5.3. Myograph—Vasoactivity

Biopsied blood vessels from both experiments were used in vitro to evaluate changes in contractile response to increasing concentrations of adrenergic and serotonergic agonists and ergot alkaloids. Contractile response experiments were run the same day as biopsy. Tissue processing for all vein segments followed methods validated by Klotz et al. [43]. Tissue preparation for experiments consisted of removal of excess fat and connective tissue. Cleaned segments were sliced into 2- to 3-mm cross sections. Cross-sections were examined under a dissecting microscope (Stemi 2000-C, Carl Zeiss Inc., Oberkochen, Germany) at 12.5× magnification to measure dimensions for assurance of consistent segment size and to verify physical integrity of tissue. Cross-sections were suspended horizontally in a 5-mL tissue bath (DMT610M Multichamber myograph, Danish Myo Technologies, Atlanta, GA, USA) containing continuously oxygenated modified-Krebs Henseleit buffer (95% O₂/5% CO₂; pH = 7.4; 37 °C), with 3 × 10⁻⁵ M desipramine and 1 × 10⁻⁶ M propranolol (Sigma Chemical Co.) to inactivate catecholamine-neuronal uptake and β-adrenergic receptors, respectively. After equilibration to 1 g of tension (~1.5 h), tissues were exposed to the α-adrenergic agonist norepinephrine (1 × 10⁻⁴ M; Sigma Chemical Co.) to verify tissue viability and as a reference for normalization of the corresponding contractile responses.

Cross-sections of lateral saphenous veins were run in duplicate from each steer for each contractile response treatment. After recovery from the 1 × 10⁻⁴ M norepinephrine addition (45 to 60 min) and the reestablishment of the 1-g baseline tension, alkaloid additions occurred in 15-min intervals. Each 15-min interval consisted of a 9-min treatment incubation period, followed by a washout period during which duplicate aliquots of buffer minus the treatment were incubated with the vein segment for two 2.5-min periods, followed by a final buffer replacement and 1-min incubation before the next addition.

For Experiment 1, the treatment additions were an ergot alkaloid ergovaline tartrate (93% purity; supplied by Forrest T. Smith, Auburn University, Auburn, AL, USA), a serotonin (5HT) receptor agonist selective for the 5HT_2A receptor (4-bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine HCl (TCB2; Tocris Biosciences, Ellisville, MO, USA), a selective α_2A-adrenergic receptor agonist guanfacine HCl (GF; Tocris Biosciences), and a α_2C-adrenergic receptor agonist (R)-(+)-m-nitrobiphenyline oxalate (NBP; Tocris Biosciences). The contractile response curves were constructed with 8 consecutive additions every 15 min at fixed concentrations ranging from 1 × 10⁻¹¹ to 1 × 10⁻⁴ M in the tissue bath. For Experiment 2, the ergot alkaloid ergotamine tartrate (≥97% purity, Fluka, as distributed by Sigma Chemical Co.) was used in the same dilutions as Experiment 1, because of a shortage of purified ergovaline. Ergotamine was selected because Klotz et al. [28] demonstrated that ergovaline and ergotamine produce similar contractile responses in the bovine lateral saphenous vein bioassay. The receptor agonists TCB2, GF, and NBP were used as in Experiment 1, however the range of concentrations tested were modified to 5 × 10⁻⁸ to 1 × 10⁻⁴ M to better characterize the response curve.

In both experiments, isometric contractions of lateral saphenous vein were recorded as grams of tension in response to exposure to norepinephrine and the myograph treatments. The data were digitally recorded using a Powerlab/8sp (ADInstruments, Colorado Springs, CO, USA) and Chart software (Version 7.2, ADInstruments). Contractile response was recorded as the greatest contractile response, in grams, within the 9-minute incubation after a treatment addition and corrected by the baseline tension recorded just before the addition of 1 × 10⁻⁴ M norepinephrine. Response data were normalized as a percentage of the maximal contraction produced by norepinephrine (1 × 10⁻⁴ M). Normalization compensated for variation of the tissue responsiveness due to differences in tissue size across individual cattle. To construct the concentration-response curves, the normalized data were plotted with GraphPad Prism (version 5.0f; San Diego, CA, USA). This graphical presentation used a nonlinear regression (sigmoidal concentration-response curve) to fit a line to contractile response data points for a treatment using the four-parameter equation:

where the top and bottom are plateaus in the units of the y-axis and the Hill slope is the numerical determination of the steepness of the curve. This calculation also allowed for the calculation of a compound’s potency in the bovine lateral saphenous vein bioassay expressed as the concentration the compound required to produce 50% of the observed contractile response (EC₅₀).