Introduction

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Invasion of the body by infectious bacteria activates a series of mechanisms to defend against the incursion, resulting in a localized inflammatory response. When this defense response fails, and bacteria or their products reach the vasculature, septic shock can ensue, producing hypotension and death. In the United States, approximately 400,000 hospitalized patients per year are diagnosed with septicemia (Munford, 1996). The mechanisms contributing to the pathology of septic shock have received much attention in recent years (Cusumano et al., 1997; Makhlouf et al., 1997). Lipopolysaccharide (LPS, endotoxin), a constituent of the external membrane of Gram negative bacteria, is one of the most potent and widely studied signal molecules involved in the initiation of septic shock. Cellular responses to exposure to Lipid A, the signaling moiety of LPS, include the release of a variety of endogenous substances, such as cytokines (tumor necrosis factor-, interleukin-1, interleukin-6, interferon-, etc.), metabolites of arachidonic acid, coagulation factors, nitric oxide, and platelet-activating factor (Wheeler and Bernard, 1999). In addition, activation of the kininogen-kallikrein-kinin system with release of the kinins has been demonstrated (Miller and Margolius, 1997). These mediators contribute to the subsequent cellular responses, including increased vascular permeability, generation of toxic oxygen metabolites, generation of microthrombi, systemic hypotension, and organ failure (Krzanowski, 1994; Munford, 1996). Because kinins are potent vasodepressors and stimulate the release of cytokines, arachidonic acid metabolites, and nitric oxide production, their rapid generation by LPS is considered an important initial stimulus for the cardiovascular collapse seen in septic shock (Miller and Margolius, 1997).

Bradykinin (BK) or kallidin (LBK) are the cleavage products of the action of specific kallikreins upon specific substrate kininogens (Bhoola et al., 1992). The principal cardiovascular effect of kinins is endothelial-dependent vasodilation, a well documented effect in the pathological hypotension associated with septic (endotoxin) shock (Whalley et al., 1992; Lu et al., 1996). The initial digestion of kinins by kininase II results in the removal of the terminal phenylalanine-arginine, leaving des-R⁹-F⁸-BK or des-R⁹-F⁸-LBK. Subsequent cleavages result in additional kinin fragments, including the pentapeptide RPPGF (Shima et al., 1992; Majima et al., 1996). All proteolytic fragments of kinins, including RPPGF, have been considered for decades to be inactive, with the exception of des-R⁹-F⁸-BK or des-R⁹-F⁸-LBK (Regoli and Barabe, 1980). However, some recent evidence suggests that RPPGF interacts with the platelet thrombin receptor at relatively high concentrations (~1 mM), inhibiting the activation of platelets by thrombin (Hasan et al., 1996) as well as inhibiting coronary occlusion in dogs (Hasan et al., 1999). In addition to RPPGF having direct physiological actions of its own, it also has been shown to act as substrate for a novel neuronal nitric-oxide synthase (nNOS) (Chen and Rosazza, 1996), releasing nitric oxide and citrulline from the N-terminal arginine.

The generation and degradation of the kinins can occur very rapidly during episodes of local tissue damage, allergic reactions, and inflammatory responses (Bhoola et al., 1992). In consideration of the central role of bradykinin in endotoxin shock, we chose in the present study to examine the pharmacological activity of RPPGF, a stable metabolic fragment of BK, in a rat model of endotoxin shock. As a result, we have uncovered a potent activity of RPPGF, an activity that protects against the deleterious effects of LPS.

	Materials and Methods

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Rat Endotoxin Survival Studies. Male Sprague-Dawley rats (220-480 g) were anesthetized with Inactin (120 mg/kg i.p.). The trachea was cannulated and the animal was allowed to breathe unaided 100% O₂ provided by an oxygen hose. Polyvinyl cannulas (Norton Plastics, Akron, OH) were inserted into the jugular vein and the femoral artery for the purposes of infusion of saline and test compounds and for monitoring of blood pressure, respectively. Heart rate was monitored by tachograph and body temperature was maintained at 37°C. After a 2-h period of equilibration with saline infusion (0.25 ml/h), peptides [1 µg/kg followed by 143 ng/kg/h; amounts that should provide circulating concentrations of RPPGF of approximately 1 nM, based upon a reported half-life of 5.5 h (Majima et al., 1996)], or saline was administered. One hour later a bolus i.v. injection of LPS at a concentration approximating its LD₅₀ (Armstrong et al., 1986; Etemadi et al., 1987; Wilson et al., 1989; Whalley et al., 1992; 12 mg/kg, dissolved in saline) was administered. Mean blood pressure [LPS only, 115 ± 2.7 mm Hg, n = 39; RPPGF/LPS, 116 ± 5.2 mm Hg, n = 10; bradykinin/LPS, 118 ± 11 mm Hg, n = 5; Pro-Arg-Gly-Phe-Pro (PRGFP)/LPS, 118 ± 4.9 mm Hg, n = 5] and heart rates (LPS only, 409 ± 8.3 bpm, n = 39; RPPGF/LPS, 385 ± 10 bpm, n = 10; bradykinin/LPS, 436 ± 10 bpm, n = 5; PRGFP/LPS, 400 ± 9.3 bpm, n = 5) were not different between the tested groups of animals before the administration of LPS. In the studies in which bradykinin was administered in place of RPPGF, captopril (1 mg/kg i.p.) was administered 15 min before bradykinin, or saline, for the LPS only rats. Animals were monitored until they expired. Time of death was taken as time after administration of LPS when there was no longer a detectable electrocardiogram.

Rat Aortic Contraction Studies. Measurements of aortic contractions in response to phenylephrine (PE) were performed as previously described (Brizzolara-Gourdie and Webb, 1997). Briefly, aortae were removed from ether-anesthetized male Sprague-Dawley rats (250-450 g), cleaned of adventitia, cut into 5-mm segments, and hung under 1.5 g of tension in a 10-ml organ bath. The segments were equilibrated for 1 h, 37°C (95% O₂, 5% CO₂) in Krebs-Henseleit solution (1.5 mM NaH₂PO₄, 16 mM NaHCO₃, 133 mM NaCl, 4.6 mM KCl, 1.2 mM MgSO₄, 2.5 mM CaCl₂, 7.8 mM glucose) followed by stimulation by potassium chloride (50 mM), washout, and a return to baseline conditions. Forty-five minutes later, an initial concentration-response curve to PE was performed on each segment. Integrity of the endothelium was tested by the presence of relaxation in response to acetylcholine (10 µM) subsequent to contraction by PE. The agents were washed out of the organ bath and segments were allowed to reequilibrate for 1 h. RPPGF, or saline, was added 15 min before the addition of LPS. The segments were incubated for 5 h with two changes of buffer and readdition of agents. A concentration-response curve was then performed with PE. Tension produced in response to the addition of PE was corrected for the dry weight of each segment. The Medical University of South Carolina's Institutional Animal Care and Use Committee approved all studies that involved animals.

Chemicals. The agents used in these studies were from the following sources: bradykinin, RPPGF, phenylephrine, acetylcholine, lipopolysaccharide (Salmonella enteritidis, lyophilized powder), and all reagent grade chemicals were from Sigma Chemical Co. (St. Louis, MO). PRGFP (scrambled-RPPGF) was synthesized by the Peptide Synthesis Facility, Medical University of South Carolina (Charleston, SC).

Statistics. In contraction studies, EC₅₀ values, i.e., the concentration of agonist that produced a 50% maximum response, were derived from log-logit transformations of concentration-response curves. Values expressed are the mean ± S.E.M. and were compared using ANOVA with a Fisher's test for significance using Statview 4.5 for Macintosh. Significance was indicated at the 95% level.

	Results

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BK1-5 (RPPGF) increases survival time of LPS-treated anesthetized rats. LPS treatment of anesthetized rats is an accepted screening model for endotoxin shock (Wilson et al., 1989; Whalley et al., 1992; Paya and Stoclet, 1995). RPPGF was evaluated for its ability to alter survival time in this model. Initial responses to LPS, in both RPPGF-treated and saline-treated rats, showed a biphasic change in mean arterial blood pressure (MABP), namely, a rapid, short-lived drop in pressure, followed by a trend toward stabilization with a subsequent drop in pressure (Fig. 1A). Subsequent to this initial response, rats receiving only LPS, i.e., without addition of RPPGF, responded with a more rapid progressive drop in blood pressure. In comparison, rats pretreated with RPPGF and then administered LPS showed a tendency toward stabilization followed by a more gradual drop in pressure. Initial changes in heart rate in the two groups of rats also showed similar trends. In each group, heart rate increased during the early phase after LPS administration and then dropped rapidly in the LPS-only-treated rats, with a more gradual decrease in the RPPGF/LPS-treated rats (Fig. 1B). Rats treated with RPPGF/LPS showed a 56% increase in survival time over those animals receiving saline/LPS treatment (140.3 ± 15.9 min, n = 10 versus 93.2 ± 8.1 min, n = 39, respectively; p < 0.05, Fig. 2).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Time course for changes in MABP (A) and heart rate (B) in anesthetized male Sprague-Dawley rats after treatment with saline and LPS or with RPPGF plus LPS. Values shown are the mean ± S.E.M. from selected time points; LPS, n = 39; RPPGF/LPS, n = 10.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Effect of various peptides on survival time of rats treated with LPS. Anesthetized rats were treated with saline (LPS alone, n = 39) or with one of the following peptides plus LPS: RPPGF, n = 10; PRGFP (scrambled), n = 5; or BK, n = 4. Survival time (min) for each peptide/LPS treatment is shown. Values are the means ± S.E.M. *p < 0.05 versus LPS only; +p < 0.05 versus bradykinin/LPS.

To test for the specificity of the protective effects of RPPGF against LPS, additional studies were performed in which an analog of RPPGF, or the parent hormone BK, was infused into the animals in place of RPPGF. Infusion of the parent hormone BK followed by LPS did not prolong survival time of the rats compared with matched rats receiving LPS alone (Fig. 2), nor did it significantly shorten the survival time. Similarly, treatment with the "scrambled" sequence peptide PRGFP did not significantly alter survival time after treatment with LPS. Infusion of RPPGF, its analog, or BK did not produce significant changes in MABP or heart rate before the administration of LPS (data not shown). Sham-treated rats in these studies, i.e., rats receiving saline only instead of peptides or LPS, survived without significant alterations in MABP or heart rate for greater than 3.5 h (n = 2, data not shown).

RPPGF protects against the deleterious effects of LPS on isolated rat aortic rings. Having seen that RPPGF counteracted the deleterious effects of LPS in vivo, we next investigated whether the same phenomenon would occur in isolated segments of rat aorta. LPS treatment of aortic segments is known to result in decreased responsiveness of the aortae to various contractile agents (Zelenkov et al., 1993; Kiff et al., 1994; Takakura et al., 1994). Isolated segments of aorta were exposed to LPS (30 µg/ml), RPPGF (1 nM) plus LPS, or saline (control) for 5 h, as described above. At the end of the incubation period, contractile responsiveness of the segments to PE, an -adrenergic agonist, was measured. Treatment with LPS alone resulted in significant deterioration of the contractile response to PE (Fig. 3). However, pretreatment of segments with RPPGF for 15 min followed by LPS protected contractile integrity, as assessed by determining the extent of contraction to increasing concentrations of PE. The PE concentration-response curve of segments pretreated with RPPGF before exposure to LPS was significantly different from that seen with segments exposed to LPS alone. Segments treated with RPPGF alone responded identically to control segments (data not shown). A similar preservation of function was seen when the contractile agent was U46619, a stable mimetic of the vasopressor thromboxane A₂, rather than phenylephrine (data not shown).

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Effects of RPPGF on the LPS-induced deterioration of contraction of rat aortic segments in response to stimulation by phenylephrine. Isolated segments of endothelial-intact rat aorta were treated as described with saline alone (control, n = 9), saline and LPS (30 µg/ml, n = 10), or RPPGF (1 nM) and LPS (RPPGF/LPS, n = 10). Force of contraction (g) was measured in response to increasing concentrations of phenylephrine and is expressed as a function of the dried weight of aortic segments (g/mg). Values shown are the mean ± S.E.M. from the indicated number of segments. Average curves were compared using ANOVA with Fisher's protected least-significant difference post hoc test *p < 0.05 versus LPS alone; +p < 0.05 versus RPPGF/LPS.

Comparison of EC₅₀ values indicated that treatment of the segments with LPS significantly reduced the affinity of PE for its receptor. Pretreatment of the segments with RPPGF did not significantly effect the decrease in the phenylephrine EC₅₀ value produced by LPS. Nor did RPPGF pretreatment yield values that were different than control segments [control, 235.2 ± 33 nM (n = 16); LPS alone, 405.3 ± 70 nM* (n = 12); RPPGF/LPS, 361 ± 69 nM (n = 4); *p < 0.05 versus control].

To assess the contribution of an intact endothelium in the protective actions of RPPGF, additional contraction studies were performed on aortae lacking a functional endothelial layer. The intima of aortic segments was rubbed with a cotton swab to remove the endothelial layer. The lack of a relaxation response to addition of acetylcholine (10 µM) of PE-contracted segments was confirmation of successful removal of the endothelial lining. Segments of aorta were pretreated with RPPGF (1 nM) for 15 min, followed by exposure to LPS (30 µg/ml) for 4 h, and then stimulated by PE (1 µM). Removal of a functional endothelial layer, combined with a shorter incubation time (4 versus 5 h), resulted in contraction in response to 1 µM PE of control segments that was significantly greater than in those vessels possessing a functional endothelial lining (0.63 ± 0.2 g/mg, n = 4 versus 0.31 ± 0.04 g/mg, n = 14, respectively; p < 0.05). In these studies, LPS treatment produced a significant deterioration in the contractile response to PE, reducing the contractile response to 29.3 ± 8% (0.17 ± 0.06 g/mg, n = 4) of control segments. Additionally, the contractions seen in response to PE (1 µM) after LPS treatment were significantly greater subsequent to the removal of the endothelial lining compared with those seen with the lining intact (0.17 ± 0.06 g/mg, n = 4 versus 0.07 ± 0.01 g/mg, n = 13, respectively; p < 0.05). As in the previous studies, pretreatment of the de-endothelialized segments with RPPGF significantly protected the segments against the deleterious effects of LPS, returning the contractile response to 88 ± 9% of control segments (0.57 ± 0.23 g/mg, n = 4; p < 0.05). In all treatments, those segments lacking a functional endothelial lining demonstrated PE-induced contractions that were significantly greater than did those with a functional endothelial lining.

	Discussion

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During the septic response, exposure of the internal environment to exogenous bacterial membrane proteins produces a cascade of physiological and biochemical responses. Among the plethora of mediators released is the potent vasodilatory hormone bradykinin. The short half-life of bradykinin results in the generation of several metabolites, including the stable end product RPPGF. Several studies have documented the role of bradykinin in the early events of the septic response (Wilson et al., 1989; Whalley et al., 1992; Otterbein et al., 1993; Paya and Stoclet, 1995) without examining the potential effects of the metabolic products of bradykinin. Administration of bradykinin B2 receptor antagonists have demonstrated beneficial effects, i.e., increased survival rates and improved hemodynamic parameters, suggesting a pathological role for bradykinin in this condition (Wilson et al., 1989; Whalley et al., 1992; Otterbein et al., 1993; Paya and Stoclet, 1995).

The findings presented in the current study provide new and exciting evidence for a previously unidentified activity of a fragment of bradykinin. RPPGF (BK1-5), when administered to anesthetized rats, prolonged survival time of the rats after exposure to the toxic agent lipopolysaccharide. Administration of the scrambled peptide PRGFP did not produce an increase in survival time, as neither did the parent hormone bradykinin. Considering that the parent hormone bradykinin and the scrambled amino acid peptide PRGFP did not have this beneficial effect in this model system points to a specific ability of RPPGF to counteract the effects of LPS.

Currently, the levels of endogenous RPPGF found in rats or humans have not been reported. However, several studies using either an enzyme-linked immunosorbent assay or mass spectrophotometric techniques have quantified levels of RPPGF under various experimental and pathological settings. In these studies, the levels of generated RPPGF have ranged from 350 nM in glass-activated rat plasma (Majima et al., 1996) to 3 µM in human blood from patients receiving infusions of 1 mg of bradykinin (Murphy et al., 2000). Because of its short half-life, circulating concentrations of bradykinin are also difficult to quantitate. However, it is thought that under basal conditions levels of bradykinin may reach 100 pM (Miller and Margolius, 1997). For these reasons, we chose to use concentrations of RPPGF that might exist under conditions in which bradykinin is released. Using current technology, it is uncertain what the levels of RPPGF may be under conditions such as septicemia. Therefore, whether one can conclude that RPPGF may act as an endogenous protective compound in this circumstance is problematic.

Having demonstrated the protective effects of RPPGF in whole animals, we next chose to probe for a potential mechanism for these effects by examining the ability of RPPGF to alter the deleterious effects of LPS on the in vitro isolated blood vessel model of vascular contractility. In this system, LPS has been demonstrated to reduce the ability of vascular smooth muscle to contract in response to vasoconstrictive agents (Zelenkov et al., 1993; Takakura et al., 1994; Loegering et al., 1995; Villamor et al., 1995). Treating isolated segments of rat aorta with RPPGF "protected" the aorta against the deleterious effects of LPS on PE-induced contraction. Treatment of the segments with this peptide before addition of LPS resulted in PE-induced contractile responses that were significantly enhanced compared with those seen in segments exposed to LPS alone. However, the deleterious effects of LPS were not completely overcome by the concentration of RPPGF used in this study, in that the concentration-response curve for the RPPGF/LPS segments was significantly different than that for the control segments. This beneficial effect of the peptide occurred without significantly reversing the loss of affinity to PE that was caused by LPS, yielding EC₅₀ values for RPPGF/LPS-treated segments that were not significantly different from LPS alone, nor from control values. In addition, the protection was not dependent upon a functional endothelial lining. This suggests that this protective action of the peptide may reside within the contractile machinery of the smooth muscle itself rather than at the receptor for phenylephrine or with an ability of the peptide to interfere with release of vasodilatory agents by the endothelium. Further support for this potential mechanism is the fact that in all cases in which the functional endothelium was removed, the contractile response to PE was enhanced and RPPGF pretreatment still produced a reversal of the effects of LPS.

It is also unlikely that involvement of either the bradykinin B2 or even the LPS-inducible B1 receptor can be invoked. RPPGF, at concentrations used in the present studies, did not significantly displace the binding of [³H]bradykinin from the bradykinin B2 receptors in cultured vascular smooth muscle cells (data not shown). Additionally, induction of vascular bradykinin B1 receptors, which has been shown to occur within 18 to 24 h in LPS-treated rats (Nicolau et al., 1996), or in cultured rabbit aorta vascular smooth muscle cells treated with epidermal growth factor (Schneck et al., 1994), is unlikely to occur within the time frame of these studies.

Recently, Hasan et al. (1996, 1999) have demonstrated that RPPGF has the ability to inhibit thrombin-induced platelet activation and inhibit electrolytic-induced coronary thrombosis in dogs. However, it is unlikely that these effects of RPPGF occur by the same mechanism for LPS protection because the concentrations required to see the antithrombin activities of RPPGF are approximately 1000-fold greater than that used in the present study (~1 µM versus 1 nM, respectively). Chen and Rosazzo (1996) have shown that this peptide, RPPGF, as well as the parent hormone BK, have the potential to act as a substrate for a novel noncalmodulin-dependent constitutive nNOS-II. This nNOS-II used both the N- and C-terminal arginine of bradykinin and the N-terminal arginine of RPPGF as a source for the generation of nitric oxide. However, this potential role for RPPGF, i.e., to act as a substrate for a form of nitric-oxide synthase found in the central nervous system, would not be a likely mechanism of action for the activities seen in our present study because the effects we see for RPPGF are found not only in the whole animal model but also in the isolated tissue studies. Additionally, if RPPGF were a source for generation of nitric oxide, its presence would likely augment, rather than prevent, the LPS-induced reduction in contractile response to PE found in the isolated blood vessel studies. Further studies will address the potential mechanism for this protective action, including studies aimed at the role of RPPGF in the LPS-induced release of cytokines.

In the present study, we have shown that a peptide fragment of an endogenous hormone demonstrates activities that, in the case of LPS-induced cardiovascular collapse, act in a manner different than the parent hormone. Recently, several studies have demonstrated similar activities in other vasoactive hormones. These vasoactive hormones are generally synthesized as inactive prohormones, or as components of larger substrate molecules that are acted upon by specific proteases, and released from their storage sites in response to various changes in the internal milieu. The released hormone affects its target tissues to sustain a biochemical or physiological function. Formerly ignored fragments of peptide hormones appear to possess some striking biological activities (Pavoine et al., 1991; Ido et al., 1997). In general, their activities have been found to be similar to, or to potentiate those of, the parent hormone. For example, a fragment of endothelin-1 (ET 16-21) produces arterial constriction similar to that of the parent hormone (Maggi et al., 1989; Douglas and Hiley, 1991). Fragments of vasopressin, like the parent hormone, produced a paradoxical centrally mediated hypotension (Brattstrom et al., 1989). Such results suggest that hormonal catabolism can result in products that sustain the homeostatic response produced by the parent. In contrast, a few recent studies have shown that some fragments of peptide hormones possess properties opposite to that of the parent. Metabolism of glucagon produces a fragment called miniglucagon, which suppresses insulin secretion, an action opposite that of glucagon (Pavoine et al., 1991). Angiotensin 1-7, a metabolite of the octapeptide vasoconstrictor angiotensin II, produces vasodilation in a number of animal models (Ferrario et al., 1991; Porsti et al., 1994; Brosnihan et al., 1995), suggesting that peptide hormone fragments also serve to modulate or dampen the actions of the parent.

Our recent discovery of a very potent pharmacological activity in an "inert" fragment of BK, which appears opposite to the generally considered, deleterious effects of kinins in shock-like states, might allow for the development of new therapeutic strategies based upon stable mimetics of this metabolite as drugs. The spectrum of activity of these entities also needs to be explored because the parent kinins have been long known for their extremely broad array of cellular and tissue responsiveness in sites as diverse as afferent nerves, glia, osteoclasts, and renal tubular epithelial cells.