Vasoconstrictive effects of tall fescue alkaloids on equine vasculature

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VASOCONSTRICTIVEEFFECTSOF TALL FESCUEALKALOIDS ON EQUINEVASCULATURE L. Kim Abney, BA, J.W. Oliver, DVM, PhD; and C. R. Reinemeyer, DVM, PhD

The in vitro vasoconstrictive effects of equine arteriovenous tissues to alkaloids found in Acremonium coenophialum-infested rescue grass (ergotamine tartrate, ergonovine maleate, and N-acetyl loline) were compared to those of various biogenie amines, including norepinephrine, phenylephrine, BHT-920, and serotonin. In this initial study, both the loline and the ergot compounds were vasoactive, although the contractile effect was less than that of the biogenic amine compounds. The study demonstrates the potential for decreased perfusion of peripheral tissues in horses consumingA, coenophialum-infested rescue grass or hay.

are much less able to withstand environmental extremes. 12 Reproductive dysfunction has been the primary disorder seen in mares grazing endophyte-infected pastures.3,6,9 In addition, results of feeding trials suggest that consumption of A. coenophialum-infested forage reduces feed efficiency, causing decreased weight gain.1oWhile horses are not visibly affected by peripheral vasoconstriction similar to that occurring in cattle, 4's the potential still exists for vascular involvement. Studies on potential peripheral vascular effects in horses consuming toxic rescue have not been done. The purpose of the study reported here was to compare the vasoconstrictive effects of the biogenic amines norepinephrine, phenylephrine, BHT-920 and serotonin to those of ergotamine tartrate, ergonovine maleate, and Nacetyle loline in isolated equine lateral saphenous vein and dorsal metatarsal artery.

INTRODUCTION

MATERIALS AND METHODS

Tall fescue occupies approximately 15 million hectares inthe United States.2 The grass is an excellent forage in many respects, due to the presence of the endophytic fungus Acremonium coenophialum} 2 However, this endophytic fungus has been causally related to considerable adverse health effects in horses. 9 The prevalence of the endophyte fungus in the grass is conservatively estimated to be 75%. 1 Fungus-free strains of fescue, although less toxic to animals,

Tissues were collected immediately after euthanasia from 6 normal, mixed-breed quarter horses, ranging in age from 8-14 months. The horses represented control animals from a separate study, and were fed 2 pounds of grain twice a day as well as good quality alfalfa hay ad libitum. The animals were euthanized over a 4 week period of time during the summer months. Tissues were collected and handled similar to those in previous studies in our laboratory.TM Five centimeter segments of the dorsal metatarsal artery and the lateral saphenous vein were dissected from both distal hind limbs. The tissues were placedin a modified-Krebs oxygenated-

SUMMARY

Authors' address: Department of Environmental Practice, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37901 Acknowledgement: Supported in part by The University of Tennessee, College of Veterinary Medicine, Center of Excellence in Livestock Diseases and Human Health.

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buffer solution (95% 02/5% CO2) and kept on ice until used. Both arteries and veins were trimmed of excessive fat and connective tissues, and cross-sections 2 mm wide were suspended vertically in a 10-ml organ batha containing the oxygenated buffer (95% 02/5% CO2; 37 ° C; pH 7.4). Crosssections of vessels were used (rather than helical preparation) because of the small size of vessels. Desipramine HC1 (3x10" 5M) and propranolol (lx10-6 M) were included in the buffer solution to inactivate cathecholamine-neuronal uptake and beta-adrenergic receptors, respectively. Each tissue section was suspended between a stationary tissue holder and an isometric force transducerb (linear range of 0-60 grams). The tissue was allowed to equilibrate for 60 minutes prior to experimentation under a resting tension of 1 gram. The bath solution was replaced every 15 minutes during the equilibration period. Following equilibration, the tissues were maximally contracted with an alpha-adrenergie agonist (norepinephrine, 2x10"4M) in order to assure tissue responsiveness. Tissues then were washed every 15 minutes until the original resting tension was restored, followed by exposure to the biogenic amines or alkaloids. Isometric vascular contractions were recorded as change in grams of tension with a polygraph,h Values reported represent data derived from tissue sections from each of the horses. Twentyfour tissue sections per animal were available; however, since some tissues failed to respond to initial norepinephrine stimulus and were thus discarded, uneven sample numbers resulted. Cumulative dose-response curves were determined in both artery and vein for norepinephrine (non-selective alpha adrenergic agonist), phenylephrine (selective alpha-1 adrenergic agonist), the experimental drug BHT-920 e (selective alpha-2 adrenergic agonist), serotonin (mixed serotonergic activity), N-acetyl loline d (pyrrolizidine alkaloid; a selective agonist for alpha-2 adrenergic receptors in cattle; r ergotamine tartrate (ergopeptine alkaloid), and ergonovine maleate (simple lysergic acid amide alkaloid). Isometric contraction was recorded as grams of tension produced. In order to compensate for differences in weights of tissues and/or individual variation in tissue responsiveness, values were expressed as a percent of the maximal contraction produced by each agonist. The concentration of each compound that was required to produce 50% of the maximal tissue contraction (Ee50 values) was determined from the dose-response curve, according to a double reciprocal plot methods, la Results

All of the alkaloids and biogenic amines tested caused concentration-dependent vascular contractions in equine aHarvard Biomedical Instruments. Boston, MA. bNarco Biosystems, Inc. Houston, "IX CGift of Boeringer Ingelheim, Ridgefield, CT. dGift of Bioactive Constituents Research Group, R. B. Powell, Director. USDA/ARS, Peoria, IL.

Volume 13, Number 6, 1993

lateral saphenous vein and dorsal metatarsal artery, except for N-acetyl loline which did not cause contractile effects in the latter. Receptor sensitivity (the concentration of agonist at which contraction begins) and mean maximal contractile response (the largest tension in grams produced in response to the highest concentration of agonist) of veins, was similar for norepinephrine, phenylephrine and serotonin (lx10-8,1 xl0 -8 and 3x10-9 M and 16.1+3.0, 16.9+2.2, and 18.3+4.2 grams, respectively), while the values for BHT-920 were lx10-8 M and 6.7+1.3 grams (Figure 1). The receptor sensitivity in response to ergotamine tartrate and ergonovine maleate was similar to that of biogenic amines (3x10 "9 and l x l 0 "8 M, respectively, but the maximal contractile response for each alkaloid was considerably less (6.2 + 0.8 grams and 5.5 + 4.0 grams for Ergotamine tartrate and Ergonovine Maleate) (Figure 2). In veins, N-acetyl loline induced a mean maximal contractile response of 1.4 +0.4 grams with a receptor sensitivity of 3x10 -9 M (Figure 2). Receptor sensitivity and mean maximal contractile response of arteries was similar for norepinephrine, phenylephfine and serotonin (lxl0 "8, 3x10 -7 and l x l 0 "7 M, and 5.5+0.7, 5.9+0.8 and 5.1+0.9 grams, respectively), while the values for BHT-920 were 3x10 -8 M and 4.8+0.8 grams of tension (Figure 3.). Receptor sensitivity for ergotamine tartrate and ergonovine maleate were similar to that of the biogenic amines at l x l 0 "7M and 3x10 "7M, but the contractile response was considerably less (0.8+0.2 and 1.5+0.4 grams of tension respectively, (Figure 4). N-acetyl loline gave no contractile response in equine arteries in this study. Halfmaximal (Ec50) contractile values for biogenic amines were similar in arteries and veins, while the Ec50 values for the alkaloids occurred at lower concentrations in veins than in arteries.

DISCUSSION

Vascular tissues in the equine (lateral saphenous vein, dorsal metatarsal artery) were responsive to norepinephrine, phenylephrine, BHT-920 and serotonin. Thus, populations of alpha-adrenoceptors and serotonin receptors are present in these equine vessels that mediate the contractile responses. Both artery and vein tissues exhibited strong vasoconstriction in response to the ergot alkaloids ergotamine and ergonovine. Likewise, the loline alkaloid N-acetyl loline caused venoconstriction, but not arterioconstriction. Despite the vasoactive response to the three types of alkaloids used in this study, horses do not appear to develop severe peripheral vasoconstriction similar to that seen in cattle 4 where gangrenous changes of the feet can occur. The reason for this difference in severity of pathophysiologic response between the two species may be related to anatomical difference in blood supply to the feet, or perhaps differences in management

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Log MolarConcentration Figure 4, Mean contractile dose-response, of equine dorsal metatarsal artery in vitro, to norepinephrine (n=7), N-acetyl Ioline (n=15), ergotamine (n=7), and ergonovine (n-7) expressed as a percent of norepinephrine's maximal contractile response. * Different (P<.01) than corresponding concentration of norepinephrine. ** Different (P<.05) than corresponding concentration of norepinephrine. Ec50 values and confidence levels for norepfnephrine, N-acetyl Ioline, ergotamine tartrate and ergonovine maleate were, respectively: 1.6x10 "5 M, 6.2xl 0 .6 and 4.0x105; 0; 5.0x10 "6 M, 4.0xl 0 .6 and 6.3xl 06; 2.6x10 "6 M, 5.9x10 .7 and 1.2xl 0 -5. V o l u m e 13, N u m b e r 6, 1993

339

and nutritional handling. Results of the present study indicate that tall fescue infested with the endophytic fungus A. coenophialum, and thus ergot alkaloids may cause vasoconstriction in the extremities of horses,s,14 The reduced blood flow may predispose affected animals to chronic foot and leg disorders.

REFERENCES 1, Bacon, C.W., Lyons, P.C., Porter, J.K. and Robbins, J.D. (1986) Ergot toxicity from endophyte-infected grasses: a review. Agron. J. 78, 106-116. 2. Burns, J.C. and Chamblee, D.S. (1979) Adaptation. In: Tall fescue. Ed: L.P. Bush and R.C. Buckner. Am. Soc. Agron, Madison, WI. Agronomy 20, pp 9-30. 3. Earle, W.F., Cross, D.L., Hudson, L.W., Redmon, L.M. and Kennedy, S.W. (1990) Effect of energy supplementation on gravid mares grazing endophyte-infected fescue. J. Equine. Vet. Sci. 10 (2), 126-130. 4. Garner, GB. and CornelI,C.R. (1978) Fescue Foot in Cattle. In: Mycotoxic Fungi, Mycotoxins, and Mycotoxicoses. Ed: T.D. Wyllie and LG. Morehouse. New York: Marcel Dekker, pp 4562. 5. Hammond, A.C., Bond, J. and Stroud, B. (1982) Tall Fescue summer toxicosis in cattle. Bovine Pract. 17, 137-142. 6. Monroe, J.L., Cross, D.L., Judson, L.W., Hendricks, D.M., Kennedy, S.W., Bridges, W.C. Jr. (1988) Effect of selenium and endophyte-contaminated fescue on performance and reproduction in mares. J. Equine Vet Sci. 8, 148-153.

7. Oliver, d.W., Robinson, A.J., Abney, L.K. and Linnabary, RD. (1992) Effects of phenothiazine and thiabendazole on bovine dorsal pedal vein contractility induced by ergonovine and serotonin; potential for alleviation of fescue toxicity. J. Vet. Pharmacol. Therap. 15, 661-665. 8. Petroski, R.J. and Powell, R.G. (1991) Preparative separation of complex alkaloid mixture by high-speed countercurrent chromatography. Naturally Occurring Pest Bioregulators. pp 426-434. 9. Propenga, R.H., Mostrum, M.S., Haschek, W.M., Lock, T.F., Buck, W.B. and Beasley, V.R. (1984) Mare agalactia, placental thickening and high goal mortality associated with the grazing of tall fescue: a case report. Amer. Assn. Vet Lab Diagnost. (27 Ann. Proc), 325-336. 10. Redmond, L.M., Cross, D .L., Jenkins,T.C. and Kennedy, S.W. (1991) The effect of Acremonium coenophialum on intake and digestibility of tall fescue hay in horses. J. Equine Vet. ScL 11 (4), 15219. 11. Solomons, R.N., Oliver, J.W. and Linnabary, R.D. (1989) Reactivity of dorsal pedal vein of cattle to selected alkaloids associated with Acremonium coenophialum-infested fascue grass. Am. J. Vet Res. 50, 235-238. 12. Stuedemann, J.A. and Hoveland, C.S. (1988) Fescue endophyte: history and impact on animal agriculture. J. Prod. Agric. 1, 39-44. 13. Tallarida, R.J. and Murray, R.B. (1987) Manual of pharmacologic calculations with computer programs, 2nd ed. Springer-Verlad, New York. pp 3-63. 14. Yates, S.G. and Powell, R.G. (1988) Analysis of ergopeptine alkaloids in endophyte-infected tall rescue. J. Agric. Food Chem. 36, 337-340.

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