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. 2005 Apr;69(2):116–122.

Role of endothelium and nitric oxide in modulating in vitro responses of colonic arterial and venous rings to vasodilatory neuropeptides in horses

Rustin M Moore 1,✉, Steven A Sedrish 1, Earnestine P Holmes 1, Catherine E Koch 1, Changaram S Venugopal 1
PMCID: PMC1142178  PMID: 15971675

Abstract

The objective of this study was to determine and compare the in vitro responses of equine large colon arterial and venous rings to vasodilatory neuropeptides; calcitonin gene-related peptide (CGRP); substance P (SP); vasoactive intestinal polypeptide (VIP); and acetylcholine (ACh), a standard nonpeptide endothelium-dependent vasodilator. Responses of vessel rings to graded concentrations (10−11 M to 10−5 M) of each drug were determined in endothelium-intact, denuded, and Nω-nitro-L-arginine methyl ester (L-NAME, 10−5 M)-treated rings that were pre-contracted with norepinephrine. Percentage maximal relaxation (PMR), defined as the % decrease from the contracted state, was determined. Because all rings did not relax at least 50%, EC50 values could not be consistently calculated. Arterial rings with intact endothelium were more sensitive to CGRP, compared with VIP and SP, and venous rings of all conditions were more sensitive to VIP than CGRP or SP. Overall, arteries had a greater PMR for ACh compared with SP and VIP. Intact and L-NAME treated arteries had a greater PMR than denuded arteries; there were no differences in PMR of intact and L-NAME treated arteries. Veins had a greater PMR for VIP than CGRP, SP, or ACh. Calcitonin gene-related peptide caused greater relaxation in intact arteries, whereas VIP causes greater relaxation in veins. Arterial relaxation was dependent upon the presence of intact endothelium. The response of veins to VIP among the conditions tested was not different, suggesting VIP has direct actions on venous smooth muscle. These neuropeptides modulate vasomotor tone via vasorelaxation in colonic arteries and veins.

Introduction

Restoration of blood flow after intestinal ischemia frequently results in reactive hyperemia, an overshoot in blood flow above that required to repay the oxygen debt incurred during ischemia (1). Reactive hyperemia is profound in the large colon of horses after low-flow arterial ischemia (2,3). This hyperemia is equally distributed to the mucosal and seromuscular layers and is temporally related to the mucosal necrosis that develops during reperfusion (3,4). Colonic arterial resistance decreases during low flow arterial ischemia and remains decreased throughout reperfusion, suggesting the hyperemia observed during reperfusion occurs due to vascular smooth muscle relaxation (2). According to the metabolic theory of blood flow regulation, endogenous vasoactive substances accumulate during ischemia leading to vascular smooth muscle relaxation and a resultant hyperemia upon restoration of blood flow (1). This hyperemia has been shown to occur with low-flow arterial occlusion; however, colonic blood flow remains significantly decreased after correction of experimentally induced complete arteriovenous occlusion in ponies (5). Therefore, the type of ischemia, among other factors, likely plays a role in the blood flow response that occurs upon correction of the ischemic insult.

Endothelium, a metabolically active layer of cells lining blood vessels, has specialized functions in regulating vascular permeability, vasomotor tone, inflammatory cell adhesion, coagulation, and platelet aggregation (6). Intact functional endothelium is essential for acetylcholine (ACh) and other vasodilator substances to cause vascular smooth muscle relaxation through the release of nitric oxide (NO) (7,8). Endothelial damage can occur in the colonic vasculature in horses subsequent to large-colon volvulus due to ischemia (9). Endothelial damage may contribute to the colonic mucosal injury occurring secondary to volvulus, causing complete arteriovenous occlusion and subsequent alterations of vasomotor tone and reduced blood flow. Experimentally, the effect of endothelial damage on vasomotor tone can be partially mimicked by blocking NO synthesis in vivo or in vitro, using inhibitors of NO synthase (NOS), such as Nω-nitro-L-arginine methyl ester (L-NAME) or in vitro by mechanical removal of endothelium (10,11). The endothelial isoform of NOS is more abundant in the arterial circulation, compared with the venous circulation (8), and thus, endothelium-dependent relaxation should be greater in arteries than veins.

Calcitonin gene-related peptide (CGRP), substance P (SP), and vasoactive intestinal polypeptide (VIP) are 3 neuropeptides in the enteric nervous system with potent vasodilatory properties (12,13). Calcitonin gene-related peptide, SP, and VIP immunoreactive neurons are localized in both the myenteric and especially the submucosal plexus in the large colon of horses (14). An extensive network of these peptidergic immunoreactive fibers is distributed in the myenteric and submucosal ganglia, around blood vessels, in the longitudinal and circular muscle layers, and in the mucosa of the equine colon (14). Because these neuropeptides are contained within the enteric nervous system, which constitutes an abundant perivascular network, they are implicated in the regulation of intestinal blood flow (15). These neuropeptides reportedly exert their vasorelaxant effects by causing the release of NO from endothelium (16). Both VIP and CGRP appear to increase in the circulation system and in neuronal tissue subsequent to ischemia or ischemia-reperfusion of various tissues, suggesting they may play a protective role in these conditions (17,18).

Colonic venous plasma VIP concentrations significantly increase during low-flow ischemia of the large colon in horses, and both colonic arterial and venous plasma CGRP concentrations increase during early reperfusion after low-flow ischemia (19). However, SP did not change in colonic arterial or venous plasma subsequent to ischemia-reperfusion (I-R). Increased concentrations of these neuropeptides in colonic arterial and venous plasma suggests possible involvement in the decreased colonic arterial resistance and associated reactive hyperemia observed after restoration of blood flow following low-flow arterial ischemia (2,3,19).

We hypothesized that large colon arterial and venous smooth muscle would relax in response to CGRP, SP, and VIP in vessels with intact endothelium, but not in vessels with intact endothelium pre-treated with L-NAME or in vessels with denuded endothelium. Responsiveness of vessels with intact endothelium, but not L-NAME pretreated vessels or vessels denuded of endothelium, will support the role of NO in mediating this vasodilatation. All of the neuropeptides tested in this study were believed to cause some vasodilatation, however, because VIP is one of the most potent vasodilator substances known, it was theorized that it would cause the most pro-found vasodilatation in colonic vessels. The relaxant effect of these substances was anticipated to be greater on colonic arteries than on colonic veins in vessels with intact endothelium.

The purpose of the study reported here was to determine and compare the relative potency and efficacy of CGRP, SP, and VIP in causing in vitro relaxation of colonic arterial and venous rings of horses, and to determine the role of endothelium and NO in modulating these responses.

Materials and methods

Horses

This study was approved by the Institutional Laboratory Animal Care and Use Committee of Louisiana State University. Six adult horses destined for euthanasia for reasons unrelated to the cardio-vascular system or gastrointestinal tract were used. A catheter was inserted into the left jugular vein and horses were anesthetized with intravenous administration of thiopental sodium (Abbott Laboratories, Chicago, Illinois, USA), 10 mg/kg bodyweight (BW), and sodium pentobarbital (The Butler Company, Columbus, Ohio, USA), 7.5 mg/kg BW. A ventral median celiotomy was performed and the colonic artery and vein were harvested from the left ventral colon 10 cm from the pelvic flexure and placed in oxygenated (95% O2 and 5% CO2) Tyrode’s solution. Vessels were maintained in Tyrode’s solution until they were cut into rings and mounted in the organ baths. Horses were then euthanatized with an intravenous overdose of sodium pentobarbital (Beuthanasia-D Special; Schering- Plough Animal Health, Kenilworth, New Jersey, USA), 100 mg/kg BW.

Tissues

Vessels were gently cleansed of excess connective and adipose tissue and cut into 4 mm wide rings. One side of the vessel ring was fixed to the floor of a 10-mL organ bath containing oxygenated Tyrode’s solution at 37°C and the other side was attached to a force displacement transducer (Model FT03 force-displacement transducer; Grass Medical Instruments, Braintree, Massachusetts, USA) interfaced with a polygraph (Model 7D polygraph; Grass Medical Instruments). An initial tension of 2 grams was applied to each ring, which has been shown in our laboratory to be the optimal tension for these vessel rings (20). Vessel rings were allowed to equilibrate for 45 min; during this period, the bath solution was gently replenished with fresh Tyrode’s solution at 15-minute intervals and the tension was readjusted to 2 grams (20). No tension was reapplied after the last bath solution change.

After equilibration, vessel rings were pre-contracted with an EC25 concentration, the concentration that yielded a response of 25% of the maximal contraction, of norepinephrine (NE; Sigma Chemical Company, St. Louis, Missouri, USA), and were allowed to equilibrate for approximately 3 to 5 min after reaching a plateau; this concentration of NE was shown to yield a consistent and sustained contraction of colonic vessels (20). Cumulative concentration-response (C-R) relationships were then determined for CGRP (Sigma Chemical Company); SP (Sigma Chemical Company); VIP (Sigma Chemical Company); and ACh (Sigma Chemical Company), a standard non-peptide endothelium-dependent vasodilator agent. All agents were dissolved following manufacturer’s recommendations and were frozen in aliquots at −80°C. Aliquots were thawed immediately prior to use and were further diluted with Tyrode’s solution so that 100 μL added to the 10-mL organ bath yielded the desired molar concentration. The range of concentrations used for each agent was 10−11 M to 10−5 M.

Three different vessel groups were evaluated for both colonic arteries and veins. The 1st group consisted of colonic vessels with intact endothelium. In the 2nd group, endothelium was removed by mechanical debridement, by gently rotating the vessel segment about a wooden dowel inserted through the lumen of the vessel ring (20). The 3rd group consisted of vessels with intact endothelium that were incubated with freshly prepared 10−5 M L-NAME (hydrochloride) (Sigma Chemical Company), a NO synthase inhibitor, for 30 min before the concentration-response relationships were determined (20). Only 1 C-R relationship was determined on each ring.

When the rings were cut, 4 additional rings (2 arteries and 2 veins) were collected and endothelium was removed from 1 arterial and 1 venous ring, similar to the rings used in the in vitro studies. Histological examination of these representative vessels was performed to document complete endothelial removal and to verify integrity of the vascular smooth muscle.

The change in isometric tension of each ring was measured continuously on the polygraph as change in grams of force. The response to each drug was determined on arterial and venous rings with and without intact endothelium and those incubated with L-NAME from each horse.

Relaxation of the tissues from the norepinephrine-induced contracted level to the initial baseline (before contraction) was considered a maximum (100%) response. Lesser responses of vessel rings to these drugs were considered as a percentage of this complete response. The concentration of the drug required to relax the tissue 50% of the contracted state (EC50), was determined for each drug from the cumulative C-R curves. If the tissue did not relax at least 50% of the contracted state, the EC50 value could not be determined. The maximum relaxation response of vessels to each drug was expressed as the percentage decrease from the contracted state and was defined as the percentage maximal relaxation (PMR).

Statistical analyses

The PMR values for each vessel ring were compared, using a two-way analysis of variance (ANOVA) for repeated measures. Comparisons of arteries versus veins and vessels with intact endothelium, denuded endothelium, and those incubated with L-NAME were also made using two-way ANOVA. Post-hoc comparisons were made, using Tukey’s test. Statistical significance was set at P < 0.05 for all tests. Because EC50 values could not be calculated on many of the tissues, no statistics could be performed.

Results

Colonic arteries with intact endothelium were more sensitive and relaxed the most when treated with CGRP, compared with VIP and SP. Overall, there was a significant difference in the PMR among agents on colonic arteries (Figures 1 to 3). The PMR was significantly lower for SP and VIP, compared with ACh, the relaxant standard. There was no difference in the relaxation response between ACh and CGRP. The PMR was greater in endothelium intact and L-NAME treated arteries, compared with endothelium-denuded arteries. There was no difference in the PMR between endothelium intact and L-NAME treated arteries. There were no differences among the drugs for PMR in endothelium-denuded arteries. In ACh treated arteries, the PMR was significantly greater in endothelium intact and L-NAME treated vessels, compared with endothelium-denuded vessels; there was no difference between endothelium intact and L-NAME treated arteries.

Figure 1.

Figure 1

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In vitro concentration-response (% relaxation) curves of colonic arterial (A) and venous (V) rings with intact endothelium to vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), substance P (SP), and acetylcholine (ACh) in horses. Different letters (a, b) denote a significant (P < 0.05) difference between agents. If the same letter appears on an agent, there is no difference in maximal relaxation among the agents. There were no significant differences between agents on veins.

Figure 3.

Figure 3

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In vitro concentration-response (% relaxation) curves of colonic arterial (A) and venous (V) rings incubated with 10−5 M Nω-nitro-L-arginine methyl ester (L-NAME) to vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), substance P (SP), and acetylcholine (ACh). Different letters (a, b) denote a significant (P < 0.05) difference between agents. If the same letter appears on an agent, there is no difference in maximal relaxation between the agents. There were no significant differences between agents on arteries.

The EC50 values could only be calculated for colonic veins treated with VIP or ACh. Colonic veins with intact endothelium, denuded endothelium, or those treated with L-NAME were more sensitive and relaxed the most when exposed to VIP. There was no difference in the response of the colonic veins of the 3 conditions to VIP. There was a significant difference in the PMR among agents on colonic veins; PMR was significantly greater for VIP and SP, compared with CGRP. Overall, there were no differences in the PMR among endothelium intact, endothelium denuded, and L-NAME treated veins when all agents were considered. Colonic veins relaxed the most when exposed to VIP; this VIP-mediated relaxation was significantly greater than that caused by CGRP, SP, or ACh in endothelium-denuded or L-NAME-treated colonic venous rings.

The PMR was significantly greater for intact arteries than corresponding veins; there was a significantly greater response in arteries for CGRP and ACh than veins. Overall, the PMR was significantly greater in endothelium-denuded veins, compared with endothelium-denuded arteries; the PMR was significantly greater for VIP in veins, compared with arteries; and the PMR was significantly greater in L-NAME treated arteries, compared with veins; arteries relaxed significantly more when exposed to CGRP, compared with corresponding veins.

Histologic examination of representative vessel rings revealed complete endothelial removal and intact smooth muscle in all endothelium- denuded vessels. Morphologically, endothelium was unaltered in all endothelium intact vessels.

Discussion

The major findings of this study included: 1) neuropeptides (VIP, CGRP, SP) evaluated in this study exert vasorelaxant effects on both colonic arteries and veins of horses; 2) the sensitivity and magnitude of the response of colonic arteries to some neuropeptides was altered by the removal of the endothelium and L-NAME treatment; 3) colonic arteries became less sensitive and relaxed to a lower magnitude with these conditions, suggesting that endothelium and NO modulate the vasorelaxant responses of colonic arteries to these neuropeptides; 4) colonic veins relaxed in response to these agents, but removal of endothelium or treatment with L-NAME did not alter the sensitivity or magnitude of the vasorelaxant response of veins to these agents, indicating endothelium and NO do not appear to modulate the vasorelaxant effect of neuropeptides on colonic veins; 5) VIP is a potent relaxant in colonic veins, and this response is not altered by endothelial removal or L-NAME treatment, suggesting a direct action on venous smooth muscle; and 6) CGRP appears to be a more effective relaxant of colonic arteries, this response is diminished by endothelial removal and L-NAME treatment. Because the endothelium of the colonic vasculature can be damaged or disrupted subsequent to an ischemic insult resulting from strangulation obstruction, the vasorelaxant responses of vessel rings with endothelium experimentally removed by mechanical denudation are relevant and may reflect the anticipated response of colonic vessels in horses with strangulating large colon volvulus. From these findings, it appears that neuropeptides could play a role in blood flow regulation in the large colon of horses, which can be disrupted secondary to experimental or naturally acquired ischemia and reperfusion.

Acetylcholine, a nonpeptide neurotransmitter released in the parasympathetic nervous system, causes vascular smooth muscle relaxation of both large conduit vessels and smaller resistance vessels (10,21). It is the prototypic endothelium-dependent vasorelaxant in the arterial circulation and is often used as the standard to compare other substances or to confirm the functional absence of the endothelium. The relaxant effect of ACh is much less appreciable in the venous circulation. The vasorelaxant effect of ACh is only observed in vessels with intact endothelium; this endothelium-dependent phenomenon is due to the release of NO upon stimulation of muscarinic receptors located on the endothelium (10). Acetylcholine has been shown to induce endothelium-dependent and NO-mediated relaxation of palmar digital and large colon arteries and veins in horses (11,20). Although it has been reported that the response of equine colonic vessels to ACh is biphasic, whereby vessels relaxed at lower concentrations and contracted with greater concentrations (20), we did not evaluate neuropeptides concentrations above 10−5 M because they were cost prohibitive.

Calcitonin gene-related peptide is a 37 amino acid peptide, which is widely distributed in both the central and peripheral nervous system (22). Calcitonin gene-related peptide is an extremely powerful vasodilator (13). The vasodilator effect of CGRP in the gastrointestinal tract is consistent with the presence of abundant receptors both on the smooth muscle and endothelium of arteries and arterioles (23). Perfusion of isolated mesenteric vessels of rats with CGRP caused marked vasodilatation (24). The hyperemic effect of CGRP on rat gastric mucosa and left gastric artery flow is decreased by treatment with a NO synthase inhibitor indicating NO contributes to CGRP-evoked vasodilatation of both small and large resistance vessels in the rat stomach (25). However, larger doses of CGRP will often overcome the effects of NO synthase blockade and cause gastric hyperemia, indicating that CGRP is capable of dilating the rat gastric arterial system by NO-independent mechanisms (25). In general, these findings are in agreement with what we found in this study for the effect of CGRP on colonic arterial and venous rings of horses.

Vasoactive intestinal polypeptide is a peptide made up of 28 amino acids, originally isolated from porcine intestine and it possesses potent vasodilator properties (12). There is some evidence that plasma VIP concentrations may increase after ingestion of a meal, and may contribute to postprandial hyperemia (1). Increased splanchnic concentrations of VIP have been observed during reperfusion of ischemic intestine in dogs, pigs, and sheep (26,27). There is a large release of VIP in response to decreased mesenteric arterial blood flow and during reperfusion of ischemic small intestine (26). The physiologic vasodilatory effects of VIP released during low-flow ischemia may be beneficial in improving blood flow to the poorly perfused intestine (26). Local infusion of VIP into the intestinal arterial circulation of cats leads to an immediate decreased vascular resistance and increased blood flow during the infusion (28). This hyperemia persists during the entire infusion period, but intestinal blood flow rapidly returns to a control value once the VIP infusion is discontinued.

Although VIP has been reported to be one of the most potent vasodilator substances known, at least at the concentrations used in this study, it was not as potent or efficacious as ACh in causing relaxation of colonic arteries or veins. This could be a difference among species or regional vascular beds. In general, at greater concentrations, ACh begins to cause contraction, whereas VIP would probably continue to cause relaxation (20).

Substance P is a peptide distributed in the brain and spinal cord in addition to the enteric nervous system (12). Substance P causes vasodilation, which results in increased blood flow in hepatic, mesenteric and portal vascular beds, and inhibits intestinal acid secretion and intestinal absorption (12,29). The fact that there was minimal vasorelaxation of the colonic vessel rings in this study combined with the lack of a measurable increase in colonic arterial or venous plasma during low-flow ischemia and reperfusion (19) suggests that SP probably has a minor role in regulation of the colonic vasculature under physiologic or pathophysiologic conditions in horses.

Overall, colonic arteries with intact endothelium were more sensitive and responded to the neuropeptides evaluated in this study with a greater magnitude of relaxation than those without endothelium. This indicates that the relaxant effects of these neuropeptides on colonic arteries are dependent upon intact, functional endothelium. Lack of a significant difference in the percentage maximal response of colonic arteries with intact endothelium and those incubated with L-NAME may indicate that other endothelium-dependent relaxant mechanisms are involved. Although the concentration of L-NAME in the bath may have been insufficient to completely abolish the release of NO, we believe this is unlikely since a 10−5 M, a relatively high concentration, was used.

There was no difference among veins with intact endothelium, denuded endothelium, or those incubated with L-NAME regarding the sensitivity (EC50) or magnitude (PMR) of relaxation in response to neuropeptides. This finding is similar to that observed with other substances with vasodilatory properties (20), and indicates that endothelium and NO play little or no role in modulating relaxation of colonic veins. Vasoactive intestinal peptide caused more colonic venous relaxation whereas the other neuropeptides caused more of an effect on arteries, indicating the released neuropeptides may exert their action on specific regions of the colonic circulation. Additionally, VIP appears to have a direct action on venous smooth muscle, considering there was no difference in the response of colonic veins with intact endothelium, denuded endothelium, or L-NAME treatment.

Colonic arteries with intact endothelium were more sensitive and relaxed to a greater magnitude than corresponding colonic veins. This was predicted based upon the relaxation response of arteries from other vascular beds in other species, whereby endothelium-dependent relaxation is more pronounced on the arterial side of the circulation (8,16,21). Colonic veins without endothelium were more sensitive and responded by a greater magnitude than corresponding colonic arteries. This further highlights the absence of the role of the endothelium in modulating relaxation of colonic veins and the integral role it plays in colonic arterial relaxation. The fact that colonic arteries incubated with L-NAME had a greater PMR than corresponding veins can be explained by the fact that endothelium-derived substances other than NO may play a role in colonic arterial relaxation.

The effect of endothelial removal or L-NAME treatment on colonic arteries in this study is consistent with cholinergic-mediated relaxation observed in studies in the arterial circulation of the equine digital vasculature (11,30). Nitric oxide appears to mediate the vasorelaxant effects of mechanical and numerous chemical stimuli; endothelial nitric oxide synthase (eNOS) is apparently present to a greater degree on the arterial side of the circulation (8,16,21). Activation of eNOS by these stimuli occurs via increases in intracellular calcium concentrations. Stimulation of eNOS results in immediate synthesis and release of NO, which diffuses from the endothelial cell to the underlying vascular smooth muscle where it stimulates guanylate cyclase, leading to increased cyclic GMP formation and resultant smooth muscle relaxation by sequestration of calcium (31). The lack of a modulatory effect of the endothelium and NO on relaxation of colonic venous rings in this study is similar to findings in another study in our laboratory evaluating the response of equine colonic vessel rings to ACh, bradykinin, histamine, and serotonin, all of which are believed to cause vasodilation at low concentrations via the release of NO (20).

The reactive hyperemia observed after restoration of blood flow following low-flow arterial ischemia in the large colon is likely associated with the release and accumulation of vasodilatory sub-stances during the ischemic period (2). Vasoactive intestinal poly-peptide and CGRP, 2 of the neuropeptides evaluated in this study, have been shown to increase in colonic venous and colonic arterial and venous plasma, respectively, during low-flow ischemia and early reperfusion (19). The fact that these neuropeptides increase in the colonic circulation during ischemia and reperfusion, and they exert in vitro vasorelaxant effects on colonic arteries and veins, combined with the increased blood flow (hyperemia) and decreased colonic vascular resistance during reperfusion, suggests that these neuropeptides probably play some role in the observed blood flow responses (2,3,19).

The studies reported here were conducted on mesenteric colonic arterial and venous rings. The fact that these neuropeptides exerted vasorelaxant effects on these conduit type vessels suggests that they may have an even greater effect on the colonic microcirculation, particularly on the resistance vessels, which are the predominant segment responsible for blood flow regulation (1,32,33).

Endothelial damage is commonly observed in the splanchnic vasculature of horses affected with acute gastrointestinal tract disease (9,34). Endothelial damage can alter gastrointestinal tract blood flow by disrupting normal regulatory mechanisms. Because many vasoactive substances exert their effect through an endothelium-dependent mechanism and since the endothelium of the colonic vasculature sustains injury associated with ischemia and reperfusion, it is important to determine the effect of vasoactive substances on vessels with and without intact, functional endothelium. These pathologic conditions involving the endothelium can be mimicked in vitro by either mechanically removing the endothelium or by incubating the vessel rings with L-NAME, a NOS inhibitor (11,20).

In summary, CGRP, SP, and VIP cause in vitro relaxation of colonic arterial and venous rings. In intact colonic vessels, CGRP causes greater relaxation in arteries whereas VIP causes greater relaxation in veins. In general, SP caused the least relaxation in arteries and veins. Unlike in colonic veins, colonic arterial relaxation was dependent upon the presence of intact, functional endothelium. The VIP appears to mediate its relaxation via a direct effect on colonic venous smooth muscle. These vasodilatory neuropeptides appear to modulate vasomotor tone in colonic vessels of horses. Neuropeptides may play a role in colonic blood flow regulation in horses, which can be disrupted secondary to experimental or naturally acquired ischemia and reperfusion.

Figure 2.

Figure 2

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In vitro concentration-response (% relaxation) curves of colonic arterial (A) and venous (V) rings with denuded endothelium to vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), sub-stance P (SP), and acetylcholine (ACh). Different letters (a, b) denote a significant (P < 0.05) difference between agents. If the same letter appears on an agent, there is no difference in maximal relaxation between the agents. There were no significant differences between agents on arteries.

Acknowledgments

This study was supported by grants from the American Quarter Horse Association and the Louisiana State University School of Veterinary Medicine Equine Health Studies Program. The authors thank Dr. Lee Ann Fugler for technical assistance.

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