Scand J Thor Cardiovasc Surg 26: 135-141, 1992
IN VITRO RESPONSES TO ATRIAL NATRIURETIC POLYPEPTIDE IN HUMAN VESSELS COMMONLY USED AS AORTOCORONARY BYPASS GRAFTS Sidsel Aardal, Karen B. Helle and Einar Svendsen' From the Department of Physiology and the 'Department of Pathology, University of Bergen, Norway
Scand Cardiovasc J Downloaded from informahealthcare.com by McMaster University on 02/15/15 For personal use only.
(Accepted for publication November 14, 1991)
Abstract. Vascular effects of atrial natriuretic polypeptide (APII), i.e. the peptide hormone released from the atrial myocardium, were investigated in segments of the human internal thoracic artery (ITA) and saphenus vein (SV) with intact (+E) or injured (-E) endothelium. All segments were subject to several cycles of agonists in order to detect tachyphylactic or facilitatory responses. Opposite, indirect effects on the noradrenaline contracted ITA and SV were obtained in response to APII at a supranormal concentration (50 nM) which had no direct relaxing action on the isolated segments in vitro. In ITA the noradrenaline contractures in subsequent cycles were reduced to 41 2 2 1 % (+E) and 2 8 5 9 % (-E), but in SV they were enhanced to 21 1 2 115O h (+E) and 483-+242% (-E) of those before APII exposure. Thus under in vitro conditions ITA could be indirectly relaxed by APII via tachyphylactic effect on the noradrenaline contracture. SV, on the other hand, was markedly potentiated by APII in its noradrenaline response. In injured endothelium these opposite effects were aggravated. Key words: endothelium, internal thoracic artery, saphenous vein, human atrial natriuretic polypeptide, glyceryl nitrate, noradrenaline, vasoactive intestinal peptide, calcitonin gene related peptide.
Both arteries and veins are now used as coronary bypass grafts. The internal thoracic artery (ITA) has become the conduit of choice, as the patency rate is higher than that of saphenous vein (SV) grafts to the same coronary bed (17), suggesting differing morphology and functional properties of the endothelium and vascular smooth muscle between arterial and venous grafts (12). An intact endothelium is essential for production of the endothelium-derived relaxing factor (EDRF) shown to be nitric oxide (14) or nitrocysteine (13), and it is assumed that this endogenous vasodilatory substance protects against postoperative vasospasms and thrombus formation ( 1 5).
Atriopeptin (AP) is another endogenous vasodilator, first referred to as atrial natriuretic factor. AP is synthesized and stored in preeursor form in the atrial myocardium (2) from which it is released into the circulation upon volume load or tachycardia as a smaller peptide, atriopeptin I1 (APII). This hormone is a potent relaxant of some, but not all, regions of vascular smooth muscle (4). Plasma levels are elevated from 0.02-0.1 nM to 0.2-2.5 nM in patients with a wide range of cardiac dysfunctions (3, 4, 18). Thus after cardiopulmonary bypass operations both the in situ and free grafts will be exposed to equally high, often nM concentrations of APII. It is not yet known to what extent the human arterial and venous segments used as grafts respond directly or indirectly to APII compared with other vasodilating peptides released from perivascular nerves, such as vasoactive intestinal peptide (VIP) and calcitonin gene related peptide (CGRP). These neuronal peptides are potent relaxants of gastric, splenic, hepatic and cerebral arteries (8). We now report significant differences found in the effects of APII on noradrenaline-induced u2-contractures in isolated ITA and SV segments. The influence of endothelial integrity on these differences was also elucidated. MATERIALS AND METHODS Tissue collection. Segments of ITA and SV which are routinely cut away and discarded in the course of coronary bypass operations were obtained from patients with angina pectoris and, with institutional approval, used for in vitro experiments. Patient premedication included diazepam, droperidol and fentanyl and anaesthesia was induced with diazepam, fentanyl and pancuronium. The patients were ventilated with 50% O2 and isoflurane 0.2-1.0%. Glyceryl Scand J Thoracic 26
Scand Cardiovasc J Downloaded from informahealthcare.com by McMaster University on 02/15/15 For personal use only.
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nitrate infusion was begun before the induction of anaesthesia and maintained until the start of cardiopulmonary bypass. For the in vitro experiments 8-12 mm long ITA segments were obtained from the distal end with a portion of the adjacent transverse thoracic muscle still attatched, while 10-15 mm long SV segments were taken from the proximal end of the vessel. Isometric recordings of contractile tension. The segments were subdivided into 3 mm long rings and mounted between stainless steel bars in thermostatically controlled organ baths containing 2.5 ml oxygenated Krebs Henseleits medium (95% O2and 5% COJ at 37°C. In one segment the endothelium was injured by probing, and the other was left intact. The time from surgical removal of the segments until mounted and stretched was < 1 h. The medium contained (mM) NaCl 118, KC1 4.7, CaCl, 2.5, MgSO, 1.2, NaHCO, 25, KH2P04 1.2 and glucose 10.1. Tension was recorded isometrically as previously described (1). The half maximally effective (ED,,) concentration of noradrenaline (NA) at different loads was 2 . 6 ~ M for ITA and 2 . 6 ~ M for SV at 15 mN. This preload corresponded to the optimum on the length-tension curves for both ITA and SV. The stretched preparations were repeatedly washed for 60 min before the analyses of agonists. Relaxation of the contracted ITA was quantified as the decline in tension 4 min after addition of the agonist and expressed in O/o of the maximal relaxation, i.e. back to the level of the resting tension (100%). Relaxation of the NA-contracted SV was quantified as the decline from T,,, measured 12 and 18 min (T,2 and T,& after the onset of the tension rise (To). Agonists were added as indicated in cumulative concentrations. Potency was expressed as the negative logarithm to the agonist concentration giving half-maximal inhibitory effect (pD,). The intrinsic activity (a) was calculated as the ratio of the maximal effect to the value for maximally obtainable relaxation. After each cycle the segments were washed every 5 min until the tension had returned to resting level and at least twice thereafter. Each segment was exposed to 4-5 cycles of agonist additions in order to detect tachyphylactic or potentiating effects or receptor activation. In control experiments the contractile activity could be maintained for 5-8 hours. Morphology. In order to verify the degree of endothelial injury, the probed and unprobed ITA and SV segments were carefully removed from the organ baths after the experiments and fixed by overnight immersion in 2 Yo buffered glutaraldehyde. The segments were cut into halves with a razor blade and processed for scanning electron microscopy according to conventional procedures. They were further dried by critical point, using COz as drying solvent. The intimal surfaces were coated with gold in a Polaron sputtering coating unit, and viewed in a Phillips 500 SEM electron microscope at an accelerating voltage of 25 kV. Statistical analyses. The results are expressed as means 2 SD for segments from (n) patients. Significance of differences between means was calculated Scand J Thoracic 26
with the Wilcoxon rank test for nonparametric, dependent and independent groups. Statistical significance was assumed when p
IN VITRO RESPONSES TO ATRIAL NATRIURETIC POLYPEPTIDE IN HUMAN VESSELS COMMONLY USED AS AORTOCORONARY BYPASS GRAFTS Sidsel Aardal, Karen B. Helle and Einar Svendsen' From the Department of Physiology and the 'Department of Pathology, University of Bergen, Norway
Scand Cardiovasc J Downloaded from informahealthcare.com by McMaster University on 02/15/15 For personal use only.
(Accepted for publication November 14, 1991)
Abstract. Vascular effects of atrial natriuretic polypeptide (APII), i.e. the peptide hormone released from the atrial myocardium, were investigated in segments of the human internal thoracic artery (ITA) and saphenus vein (SV) with intact (+E) or injured (-E) endothelium. All segments were subject to several cycles of agonists in order to detect tachyphylactic or facilitatory responses. Opposite, indirect effects on the noradrenaline contracted ITA and SV were obtained in response to APII at a supranormal concentration (50 nM) which had no direct relaxing action on the isolated segments in vitro. In ITA the noradrenaline contractures in subsequent cycles were reduced to 41 2 2 1 % (+E) and 2 8 5 9 % (-E), but in SV they were enhanced to 21 1 2 115O h (+E) and 483-+242% (-E) of those before APII exposure. Thus under in vitro conditions ITA could be indirectly relaxed by APII via tachyphylactic effect on the noradrenaline contracture. SV, on the other hand, was markedly potentiated by APII in its noradrenaline response. In injured endothelium these opposite effects were aggravated. Key words: endothelium, internal thoracic artery, saphenous vein, human atrial natriuretic polypeptide, glyceryl nitrate, noradrenaline, vasoactive intestinal peptide, calcitonin gene related peptide.
Both arteries and veins are now used as coronary bypass grafts. The internal thoracic artery (ITA) has become the conduit of choice, as the patency rate is higher than that of saphenous vein (SV) grafts to the same coronary bed (17), suggesting differing morphology and functional properties of the endothelium and vascular smooth muscle between arterial and venous grafts (12). An intact endothelium is essential for production of the endothelium-derived relaxing factor (EDRF) shown to be nitric oxide (14) or nitrocysteine (13), and it is assumed that this endogenous vasodilatory substance protects against postoperative vasospasms and thrombus formation ( 1 5).
Atriopeptin (AP) is another endogenous vasodilator, first referred to as atrial natriuretic factor. AP is synthesized and stored in preeursor form in the atrial myocardium (2) from which it is released into the circulation upon volume load or tachycardia as a smaller peptide, atriopeptin I1 (APII). This hormone is a potent relaxant of some, but not all, regions of vascular smooth muscle (4). Plasma levels are elevated from 0.02-0.1 nM to 0.2-2.5 nM in patients with a wide range of cardiac dysfunctions (3, 4, 18). Thus after cardiopulmonary bypass operations both the in situ and free grafts will be exposed to equally high, often nM concentrations of APII. It is not yet known to what extent the human arterial and venous segments used as grafts respond directly or indirectly to APII compared with other vasodilating peptides released from perivascular nerves, such as vasoactive intestinal peptide (VIP) and calcitonin gene related peptide (CGRP). These neuronal peptides are potent relaxants of gastric, splenic, hepatic and cerebral arteries (8). We now report significant differences found in the effects of APII on noradrenaline-induced u2-contractures in isolated ITA and SV segments. The influence of endothelial integrity on these differences was also elucidated. MATERIALS AND METHODS Tissue collection. Segments of ITA and SV which are routinely cut away and discarded in the course of coronary bypass operations were obtained from patients with angina pectoris and, with institutional approval, used for in vitro experiments. Patient premedication included diazepam, droperidol and fentanyl and anaesthesia was induced with diazepam, fentanyl and pancuronium. The patients were ventilated with 50% O2 and isoflurane 0.2-1.0%. Glyceryl Scand J Thoracic 26
Scand Cardiovasc J Downloaded from informahealthcare.com by McMaster University on 02/15/15 For personal use only.
136
S. Aardal et al.
nitrate infusion was begun before the induction of anaesthesia and maintained until the start of cardiopulmonary bypass. For the in vitro experiments 8-12 mm long ITA segments were obtained from the distal end with a portion of the adjacent transverse thoracic muscle still attatched, while 10-15 mm long SV segments were taken from the proximal end of the vessel. Isometric recordings of contractile tension. The segments were subdivided into 3 mm long rings and mounted between stainless steel bars in thermostatically controlled organ baths containing 2.5 ml oxygenated Krebs Henseleits medium (95% O2and 5% COJ at 37°C. In one segment the endothelium was injured by probing, and the other was left intact. The time from surgical removal of the segments until mounted and stretched was < 1 h. The medium contained (mM) NaCl 118, KC1 4.7, CaCl, 2.5, MgSO, 1.2, NaHCO, 25, KH2P04 1.2 and glucose 10.1. Tension was recorded isometrically as previously described (1). The half maximally effective (ED,,) concentration of noradrenaline (NA) at different loads was 2 . 6 ~ M for ITA and 2 . 6 ~ M for SV at 15 mN. This preload corresponded to the optimum on the length-tension curves for both ITA and SV. The stretched preparations were repeatedly washed for 60 min before the analyses of agonists. Relaxation of the contracted ITA was quantified as the decline in tension 4 min after addition of the agonist and expressed in O/o of the maximal relaxation, i.e. back to the level of the resting tension (100%). Relaxation of the NA-contracted SV was quantified as the decline from T,,, measured 12 and 18 min (T,2 and T,& after the onset of the tension rise (To). Agonists were added as indicated in cumulative concentrations. Potency was expressed as the negative logarithm to the agonist concentration giving half-maximal inhibitory effect (pD,). The intrinsic activity (a) was calculated as the ratio of the maximal effect to the value for maximally obtainable relaxation. After each cycle the segments were washed every 5 min until the tension had returned to resting level and at least twice thereafter. Each segment was exposed to 4-5 cycles of agonist additions in order to detect tachyphylactic or potentiating effects or receptor activation. In control experiments the contractile activity could be maintained for 5-8 hours. Morphology. In order to verify the degree of endothelial injury, the probed and unprobed ITA and SV segments were carefully removed from the organ baths after the experiments and fixed by overnight immersion in 2 Yo buffered glutaraldehyde. The segments were cut into halves with a razor blade and processed for scanning electron microscopy according to conventional procedures. They were further dried by critical point, using COz as drying solvent. The intimal surfaces were coated with gold in a Polaron sputtering coating unit, and viewed in a Phillips 500 SEM electron microscope at an accelerating voltage of 25 kV. Statistical analyses. The results are expressed as means 2 SD for segments from (n) patients. Significance of differences between means was calculated Scand J Thoracic 26
with the Wilcoxon rank test for nonparametric, dependent and independent groups. Statistical significance was assumed when p
