Isolated vessel preparation

The experimental protocols used in this study were approved by the University of Calgary Animal Care Committee, and conform to the guidelines for the care and use of laboratory animals established by the Canadian Council on Animal Care and the Guide for the Care and Use of Laboratory Animals (NIH, 8th edition, 2011). Male Sprague‐Dawley rats (225–250 g body weight) were obtained from Charles River Laboratories and housed under standard conditions (i.e., 12 h day/light cycle, 22°C) with continuous access to food and water. Rats were injected intraperioneally with sodium pentobarbital (50 mg/kg) to induce surgical anesthesia (i.e., stage 3, loss of blink reflex), and cremaster muscles were then surgically removed and placed in a cooled (4°C) dissection chamber containing Krebs’ buffer (115 NaCl, 5 mmol/L KCl, 25 mmol/L NaHCO₃, 1.2 mmol/L MgCl₂, 2.5 mmol/L CaCl₂, 1.2 mmol/L KH₂PO₄ and 10 mmol/L d‐glucose); pH was adjusted to 7.4 with 1 N NaOH (Meininger et al. 1991; Sheng et al. 2009). Euthanasia was completed by an overdose administration of sodium pentobarbital (150 mg/kg, I.P.). Following isolation, cremaster 1A arteries were cannulated on glass pipettes fitted in a pressure myography chamber (Living Systems, Burlington, VT) and the vessel lumen was filled with Krebs’ buffer containing 1% bovine serum albumin, pH was adjusted to 7.4. The cannulated vessel/chamber apparatus was placed on the stage of an inverted microscope, and one cannula end was closed and the other connected to a hydrostatic pressure column. The vessel was superfused with Krebs’ buffer at a constant flow of 7 mL/min, using a peristaltic pump and suction line. Bath solution was gassed with 95% air/5% CO₂ and maintained at 34°C. The intraluminal pressure of cannulated vessels was increased in a step‐wise manner under no‐flow conditions and then maintained at 70 mmHg; vessels typically developed myogenic tone within 15–20 min. Intraluminal vessel diameter was continuously tracked using a video camera‐based imaging system (IonOptix, Milton, MA). Drugs were added to the bath via the peristaltic pump. Note that changing the experimental order in which vasodilators were applied to arteries did not change the magnitude of observed responses to a given dilator.

Drug‐induced changes in arterial intraluminal diameter (i.e., inhibition of myogenic tone, expressed in microns) were calculated as the difference in steady‐state diameter in the presence of a given vasodilator (D_drug) and the intraluminal diameter under baseline conditions (D_basal) measured immediately prior to a given drug exposure. Thus,

where D_drug = intraluminal diameter in the presence of a given vasodilator (e.g. ACh or SNP), D_basal = intraluminal diameter under basal myogenic tone at 70 mmHg. When vasodilatory responses were measured in the presence of myogenic tone + a constrictor agent (i.e. PE, NE or U46619), the D_basal value was calculated at the new baseline diameter observed in the presence of the constrictor, immediately prior to each vasodilatory response.