Tall fescue ergot alkaloids are vasoactive in equine vasculature1,2

Abstract

Mares grazing endophyte-infected () tall fescue () typically exhibit reproductive dysfunction rather than problems associated with peripheral vasoconstriction as a primary sign of the fescue toxicosis syndrome. Research using Doppler ultrasonography demonstrated that consumption of endophyte-infected tall fescue seed causes measurable vasoconstriction in the medial palmar artery. The objective of this study was to evaluate contractile responses of medial palmar artery and vein to increasing concentrations of various tall fescue alkaloids. Medial palmar arteries and veins were collected immediately following euthanasia from 23 horses of mixed breed, age, and gender from both forelimbs, and uterine arteries were collected from females ( = 12). Vessels were separated, cleaned of excess connective and adipose tissue, divided into 2- to 3-mm cross-sections, and suspended in a multimyograph chamber with continuously oxygenated Krebs-Henseleit buffer (95% O/5% CO; pH 7.4; 37°C). Following a 90-min equilibration and recovery from reference compound exposure, increasing concentrations of norepinephrine, 5-hydroxytryptamine, ergotamine, and ergonovine for the palmar artery and vein and uterine artery and ergovaline, ergocryptine, ergocristine, ergocornine, and lysergic acid for the palmar artery and vein were added to assess vasoactivity. Data were normalized as a percentage of contractile response induced by the reference compound addition and analyzed as a completely randomized design. Both norepinephrine and serotonin were vasoactive in all 3 types of blood vessels. Neither ergotamine nor ergonovine were vasoactive in the uterine artery. All alkaloids tested with the palmar artery and vein produced a contractile response, except that neither the palmar artery nor the palmar vein responded to lysergic acid ( > 0.05). Ergovaline was the most vasoactive ergot alkaloid in both the palmar artery and the palmar vein ( < 0.05) followed by ergonovine, whereas out of the 4 remaining ergopeptine alkaloids tested, ergocristine induced the lowest contractile response. Although horses do not outwardly appear to be affected by peripheral vasoconstriction as observed in cattle, these data indicate that tall fescue alkaloids are vasoactive and suggest that potential exists for peripheral vascular effects of tall fescue alkaloids in horses. This does not appear to be the case for the uterine artery, and future research should be directed at understanding how ergot alkaloids cause equine reproductive dysfunction.

Assessment of the vasoactive effects of the (S)-epimers of ergot alkaloids in vitro

“…From each foot limb, muscle, nerves, and tissues were removed to access a 15-cm segment of the dorsal metatarsal artery that was located directly next to the bone. Once the segment was carefully removed using surgical scissors and a scalpel, it was placed in a small container with a lid containing modified Krebs–Henseleit oxygenated buffer, (95% oxygen/5% carbon dioxide mixture; pH = 7.4; composition in g/2 liters deionized water: 13.68 NaCl, 0.7 KCL, 0.28 MgSO 4 , 0.32 KH 2 PO 4 , 3.6 NaHCO 3 , 1.8 glucose, and CaCl 2 , made in house) and on ice for transport to the laboratory ( Klotz and McDowell, 2017 ). Upon arrival to the laboratory, excess fat and connective tissue were removed from each artery segment ( Klotz and Barnes, 2014 ), which were sliced into 3-mm arterial cross sections ( Klotz and McDowell, 2017 ).…”

“…Once the segment was carefully removed using surgical scissors and a scalpel, it was placed in a small container with a lid containing modified Krebs–Henseleit oxygenated buffer, (95% oxygen/5% carbon dioxide mixture; pH = 7.4; composition in g/2 liters deionized water: 13.68 NaCl, 0.7 KCL, 0.28 MgSO 4 , 0.32 KH 2 PO 4 , 3.6 NaHCO 3 , 1.8 glucose, and CaCl 2 , made in house) and on ice for transport to the laboratory ( Klotz and McDowell, 2017 ). Upon arrival to the laboratory, excess fat and connective tissue were removed from each artery segment ( Klotz and Barnes, 2014 ), which were sliced into 3-mm arterial cross sections ( Klotz and McDowell, 2017 ). Four arterial cross sections were suspended horizontally in a four-chamber (one arterial cross section per chamber) tissue bath (Radnoti [159906], Monrovia, CA, USA) containing 15 mL of continuously oxygenated (95% oxygen/5% carbon dioxide) modified Krebs–Henseleit buffer, same as above, at 37 °C.…”

“…Twelve ( n = 12) arteries (artery segments) in total were used with six ( n = 6) exposed to ergocryptinine and ergocorninine, and the other six ( n = 6) exposed to ergocristinine and ergotaminine ( n = 6 arteries/( S )-epimer). After resting tension was restored, ( S )-epimers were added separately to each chamber in the tissue bath from lowest concentration (1 × 10 –11 M) to highest (1 × 10 –6 M) for an incubation period of 29 min, with one buffer replacement, followed by a 2.5-min washout period and 1 min recovery before the next ( S )-epimer addition ( Klotz and McDowell, 2017 , with slight modification). Preliminary investigation indicated that the arteries do not relax after the ( S )-epimers were removed from the buffer within the tissue bath.…”

“…In comparison, when PE was washed out from the buffer solution, the contraction abated almost immediately. A maximal response was not achieved in a 9- or 14-min incubation period, which had been used in previous validated assays ( Klotz et al, 2006 ; Klotz and McDowell, 2017 ); therefore, a longer incubation period for each ( S )-epimer was employed to ensure contraction was not overlooked. After the last concentration, PE (1 × 10 –4 M) was added again to ensure viability of all arterial cross sections.…”

“…The bioactivity, in terms of vasoactivity, of ( R )-epimers is commonly assessed using an in vitro tissue bath system where the contractile response of dissected arteries is monitored after incubation with increasing concentrations of a particular ( R )-epimer (Klotz et al, 2010; Foote et al, 2011 ; Klotz and McDowell, 2017 ). Specifically, cattle arteries ( Oliver et al, 1993 , Klotz et al, 2010 , Foote et al, 2011 ), such as the dorsal metatarsal artery ( Oliver et al, 1993 ; Yonipam, 2018 ), have been utilized.…”