Lipoteichoic Acid from Staphylococcus aweus Depresses Contractile Function of Human Arteries In Vitro Due to the Induction of Nitric Oxide Synthase

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1 Lipoteichoic Acid from Staphylococcus aweus Depresses Contractile Function of Human Arteries In Vitro Due to the Induction of Nitric Oxide Synthase Isao Tsuneyoshi, MD, Yuichi Kanmura, MD, PhD, and Nozomu Yoshimura, MD, PhD Department of Anesthesiology and Critical Care Medicine, Kagoshima University School of Medicine, Kagoshima, Japan The aim of this study was to clarify the role of Grampositive organisms in the genesis of sepsis. In the present study, we investigated the effect of lipoteichoic acid (LTA) from the cell wall of Staphylococcus aweus on contractions elicited by norepinephrine (NE) in rings cut from human gastroepiploic arteries. LTA diminished the contractile response to NE. This attenuation began after several hours exposure to LTA, and reached its maximum after 10 h of exposure, whether or not endothelium was present. The cyclic guanosine monophosphate content of LTA-treated rings was higher than that of control rings, whether there was a functional endothelium. These LTA-mediated responses were reduced significantly by inhibitors of nitric oxide (NO) synthase and guanylate cyclase. All of this indicates that the main underlying cause of the vascular hyporeactivity to NE was a massive generation of NO. In addition, cycloheximide, an inhibitor of inducible NO synthase, prevented the attenuation of NE-induced contractions caused by LTA. Thus, our results offer strong supporting evidence that the important factor in the genesis by Gram-positive organisms of a diminished contractile response to pressor drugs is their induction of inducible NO synthase in smooth muscle. (Anesth Analg 1996;82:948-53) 0 ne important cause of the high mortality associated with septic shock is the depression of contractile sensitivity to pressor drugs (1). Recent reports suggest that this desensitization of blood vessels is not mediated by changes in the number of receptors or their affinity for agonists, but is possibly caused by an excessive production of endogenous nitric oxide (NO) (2,3). Bacterial pathogens and inflammatory products, such as cytokines, activate NO synthase in rodent vascular smooth muscle cells, suggesting that this intracellular source of NO may play a major role in vascular desensitization to pressor drugs during sepsis (4). Indeed, the administration of NO synthase inhibitors reversed the hypotension in patients with septic shock (5,6). A report on septic shock revealed a surprisingly high percentage of patients with Gram-positive bacterial sepsis (-40% of the total); these patients demonstrated various biological dysfunctions and multiple organ failure, with mortality rates as high as those in a Gram-negative sepsis group (7). However, the Accepted for publication December 20, Address correspondence and reprint requests to Isao Tsuneyoshi, MD, Department of Anesthesiology and Critical Care Medicine, Kagoshima University School of Medicine, 8-35-l Sakuragaoka, Kagoshima 890, Japan. pathogenic mechanisms involved in Gram-positive sepsis are poorly understood. There have already been several studies using Gram-negative, rather than Gram-positive, pathogens, such as endotoxins, in animals and humans designed to elucidate the sequence of the pathophysiologic events that lead to severe sepsis and ultimate circulatory shock (S-10). However, these studies provide little, if any, insight into Gram-positive shock, because Gram-positive organisms do not contain endotoxin. Some recent studies have indicated that lipoteichoic acid (LTA), a cell-wall component from Gram-positive bacteria (ll), may promote the release of NO and thus reduce sensitivity to pressor drugs in both in vitro and e;y viva animal experiments (12,13). Thus, we hypothesized that sepsis, whether caused by Gram-positive or Gram-negative organisms, might involve similar hemodynamic dysfunctions, the final common pathway of which might involve NO synthase. However, in human blood vessels, there are no experimental studies in which LTA has been shown to play a role in the depression of the contractile response to pressor drugs that occurs in the absence of endotoxin. In this study, we sought to determine the effects of LTA derived from a Gram-positive bacterium, Staphy2ococcus aweus, on the contractile function of human 948 Anesth Analg 1996;82: by the International Anesthesia Research Society /96/55.00

2 ANESTH ANALG 1996;82: TSUNEYOSHI ET AL. 949 EFFECT OF LIPOTEICHOIC ACID ON HUMAN ARTERIES arteries. To this end, we investigated the timedependent effects of LTA on the contractile responses to norepinephrine (NE) in human gastroepiploic arterial rings. In addition, we measured cyclic guanosine monophosphate (cgmp) levels in the same vascular tissue as an index of NO synthase activity. We also tried to evaluate the role of NO synthase on these responses using NO synthase inhibitors. Methods With approval from the ethics committee of Kagoshima University School of Medicine, the omentum was resected from the stomach immediately after gastrectomy had been performed under general anesthesia. The patients, who were all less than 70 yr (mean = 58.9 yr, range = 46-69, n = 241, had no vascular disease or other complications. They or their family gave informed consent. Ten minutes or less after the omentum had been resected, the normal sites of gastroepiploic arteries were isolated from it in a dissecting chamber filled with Krebs-Ringer solution and fat, and connective tissues were carefully removed under a binocular microscope. Two vascular rings of 2-mm length were prepared from each artery for tension recording. One ring was carefully denuded of endothelium by inserting small forceps into the lumen and gently rolling the ring backward and forward in the dissecting chamber. When a functional endothelium is present, but not when it is absent, NE-induced contractions can be relaxed by acetylcholine (ACh). In our rings with intact endothelium, ACh (5 X 10e6 PM) relaxed the 1.0 JLLM NE-induced contraction to 61%? 5% of control (P < 0.01, YI = 24; with the amplitude of the 1.0 PM NE-induced maximal contraction being normalized as 100%). On the other hand, in our nominally endothelium-denuded rings, an ACh-induced relaxation was not observed (98% 2 8%, n = 24). Mechanical activity was assessed using a strain gauge with the rings in a tissue bath filled with Krebs- Ringer solution continuously bubbling with 95% 0,:5% CO,. Krebs-Ringer solution maintained at 37 C was continuously infused from one end of the bath at a rate of 1 ml/min by a pump and simultaneously aspirated by a water pump from the other. The resting tension was set at a value (2 g) shown by the lengthtension relationship to allow maximal active tension to be induced by 1.0 PM NE. After 2-h equilibrium in Krebs-Ringer solution, 1.0 PM NE was applied to the vascular rings for 7 min every 30 min. After amplitude of the contraction induced by 1.0 PM NE became constant, LTA (10 pg/ml) was applied to the tissue by adding it to the Krebs-Ringer solution. Other agents (fl-nitro-l-arginine-methyl ester [L-NAME] and methylene blue [MB]) were infused in Krebs-Ringer solution so that the tissue was exposed to the dose indicated in the text. The amount of cgmp in the tissue was measured using a cgmp enzyme-immunoassay kit from Amersham International (Little Chalfont, UK). After fat and connective tissue had been removed, gastroepiploic artery rings (weight = mg) were allowed to equilibrate in beakers containing Krebs-Ringer solution. After 12-h equilibration with or without 10 pg/ml LTA, the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (100 PM), was added to each solution to inhibit phosphodiesterase 30 min before cgmp determination. Then, cgmp was extracted from a homogenate made from the rings using trichloroacetic acid. Composition of the Krebs-Ringer solution was as follows (mm concentrations): Na+, 137.4; K+, 5.9; Mg +, 1.2; Ca +, 2.6; HCOs-, 15.5; H,PO,-, 1.2; Cl-, 134.4; and glucose, All solutions also contained 10 FM indomethacin to prevent the effects of prostaglandins or their intermediates. To exclude the possibility that any endotoxin present might play a causal role in the vascular reactions, all solutions contained an endotoxin-neutralizing drug, polymyxin B (100 PM). Chemicals used were NE HCI, LTA (derived from S. uureus), MB, polymyxin B, fl-nitro-l-arginine (L-NoArg) and indomethacin from Sigma Chemical Co. (St. Louis, MO), and L-NAME from Funakoshi (Tokyo, Japan). The amplitude of contraction induced by NE at the start of the experiment was normalized as a relative tension of 1.0 for each ring. Values were expressed as mean? SD (n = number of observations). Statistical analysis was performed by a one- or two-factor analysis of variance with repeated measures, followed by Scheffe s test (for multiple comparisons) or a twotailed, unpaired Student s t-test (for comparison between two treatments). P values were two-tailed, and values less than 0.05 were considered significant. Results After 2-h equilibration of the rings in Krebs-Ringer solution, 1.0 PM NE was applied for 7 min every 30 min. After the contractile response to NE had become stable, 10 pg/ml LTA was applied for 12 h. In the absence of LTA, the amplitude of the contractions induced by NE in endothelium-intact rings or in endothelium-denuded rings remained more or less constant for 12 h (Table 1). LTA (10 pg/ml) gradually attenuated the NE-induced contractions over a long time course, so that their amplitude was significantly decreased after 12 h, both in endothelium-denuded rings and in endothelium-intact rings (Figs. 1 and 2; Table 1). At 12.5 h after the start of the experiment, an inhibitor of NO synthase (L-NAME, 1.0 mm) and an

3 950 TSUNEYOSHI ET AL. ANESTH ANALG EFFECT OF LII OTEICHOIC ACID ON HUMAN ARTERIES 1996;82: Table 1. Depression of Sensitivity to Norepinephrine Caused by 12-H Exposure to Lipoteichoic Acid (LTA) and Its Inhibition by p-nitro-l-a&nine Methyl Ester (L-NAME) or Methylene Blue (MB) Control (n = 6) Endothelium-intact Endothelium-denuded 12 h after 1.05 (0.17) 1.10 (0.21) 1.16 (0.20) 1.28 (0.17) Relative tension 12.5 h after L-NAME (1 mm) 12 h after 12.5 h after MB (10 PM) 1.05 (0.13) 1.05 (0.13) 1.04 (0.15) 1.15 (0.09) LTA (10 pg/ml) added (n = 6) Endothelium-intact Endothelium-denuded 0.50 (0.13) 1.28 (0.19) 0.51 (0.18) 1.39 (0.27) 0.55 (0.13) 1.04 (o.lo)t 0.58 (0.13) 1.18 (0.07)t Values are means (? SD). Initial maximum contraction induced by norepinephrine is given the value of 1.0 * Slgniflcantly different (P < 0.05) from value after 12 h. t Significantly different (P < 0.01) from value after 12 h. A Control B 1Ouplml LTA endothelium-intuct Ohr 6hr 12hr 12.5hr b.l 7min I I -w-- +(1(1;h4) L-NAME 1g T (ImM) endothelium-intact Ohr 6hr 12hr 12.5hr t$t (1;M )- L-NAME (1 mm) endothelium-denuded Ohr 6hr 12hr 12.5hr endothelium-denuded Ohr 6hr 12hr 12.5hr 7min 7min T 1g 1g GE U;;M )- L-NAME (1mW L-NAME (1mM) Figure 1. Representative recording of contractions elicited by norepinephrine (NE, 1.0 PM) in endothelium-intact and endothelium-denuded human gastroepi loic arteries incubated with lipoteichoic acid (LTA, 10 pg/ml). A, Control contractions induced by 1.0 /.LM NE. After 12-h measurement, J mtro - L - argmme methyl ester (L-NAME, 1 mm) was applied where indicated. B, Effect of LTA (10 pg/ml) on contractions induced by NE (1.0 PM). LTA treatment was continued for 12 h, and then L-NAME (1 mm) was applied where indicated. inhibitor of guanylate cyclase (MB, 10 PM), used sep- whether in the presence or absence of endothelium arately, each restored the NE-induced contraction to (P > 0.05 for comparison with control contractions; the control amplitude seen before LTA (Table 1). Fig. 2). To determine whether the attenuation of the NE- Whether in the presence or absence of endothelium, induced contraction evoked by LTA was provoked via 12-h incubation with LTA increased the amount of inducible NO synthase (inos), the effect of cyclohex- cgmp in human gastroepiploic arteries (P < 0.05; Fig. imide, an inhibitor of inos, was examined in arterial 3). In these data, there was no significant difference rings treated with LTA. Cycloheximide (10 pg/ml) between the groups with or without endothelium. The was applied for 2 h before and was present during the LTA-induced increase in cgmp content was blocked application of LTA. In such rings, LTA failed to exert both by 1.0 mm L-NoArg and 1.0 mm L-NAME, its inhibitory effect on NE-induced contractions, whether in the presence or absence of endothelium.

4 ANESTH ANALG TSUNEYOSHI ET AL ;82: EFFECT OF LIPOTEICHOIC ACID ON HUMAN ARTERIES A, endothelium-intact Endothelium-intact I IO 12 Time (hr) B, endothelium-denuded O 12hr after exposure to LTA ImM ImM L-NoArg L-NAME Endothelium-denuded 0 4 I 1 I I I IO 12 Time (ht) Figure 2. Effect of 10 pg/ml lipoteichoic acid (LTA) on 1.0 PM norepinephrine (NE)-induced contractions in endothelium-intact (A) and endothelium-denuded (B) human gastroepiploic arteries. Maximum amplitude of contraction at the starting point of the experiment was normalized as a relative tension of 1.0 for each arterial ring. Polymyxin B (100 PM) was present in Krebs solution during the application of LTA to prevent the influence of endotoxin. In some LTA-treated rings, cycloheximide (10 PM) was present in solution for 2 h before and throughout the experiment. Vertical bars indicate SD (*P < 0.05, n = 6). A, NE-induced contraction with (0) or without (W) 10 pg/ml LTA treatment in endothelium-intact arteries. In some LTA-treated rings (A), cycloheximide was used, and it prevented the attenuation of the NE-induced contraction caused by LTA. B, In endothelium-denuded arteries, NE-induced contraction with (0) or without (0) 10 pg/ml LTA treatment. Cycloheximide treatment (a ) prevented LTA-induced hyporeactivity to NE. Discussion This study indicates that LTA depresses the sensitivity to NE of human smooth muscle via activation of the L-arginine pathway and the cgmp signal cascade. These results suggest that this intracellular source of NO may play a major role in vascular relaxation and in the reduction of the contractile response to pressor drugs during Gram-positive sepsis. Furthermore, present observations have important implications for the possible prophylactic use of NO synthase inhibitors during Gram-positive sepsis. They also help to E j n O 12hr after exposure to LTA ImM ImM L-NoArg L-NAME Figure 3. Effects of 10 wg/ml lipoteichoic acid (LTA, solid columns), 1.0 mm fl-nitro-l-arginine methyl ester (L-NAME, hatched columns), 1.0 mm $J -nitro-l-arginine (L-NoArg, stippled columns) on cyclic guanosine monophosphate (cgmp) content in human gastroepiploic arteries with (top) or without (bottom) intact endothelium. Before (open column) and after 12-h treatment with 10 kg/ml LTA, 3-isobutyl-1-methylxanthine (100 PM) was added to each solution 1 h before cgmp determination and then either methylene blue (10 FM), 1.0 mm L-NAME, or 1.0 mm L-NoArg was added 30 min before freezing the rings. Polymyxin B (100 PM) was applied immediately after isolation and was present throughout the experiment. Vertical bars indicate SD (n = 6). Asterisks indicate significant difference from the amount of cgmp at 0 h (*P < 0.05).

5 952 TSUNEYOSHI ET AL. EFFECT OF LIPOTEICHOIC ACID ON HUMAN ARTERIES ANESTH ANALG 1996;82: improve our understanding of the complex mechanisms by which vasodilation is induced in septic patients without detectable levels of endotoxin. In this study, direct incubation of isolated human gastroepiploic arterial rings with 10 pg/ml LTA attenuated the NE-induced contraction, whether in the presence or absence of endothelium. This attenuation of contraction took several hours to become apparent and reached its maximum after 10-h exposure to LTA. The depression of sensitivity to NE after 12-h incubation with LTA was reversed by both MB, an inhibitor of guanylate cyclase, and L-NAME, an inhibitor of NO synthase. Furthermore, in our experiments, the cgmp content of rings treated with LTA for 12 h was significantly greater than that of nontreated rings, whether or not endothelium was present. This effect was blocked by both L-NoArg and L-NAME, which are NO synthase inhibitors. Consequently, it seems that the release of NO from smooth muscle on exposure to LTA activates guanylate cyclase and produces cgmp, which then induces vascular desensitization to NE. In animal blood vessels, two types of NO synthase have been documented on the basis of calcium/ calmodulin dependence, inhibitor specificity, and immunogenicity (2). One is a constitutive NO synthase that is localized in the endothelium, and the other is an inos found mainly in vascular smooth muscle. Inflammatory products, such as endotoxin or cytokines, activate inos in vascular smooth muscle (2). In our study, the depression of sensitivity to NE and the accumulation of cgmp were both induced by LTA independently of the presence of a functional endothelium. In addition, cycloheximide, an inhibitor of inos (14), prevented the attenuation of contraction caused by LTA. Thus, we conclude that this effect was caused by the expression of inos, which is present in the smooth muscle layer. Sepsis is believed to result from a complex mechanism involving activation of a number of cells, most notably monocytes, T cells, neutrophils, and platelets. Some studies suggest that Gram-positive organisms can prompt release of the inflammatory mediators that underly sepsis and can also reproduce its clinical signs and symptoms (15,16). Our results indicate that LTA directly inhibits the NE-induced contraction of human vascular smooth muscle in vitro and may thus play a important role in vascular pathophysiology. The mechanism appears to be independent of cytokines, such as tumor necrosis factor (TNF) and interleukin-1 (IL-l), which are thought to mediate many of the pathophysiologic changes induced by Gram-positive bacteremia (16). Concentration of LTA used in the present study (10 pg/ml) may or may not exceed the plasma concentrations that occur in patients with Gram-positive sepsis: concentration of LTA that circulates in humans is not known. Previous studies have revealed that LTA-induced cgmp accumulation in cultured smooth muscle cells of the rat aorta increased concentrationdependently between 0.01 and 100 pg/ml (12). Recent emphasis has been placed on the role of TNF as a inducer of inos during Gram-positive sepsis (13). Indeed, levels of cytokines, such as IL-l and TNF, correlated well with the degree of hypotension induced via a Gram-positive pathogen such as Staphylococcus epidermidis (16). Thus, clinically, much lower doses of LTA than that used in our study may well cause vascular hyporeactivity, because TNF and IL-l, which may be present independently of any stimulation with LTA, have a synergistic effect on the induction of inos. Our studies using Gram-positive organisms give us the opportunity to study both NO production and the depression of sensitivity to pressor drugs in human arteries that occur independently of endotoxin. Indeed, by no means do all septic or hypotensive patients have Gram-negative infections (17), suggesting that endotoxin is not the only factor determining blood pressure during sepsis. It appears that Grampositive organisms may be becoming more virulent, as evidenced by the emergence of streptococcal toxic shock syndrome (18) and the resurgence of acute rheumatic fever (19). In addition, several species of streptococci, including Strepfococcus ugulactiu (20), are believed to play a major role in the pathogenesis of neonatal sepsis and meningitis. In conclusion, we have emphasized the importance of Gram-positive organisms in the diminution of agonist-induced contractile responses in vascular smooth muscle in humans. The attenuation of contractions was probably caused by activation of the soluble guanylate cyclase effector system in the smooth muscle layer, since it was associated with an increase in the intracellular cgmp content of smooth muscle cells. Our results support the idea that, during Grampositive sepsis, LTA-induced activation of inos located in the smooth muscle layer of human blood vessels may contribute to the decreased sensitivity to pressor drugs and thus to hypotension. The authors with to thank Professor T. Itoh (Department of Pharmacology, Nagoya City University Medical School) for his pertinent advice on this work. We also thank T. Sameshima and J. Miyao (Department of Anesthesiology and Critical Care Medicine, Kagoshima University School of Medicine) for their help with this work, and Dr. R. J. Timms for his help in preparing the manuscript. References 1. Lowry SF. Sepsis and complications: clinical definitions and therapeutic prospects. Crit Care Med 1994;22:Sl Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol Rev 1991;43:

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