The aim of this study was to determine the role of calcitonin gene-related peptide (CGRP) in the postischemic anti-inflammatory effects of antecedent ethanol ingestion. Ethanol was administered to wild-type C57BL/6 mice on day 1 as a bolus by gavage at a dose that produces a peak plasma ethanol of 45 mg/dl 30 min after administration. Twenty-four hours later (day 2), the superior mesenteric artery was occluded for 45 min followed by 70 min of reperfusion (I/R). Intravital fluorescence microscopy was used to quantify the numbers of rolling (LR) and adherent (LA) leukocytes labeled with carboxyfluorescein diacetate succinimidyl ester in postcapillary venules of the small intestine. I/R increased LR and LA, effects that were prevented by antecedent ethanol. The postischemic anti-inflammatory effects of ethanol consumption were abolished by administration of a specific CGRP receptor antagonist [CGRP-(8–37)] or after sensory nerve neurotransmitter depletion using capsaicin administered 4 days before ethanol ingestion, which initially induces rapid release of CGRP from sensory nerves, thereby depleting stored neuropeptide. Administration of exogenous CGRP or induction of endogenous CGRP release by treatment with capsaicin 24 h before I/R mimicked the postischemic anti-inflammatory effects of antecedent ethanol ingestion. Preconditioning with capsaicin 24 h before I/R was prevented by coincident treatment with CGRP-(8–37), while exogenous CGRP induced an anti-inflammatory phenotype in mice depleted of CGRP by capsaicin administration 4 days earlier. Our results indicate that the effect of antecedent ethanol ingestion to prevent postischemic LR and LA is initiated by a CGRP-dependent mechanism.
- leukocyte rolling
- leukocyte adhesion
the results of a large number of epidemiological studies demonstrate that low to moderate consumption of alcoholic beverages reduces the risk for coronary artery disease and decreases mortality rates due to myocardial infarction (11). While the protective effects induced by ethanol consumption have been attributed to its effects on plasma lipids, platelet function, and fibrinolytic activity (14), more recent work indicates that antecedent ethanol ingestion induces the development of an anti-inflammatory phenotype in postcapillary venules such that these vessels fail to support leukocyte adhesion and emigration in tissues exposed to ischemia-reperfusion (I/R) (18, 41, 42). The molecular basis for the powerful antiadhesive effects induced by ethanol ingestion appears to involve prevention of adhesion molecule expression by the endothelium (7, 8, 33). Given the critical importance of infiltrating leukocytes in the pathogenesis of atherosclerosis and ischemia-reperfusion (I/R) injury (12, 20), these studies provide important insight regarding the mechanisms whereby ingestion of alcoholic beverages reduces the incidence and severity of cardiovascular disease.
The temporal expression of the protected or preconditioned state that develops in postcapillary venules in response to antecedent ethanol ingestion is biphasic (41). The first or acute phase is short-lived, first becoming apparent within 1 h after ingestion, with peak anti-inflammatory effects occurring 2 to 3 h after intake and then disappearing 4 h after consumption of ethanol (41). The second or delayed phase of ischemic tolerance becomes evident 24 h after ethanol ingestion and is notable for its magnitude of protection (41). In view of the fact that the anti-inflammatory effects induced by the late phase are much more powerful than those induced in the early phase, most attention has focused on elucidating the mechanistic underpinnings for delayed ethanol preconditioning. This work supports the concept that development of the anti-inflammatory phenotype expressed by postcapillary venules 24 h after the ingestion of ethanol is triggered by adenosine A2-receptor-dependent nitric oxide (NO) production that is derived from endothelial NO synthase (eNOS) (7, 41).
Although the downstream signaling events that are activated by ethanol-induced eNOS activation are unknown, it is well-established that both ethanol and NO can activate capsaicin-sensitive neurons to release calcitonin gene-related peptide (CGRP) (3, 22, 38). Moreover, this neuropeptide is contained within neurons of both the myenteric and submucosal plexi and is also found in perivascular peptidergic nerves supplying the intestinal and other vasculatures (9, 19). In addition, administration of exogenous CGRP has been shown to be protective in I/R (22, 23, 28). Taken together, these observations led us to postulate a role for CGRP as a trigger for ethanol preconditioning. To address this hypothesis, we observed postischemic leukocyte rolling and adhesion in postcapillary venules in mice that ingested ethanol coincident with administration of a specific CGRP receptor antagonist and in animals depleted of CGRP by treatment with capsaicin 4 days before ethanol consumption. We also exploited the fact that capsaicin initially induces rapid release of stored CGRP (which ultimately results in depletion of the neuropeptide), thereby allowing its use as a potential preconditioning stimulus when administered 24 h before I/R. Thus we hypothesized that release of endogenous CGRP as a result of capsaicin treatment 24 h before I/R would prevent postischemic leukocyte rolling and adhesion. Finally, we evaluated the ability of exogenously applied CGRP to induce the development of an anti-inflammatory phenotype to further substantiate a role for this neuropeptide as a preconditioning stimulus.
MATERIALS AND METHODS
Wild-type male C57BL/6 mice (6–7 wk of age) were obtained from the Jackson Laboratories (Bar Harbor, ME). All mice were maintained on standard mouse chow and used at 8–10 wk of age. The experimental procedures described herein were performed according to the criteria outlined in the National Institutes of Health guidelines and were approved by the Louisiana State University Health Sciences Center-Shreveport and the University of Missouri Institutional Animal Care and Use Committees.
Ethanol was instilled into the stomachs of conscious mice by gavage 24 h before the experiments. The volume of 95% ethanol to be instilled (in μl) was calculated as follows: [body wt (in g) × 0.6] + 0.3. This volume of ethanol was mixed in 0.3 ml of sterile distilled water just before administration. We previously demonstrated that this dosing protocol produced a peak plasma ethanol concentration of ∼45 mg/dl 30 min after administration, a level equivalent to that achieved in humans consuming 1–2 alcoholic beverages (41). Mice in the sham control [no I/R, no ethanol preconditioning (no EtOH-PC)] and I/R alone (no EtOH-PC) groups received sterile distilled water without ethanol by gavage. In addition, to assess the effects of the pharmacological agents described below on the ability of ethanol to initiate entrance into an anti-inflammatory phenotype, we used an intraperitoneal injection/lavage procedure described earlier (41, 42). Briefly, vehicle (sterile saline) or the pharmacological agent of interest was administered by intraperitoneal injection 10 min before ethanol gavage. Fifty-five minutes later, the animals were lightly anesthetized by administration of a mixture of ketamine (135 mg/kg body wt im) and xylazine (6.8 mg/kg body wt im). Fifteen minutes later, the peritoneal cavity was slowly flushed three times with 0.5 ml warmed (37°C) saline via a syringe attached to a 16-gauge needle that was introduced through the abdominal wall at the midline. Before each flush was aspirated, the abdomen was gently massaged to ensure adequate mixing, with care taken to avoid injury to the intestine by the needle. On completion of the lavage cycles, the needle was removed and the incision closed with 6-0 nylon sutures. After recovery from anesthesia (2 h after the flush), the animals were allowed free access to standard mouse chow and water until surgical procedures on day 2. We previously demonstrated that this procedure does not influence baseline or postischemic leukocyte/endothelial cell adhesive interactions (41, 42).
Surgical Procedures and Induction of I/R
minutes 30–40 and 60–70 of reperfusion.
Intravital Fluorescence Microscopy
The mice were positioned on a 20 × 30-cm Plexiglas board in a manner that allowed a selected section of the jejunum to be exteriorized and placed carefully and gently over a glass slide covering a 4 × 3-cm hole centered in the Plexiglas. The exposed small intestine was superfused with warmed (37°C) bicarbonate-buffered saline (BBS, pH 7.4) at 1.5 ml/min using a peristaltic pump (model M312; Gilson). The exteriorized region of the small bowel was covered with BBS-soaked gauze and cellophane to minimize the tissue dehydration, temperature changes, and the influence of respiratory movements. The superfusate was maintained at 37 ± 0.5°C by pumping the solution through a heat exchanger warmed by a constant-temperature circulator (model 1130; VWR). Body temperature of the mouse was maintained between 36.5 and 37.5°C by use of a thermostatically controlled heat lamp. The board was mounted on the stage of an inverted microscope (Diaphot TMD-EF; Nikon), and the intestinal microcirculation was observed through a ×20 objective lens. Fluorescence images of the microcirculation (excitation wavelength 420–490 nm; emission wavelength 520 nm) were detected with a charge-coupled device (CCD) camera (XC-77; Hamamatsu Photonics), a CCD camera control unit (C2400; Hamamatsu Photonics), and an intensifier head (M4314; Hamamatsu Photonics) attached to the camera. Microfluorographs were projected on a television monitor (PVM-1953MD; Sony) and recorded on videotape using a videocassette recorder (HR-S4600U; JVC) for off-line quantification of measured variables during playback of the videotaped image. A video time-date generator (WJ810; Panasonic) displayed the stopwatch function onto the monitor.
The intravital microscopic measurements described below were obtained over minutes 30–40 and 60–70 of reperfusion or at equivalent time points in the control groups. The intestinal segment was scanned from the oral to aboral section, and 10 single, unbranched venules (20–50 μm diameter, 100 μm length) were observed, each for at least 30 s. Leukocyte-endothelial cell interactions (the numbers of rolling and firmly adherent leukocytes) were quantified in each of the 10 venules, followed by calculation of the mean value, which was used in the statistical analysis of the data. Circulating leukocytes were considered to be firmly adherent if they did not move or detach from the venular wall for a period equal to or greater than 30 s. Rolling cells are defined as cells crossing an imaginary line in the microvessel at a velocity that is significantly lower than centerline velocity; their numbers were expressed as rolling cells per minute. The numbers of rolling or adherent leukocytes were normalized by expressing each as the number of cells per square millimeter vessel area.
Figure 1 illustrates the general design of the experimental protocols for the study. Six animals were used in each group described below. Drug doses were selected based on reports in the literature (15, 23, 27–29, 35, 44, 45).
Group 1: Sham.
As a time control for the effects of experimental duration, mice in this group received an intraperitoneal injection of 0.5 ml saline, which was used as a vehicle for CGRP-(8–37) or exogenous CGRP in groups outlined below, 10 min before administration of sterile distilled water alone (ethanol vehicle) by gavage, followed by the lavage procedure 60 min later on day 1. Twenty-four hours later, the superior mesenteric artery was exposed but not subjected to occlusion, with leukocyte/endothelial cell adhesive interactions quantified at time points comparable to those described for mice subjected to 45 min of intestinal ischemia followed by 70 min reperfusion (group 2, below).
Group 2: I/R alone.
Mice in this group were treated as described for group 1 above except that I/R was induced 24 h after the gavage-lavage procedure on day 1. Leukocyte rolling and adhesion were quantified during minutes 30–40 and 60–70 of reperfusion.
Group 3: EtOH-PC + I/R.
To confirm the effects of EtOH-PC on I/R-induced leukocyte rolling and adhesion that we reported in earlier studies (41, 42), mice in this group were treated as described for group 2, except that they received ethanol by gavage 24 h before induction of I/R.
Group 4: CGRP-(8–37) + EtOH-PC + I/R.
To determine whether CGRP was involved as an initiator of the protective effects of ethanol to prevent postischemic leukocyte rolling and adhesion on exposure to I/R 24 h later, mice in this group were treated with CGRP-(8–37) (Sigma; St. Louis, MO) (100 nM, 0.5 ml, intraperitoneal injection), a specific CGRP receptor antagonist, 10 min before ethanol gavage. Peritoneal lavage was performed 60 min after ethanol to reduce peritoneal CGRP-(8–37) concentrations to minimal levels. Twenty-four hours later, the intestine was exposed to I/R, and leukocyte rolling and adhesion were quantified as described above.
Group 5: CGRP + I/R.
The aim of the studies outlined for this group was to determine whether administration of exogenous CGRP (Sigma; 5 nM, 0.5 ml, intraperitoneal injection), in lieu of ethanol, would induce anti-inflammatory effects similar to that produced by antecedent ethanol ingestion. Peritoneal lavage was performed 60 min after CGRP to reduce peritoneal CGRP concentrations to minimal levels. Twenty-four hours later, the intestine was exposed to I/R, and leukocyte rolling and adhesion were quantified as described above.
Group 6: Capsaicin (24 h) + I/R.
To determine whether capsaicin, a neurotoxin that induces the release of CGRP from sensory nerves, would trigger the development of a preconditioned state similar to that induced by ethanol or exogenous CGRP administration, mice in this group were treated as described for group 5, except that they were administered a single dose of capsaicin (Sigma) (30 mg/kg, dissolved in a mixture of 10% DMSO, 10% Tween 80, and 80% saline) by intramuscular injection while under light anesthesia (using the ketamine-xylazine mixture described above) 24 h before I/R.
Group 7: CGRP-(8–37) + capsaicin + I/R.
To verify that the preconditioning effect of capsaicin to prevent postischemic leukocyte adhesion 24 h later was due to CGRP, an additional group of six mice were treated with CGRP-(8–37) by intraperitoneal injection coincident with capsaicin administration, followed by I/R 24 h later.
Group 8: Capsaicin (4 days) + EtOH-PC + I/R.
Mice in this group were treated with a single dose of capsaicin (30 mg/kg sc) 4 days before ethanol administration, a temporal treatment regimen that induces rapid CGRP release from sensory nerves, resulting in a depletion of stored neuropeptide. It is important to note that the preconditioning effect caused by the initial release of CGRP wanes by this time. Animals were subjected to intestinal I/R 24 h after ethanol ingestion.
Group 9: Capsaicin (4 days) + CGRP + I/R.
To determine whether exogenous CGRP would remain effective as a preconditioning stimulus in mice treated with capsaicin 4 days before preconditioning, mice in this group were treated as described for group 7, except that exogenous CGRP (using the preconditioning protocol described for group 5 above) was administered in lieu of ethanol 24 h before intestinal I/R.
The data were analyzed with standard statistical analysis, i.e., one-way ANOVA with Scheffé’s (post hoc) test for multiple comparisons. All values are expressed as means ± SE. Statistical significance was defined at P < 0.05.
Figure 2 illustrates the average numbers of rolling and adherent leukocytes in postcapillary venules of small intestine exposed to I/R 24 h after gastric instillation of distilled water (I/R alone, group 2) or ethanol (EtOH-PC + I/R, group 3) alone or coincident with intraperitoneal injection of the specific CGRP receptor antagonist CGRP-(8–37) [CGRP-(8–37) + EtOH-PC + I/R, group 5] and in mice preconditioned with exogenous CGRP (CGRP + I/R, group 4), in lieu of ethanol. These values were compared with those obtained at corresponding time points in sham control group (no EtOH, no I/R, group 1). I/R induced marked increases in the numbers of rolling and firmly adherent leukocytes after 30 and 60 min of reperfusion, effects that were largely abolished by antecedent ethanol ingestion. The antiadhesive effects of EtOH-PC that become apparent during I/R 24 h later (day 2) were abolished in mice pretreated with CGRP-(8–37) 10 min before ethanol gavage on day 1. On the other hand, preconditioning with exogenous CGRP 24 h before I/R was as effective as antecedent ethanol in preventing postischemic leukocyte endothelial cell adhesive interactions.
As shown in Fig. 3, capsaicin administration (capsaicin + I/R, group 6) in lieu of ethanol 24 h before I/R produced a preconditioned phenotype similar to that initiated by antecedent ethanol or exogenous CGRP administration (Fig. 2), preventing postischemic leukocyte rolling and adhesion. Because the antiadhesive effects of capsaicin treatment by this protocol were prevented by coincident administration of the CGRP receptor antagonist CGRP-(8–37) [CGRP-(8–37) + capsaicin + I/R, group 7], our results suggest that release of endogenous CGRP accounts for the beneficial actions of capsaicin (Fig. 3). On the other hand, administration of this sensory neurotoxin 4 days before ethanol ingestion (capsaicin + EtOH-PC + I/R, group 8), as a means to deplete endogenous CGRP, abrogated the antiadhesive effects of antecedent ethanol ingestion in intestines subsequently exposed to I/R. However, provision of exogenous CGRP by intraperitoneal injection 24 h before I/R remained effective in preventing postischemic leukocyte rolling and adhesion in mice subjected to capsaicin 4 days before CGRP administration [group 9, capsaicin (4 days) + CGRP + I/R]. The latter observation suggests that capsaicin treatment does not influence signaling events that are activated downstream from CGRP receptor activation to induce the development of a preconditioned state.
The results of the present study provide the first evidence that the effect of ethanol ingestion to induce entrance into an anti-inflammatory phenotype, such that postcapillary venules fail to support leukocyte rolling and adhesion on exposure to I/R 24 h later, is triggered by release of CGRP. Three lines of evidence support this conclusion. First, we demonstrated that the antiadhesive effects of late-phase EtOH-PC were abolished by pretreatment with a selective CGRP receptor antagonist [CGRP-(8–37)] 10 min before ethanol ingestion (Fig. 2). In addition, treatment with capsaicin 4 days before ethanol ingestion, an intervention protocol that has been shown to deplete endogenous CGRP, was as effective as treatment with CGRP-(8–37) in abrogating the postischemic antiadhesive effects of late phase EtOH-PC (Fig. 3). Thus two mechanistically distinct interventions directed at interfering with the actions of CGRP prevented entrance into a preconditioned state by ethanol. A third line of evidence supporting our conclusions is drawn from experiments demonstrating that exogenous administration of CGRP 24 h before I/R mimicked the protective effects of antecedent ethanol ingestion to prevent postischemic leukocyte rolling and adhesion (Fig. 2). Capsaicin administration 24 h before I/R, a treatment protocol that induces the acute release of endogenous CGRP from sensory neurons, produced similar effects (Fig. 3). Because the antiadhesive effects of capsaicin administered by this protocol were abrogated by coincident administration of CGRP-(8–37) (Fig. 3), it appears that release of endogenous CGRP induces entrance into a protected phenotype in response to capsaicin administered 24 h before I/R.
Our interest in CGRP as an initiator of ethanol preconditioning was initially derived from the observation that ethanol activates capsaicin-sensitive neurons to release CGRP, and to a lesser extent, substance P and 5-hydroxytryptamine (22, 38). Of these, CGRP was of the most interest as a trigger for ethanol preconditioning because exogenous administration of this neuropeptide has been shown to be protective in I/R models (4, 22, 23). CGRP is a 37-amino acid neuropeptide that exists in two isoforms, CGRPα and CGRPβ, which differ by only one amino acid in rodents and three amino acids in humans (4). CGRP interacts with specific high-affinity receptors that require a single transmembrane domain protein, termed receptor activity modifying protein-1 (RAMP-1), to function as a CGRP receptor.
In addition to producing intestinal vasodilation, CGRP also induces leukocyte adhesion via an effect on leukocytes, not the endothelium (36, 46). This latter observation may seem counterintuitive to our conclusions that ethanol-induced CGRP release induces development of an anti-inflammatory phenotype. Indeed, the fact that acute CGRP exposure induces leukocyte/endothelial cell adhesive interactions suggests that if CGRP plays a role in producing an anti-inflammatory phenotype that becomes manifest 24 h after ethanol ingestion, it does so by acting as a trigger, rather than as an end effector, of this response. This notion is supported by our observation that coincident treatment with a CGRP receptor antagonist during the period of ethanol exposure prevents the antiadhesive effects of antecedent ethanol that normally occur on exposure of the intestine to I/R 24 h later (Fig. 2). In this regard, it is particularly interesting to note that nitric oxide, which we have implicated as an important trigger for the development of ethanol preconditioning (41), also promotes CGRP release (3).
In addition to its role in inaugurating entrance into the anti-inflammatory phenotype induced by antecedent ethanol ingestion, CGRP release has also been implicated as a trigger for the infarct-sparing effects of both early- and late-phase ischemic preconditioning (IPC) (23, 27, 28, 37, 40) and in the development of a protected phenotype in response to antecedent treatment with nitroglycerin, monophosphoryl lipid A, or heat stress 24 h before index I/R (15, 35, 45). A role for CGRP in the beneficial actions of IPC has also been noted in gastrointestinal organs (27), as well as in remote protection of the myocardium against I/R injury induced by subjecting the small intestine to brief periods of ischemia 24 h before coronary occlusion (37). Our results and the aforementioned observations indicate that CGRP plays an essential role in the development of preconditioned states induced by a wide variety of preconditioning stimuli, including antecedent local and remote ischemia, heat shock, or pharmacological preconditioning with ethanol, NO donors, and monophosphoryl lipid A.
Our data indicate that provision of exogenous CGRP or induction of endogenous CGRP release (by antecedent capsaicin or ethanol administration) 24 h before I/R prevents postischemic leukocyte rolling and adhesion. However, the downstream mechanisms whereby CGRP induces the development of this anti-inflammatory phenotype in postcapillary venules are unclear. Because the potent vasodilatory effects of CGRP occur by an adenylyl cyclase/cAMP/protein kinase A (PKA)-dependent mechanism that also involves activation of ATP-sensitive potassium (KATP) channels (5, 10, 39), it is tempting to speculate that these events may serve as downstream signals to initiate entrance into the preconditioned anti-inflammatory state induced by antecedent ethanol ingestion or CGRP exposure. However, others have demonstrated that activation of PKA by cAMP-elevating agents results in CGRP release (32), suggesting the adenylyl cyclase/cAMP/PKA-dependent signaling may occur upstream from CGRP release. It has also been reported that CGRP increases the activity of protein kinase C (PKC) (2, 31) and induces the expression of heme oxygenase-1 (HO-1) (29, 33), signaling events that we and others have implicated as obligatory downstream effectors in early- and late-phase ethanol and ischemic preconditioning (1, 6, 8, 14, 18, 24, 26, 29, 31, 41, 42). Additionally, interventions that raise intracellular cAMP levels induce HO-1 via a PKA-dependent pathway (16, 30). Delayed preconditioning induced by NO donors, short bouts of ischemia, and chronic ethanol exposure is also abolished by PKC inhibition and KATP channel inactivation (18, 21, 24, 26). Taken together, these data suggest that ethanol-induced, CGRP-dependent preconditioning may occur by an adenylyl cyclase/cAMP/PKA-dependent triggering mechanism that involves activation of PKC and KATP channels as obligatory downstream signaling elements and is mediated by expression of HO-1 during I/R. It is clear that much additional work will be required to determine the role of these signaling elements in the transformation of postcapillary venular endothelium to an anti-inflammatory phenotype by antecedent ethanol ingestion.
In summary, the results of the present study provide the first evidence that antecedent ethanol ingestion induces the development of an anti-inflammatory phenotype in postcapillary venules, such that these vessels fail to support leukocyte rolling and adhesion on exposure of the small intestine to I/R 24 h after ingestion by a CGRP-dependent mechanism. In addition, exposing the small bowel to CGRP by exogenous administration or to endogenous CGRP released in response to capsaicin treatment was as effective as ethanol in preventing postischemic leukocyte-endothelial cell adhesive interactions when I/R was induced 24 h later.
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-43785.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2006 by the American Physiological Society