expression or Ca2+ sensitization. Therefore, Ca2+ homeostasis could be important in controlling systemic vasomotor complications in sepsis.
This work was supported by a grant from the Egyptian Government (PhD studentship MM31/04 to M. S. H. El-Awady).
We gratefully acknowledge the technical assistance and helpful advice of Dr. Adrian T. Rogers (calcium measurement) and Dr. Mike Storm (RT-PCR).
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