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J Thorac Cardiovasc Surg 1999;118:173-180
© 1999 Mosby, Inc.
CARDIOPULMONARY SUPPORT AND PHYSIOLOGY |
From the Cardiovascular Research Laboratory, Grantham Hospital, and Division of Cardiothoracic Surgery, Department of Surgery, University of Hong Kong, Aberdeen, Hong Kong.
Supported in part by Hong Kong Research Grants Council grant (HKU7280/97M), the University of Hong Kong Committee of Research and Conference Grants (337/048/0018, 335/048/0079), and the University of Hong Kong Grants (014/048/9602, 344/048/0001).
Address for reprints: Professor Guo-Wei He, MD, PhD, Chair of Cardiothoracic Surgery, University of Hong Kong, Grantham Hospital, 125 Wong Chuk Hang Rd, Aberdeen, Hong Kong.
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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Introduction |
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1,2 In addition, attention has been paid to the effect of this solution on coronary endothelial function. 3,4
Vascular endothelium plays a key control role in the regulation of vascular tone through releasing diverse vasoactive substances. Among those, endothelium-derived relaxing factors (EDRFs) are responsible for endothelium-dependent relaxation. 5 The major components of EDRFs have been shown to be endothelium-derived nitric oxide (EDNO), epoprostenol (prostacyclin [PGI2]), and endothelium-derived hyperpolarizing factor (EDHF). 6,7 It is generally considered that EDHF plays a significant role in the regulation of microvasculature and thus of blood flow distribution. 8 Because the coronary microvascular system plays the key role in the regulation of myocardial perfusion, the investigation on the effect of STS on EDHF-mediated function in the coronary microvascular system is particularly important during cardioplegic arrest. 9 However, this has not been studied.
Potassium (K+) at high concentrations (hyperkalemia) is a known vascular irritant. 10,11 Hyperkalemia has been shown to damage endothelium-dependent relaxation. 4,12,13 In those studies, the K+ concentrations ranged from 16 to 125 mmol/L. Recently, we have performed a series of experiments that show that hyperkalemia impairs EDHF-mediated endothelial function in large conduit coronary arteries. 7,14-16 However, the components of STS are complex; the effect of other components in STS, such as magnesium and procaine, and the effect of STS as a whole remain unknown. Further, the previous studies regarding the effect of hyperkalemia on the EDHF-mediated function were performed in large coronary arteries and whether this is true in the more important resistant arteries (microarteries) is unknown.
In addition, 2 other factors (ie, temperature and washout time) may also affect the possible effect of STS on the endothelial function.
The present study was therefore designed to examine the effect of STS on EDHF-mediated endothelial function when the EDNO and PGI2 were inhibited by specific inhibitors in porcine coronary microarteries, with emphasis on the effect of temperature and washout time.
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Materials and methods |
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,17 and another wire was passed lightly through the vascular lumen and anchored another jaw of the same chamber. These 2 wires were attached to a force transducer or to a micrometer, respectively. An adjustable micrometer can pull the jaws apart and stretch the artery between the 2 parallel wires. A calibrated force transducer was used to measure the force, with the output shown on a computer screen and printed in a printer. Data were digitized and stored in the computer. The Krebs solution had the following composition: Na+, 144 mmol/L; K+, 5.9 mmol/L; Ca2+, 2.5 mmol/L; Mg2+ ,1.2 mmol/L; Cl–, 128.7 mmol/L; HCO3 –, 25 mmol/L; SO42–, 1.2 mmol/L; H2PO4 –, 1.2 mmol/L; and glucose, 11.0 mmol/L. During normalization or relaxation studies, the solution was aerated with a gas mixture of 95% oxygen and 5% carbon dioxide at 37°C.
Two organ chamber arrangements in the same myograph were run concurrently.
Normalization
The vessels were equilibrated for 45 minutes. The arterial rings were progressively stretched until the passive transmural pressure reached 100 mm Hg, and then the pressure was immediately released. The computerized program determines the length-tension exponential curve for each ring and gives the internal circumference and diameter at a pressure of 100 mm Hg. 17,18 The internal circumference was then set to the value, estimated to be equivalent to 90% of the circumference at a passive transmural pressure of 100 mm Hg. 17 Only arterial rings in internal diameter of 200 to 450 µm at the pressure of 100 mm Hg were used in the present study. The experiment was performed at the internal diameter set up at 90% of the diameter (D0.9) under a pressure of 100 mm Hg that is determined by the computer program. 17,18 The transluminal pressure, read from the computer recording, at this diameter (D0.9) was between 55 and 70 mm Hg in our experiment. The whole experiment was then performed under this passive pressure (tension) on the vessel. The isometric force at this setting is the "passive" or "resting" force in the absence of constrictor tone. This method allows one to set the vessel in vitro at a physiologic pressure and has been shown to allow maximum tension development. In the myograph (model 500A), the normalization procedure was performed automatically. The diameter of the vessel at 100 mm Hg was determined by the length-tension curve during the normalization procedure.
Protocol
Precontraction. The vessel was contracted with thromboxane A2 mimetic U46619 (–8.5 log M). This dose was chosen because, in our experiments, the median effective concentration EC50 for U46619-induced contraction was –8.78 ± 0.14 log M (n = 9 taken from 9 pig hearts), calculated from the concentration-response curves in porcine coronary microarteries. U46619 at –8.5 log M caused a stable submaximal contraction of all rings.
EDHF-mediated (indomethacin and L-NNA–resistant) relaxation. With the presence of the specific inhibitor for cyclooxygenase indomethacin (7 µm) and the EDNO synthase inhibitor NG-nitro-L-arginine (L-NNA, 300 µmol/L), concentration-relaxation curves to bradykinin (–10 to –5.5 log M) were established when the U46619-induced contraction reached a plateau. This relaxation is considered to be related to the EDHF function because EDNO and PGI2 are blocked by indomethacin and L-NNA. The following protocol was used.
EXPERIMENTAL GROUP I: PROFOUND HYPOTHERMIA (4°C)
GROUP IA. The washout time was 45 minutes. In cold Krebs solution, 1 coronary arterial segment was cut into 2 pieces, each 2 mm long. Two chambers of the duel-chamber myograph was filled with either STS or Krebs solution (as the control) at 4°C, respectively. Two rings were separately mounted in the 2 chambers, and the myograph was then placed in a refrigerator at 4°C for 4 hours. After storage for 4 hours, both rings were repeatedly washed with Krebs solution of 37.0° ± 0.1°C that had been aerated with 95% oxygen and 5% carbon dioxide gas. The normalization procedure was then performed, and 7 µmol/L indomethacin and 300 µmol/L L-NNA were added to the chambers. Twenty minutes later, the rings were precontracted with U46619 (–8.5 log M). Cumulative concentration-response curves to bradykinin were then established. The washout time (the time between the washout of the cold STS or Krebs solution with warm Krebs solution and the start of the concentration-relaxation curve to bradykinin) was controlled at 45 minutes.
The composition of STS is Na+, 138 mmol/L; K+, 20 mmol/L; Ca2+, 2.7 mmol/L; Mg2+, 16 mmol/L; Cl –, 157 mmol/L; HCO3–, 8 mmol/L; lactate, 28 mmol/L; and procaine, 1 mmol/L. The osmolarity is 370.
GROUP IB. The washout time was 90 minutes. In this group of experiments, 2 arterial rings (2 mm long) obtained from the same coronary arterial segment were immersed in either STS or Krebs solution (as the control) at 4°C (in a refrigerator) for 4 hours. The rings were then taken from the refrigerator, repeatedly washed with Krebs solution of 37° ± 0.1°C, and mounted on the dual-chamber myograph. After equilibration for 20 minutes, the arterial rings were normalized. Indomethacin (7 µmol/L) and L-NNA (300 µmol/L) were added into the chambers. Twenty minutes later, the rings were contracted with U46619 (–8.5 log M). Cumulative concentration-response curves to bradykinin were then established. The washout time was controlled at 90 minutes.
EXPERIMENTAL GROUP II: MODERATE HYPOTHERMIA (22°C).
The arteries were incubated in either STS or Kreb's solution at room temperature (22°C). The exposure time to either STS or Krebs solution (as the control) was 1 hour. Similar to that mentioned earlier, the washout time was either 45 (group IIa) or 90 minutes (group IIb).
In all experiments, only 1 concentration-relaxation curve was obtained from each coronary ring. A mean concentration-relaxation curve was established from 7 to 9 rings in each group of experiments. During the experiments, the Krebs solution in the myograph chamber was continuously bubbled with a gas mixture of 95% oxygen and 5% carbon dioxide.
Data analysis
Mean maximal relaxation for each group was calculated from the maximal relaxation of different rings induced by bradykinin. The effective concentration of the relaxation agent that caused 50% of maximal relaxation was defined as EC50. The EC50 was determined from each concentration-relaxation curve by a logistic, curve-fitting equation:
E = MAP/(AP + KP)
where E is response, M is maximal relaxation, A is concentration, K is EC50 concentration, and P is the slope parameter. 18 From this fitted equation, the mean EC50 value ± the SEM was calculated for each group.
Statistical analysis
All statistical analysis was performed with SPSS software (SPSS, Inc, Chicago, Ill). The significance of the difference between mean values was calculated by the paired Student t test. Results are expressed as mean ± SEM for observations of "n," where "n" equals the number of coronary arterial rings.
Drugs
Chemicals used and their sources were as follows: bradykinin, L-NNA, and indomethacin (Sigma Chemical Co, St Louis, Mo); U46619 (Cayman Chemical, Ann Arbor, Mich). L-NNA (dissolved in distilled water) and indomethacin (dissolved in ethanol) were stored at 4°C. The solution of U46619 was held frozen until required. The STS was purchased from David Bull Laboratories, Mulgrave, Victoria, Australia.
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Results |
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U46619-induced contraction force
In all coronary arterial rings, U46619 (–8.5 log M) induced a stable and rapidly developing tension. This contraction force was reduced by exposure to STS when measured at washout for 45 minutes (group Ia, 9.5 ± 0.9 mN vs 14.6 ± 0.7 mN; n = 8; 95% CI, 3.1 to 7.2 mN; P = .000; group IIa, 8.9 ± 0.1 mN vs 13.0 ± 0.1 mN; n = 8; 95% CI, 2.4 to 5.9 mN; P = .001). However, when washout period was prolonged to 90 minutes, the contraction to U46619 had no difference compared with the control (group Ib, 14.1 ± 1.7 mN vs 14.6 ± 1.5 mN; n = 7; 95% CI, –2.8 to 3.8 mN; P = .7; and group IIb, 13.3 ± 0.1 mN vs 13.5 ± 0.1 mN; n = 9; 95% CI, –2.2 to 2.5 mN; P = 0.9). These results demonstrate that the smooth muscle contractility was impaired by exposure to STS within 45 minutes after exposure but fully recovered after 90 minutes (Fig. 1).
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GROUP IA. With the presence of indomethacin (7 µmol/L) and L-NNA (300 µmol/L) in control rings incubated in Krebs solution, bradykinin induced a maximal relaxation of 69.0% ± 5.3% with EC50 of –7.24 ± 0.09 log M (Fig 2). Treatment with STS at 4°C for 4 hours followed by washout for 45 minutes significantly reduced bradykinin-induced maximal relaxation to 42.7% ± 3.5% (n = 9; 95% CI, 17.8% to 34.8%; P = .000; Fig 2, A) although there was no change on the EC 50 (–7.12 ± 0.15 log M; n = 9; 95% CI, –0.22 to 0.46 log M; P = .44) (Table II).
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). Similarly, there was no change on the EC50 (–7.23 ± 0.08 log M vs –7.35 ± 0.06 log M; n = 7; 95% CI, –0.12 to 0.38 log M; P = .25). These results demonstrate that the bradykinin-induced EDHF-mediated relaxation was impaired by exposure to STS for 4 hours at 4°C within 45 minutes after exposure but fully recovered after 90 minutes. Experimental group II: moderate hypothermia (22°C) . The experiments were performed at room temperature (22°C). The exposure time to either STS or Krebs solution (as the control) was 1 hour. Similar to that mentioned earlier, the washout time was either 45 (group IIa) or 90 (group IIb) minutes.
GROUP IIA. STS exposure at 22°C for 1 hour followed by washout for 45 minutes had a profound effect on bradykinin-induced maximal relaxation (12.3% ± 1.6% vs 56.1% ± 4.4% in the control; n = 8; 95% CI, 36.0% to 51.8%; P = .000; Fig 3, A) although the EC50 was not significantly changed (–7.02 ± 0.17 log M vs –7.06 ± 0.13 log M; n = 8; 95% CI, –0.43 to 0.52 log M; P = .82) (Table II).
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Discussion |
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Indomethacin- and L-NNA–resistant (EDHF-mediated) endothelial function in the coronary microarteries
Endothelial cells derive 3 major EDRFs. Compared with EDNO and PGI 2 that have been well studied, the nature of EDHF has not been finally identified, although most recently the cytochrome P450–mono-oxygenase metabolite of arachidonic acid has been suggested to be EDHF. 19-21 EDHF induces vascular smooth muscle relaxation by hyperpolarization of the smooth muscle cells, which may involve potassium (K+) channels. 7,22-24 In contrast, EDNO relaxes blood vessels through the cyclic guanosine monophosphate pathway. However, all of these EDRFs are released in response to the increase of intracellular (cytosolic free) calcium concentration in the endothelial cell. 6,7
Although the exact role of EDHF in regulating vascular tone and in the development of vascular diseases is still unclear, there is evidence that shows that EDNO and EDHF are 2 primary mechanisms of endothelium-dependent relaxation. 25,26 We have previously reported that EDHF plays a role in large conductance coronary arteries. 7,14-16 It has been suggested that EDHF plays an even more important role in the regulation of the vascular tone in microcirculation than in the large conductance arteries. 8 In the present study, all experiments were performed in the presence of the inhibitors of EDNO and PGI2 (L-NNA and indomethacin) and therefore the bradykinin-induced relaxation is EDHF mediated. We have previously reported that this relaxation is linked with a membrane hyperpolarization in the coronary conductance arteries 16 and in microarteries. 27 In our experiments, the EDHF-stimulus bradykinin induced 62.2% relaxation of the U46619-induced precontraction, which reveals the role of EDHF in the regulation of the tone in the microarteries. Studies have also shown that EDHF may back up or enhance the relaxing action of EDNO, particularly when EDNO-mediated relaxation is impaired 28 as seen in some pathologic status such as hypercholesterolemia, hypertension, and diabetes mellitus. 28 It may be true that in the coronary microcirculation during ischemia and reperfusion period, when the EDNO mechanism is impaired, 29 the EDHF-mechanism may play an important role in regulating the coronary circulation.
EDHF-related relaxation after STS exposure and possible mechanism
The effect of STS on the EDHF-mediated endothelial function is shown by the reduced relaxation induced by bradykinin after exposure to STS under profound hypothermia for 4 hours (group Ia) or under moderate hypothermia for 1 hour (group IIa), followed by washout for 45 minutes. These results clearly demonstrate that, during the initial reperfusion period, the EDHF-mediated relaxation in the microvessels is altered. We have previously reported that hyperkalemia damages the EDHF-mediated relaxation. 7,14-16 Because STS is hyperkalemic (20 mmol/L K +), this alteration is most likely related to the high K+. Hyperkalemia reduces the EDHF-mediated relaxation through the prolonged partial membrane depolarization and affecting K-channels. 14-16 This is also the likely mechanism in the microvessels.
Effect of washout time on EDHF-related relaxation
We previously observed that, as mentioned, the reduced EDHF-mediated relaxation is related to the prolonged partial depolarization of the smooth muscle membrane potential. 14-16 Logically, such an effect may be possibly reduced with prolonged washout time. We therefore designed 2 different washout periods to examine the EDHF-mediated relaxation. In this study, we are able to support our hypothesis because, in our experiments, it was demonstrated that with a prolonged washout time (90 minutes) the effect of STS on the EDHF-mediated relaxation was eliminated. However, in cardiac operations, the first 45-minute reperfusion period is probably the most critical time when the heart is just resuscitated from ischemia as the result of total arrest. Coronary dysfunction during this period may predispose the heart to reduced myocardial perfusion that is detrimental to the myocardial function.
Contractility of coronary artery after exposure to STS
Under both profound and moderate hypothermia, after exposure to STS, the contraction to U46619 was reduced (Fig. 1). This suggests that the coronary artery is still at a partially plegic status at 45 minutes after exposure to STS. Similar to the EDHF-mediated relaxation, the reduced contraction was fully recovered after 90 minutes. This shows that the reduced contraction after exposure to STS is also time dependent.
Clinical implications
The present study suggests that the EDHF-mediated endothelial function in the coronary microarteries is reduced after incubation with STS for either 4 hours at 4°C or 1 hour at 22°C. This effect is seen within the initial 45-minute reperfusion period and is eliminated at 90 minutes. Therefore during the initial reperfusion period, the altered EDHF-mediated relaxation in microcirculation may cause coronary endothelial dysfunction and have a detrimental effect on the myocardial perfusion.
Moderate hypothermia is commonly used in cardiac operations and 4-hour cold (4°C ) storage with STS is used for the donor heart preservation for transplantation operations. Therefore the present study has strong clinical implications with regard to the coronary dysfunction in the microcirculation in cardiac operations and heart transplantation.
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