The use of Mg2+-supplemented hyperkalemic cardioplegia preserves microvascular function. However, the mechanism of this beneficial action remains to be elucidated. We investigated the effects of Mg2+ supplementation on the regulation of intracellular calcium concentration ([Ca2+]i) and vascular function using an in vitro microvascular model. Ferret coronary arterioles (80–150 μm in diameter) were studied in a pressurized (40 mmHg) no-flow, normothermic (37°C) state. Simultaneous monitoring of internal luminal diameter and [Ca2+]iusing fura 2 were made with microscopic image analysis. The microvessels (n = 6 each group) were divided into four groups according to the content of MgCl2 (nominally 0, 1.2, 5.0, and 25.0 mM) in a hyperkalemic cardioplegic solution ([K+] 25.0 mM). After baseline measurements, vessels were subjected to 60 min of hypoxia with hyperkalemic cardioplegia (equilibrated with 95% N2-5% CO2) containing each concentration of Mg2+([Mg2+]) and were then reoxygenated. During hyperkalemic cardioplegia, [Ca2+]iincreased in a time-dependent manner in all groups. In the lower [Mg2+] cardioplegia groups, [Ca2+]iwas significantly increased at the end of the 60-min cardioplegic period (247 ± 44 nM and 236 ± 49 nM in [Mg2+] 0 and 1.2 mM groups, respectively; both P < 0.05 vs. baseline) with 19.6–17.2% vascular contraction. Conversely, there was no significant [Ca2+]iincrease in the higher [Mg2+] cardioplegia groups and less vascular contraction (5.4–4.1%, bothP < 0.05 vs. [Mg2+] 1.2 mM group). After reperfusion, agonist (U-46619, thromboxane A2 analog)-induced vascular contraction was significantly enhanced in the lower [Mg2+] cardioplegia groups (both P < 0.05 vs. control) but was normalized in the higher [Mg2+] cardioplegia groups. Intrinsic myogenic contraction was significantly decreased in the lower [Mg2+] cardioplegia groups (both P < 0.05 vs. control) but was preserved in the higher [Mg2+] cardioplegia groups. These results suggest that supplementation of the solution with >5.0 mM [Mg2+] may prevent hyperkalemic cardioplegia-related intracellular Ca2+ overloading and preserve vascular contractile function in coronary microvessels.
- coronary microvessel
the coronary microcirculation plays a central role in the regulation of myocardial perfusion, which in turnmay affect myocardial contractile function. Recently, the influence of various surgical cardioplegic solutions on coronary microvascular function has received attention. Hyperkalemic cardioplegic solutions have been used to achieve cardiac arrest and to protect the myocardium during cardiac operations. However, there is abundant evidence that ischemic cardiac arrest using a hyperkalemic cardioplegic solution significantly changes the response of coronary microvessels to various vasoactive agents (13,15, 20, 25). The pathophysiology underlying this hyperkalemic cardioplegia-related microvascular dysfunction is likely to be multifactorial and, at present, not fully understood. However, intracellular calcium ([Ca2+]i) overloading in coronary vascular smooth muscle could play a critical role in the development of microvascular dysfunction. The cellular mechanism of Ca2+ accumulation has been related, in part, to the high K+ concentration and insufficient oxygen supply in conventional crystalloid cardioplegic solutions.
It is well known that magnesium is an important ionic modulator of blood vessel tone. Indeed, Mg2+has been characterized as an endogenous calcium channel blocker that relaxes vascular smooth muscle and attenuates the vasoconstriction induced by several vasoactive drugs (1). The importance of Mg2+ in cardioplegic solutions has been increasingly recognized in the maintenance of myocardial function after surgical ischemia (5,8, 24). Recently, we demonstrated that hypermagnesium cardioplegia can preserve coronary microvascular function after surgical cardiac arrest compared with a magnesium-free hyperkalemic cardioplegic solution (23). However, the molecular basis for the action of magnesium in the vascular system is not well known. The relationship between extracellular Mg2+ concentration ([Mg2+]) and [Ca2+]iduring or after hyperkalemic cardioplegia has not yet been well characterized.
To simultaneously assess both intracellular Ca2+ changes in coronary smooth muscle cells and microvascular function, we established an in vitro microvascular model that could simulate the regulation of coronary microcirculation under reduced oxygen supply with crystalloid cardioplegia in the operating room. The aim of the present study was to investigate the effects of Mg2+ supplementation on [Ca2+]iregulation in coronary microvessels and vascular function during and after hyperkalemic cardioplegia.
METHODS AND MATERIALS
Isolated Microvessel Preparations
The methods for isolation of coronary microvessels were described previously (20). Briefly, male ferrets (8–12 wk old) were anesthetized with chloroform, and their hearts were removed into cold (4°C) Krebs-physiological saline solution (Krebs-PSS), which consisted of the following ionic concentrations (in mM): 119.0 NaCl, 25.0 NaHCO3, 4.6 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1.8 CaCl2, and 11.0 glucose. Coronary arterial microvessels (80–150 μm ID) were dissected from the left anterior descending artery-dependent subepicardial region in the left ventricle using a ×10–60 dissecting microscope (Olympus Optical, Tokyo, Japan). During dissection, care was taken to remove as much of the surrounding myocardium as possible and to avoid stretching and rubbing the intimal surface against foreign material. Microvessels were transferred to an experimental chamber in which both ends of the microvessel were cannulated with dual glass micropipettes (tip interior diameter, ∼60 μm) and secured with 10-0 nylon monofilament suture (Ethicon, Somerville, NJ). The chamber was mounted on a transillumination system and oxygenated (95% O2-5% CO2). Krebs-PSS (37°C) was continuously circulated through the tissue chamber. In all experiments, the presence of intact endothelium was confirmed by determining the vasodilative response to acetylcholine (10−6 M) in microvessels precontracted with potassium ions (20 mM).
All of the animals received humane care in compliance with theGuide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85–23, Revised 1985).
Intraluminal diameter measurement. The vessels were pressurized to 40 mmHg in a no-flow state using a burette manometer filled with Krebs-PSS. The internal luminal diameter was measured with a video-monitored microscopic system (model KP-115, Zeiss IM35 and Hitachi CCD TV camera). The calibration of the measurement was performed using an 80-μm tungsten wire. The minimum resolution of the system was 1.5 μm.
[Ca2+]iof coronary microvascular smooth muscle was measured using calcium-sensitive fluorescent dye fura 2. Coronary microvessels in the tissue chamber were loaded with 5 μm acetoxymethyl ester of fura 2 (fura 2-AM) in Krebs-PSS containing 0.05% dimethyl sulfoxide and 0.01% Pluronic F-127. The loading time was 45 min followed by a 30-min wash period at 37°C. The objective lens used was a Nikon Fluor ×40 (NA 0.8). To avoid the influence of fluorescent signals from the endothelium, optimal focus was adjusted to the middle of the microvascular smooth muscle layer by viewing the microvascular wall under a bright microscopic field. Excitation light at 350 ± 5 and 390 ± 6 nm was used. Emission at 510 ± 24 nm was monitored with a photomultiplier tube (Hamamatsu R928) and digitized by a Data Acquisition-EZ A/D Converter. The digital signal of the two wavelengths was processed using a program written using the DTVee version 3.0 programming environment (Data Translation). [Ca2+]iwas estimated from the ratio (R) of measured fluorescence signals (F) elicited at two wavelengths according to the equation: R = (F350mv − F350bg)/(F390mv− F390bg), where F350mv and F390mv are the total measured fluorescence of the microvessels at wavelengths of 350 and 390 nm, respectively, and F350bg and F390bg are the background fluorescence signals at the respective wavelengths. The background signals were measured on microvessels before fura 2-AM was loaded. Particular care was taken to minimize possible photobleaching of fura 2 molecules.
Quantification of [Ca2+]i.
With the ratio method applied, intermicrovascular differences in fura 2 loading, microvascular thickness, light path length, and camera gain were canceled; therefore, the ratio values reflected true [Ca2+]idifferences. For quantification of [Ca2+]i, we used an in situ dual-wavelength calibrating equation of Grynkiewicz et al. (7): [Ca2+]i= K d [(R − Rmin)/(Rmax− R)]β, whereK d (224 nM) is the effective dissociation constant, β is the ratio of the fluorescence at 390 nm with 0 Ca2+to the 390 nm fluorescence with 1.2 mM Ca2+. The maximum fluorescence ratio (Rmax) of fura 2 is observed when the dye is completely bound by Ca2+ in a solution containing 1.2 mM CaCl2 plus 50 μm 4-bromo-A23187. After that, EGTA (5 mM) was added to achieve the minimum fluorescence ratio (Rmin). In these in situ calibration experiments, Rmin was highly consistent and reproducible with little variation; Rmax, however, was more variable. Values from the in situ microvessel calibration were Rmin = 0.35, Rmax = 1.22, and β = 1.10.
After a 60-min stabilizing period, measurements of [Ca2+]iand internal luminal diameter were taken (baseline control). Microvessels were divided into four groups according to the content of MgCl2 (nominally 0, 1.2, 5.0, and 25.0 mM) in hypoxic, hyperkalemic cardioplegic solution. Hypoxia was induced by switching bubbling gas from 95% O2-5% CO2 to 95% N2-5% CO2. The composition of hyperkalemic cardioplegic solution was (in mM) 121 NaCl, 25 KCl, 12 NaHCO3, 1.2 CaCl2, and 11.1 glucose; pH 7.45, oxygen tension 5–30 mmHg. True anoxic condition was not achieved because a small amount of oxygen continuously diffused into the hyperkalemic cardioplegia from the atmosphere. The temperature was maintained at 37°C. All microvessels were subjected to 60 min of hypoxic, hyperkalemic cardioplegic solutions with each concentration of MgCl2 and then reperfused with oxygenated Krebs-PSS for 60 min. During 60 min of hypoxic, hyperkalemic cardioplegia and after 60 min of normoxic reperfusion, [Ca2+]iand internal luminal diameter were measured every 15 min. After 60 min of reperfusion, a cumulative concentration-response curve to a stable thromboxane A2 analog (U-46619, 10−9-10−6M) was constructed to evaluate the agonist-induced vascular contractility. The vessels were washed with a drug-free Krebs-PSS for 30 min. After the vessels were equilibrated for at least 30 min, the active pressure-diameter relation was studied. Initially, the pressure was reduced to 10 mmHg to stabilize for 10 min. Then the pressure was increased in increments of 10 mmHg up to 100 mmHg. At each pressure increment, the change in internal luminal diameter was measured after microvessel diameter had stabilized (generally after 3 min). Once the determination of the active pressure-diameter relation was completed, the pressure was returned to 50 mmHg, and finally, papaverine (100 μM) was applied in the tissue chamber to normalize the vascular diameter. The normalized diameter was defined as the ratio of the diameter observed at a given transmural pressure to the diameter of the same vessel at 50 mmHg pressure in the presence of papaverine.
To test the osmotic influence of supplemented MgCl2 in the hyperkalemic cardioplegic solutions, a subset (n = 6) of experiments was performed in which adequate sucrose was added to the 1.2 mM [Mg] group to raise the osmolarity equal to that in the 25 mM [Mg] group (390–400 mosmol/l).
Fura 2-AM and Pluronic F-127 were obtained from Molecular Probes (Eugene, OR). U-46619 was purchased from Sigma Chemical (St. Louis, MO). Papaverine was obtained from Eli Lilly (Indianapolis, IN). All solutions were prepared on the day of the study.
The response of microvessels to each intervention was examined only once in each animal. Therefore, each animal served as one sample, andn refers to the number of animals from which microvessels were taken in all experiments. In the microvessel contraction experiments, vessels not having cardioplegic intervention were taken as control, and changes in internal luminal diameter were expressed as the percent contraction of the baseline diameter. Results are expressed as means ± SE. The paired Student’st-test was applied for within-group comparisons with baseline. ANOVA followed by a multiple-comparison Fisher’s test was used to test the differences among groups with different interventions (StatView 4.0; Abacus Concepts, Berkeley, CA). The probability was considered to be significant if theP value was <0.05.
Characterization of Isolated Coronary Microvessels
Before exposure of microvessels to hyperkalemic cardioplegia, there were no significant differences in either the baseline vascular diameter or [Ca2+]iin smooth muscle among the four groups (vascular diameter: 102 ± 15 to 116 ± 18 μm; [Ca2+]i: 74 ± 17 to 79 ± 22 nM). After reperfusion, there were no significant differences in the papaverine-applied vascular diameter among the groups (115 ± 19 to 126 ± 16 μm).
With respect to the osmotic influence of hyperkalemic cardioplegia ([Mg] 1.2 mM), the sucrose-added cardioplegia caused changes in microvascular diameter and [Ca2+]isimilar to the no-sucrose-added cardioplegia (peak vascular contraction during cardioplegia: 15.6 ± 2.8% vs. 17.2 ± 3.6%; peak [Ca2+]iduring cardioplegia: 218 ± 50 vs. 236 ± 49 nM; sucrose added vs. no sucrose added, respectively; bothP > 0.05). These observations effectively ruled out a nonspecific osmotic effect of increased concentration of MgCl2 in the present study protocols.
Intracellular Ca2+ Dynamics
Figure 1 shows time-course dynamics in [Ca2+]iduring and after hyperkalemic cardioplegia. On exposure to the hyperkalemic cardioplegic solution, [Ca2+]iin the lower [Mg2+] groups increased gradually in a time-dependent manner, and these increases were significantly different from the baseline level at the end of the 60-min cardioplegic period (247 ± 44 and 236 ± 49 nM, in [Mg2+] 0 and 1.2 mM groups, respectively; both P< 0.01 vs. baseline; between-group differences were not significant). However, a slight increase in the [Ca2+]ioccurred in the higher [Mg2+] groups during hyperkalemic cardioplegia, but these increases were not statistically significant compared with the baseline value (102 ± 35 and 86 ± 27 nM, in [Mg2+] 5.0 and 25.0 mM groups, respectively; both P > 0.05 vs. baseline). After reperfusion with oxygenated Krebs-PSS, the [Ca2+]ireturned to its baseline level within 15 min in all groups.
Changes in Diameter
As shown in Fig. 2, in the lower [Mg2+] groups percent contraction increased gradually during the cardioplegic period, and at the end of the 60-min cardioplegic period the percent contraction reached 19.6 ± 4.2% and 17.2 ± 3.6% in 0 and 1.2 mM [Mg2+] groups, respectively. In the higher [Mg2+] groups, percent contraction reached a maximum level within the initial 15 min of cardioplegia, which sustained during the cardioplegic period. The peak values were 5.4 ± 2.0% and 4.1 ± 1.7% in 5.0 and 25.0 mM [Mg2+] groups, respectively (both P < 0.05 vs. [Mg2+] 0 and 1.2 mM groups). After reperfusion, the vascular diameter recovered to its initial value within 15 min of reperfusion in all groups.
Vascular Contractility After Reperfusion
The agonist-induced contractile responses to U-46619 were significantly increased in the 0 and 1.2 mM [Mg2+] groups (bothP < 0.05 vs. control), whereas those in the 5.0 and 25.0 mM [Mg2+] cardioplegic groups were not altered compared with control. These differences were most pronounced at the higher concentrations of U-46619 tested (Fig.3).
Intrinsic myogenic contraction was observed to a stepwise increase in the transmural pressure >50 mmHg in control vessels. In vessels from higher [Mg2+] groups, similar myogenic contractions were observed, but cardioplegia caused an upward shift in the active pressure-diameter relation (bothP < 0.05 vs. control). However, the myogenic contractions were abolished in the lower [Mg2+] groups (bothP < 0.05 vs. [Mg2+] 5.0 and 25.0 mM; Fig. 4).
Changes in [Ca2+]iand Vascular Contraction During Hyperkalemic Cardioplegia
Despite the fact that magnesium has been shown to protect the myocardium and coronary vasculature from hyperkalemic cardioplegia-related cardiac injury, its effect on regulating intracellular Ca2+ in coronary microvessels has not been well elucidated. The present study provides experimental evidence that a hyperkalemic cardioplegia containing a physiological concentration of Mg2+ (1.2 mM) causes a marked intracellular Ca2+ accumulation and significant vascular contraction, whereas a higher concentration of Mg2+ (>5.0 mM) can prevent this Ca2+ overloading and contraction during hyperkalemic cardioplegia.
The mechanism responsible for the hyperkalemic cardioplegia-related [Ca2+]iaccumulation in vascular smooth muscle is most likely related to membrane depolarization on the basis of Nernst’s equation (17). Membrane depolarization promotes Ca2+ influx through voltage-dependent Ca2+ channels. Ca2+ influx also induces release of Ca2+ from intracellular Ca2+ stores (4). In addition, during surgical cardioplegia, especially using nonoxygenated crystalloid cardioplegic solutions, coronary microvessels are exposed to conditions of hypoxia. This is associated with a lower production of ATP compared with a normoxic state. It is widely recognized that [Ca2+]iis elevated during and after periods of hypoxia. In previous studies, it was suggested that transsarcolemmal Ca2+ influx via Na+/Ca2+exchange may play an important role in hypoxia/reoxygenation-mediated Ca2+ accumulation (12, 22). Recently, other investigators have reported that Ca2+ release from the sarcoplasmic reticulum may contribute to hypoxic pulmonary vasoconstriction (6, 10).
High Mg2+ concentration has been suggested to inhibit Ca2+ entry into the cell by displacing Ca2+from binding sites in the calcium channels and by hyperpolarization of sarcolemmal membrane (9). It has also been postulated that extracellular Mg2+ acts by raising intracellular Mg2+ concentration, thereby reducing the release of Ca2+ from the sarcoplasmic reticulum (3, 24). In addition, supplementation of Mg2+ in cardioplegic solutions may diminish the depletion of ATP stores, thereby protecting the intracellular metabolic function of microvascular smooth muscle. Accordingly, it seems that multiple cellular mechanisms may be involved in these beneficial actions of Mg2+ supplementation.
Indeed, it should be noted that the present study examined the effects of hyperkalemic cardioplegic solutions containing a physiological concentration of calcium (CaCl21.2 mM). Therefore, it is possible that a reduced Ca2+ concentration in a hyperkalemic cardioplegia might require a lower level of Mg2+ supplementation to achieve microvascular protection from intracellular Ca2+ overload. In clinical practice, however, it is more difficult to precisely regulate the cardioplegic Ca2+ concentration because of transient variables such as pH and temperature. As a result, patients are at risk of exposure to higher than originally intended Ca2+ levels, which may increase the likelihood of the Ca2+-mediated vascular injury. In addition, further decreasing the Ca2+ level may cause a calcium paradox in coronary microvessels. Therefore, we emphasize that the addition of Mg2+ may solve this dilemma by allowing for the safe use of higher cardioplegic Ca2+ concentrations, and we recommend sufficient supplementation of Mg2+ (>5.0 mM) in hyperkalemic cardioplegic solutions.
Changes in Vascular Contractility After Hyperkalemic Cardioplegia
One purpose of this study was to develop a better understanding of the pathophysiology in microvascular contractile dysfunction after hyperkalemic cardioplegia. In the present study, we demonstrated that in the lower [Mg2+] groups the agonist (U-46619)-induced vascular contraction was markedly enhanced, whereas the intrinsic myogenic contractile response was significantly diminished. Conversely, in higher [Mg2+] groups the agonist-induced and myogenic responses were preserved. (Figs. 3 and 4). Although the explanations for these phenomenon remain unclear, they may be attributed to endothelial dysfunction or altered contractile properties of vascular smooth muscle, or both.
We and others (15, 20) have previously showed a progressive deterioration of the endothelial-dependent relaxation in the coronary microcirculation after surgical cardioplegia. Furthermore, our observations in this study are in agreement with the findings of Pearson and associates (16), who showed that hypomagnesemia could impair the release of nitric oxide from the coronary endothelium and promote vasoconstriction. In addition, we have previously reported that a Mg2+-based cardioplegic solution ([Mg2+] 25.0 mM) prevents much of the impairment in endothelium-dependent relaxation observed after exposure of microvessels to a purely hyperkalemic cardioplegic solution (23). Therefore, it is likely that hyperkalemic cardioplegia without sufficient Mg2+ supplementation may impair the endothelial function, including the release of nitric oxide, and predispose the patient to vascular hypercontraction in response to a vasoconstrictive agonist. Accordingly, it would be tempting to speculate that sufficient Mg2+supplementation would afford endothelial protection, although the present study was not designed to provide direct evidence about functional implication of the endothelium.
It may also be possible that enhanced Ca2+ accumulation in the vascular smooth muscle may activate a Ca2+-dependent intracellular signaling pathway and alter the Ca2+ sensitivity of the contractile apparatus. Previous studies have demonstrated that an agonist-induced vascular tone is regulated by myosin light chain kinase, the activity of which is governed by a Ca2+-calmodulin-mediated phosphorylation (19). There is some evidence that [Ca2+]imay also directly activate the Ca2+-dependent isoforms of protein kinase C (conventional protein kinase C) and lead directly or indirectly to the phosphorylation of an entirely different subset of cellular proteins, including caldesmon, a number of intermediate filament proteins (desmin, synemin), and a few cytosolic proteins (18). Therefore, it is reasonable to propose that the [Ca2+]ioverloading during cardioplegia is a strong trigger for enhancement of agonist-induced vascular contraction after reperfusion.
Myogenic tone is a property of vascular smooth muscle manifested by contraction in response to the increase in transmural pressure and is involved in the autoregulation of coronary perfusion. Although not completely understood, the contribution of adenosine triphosphate-sensitive potassium ( ) channels to myogenic contraction was recently implicated (11). The channels open when the intracellular ATP concentration falls to <1 mM (21). Opening of the channels causes membrane hyperpolarization and relaxes the vascular smooth muscle by preventing Ca2+ entry via voltage-sensitive Ca2+ channels. In a previous study, the channel blocker glibenclamide preserved myogenic reactivity after hyperkalemic cardioplegia (26). Taken together these observations and the results of the present study strongly suggest that supplementation with a higher concentration of Mg2+ (>5.0 mM) may inhibit activation of channels by preventing intracellular ATP depletion in vascular smooth muscle and preserving the intrinsic myogenic contraction.
The present study was designed to monitor intracellular Ca2+ accumulation and vascular function during and after hyperkalemic cardioplegia. This in vitro microvascular model uses a hypoxic, hyperkalemic cardioplegic solution that simulates the insufficient oxygen supply with crystalloid cardioplegia in the operating room. In this study, we aimed to address the efficacy of Mg2+supplementation with the view of coronary microcirculatory protection. Coronary microvascular injury in stressed hearts is extremely important, because this microvascular function may be more vulnerable and sensitive to surgical hyperkalemic cardioplegia compared with myocardial function. Hyperkalemic cardioplegia-related coronary microcirculatory dysfunction in the setting of minimal changes in myocardial contractile function using a cardiopulmonary bypass model has recently been reported (23).
There are a number of Ca2+-sensitive indicators, such as aequorin, indo-1, and fura 2. In the present study, we used the fura 2 microscopic technique to monitor the real-time changes of [Ca2+]iin microvascular smooth muscle. Although fura 2 is useful for the evaluation of [Ca2+]i, it must be noted that determination of the absolute value of [Ca2+]ifrom the fura 2 fluorescence ratio is still problematic; it cannot be totally excluded that changes in background autofluorescence of microvessels, compartmentation of fura 2 into intracellular organelles such as sarcoplasmic reticulum, or the deesterized form of fura 2-AM cause fluorescence signals unrelated to [Ca2+]i. For quantification of [Ca2+]i, we used an in situ dual-wavelength calibration by obtaining Rmax and Rmin and thus estimated cytosolic [Ca2+]i. In these calibration experiments, Rmin was highly consistent and reproducible with little variation; Rmax, however, was more variable. Similar variability in Rmax values has been previously reported (14). The variation in Rmax values may be related to limited effectiveness of the calcium ionophore 4-bromo-A23187 in this microvascular model. Although the [Ca2+]ivalues in the present study were similar to those reported by other investigators (2, 14), they may have to be revised when better methods to calibrate fura 2 fluorescence become available.
In conclusion, we have demonstrated that supplementation of >5 mM [Mg2+] possibly prevents hyperkalemic cardioplegia-related intracellular Ca2+ overloading and preserves vascular function in coronary microvessels. To protect coronary microcirculation from Ca2+-related vasospasm under cardiac surgery, these findings appear to be significant and clinically relevant and require further investigation for the development and manipulation of pharmacological strategies.
The authors express gratitude to Prof. Shigetsugu Ohgi (Tottori, Japan) for the continuous encouragement.
Address for reprint requests and other correspondence: F. W. Sellke, Division of Cardiothoracic Surgery, Beth Israel Deaconess Medical Center, East Campus, Dana 905, 330 Brookline Ave., Boston, MA 02215 (E-mail:).
This study was supported by National Heart, Lung, and Blood Institute Grants HL-46716 (to F. W. Sellke) and HL-31704 (to K. G. Morgan).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
- Copyright © 1999 the American Physiological Society