QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.103.051805v1 306/2/538    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Farmer, M. R. Articles by Ralevic, V. Search for Related Content PubMed PubMed Citation Articles by Farmer, M. R. Articles by Ralevic, V.

CARDIOVASCULAR

Effects of in Vivo Lipopolysaccharide Infusion on Vasoconstrictor Function of Rat Isolated Mesentery, Kidney, and Aorta

School of Biomedical Sciences, University of Nottingham Medical School, Queen's Medical Centre, Nottingham, United Kingdom

Received March 18, 2003; accepted April 22, 2003.

	Abstract

Top Abstract Materials and Methods Results Discussion Conclusions References

 
Continuous infusion of lipopolysaccharide (LPS) into conscious rats elicits regionally selective cardiovascular disturbances. The aim of the present study was to assess contractile function in different vascular preparations (renal, mesenteric, and thoracic aorta) taken from rats infused with LPS for 2 or 24 h. Sustained responses to continuous infusion of methoxamine but not to KCl were reduced in the aorta (at 2 and 24 h LPS) and mesentery (at 24 h LPS) but not in the renal vascular bed. In contrast, transient responses to bolus doses of methoxamine were unchanged in the mesentery. In Ca²⁺-imaging experiments with fura-2, challenge with a single concentration of methoxamine (10 µM, which showed an impaired contractile response at 24 h LPS) induced a rise in intracellular Ca²⁺ in the mesenteric artery that was not different from the control. Furthermore, in the aorta, the contractile response to caffeine was attenuated only in the 2 h LPS group. These results show that there is regional heterogeneity in in vitro vascular responsiveness in preparations taken from LPS-infused rats. Thus, in mesenteric beds and aortae, but not renal beds, there is hypocontractility to methoxamine that is not due to a generalized inability of the smooth muscle to contract, which is evident with sustained but not transient application of agonist (mesentery) and which, in late endotoxemia (24 h LPS), does not appear to involve abnormalities in Ca²⁺ mobilization or entry.

An experimental model we characterized in vivo differs from many inasmuch as it involves continuous infusion of relatively low doses of LPS (150 µg kg^–1 h^–1) rather than a high single bolus (commonly 20–30 mg kg^–1). In that model, we have shown regionally selective changes in cardiovascular status (Waller et al., 1994; Gardiner et al., 1993, 1994, 1995, 1996a,b) associated with reduced mesenteric vasoconstrictor responses to methoxamine (an ₁-adrenoceptor agonist) in vivo at 2 and 24 h after the start of LPS infusion (Waller et al., 1994; Tarpey et al., 1998a). In contrast, the renal vasoconstrictor response to methoxamine in vivo was not suppressed (Waller et al., 1994). Interestingly, mesenteric bed preparations taken from animals infused with LPS and investigated in vitro did not show reduced vasoconstrictor effects of methoxamine (Tarpey and Randall, 1998), but this may have been due to the mode of administration, since others who have investigated mesenteric arteries isolated from LPS-treated animals have noted an impairment in the sustained contractile response, even though the initial response to agonist exposure was normal (Martinez et al., 1996; Mitolo-Chieppa et al., 1996). The in vitro contractile responsiveness of the renal vasculature in this model of endotoxemia has not been investigated to date.

Thus, we have now carried out a systematic comparison of responses to methoxamine, given as either bolus doses or as sustained concentrations, in perfused mesenteric vascular beds taken from rats infused with LPS for 2 or 24 h. Since the results showed impaired contractions to methoxamine under sustained conditions in the 24 h LPS-treated group, experiments were carried out to assess changes in intracellular Ca²⁺ in mesenteric arteries taken from rats given LPS for 24 h. Furthermore, we investigated, for the first time, in vitro contractile responses of the renal vasculature from this model of endotoxemia. Additionally, to investigate whether endotoxemia may affect resistance and conduit vessels differently, we assessed the vasoconstrictor function of thoracic aortae isolated from rats treated with LPS. Aortae were used to investigate further the possible role of intracellular Ca²⁺ in hypocontractility to methoxamine in endotoxemia using caffeine, which elicits contraction by acting on the ryanodine receptor on the sarcoplasmic reticulum to release Ca²⁺ (Zucci and Ronca-Testoni, 1997).

To our knowledge, the present study is the first comparison of the contractile function of three different tissues taken from the same groups of LPS-treated animals. A preliminary account of some of these findings has been reported to the British Pharmacological Society (Farmer et al., 2001).

	Materials and Methods

Top Abstract Materials and Methods Results Discussion Conclusions References

 
All experiments were performed on adult male Sprague-Dawley rats (290–430 g; Charles River, Margate, Kent, UK) with Home Office approval under the Animals (Scientific Procedures) Act 1986.

In Vivo Administration of Substances. The animals were surgically prepared to receive chronic infusion of either LPS or saline via catheters implanted in the right jugular vein under surgical anesthesia with fentanyl and medetomidine (300 µg kg^–1 of each i.p.). Following surgery, anesthesia was reversed, and analgesia was provided with atipamezole and nalbuphine, respectively (both given at1mgkg^–1 s.c.). Animals were allowed to recover overnight, during which time they received infusion of saline (0.4 ml h^–1) to maintain catheter patency. The animals were housed in individual cages and connected to a fluid-filled swivel to allow overnight intravenous infusion into the conscious animal as described previously (Gardiner et al., 1993). During this time, animals were allowed food and water ad libitum. On the following day, the animals were assigned to one of four groups and subjected to either a 2- or 24-h intravenous infusion of either LPS [Escherichia coli serotype 0127:B8 (Sigma Chemical, Poole, Dorset, UK); 150 µgkg^–1 h^–1] or saline (0.4 ml h^–1) (Gardiner et al., 1995). After 2- or 24-h infusion of saline or LPS, rats were anesthetized with sodium pentobarbitone (up to 60 mg i.v., supplemented as required) for removal of kidneys, and, subsequently, superior mesenteric arteries or mesenteric vascular beds and thoracic aortae.

Isolated Mesenteric Vascular Bed Preparation. The isolated mesenteric vascular bed preparation, based on the method of McGregor (1965), was prepared as described previously (Ralevic and Burnstock, 1988). Briefly, a midline incision was made, and the gastrointestinal tract was lifted out of the abdominal cavity and placed at the side of the animal on a paper towel soaked in Krebs' solution. The superior mesenteric artery was identified and cannulated from where it leaves the aorta with a blunted hypodermic needle. The mesenteric vascular bed was flushed with Krebs' solution, and the mesentery was carefully cut away from the gastrointestinal tract. It was placed within an organ bath and perfused at a rate of 5 ml min^–1 with gassed (95% O₂, 5% CO₂) Krebs' solution of the following composition: 106.1 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 25 mM NaHCO₃, 1.2 mM MgSO₄, 1.9 mM CaCl₂, and 10 mM glucose, maintained at 37°C. Perfusate was pumped by a peristaltic pump (Cole-Parmer Instrument Company Ltd., Saffron Walden, Essex, UK). Relaxations or constrictions of the preparation were measured as changes in perfusion pressure, monitored by a pressure transducer (BD Biosciences, Cowley, Oxford, UK) situated on a side arm proximal to the preparation. A 30-min period of equilibration was observed following preparation.

Isolated Renal Vascular Bed Preparation. A midline incision was made, and the gastrointestinal tract was lifted out of the abdominal cavity and placed at the side of the animal on a paper towel soaked in Krebs' solution. The right renal artery was identified and dissected away from connective tissue along its length from the aorta to the kidney. A loose ligature was then placed around the renal artery. Using blunt dissection, the kidney was separated from the surrounding fatty connective tissue. During this time, the kidney was kept moist with Krebs' solution. The animal was then killed by decapitation, and the renal artery was cannulated with a blunted hypodermic needle. Once the needle was securely in the artery, the preparation was flushed with 1 ml of Krebs' solution, containing heparin (500 U), until the kidney was blanched. The ligature was then tightened to secure the needle in the artery, and the kidney was lifted away with any remaining connective tissue cut away. The preparation was then placed on a metal grid within an organ bath (37°C) and perfused at a rate of 8 ml min^–1 with gassed (95% O₂, 5% CO₂) Krebs' solution of the following composition: 106.1 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 25 mM NaHCO₃, 1.2 mM MgSO₄, 1.9 mM CaCl₂, and 10 mM glucose, maintained at 37°C. Perfusate was pumped by a peristaltic pump (Cole-Parmer Instrument Company Ltd), and responses of the preparation were measured as changes in perfusion pressure, monitored by a pressure transducer (BD Biosciences) situated on a side arm proximal to the preparation. A 30-min period of equilibration was observed following preparation.

Isolation of Thoracic Aortae. A midline cut was made along the sternum, the lungs and heart were removed, and the thoracic aorta was exposed. While the aorta remained in situ, the vessel was cleaned of connective tissue on its anterior surface. A cut was made through the vessel just above the diaphragm, and the vessel was carefully cut away from its posterior connections. Following removal, the vessel was placed into a beaker of cold Krebs' solution to remove any excess blood before being placed in cold Krebs' solution in a Petri dish. A ring of thoracic aorta, 1.5-cm long, was cut from the center of the excised vessel and suspended, under 1 g of tension, in an organ bath (at 37°C) containing gassed (95% O₂,5%CO₂) Krebs' solution of the following composition: 106.1 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 25 mM NaHCO₃, 1.2 mM MgSO₄, 1.9 mM CaCl₂, and 10 mM glucose. The preparations were allowed a 1-h period of equilibration prior to the start of experimentation.

Experimental Protocol: Mesenteric Arterial Beds. Following the equilibration period, dose-response curves were performed for methoxamine (500 pmol-5 µmol), an ₁-adrenoceptor agonist. All doses were given as 50-µl bolus injections into an injection port, sited proximal to the preparation. The dose interval was determined by the time to recovery of the response to baseline. In a separate series of experiments, vasoconstrictor responses to cumulative concentrations of methoxamine were compared in the isolated mesenteric arterial vascular beds from the four groups. Methoxamine was added to the perfusate in cumulative concentrations between 1 and 100 µM. In a separate series of experiments, vasoconstrictor responses to cumulative concentrations of KCl (10–300 mM) were compared. Responses to the thromboxane A₂ mimetic U46619 [GenBank] were not investigated in the mesenteric arterial bed, since in this preparation, U46619 [GenBank] is a weak vasoconstrictor (Warner, 1990).

Experimental Protocol: Renal Arterial Beds. In the isolated perfused kidneys, following the equilibration period, a cumulative concentration-response curve was produced for methoxamine by addition of methoxamine to the perfusing Krebs' solution (10 nM-100 µM), with each addition being given when a plateau to the previous response had been achieved (usually within 5 min of administration). Methoxamine was then washed out over a 30-min period until baseline perfusion pressure was restored. Finally, a cumulative concentration-response curve was produced for KCl by addition of KCl to the perfusing Krebs' solution (10–300 mM), with each addition being given once a plateau to the previous evoked response was obtained.

Experimental Protocol: Aortae. Following the equilibration period, vessels were challenged with cumulative concentrations of methoxamine (1 µM-1 mM). The methoxamine was washed out, and a 30-min rest period was allowed prior to cumulative concentrations of KCl (10–300 mM) being added to the organ bath. In a separate series of preparations, aortae were challenged by cumulative concentrations of U46619 [GenBank] (1 nM-1 µM). A single concentration (20 mM) of caffeine was added to the organ bath in a separate series of preparations. Tension was measured at 5-s time points up to 1 min and then at 10-s time points up to 2 min.

Ca²⁺-Imaging of Mesenteric Arteries. These experiments were carried out in superior mesenteric arteries isolated from rats following infusion of either LPS or saline for 24 h. Changes in the level of intracellular Ca²⁺ during challenge with methoxamine and KCl were investigated. Superior mesenteric arteries were dissected out of the animal. They were then carefully cleaned of fat and connective tissue and dissected into 5-mm segments. These segments were incubated with 5 µM fura-2/acetoxymethyl ester in the presence of 0.02% Pluronic F-127, 0.1% Cremophor FL, and 1% dimethylsulfoxide in pregassed Krebs-Henseleit buffer for 3 h at room temperature. After this incubation period, the segments were cut open and placed lumen-side down in a heated culture dish (Bioptechs, Inc., Butler, PA) containing Krebs-Henseleit buffer. Two metal supports held the tissue flat against the bottom of the culture dish. The culture dish was then mounted on an inverted microscope (Leica, Wetzlar, Germany) equipped for dual excitation wavelength fluorescent measurements. The objective was a Nikon CF Fluor (10 x 0.5) (Nikon, Kingston upon Thames, UK). The light source was a 75 W xenon lamp. The Krebs-Henseleit buffer bathing the tissue was maintained at 37°C and constantly gassed with 95% O₂/5% CO₂. Tissues were allowed to recover for 20 min prior to Ca²⁺ measurements. Changes in intracellular Ca²⁺ levels were assessed by alternatively exciting the preparation with 340- and 380-nm wavelength light with a 3-s delay between exposures. Emitted light (measured at 510 nm) was collected by a photomultiplier (Photonic Science, Tunbridge Wells, UK). The system was controlled by an Apple Macintosh Power PC using IonVision software (Improvision, Coventry, UK). In its free form, fura-2 produces a high fluorescence at 380 nm and a low fluorescence at 340 nm. When fura-2 binds to Ca²⁺, the opposite is true, such that the amount of fluorescence at 380 nm decreases and the amount of fluorescence at 340 nm increases. The ratio of fluorescence at these wavelengths (340/380 nm) is an index of Ca²⁺ concentration. Therefore, changes in this ratio are recorded as an index of intracellular Ca²⁺. The Grynkiewicz equation (Grynkiewicz et al., 1985) can be used to calculate the absolute Ca²⁺ concentration from the ratio values. However, the constants required for the equation (the dissociation constant for fura-2, R_max, and R_min) are difficult to determine accurately. Therefore, as this study is concerned purely with the changes in intracellular Ca²⁺ levels, we have quoted the 340/380 nm fluorescence ratios, which are directly proportional to the absolute values.

Following 20 min of equilibration, the arteries were challenged with a single 10-µM concentration of methoxamine, and the response was recorded until a plateau had been reached (3–5 min). The preparations were then washed out, and an additional 30 min were allowed for equilibration. A single, 60-mM concentration of KCl was added to the perfusate, and the evoked response was recorded until a plateau had been reached (3–5 min).

Statistics. Responses of the perfused vascular beds were measured as increases in perfusion pressure above baseline. Aortic contractions were measured as a change in tension (g) over baseline values. Data are given as means ± S.E.M. Data were analyzed using one- or two-way analysis of variance, with Tukey's multiple comparison post hoc test or t tests as appropriate (GraphPad Prism, version 3.0; GraphPad Software Inc., San Diego, CA). Differences were only considered significant if p < 0.05.

Materials. LPS (from E. coli serotype 0127:B8), methoxamine, caffeine, and U46619 [GenBank] were obtained from Sigma Chemical. LPS was dissolved in sterile saline and prepared to a concentration of 150 µg ml^–1. KCl was obtained from BDH Laboratory Supplies (Poole, Dorset, UK). Anesthetic agents used were fentanyl citrate (Martindale Pharmaceuticals Ltd, Essex, UK), medetomidine (Domitor; Pfizer Ltd, Sandwich, Kent, UK), and sodium pentobarbitone (Sagatal; Rhône Mérieux Ltd, Harlow, UK). Reversing agents used were nalbuphine [HCl Nubain; DuPont (U.K.) Ltd, Stevenage, UK] and atipamezole (HCl Antisedan; Pfizer Ltd).

	Results

Top Abstract Materials and Methods Results Discussion Conclusions References

 
The experimental model of endotoxemia that is used here has been characterized extensively by us, and details of the regionally selective changes in resting cardiovascular status and responses to vasoconstrictors in vivo are available in Gardiner et al. (1993, 1994, 1995, 1996a,b), Waller et al. (1994), and Tarpey et al. (1998a,b).

Mesenteric Arterial Beds: Dose- and Concentration-Response Relationships to Methoxamine and Concentration-Response Relationships to KCl. There were no significant differences in the baseline perfusion pressures between the 2 h saline, 24 h saline, 2 h LPS, and 24 h LPS groups, and these were 14 ± 4mmHg(n = 6), 10 ± 5mmHg (n = 5), 7 ± 3 mm Hg (n = 8), and 16 ± 2 mm Hg (n = 9), respectively.

Methoxamine, when given as a bolus, elicited dose-dependent increases in perfusion pressure (p < 0.001) (Fig. 1a). No significant differences existed between the four groups in the maximal elicited rise in perfusion pressure. Interestingly, a plot of the time course of the methoxamine-elicited contraction to a dose of 1.5 µmol (Fig. 1b) showed that although there was no significant difference in the maximal attained rise in perfusion pressure, there was a trend in the 24 h LPS group for the response to decrease more rapidly; although this was not significantly different compared with the 24 h saline group, it was significantly different from the 2 h LPS and saline groups (p < 0.05).

View larger version (27K): [in this window] [in a new window]   Fig. 1. a, the dose-response relationship to methoxamine in the isolated mesenteric arterial bed. Increases in perfusion pressure of mesenteric arterial beds isolated from rats following intravenous infusion of 2 h saline (n = 6), 24 h saline (n = 5), 2 h LPS (n = 8), or 24 h LPS (n = 9) to injections of bolus doses of methoxamine (500 pmol-50 µmol). b, time course of response to a single dose of methoxamine in the isolated mesenteric arterial bed. Change in perfusion pressure with time of mesenteric arterial beds isolated from rats following intravenous infusion of 2 h saline (n = 6), 24 h saline (n = 5), 2 h LPS (n = 8), or 24 h LPS (n = 9) in response to single injections of a 1.5-µmol dose of methoxamine.

Cumulative concentrations of methoxamine elicited concentration-dependent increases in perfusion pressure (p < 0.0001) (Fig. 2a). These were significantly reduced in mesenteries from rats following 24 h LPS infusion (Fig. 2a). The maximum response was significantly depressed in 24 h LPS rats compared with 24 h saline rats, being 64.4 ± 9.1 mm Hg (n = 8) and 106.7 ± 9.6 mm Hg (n = 9), respectively (p < 0.05). No significant differences were noted between the 2 h LPS and 2 h saline groups, with maximum responses being 96.6 ± 15.6 mm Hg (n = 8) and 99.4 ± 9.3 mm Hg (n = 9) respectively. There were no significant differences in EC₅₀ values between 2 h saline, 24 h saline, 2 h LPS, or 24 h LPS groups at 14.9 ± 6.1, 7.3 ± 1.6, 7.9 ± 2.1, and 10.9 ± 2.0 µM, respectively.

View larger version (20K): [in this window] [in a new window]   Fig. 2. a, the concentration-response relationship to methoxamine in the isolated mesenteric arterial bed. Increases in perfusion pressure of mesenteric arterial beds isolated from rats following intravenous infusion of 2 h saline (n = 9), 24 h saline (n = 9), 2 h LPS (n = 8), or 24 h LPS (n = 8) to cumulative concentrations of methoxamine (1–100 µM). *, p < 0.05 (24 h LPS versus 24 h saline). b, the concentration-response relationship to KCl in the isolated mesenteric arterial bed. The increases in perfusion pressure of mesenteric arterial beds isolated from rats following intravenous infusion of 2 h saline (n = 7), 24 h saline (n = 7), 2 h LPS (n = 6), or 24 h LPS (n = 6) to cumulative concentrations of KCl (10–300 mM).

Cumulative concentrations of KCl elicited significant concentration-dependent increases in perfusion pressure (p < 0.0001) (Fig. 2b). There were no significant differences between the 2 and 24 h LPS groups and their respective controls in their response to cumulative concentrations of KCl.

Mesenteric Arteries: Methoxamine-Stimulated Ca²⁺ Responses. There was no significant difference in methoxamine-stimulated Ca²⁺ responses between arteries from 24 h saline and 24 h LPS-treated rats, the changes in the 340/380 nm ratio being 0.16 ± 0.05 (n = 5) and 0.11 ± 0.07 (n = 5), respectively (Fig. 3a). The responses were also similar when expressed as a percentage of the KCl response (Fig. 3b). The time to maximum response to methoxamine in arteries from 24 h LPS-treated rats (303 ± 107 s, n = 5) was not significantly different compared with that in the 24 h saline-treated rats (190 ± 98 s, n = 5) (Fig. 3c). There was no significant difference between the two groups in the change in 340/380 nm ratio induced by 60 mM KCl, being 0.15 ± 0.05 (n = 5) and 0.15 ± 0.04 (n = 5) in LPS-treated and saline-treated groups, respectively (Fig. 3d).

View larger version (27K): [in this window] [in a new window]   Fig. 3. Ca2+-imaging experiments with fura-2 during challenges with methoxamine and KCl. Experiments utilizing fura-2 Ca2+ imaging in superior mesenteric arteries isolated from rats following 24 h of intravenous infusion with either saline (n = 5) or LPS (n = 5). a, change in 340/380 ratio upon challenge with methoxamine (10 µM); b, change in 340/380 ratio upon challenge with methoxamine (10 µM), expressed as a percentage of the response to KCl (60 mM); c, time taken to the maximum contraction to methoxamine (10 µM) (s); and d, change in 340/380 ratio upon challenge with KCl (60 mM).

Renal Arterial Beds: Concentration-Response Relationships to Methoxamine and KCl. Experiments were performed on four experimental groups: 2 h saline, 24 h saline, 2 h LPS, and 24 h LPS. There were no significant differences between any of the groups in the measured basal renal perfusion pressures, these being 52 ± 11 mm Hg (n = 7), 45 ± 5 mm Hg (n = 8), 60 ± 13 mm Hg (n = 9), and 51 ± 4 mm Hg (n = 5), respectively.

Responses to cumulative concentrations of methoxamine in the renal arterial beds displayed concentration dependence in all four groups (P < 0.0001) (Fig. 4). There were no significant differences between 2 h saline (n = 6), 24 h saline (n = 8), 2 h LPS (n = 9), or 24 h LPS (n = 5) groups; R_Max values were 133 ± 22 mm Hg, 139 ± 18 mm Hg, 161 ± 25 mm Hg, and 190 ± 17 mm Hg, respectively, and pEC₅₀ values were 6.37 ± 0.24, 6.27 ± 0.30, 6.24 ± 0.63, and 6.24 ± 0.19, respectively.

View larger version (17K): [in this window] [in a new window]   Fig. 4. Concentration-response relationship to methoxamine in the isolated renal arterial bed of endotoxemic rats. The response to cumulative concentrations of methoxamine (10 nM-100 µM) in kidneys isolated from rats following infusion of either 2 h saline (n = 6), 24 h saline (n = 8), 2 h LPS (n = 9), or 24 h LPS (n = 5). Data are means with vertical bars indicating S.E.M.

Responses to cumulative concentrations of KCl were concentration-dependent (p < 0.0001). There were no significant differences between 2 h saline (n = 7), 24 h saline (n = 8),2h LPS (n = 9), or 24 h LPS (n = 5) groups; R_Max values were 58 ± 13 mm Hg, 64 ± 7 mm Hg, 75 ± 11 mm Hg, and 91 ± 16 mm Hg, respectively, and pEC₅₀ values were 1.51 ± 0.17, 1.60 ± 0.06, 1.69 ± 0.05, and 1.84 ± 0.06, respectively.

Aortae: Concentration-Response Relationships to Methoxamine, KCl, and U46619 [GenBank] . The increases in tension elicited by cumulative concentrations of methoxamine are shown in Fig. 5a. All groups displayed concentration-dependent contraction to methoxamine (p < 0.0001). Both the 2 h LPS (n = 6) and the 24 h LPS (n = 4) groups showed significantly lower contractility to methoxamine than either 2 h saline (n = 5) or 24 h saline (n = 6) groups (P < 0.05). This is reflected in the values of maximal response, which were 0.94 ± 0.09 g, 0.88 ± 0.10 g, 0.44 ± 0.05 g, and 0.49 ± 0.10 g for 2 h saline, 24 h saline, 2 h LPS, and 24 h LPS groups, respectively. With regard to pEC₅₀ values, the 2 h LPS group was significantly lower than the 2 h saline group, being 5.3 ± 0.1 and 5.9 ± 0.1, respectively (p < 0.001). However, there was no significant difference between the 24 h LPS group and the 24 h saline group, being 5.7 ± 0.1 and 5.7 ± 0.1, respectively.

View larger version (22K): [in this window] [in a new window]   Fig. 5. a, cumulative concentration-response curves to methoxamine (1 µM-1 mM) in isolated aortae. The tension (g) elicited upon contraction in response to cumulative concentrations of methoxamine in aortae isolated from rats following infusion of either 2 h saline (n = 5), 24 h saline (n = 5), 2 h LPS (n = 6), or 24 h LPS (n = 4). Data are means with vertical bars indicating S.E.M. *, p < 0.05 (versus control); **, p < 0.01 (versus control), and , p < 0.001 (versus control). b, cumulative concentration-response curves to U46619 [GenBank] (1 nM-1 µM) in isolated aortae. The tension (g) elicited upon contraction in response to cumulative concentrations of U46619 [GenBank] in aortae isolated from rats following infusion of either 2 h saline (n = 6), 24 h saline (n = 7), 2 h LPS (n = 6), or 24 h LPS (n = 7). Data are means with vertical bars indicating S.E.M.

Contractions elicited in response to cumulative concentrations of U46619 [GenBank] displayed concentration dependence (p < 0.0001) (Fig. 5b). There were no significant differences between the experimental groups in the contractions elicited by U46619 [GenBank] at any of the concentrations tested. Maximal attained contractions were 1.03 ± 0.17 g (n = 6), 1.16 ± 0.17 g (n = 7), 1.13 ± 0.10 g (n = 6), and 1.03 ± 0.14 g (n = 7) for 2 h saline, 24 h saline, 2 h LPS, and 24 h LPS groups, respectively. With regard to pEC₅₀ values, there were no significant differences between 2 h saline and 2 h LPS or between 24 h saline and 24 h LPS groups, being 8.1 ± 0.1, 8.2 ± 0.2, 8.2 ± 0.2, and 7.8 ± 0.1, respectively.

Contractions elicited by cumulative concentrations of KCl were not significantly different between the experimental groups but did display concentration dependence (Fig. 6). Maximal contractions were 0.56 ± 0.12 g (n = 5), 0.53 ± 0.13 g (n = 6), 0.54 ± 0.08 g (n = 6), and 0.70 ± 0.16 g (n = 4) for 2 h saline, 24 h saline, 2 h LPS, and 24 h LPS groups, respectively. In addition, pEC₅₀ values were not significantly different between the groups, being 1.68 ± 0.26, 1.43 ± 0.27, 1.54 ± 0.04, and 1.94 ± 0.13, respectively.

View larger version (18K): [in this window] [in a new window]   Fig. 6. Cumulative concentration-response curves to KCl (10–300 mM) in isolated aortae. The tension (g) elicited upon contraction in response to cumulative concentrations of KCl in aortae isolated from rats following infusion of either 2 h saline (n = 5), 24 h saline (n = 6), 2 h LPS (n = 6), or 24 h LPS (n = 4). Data are means with vertical bars indicating S.E.M.

Aortae: Contractile Response to Caffeine. The time course of contractions elicited by 20 mM caffeine is illustrated in Fig. 7. In the 2 h LPS group, there was a significant decrease (p < 0.05) in tension elicited by caffeine, between 15 s and 40 s, compared with the corresponding saline-treated group. However, in aortae taken after 24 h of LPS infusion, there were no significant differences in the response to caffeine compared with either control group.

View larger version (20K): [in this window] [in a new window]   Fig. 7. Contractions elicited by caffeine in isolated aortae. The tension (g) elicited upon contraction in response to caffeine (20 mM) in aortae isolated from rats at either 2 h (a) following infusion of either saline (n = 6) or LPS (n = 7) or at 24 h (b) following infusion of either saline (n = 7) or LPS (n = 5). Data are means with vertical bars indicating S.E.M. *, p < 0.05.

	Discussion

Top Abstract Materials and Methods Results Discussion Conclusions References

 
We have previously observed regional and temporal heterogeneity in vivo with respect to vascular responsiveness in a model of endotoxemia that involves continuous LPS infusion (Waller et al., 1994). The aim of the present study was to investigate, in the same experimental model, whether or not such regional heterogeneity is also evident in vitro and whether or not endotoxemia differentially affects the vasocontractile function of conduit and resistance vessels. Furthermore, we sought to investigate the possible role of Ca²⁺ in the hypocontractility to methoxamine (an ₁-adrenoceptor agonist) that we observed.

Hypocontractility to Methoxamine in the Mesenteric Arterial Bed, but Not the Renal Arterial Bed, in Endotoxemia. The present results in the mesenteric arterial beds are consistent with previous studies (Mitchell et al., 1993; Tarpey and Randall, 1998) in that there was no difference in responses to methoxamine given as bolus doses at either 2 or 24 h LPS. However, we now clearly show that there is hypocontractility of the mesenteric arterial beds at 24 h after the onset of LPS infusion if methoxamine is applied as cumulative concentrations. This was not due to a generalized inability of the mesenteric arterial smooth muscle to contract, as responses to KCl were unimpaired. These results are, in some respects, similar to those of others, who showed a waning of the contractile response to noradrenaline in the mesenteric arterial bed (Mitolo-Chieppa et al., 1996) and mesenteric resistance arteries (Martinez et al., 1996) isolated from rats treated with LPS.

It is interesting that hypocontractility was apparent when methoxamine was applied as sustained concentrations but not when applied as bolus doses. One possible explanation for this could be the way Ca²⁺ is utilized during the course of a contraction. Specifically, the transient contractions evoked by bolus doses of methoxamine may depend primarily on mobilization of Ca²⁺ from intracellular stores, whereas the sustained contractions elicited by prolonged exposure to methoxamine may involve, to a greater extent, entry of extracellular Ca²⁺ into the smooth muscle cells and Ca²⁺ sensitization. The possible role of Ca²⁺ in impaired responses to methoxamine was investigated in isolated superior mesenteric arteries and aortae (the choice of preparations for these experiments was largely dictated by the nature of the experiments) as discussed below.

In contrast to the impairment of vascular contractility to methoxamine in both the mesenteric arterial bed and the aorta, isolated from endotoxemic rats, the renal vascular bed isolated from these animals displayed no significant attenuation of the contractile response to cumulative concentrations of methoxamine or KCl. The lack of change in the renal response to methoxamine in endotoxemia is consistent with findings in the renal vasculature in vivo (Waller et al., 1994), although the renal vascular bed in vivo displays a greater degree of hyperemic vasodilatation after 24 h LPS than the mesentery (Waller et al., 1994). The present results indicate that renal vasodilatation is not likely to be due to a loss of -adrenoceptor-mediated vasoconstrictor tone. In a number of tissues (including the mesentery and aorta), induction of inducible nitric-oxide synthase (NOS) is maximal at 6 h after the start of LPS infusion, but this returns to control levels at 24 h (Gardiner et al., 1995; Mitchell et al., 1993), and kidney inducible NOS does not change during LPS infusion (Gardiner et al., 1995); therefore, other factors must be responsible for the vasodilatation seen at that stage. It remains to be determined what mechanisms are responsible for protecting the renal vascular bed under these conditions.

Hypocontractility to Methoxamine in Thoracic Aortae in Endotoxemia. The aortic preparations also showed hypocontractility to methoxamine, but here the difference was apparent in preparations isolated at both 2 and 24 h after the onset of LPS infusion. Thus, endotoxemia can cause hypocontractility to a given vasoconstrictor (methoxamine) in both conduit (aorta) and resistance (mesenteric arterial bed) vessels, although temporal differences in the susceptibility of these two vasculatures to impaired responsiveness were observed. In neither case was the hypocontractility to methoxamine due to a generalized impairment of smooth muscle contractile function, as responses to KCl were not affected. Thus, hypocontractility to methoxamine in this model of endotoxemia could involve one or more of the steps including, and/or subsequent to, stimulation of ₁-adrenoceptors.

Interestingly, in contrast to the pronounced impairment of contractions to methoxamine, contractions to U46619 [GenBank] in the aortae were unaffected by LPS infusion at either 2 or 24 h. Similarly, U46619 [GenBank] -induced contractions were reported to be largely maintained in rat mesenteric arteries in an in vitro model of endotoxemia, whereas those to phenylephrine were attenuated (O'Brien et al., 2001; Wylam et al., 2001). In rats rendered tolerant to lethal doses of endotoxin by repeated sublethal doses of endotoxin, the pressor response to phenylephrine was attenuated, but a higher peak response to U46619 [GenBank] was observed compared with controls (Coffee et al., 1991), although in the same model, the sensitivity of the response to U46619 [GenBank] in isolated aortic rings was reduced (Temple et al., 2001). The reason for these differences between the contractile agents is unclear, but one possibility is differences between their signaling pathways. For example, RhoA, a signaling molecule involved in sensitization of the smooth muscle contractile machinery to Ca²⁺, is activated to a greater extent by U46619 [GenBank] than by noradrenaline in rabbit aortic smooth muscle (Sakurada et al., 2001).

Role of Ca²⁺ in Hypocontractility in Endotoxemia. We attempted to explore some of the underlying mechanisms of the changes involved. Ca²⁺ measurements in the mesenteric artery were restricted to 24 h, since that was the time when significant changes in contractile responses were observed. The results showed a tendency for smaller changes in Ca²⁺ after LPS treatment, but these were not significant, so it would appear that, as shown by Martinez et al. (1996), a failure to release Ca²⁺ does not underlie hypocontractility to methoxamine. In the aortae, preparations taken at 24 h after LPS treatment also showed normal responses to caffeine, indicating that a change in mobilization of intracellular Ca²⁺ also does not underlie hypocontractility to methoxamine, pointing to changes in Ca²⁺ sensitization of contractile proteins (which can take place without large changes in Ca²⁺)or to the involvement of another factor. Others have shown an important contribution of L-arginine availability and the NO pathway in regulating the extent of mesenteric hypocontractility produced by LPS (Mitolo-Chieppa et al., 1996). We did not investigate the extent to which that was involved in the changes observed here.

In the aorta, responses to methoxamine were reduced at 2 and 24 h LPS, but the response to caffeine was reduced only at 2 h LPS. Thus, this indicates that there may be an impairment of Ca²⁺ release from internal stores at 2 h, but not at 24 h, after LPS infusion. Previous studies have shown elevated basal levels of intracellular Ca²⁺ in rat aorta caused by sepsis or endotoxin treatment, which may result from an impairment of Ca²⁺ storage in intracellular organelles (Song et al., 1993; Martinez et al., 1996). Impaired storage, and by implication, release of Ca²⁺ in intracellular organelles could explain the decreased response to caffeine observed at 2 h. Inducible NOS activity in the aorta in this model of endotoxemia has been shown to increase between 2 and 6 h, and then to be undetectable at 24 h, after the onset of LPS infusion (Gardiner et al., 1995), which is in some respects similar to the temporal change in the response to caffeine. In this regard, it is interesting that NO has been shown to reduce the rate of Ca²⁺ release from skeletal muscle sarcoplasmic reticulum and open probability of ryanodine receptors in lipid bilayers (Meszaros et al., 1996).

If changes in Ca²⁺ mobilization or entry (present study) and NO (Mitchell et al., 1993; Gardiner et al., 1995) do not underlie the mesenteric and aortic hypocontractility to methoxamine that we have observed, at least at 24 h LPS, this raises the question of what mechanisms are involved. One possibility is an involvement of K⁺ channels, as these have been shown to be up-regulated by LPS treatment (Czaika et al., 2000) and are involved in relaxation to LPS and in ex vivo aortic hypocontractility to phenylephrine and noradrenaline in LPS-treated rats (Hall et al., 1996; Sorrentino et al., 1999; Chen et al., 2000).

	Conclusions

Top Abstract Materials and Methods Results Discussion Conclusions References

 
In conclusion, the present study has demonstrated that in mesenteric arterial beds and in aortae, but not in the renal arterial bed, isolated from rats following infusion of LPS, there is an attenuation of the contractile response to methoxamine. This is not due to a generalized inability of the smooth muscle to contract and, at least in late endotoxemia (24 h LPS), does not appear to involve abnormalities in Ca²⁺ mobilization or entry. Moreover, in the mesenteric arterial beds from the endotoxemic animals, there clearly is an impairment of sustained contractile responses to continuous activation, but not to transient activation, of ₁-adrenoceptors, which may be significant for different patterns of stimulation used by sympathetic nerves and/or circulating catecholamines.

	Acknowledgements

 
We thank J. E. March and P. A. Kemp for technical assistance.

	Footnotes

 
This study was funded by a project grant from the British Heart Foundation and by the Royal Society. Their financial assistance is gratefully acknowledged.

DOI: 10.1124/jpet.103.051805.

ABBREVIATIONS: LPS, lipopolysaccharide; NO, nitric oxide.

¹ Royal Society Research Fellow.

Address correspondence to: Dr. Vera Ralevic, School of Biomedical Sciences, University of Nottingham Medical School, Queen's Medical Centre, Clifton Boulevard, Nottingham, NG7 2UH, United Kingdom. E-mail: vera.ralevic{at}nottingham.ac.uk

	References

Top Abstract Materials and Methods Results Discussion Conclusions References

 

Chen S-J, Wu C-C, Yang S-N, Lin C-I, and Yen M-H (2000) Hyperpolarization contributes to vascular hyporeactivity in rats with lipopolysaccharide-induced endotoxic shock. Life Sci 68: 659–668.[CrossRef][Medline]

Coffee KA, Halushka PV, Wise WC, Templel GE, and Cook JA (1991) Endotoxin tolerance differentially alters hemodynamic responses to a thromboxane A₂ mimetic and phenylephrine. J Cardiovasc Pharmacol 17: 20–26.[CrossRef][Medline]

Czaika G, Gingras Y, Zhu E, and Comtois AS (2000) Induction of the ATP-sensitive potassium channel (uK_ATP-1) by endotoxaemia. Muscle Nerve 23: 967–969.[CrossRef][Medline]

Farmer MR, Gardiner SM, and Ralevic V (2001) Vascular responses to methoxamine in isolated perfused mesenteric arterial beds from endotoxaemic rats. Br J Pharmacol 134: 88P.[CrossRef]

Gardiner SM, Kemp PA, and Bennett T (1994) Cardiac haemodynamic effects of chronic lipopolysaccharide (LPS) infusion in conscious rats. Br J Pharmacol 112: 27P.

Gardiner SM, Kemp PA, Bennett T, Palmer RMJ, and Moncada S (1993) Regional and cardiac haemodynamic effects of N^G,N^G,dimethyl-L-arginine and their reversibility by vasodilators in conscious rats. Br J Pharmacol 110: 1457–1464.[Medline]

Gardiner SM, Kemp PA, March JE, and Bennett T (1995) Cardiac and regional haemodynamics, inducible nitric oxide synthase (NOS) activity and the effects of NOS inhibitors in conscious, endotoxaemic rats. Br J Pharmacol 116: 2005–2016.[Medline]

Gardiner SM, Kemp PA, March JE, and Bennett T (1996a) Effects of dexamethasone and SB 209670 on the regional haemodynamic responses to lipopolysaccharide in conscious rats. Br J Pharmacol 118: 141–149.[Medline]

Gardiner SM, Kemp PA, March JE, and Bennett T (1996b) Influence of aminoguanidine and the endothelin antagonist, SB 209670 on the regional haemodynamic effects of endotoxaemia in conscious rats. Br J Pharmacol 118: 1822–1828.[Medline]

Grynkiewicz G, Poenie M, and Tsien RY (1985) A generation of Ca²⁺ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440–3450.[Abstract/Free Full Text]

Hall S, Turcato S, and Clapp L (1996) Abnormal activation of K⁺ channels underlies relaxation to bacterial lipopolysaccharide in rat aorta. Biochem Biophys Res Commun 224: 184–190.[CrossRef][Medline]

Martinez MC, Muller B, Stoclet J-C, and Andriantsitohaina R (1996) Alteration by lipopolysaccharide of the relationship between intracellular calcium levels and contraction in rat mesenteric artery. Br J Pharmacol 118: 1218–1222.[Medline]

McGregor DD (1965) The effect of sympathetic nerve stimulation on vasoconstrictor responses in perfused mesenteric blood vessels of the rat. J Physiol 177: 21–30.

Meszaros LG, Minarovic I, and Zahradnikova A (1996) Inhibition of skeletal muscle ryanodine receptor calcium release channel by nitric oxide. FEBS Lett 380: 49–52.[CrossRef][Medline]

Mitchell JA, Kohlhaas KL, Sorrentino R, Warner TD, Murad F, and Vane JR (1993) Induction by endotoxin of nitric oxide synthase in the rat mesentery: lack of effect on action of vasoconstrictors. Br J Pharmacol 109: 265–270.[Medline]

Mitolo-Chieppa D, Serio M, Potenza A, Montagnani M, Mansi G, Pece S, Jirillo E, and Stoclet J-C (1996) Hyporeactivity of mesenteric vascular bed in endotoxin-treated rats. Eur J Pharmacol 309: 175–82.[CrossRef][Medline]

O'Brien AJ, Wilson AJ, Sibbald R, Singer M, and Clapp LH (2001) Temporal variation in endotoxin-induced vascular hyporeactivity in a rat mesenteric artery organ culture model. Br J Pharmacol 133: 351–360.[CrossRef][Medline]

Pastor CM (1999) Vascular hyporesponsiveness of the renal circulation during endotoxaemia in anesthetized pigs. Crit Care Med 27: 2735–2740.[CrossRef][Medline]

Ralevic V and Burnstock G (1988) Actions mediated by P₂-purinoceptor subtypes in the isolated perfused mesenteric bed of the rat. Br J Pharmacol 95: 637–645.[Medline]

Sakurada S, Okamoto H, Takuwa N, Sugimoto N, and Takuwa Y (2001) Rho activation in excitatory agonist-stimulated vascular smooth muscle. Am J Physiol 281: C571–C578.

Song S-K, Karl IE, Ackerman JJH, and Hotchkiss RS (1993) Increased intracellular Ca²⁺: a critical link in the pathophysiology of sepsis? Proc Natl Acad Sci USA 90: 3933–3937.[Abstract/Free Full Text]

Sorrentino R, d'Emmanuele di Villa Bianca R, Lippolis L, Sorrentino L, Autore G, and Pinto A (1999) Involvement of ATP-sensitive potassium channels in a model of a delayed vascular hyporeactivity induced by lipopolysaccharide in rats. Br J Pharmacol 127: 1447–1453.[CrossRef][Medline]

Suffredini AF, Fromm RE, Parker MM, Brenner M, Kovacs JA, Wesley RA, and Parrillo JE (1989) The cardiovascular response of normal humans to the administration of endotoxin. New Engl J Med 321: 280–287.[Abstract]

Tarpey SB, Bennett T, and Gardiner SM (1998a) Differential changes in cardiovascular responses to methoxamine and noradrenaline during endotoxaemia in conscious rats. Br J Pharmacol 123: 332P.

Tarpey SB, Bennett T, Randall MD, and Gardiner SM (1998b) Differential effects of endotoxaemia on pressor and vasoconstrictor actions of angiotensin II and arginine vasopressin in conscious rats. Br J Pharmacol 123: 1367–1374.[CrossRef][Medline]

Tarpey SB and Randall MD (1998) Vascular activities of methoxamine and noradrenaline in isolated perfused mesenteric arterial beds from endotoxaemic rats. Br J Pharmacol 123: 332P.

Temple GE, Brown AN, Morinelli TA, Halushka PV, and Cook JA (2001) Changes in vascular responsiveness to a thromboxane mimetic in endotoxin tolerance. Shock 16: 389–392.[Medline]

Waller J, Gardiner SM, and Bennett T (1994) Regional haemodynamic responses to acetylcholine, methoxamine, salbutamol and bradykinin during lipopolysaccharide infusion in conscious rats. Br J Pharmacol 112: 1057–1064.[Medline]

Warner TD (1990) Simultaneous perfusion of rat isolated superior mesenteric arterial and venous beds: comparison of their vasoconstrictor and vasodilator responses to agonists. Br J Pharmacol 99: 427–433.[Medline]

Wylam ME, Metkus AP, and Umans JG (2001) Nitric oxide dependent and independent effects of in vitro incubation or endotoxin on vascular reactivity in rat aorta. Life Sci 69: 455–467.[CrossRef][Medline]

Zhou M, Arthur AJ, Ba ZF, Chaudry IH, and Wang P (2001) The small intestine plays an important role in upregulating CGRP during sepsis. Am J Physiol 280: R382–R388.

Zucci R and Ronca-Testoni S (1997) The sarcoplasmic reticulum Ca²⁺ channel/ryanodine receptor: modulation by endogenous effectors, drugs and disease states. Pharmacol Rev 49: 1–51.[Abstract/Free Full Text]

This article has been cited by other articles:

				J.-J. Boffa and W. J. Arendshorst Maintenance of Renal Vascular Reactivity Contributes to Acute Renal Failure during Endotoxemic Shock J. Am. Soc. Nephrol., January 1, 2005; 16(1): 117 - 124. [Abstract] [Full Text] [PDF]

				J.-J. Boffa, A. Just, T. M. Coffman, and W. J. Arendshorst Thromboxane Receptor Mediates Renal Vasoconstriction and Contributes to Acute Renal Failure in Endotoxemic Mice J. Am. Soc. Nephrol., September 1, 2004; 15(9): 2358 - 2365. [Abstract] [Full Text] [PDF]

				T. Bennett, R. P. Mahajan, J. E. March, P. A. Kemp, and S. M. Gardiner Regional and temporal changes in cardiovascular responses to norepinephrine and vasopressin during continuous infusion of lipopolysaccharide in conscious rats Br. J. Anaesth., September 1, 2004; 93(3): 400 - 407. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.103.051805v1 306/2/538    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Farmer, M. R. Articles by Ralevic, V. Search for Related Content PubMed PubMed Citation Articles by Farmer, M. R. Articles by Ralevic, V.