INTRODUCTION

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VASCULAR SMOOTH MUSCLE (VSM) is the primary cellular component of the arterial wall, and VSM contractility mediates changes in, and maintenance of, blood pressure. In VSM, energy provision is tightly matched to energy demand (13, 25, 26). It has been proposed that VSM metabolism may be compartmented with ATP produced by glycolysis used preferentially to support membrane ion pumps, whereas ATP produced by oxidative phosphorylation is used preferentially to support ATPases associated with contraction (13, 25, 26). However, the relative importance of the specific substrates utilized for oxidative phosphorylation in VSM is not fully characterized.

Early studies of the respiratory quotient in VSM suggested that the primary substrate for the tricarboxylic acid (TCA) cycle, and thus oxidative ATP production, was glucose (18, 19). More recent analysis of the oxidation rates of various substrates in resting and contracting VSM has used indirect measurements of ¹⁴CO₂ or ³H₂O production from ¹⁴C-enriched or ³H-enriched substrates in conjunction with polarographic methods (2, 3, 6, 24). In these studies, when glucose was the sole exogenous substrate, glucose oxidation ranged from ~60 to 75% of total oxidation. However, when exogenous lipid was included, lipid utilization was found to be the primary oxidative substrate of resting VSM. The reason for these discrepancies may be that the measurements made in previous studies of VSM metabolism did not simultaneously assess the oxidation of more than one substrate.

During the last decade, ¹³C NMR techniques have been applied to the measurement of substrate entry into the TCA cycle in the heart (see Refs. 7, 20, 23, and 29 as examples). The advantage of these techniques is that by providing substrates with ¹³C at carefully chosen positions it is possible to determine the relative input of multiple metabolic pathways (for example, glycolysis and -oxidation) to energy production via their contribution of acetyl-CoA to the TCA cycle. Although oxidation per se is not measured by this technique, entry of substrates into the TCA cycle via citrate synthase is directly assessed. In the current study we have applied this technique for the first time in VSM to determine the contribution of glucose, acetate, and endogenous substrates (which may include glycogen, lipids, and amino acids) to the TCA cycle using ¹³C isotopomer analysis of glutamate under both resting and contracting conditions. We found that exogenous acetate was a major substrate for the TCA cycle both at rest and during contraction over an acetate concentration range of 0.1 to 5 mM. Although the utilization of acetate was altered in contracting VSM compared with resting VSM, the percent entry of glucose into the TCA cycle was unchanged in contracting VSM. In addition, glucose utilization exhibited little change during contraction in the presence of acetate ranging in concentration from 0 to 5 mM. We conclude that exogenous acetate can be a primary substrate for resting and contracting VSM and that glucose utilization is remarkably constant over a range of substrate concentrations and activation conditions. Therefore, ¹³C isotopomer analysis of glutamate may provide the framework for the analysis of VSM substrate utilization under a wide variety of conditions both physiological and pathophysiological.

	MATERIALS AND METHODS

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Tissue preparation. Hog carotid arteries were obtained from local abattoirs within ~30 min of slaughter. Arteries were placed in a cold (~5-10°C) physiological saline solution (PSS), pH 7.4, preequilibrated by bubbling with a gas mixture of 95% O₂-5% CO₂. PSS was composed of (in mM) 116 NaCl, 4.6 KCl, 1.16 KH₂PO₄, 25.3 NaHCO₃, 2.5 CaCl₂, and 1.16 MgSO₄. All PSS routinely contained either gentamicin sulfate at a concentration of 40 mg/l or a mixture of penicillin (303 mg/l) and streptomycin (100 mg/l). At the laboratory, the arteries were placed into fresh PSS equilibrated with the gas mixture at 22°C. Segments were dissected free of loose fat, connective tissue, and adventitia. Throughout all manipulations, PSS was equilibrated with a gas mixture of 95% O₂-5% CO₂.

Reciprocal labeling experiments. Unmounted hog carotid artery segments (~300-600 mg each) were incubated in a solution of (5 mM each) [1-¹³C]glucose and [1,2-¹³C]acetate (n = 3) or [1,2-¹³C]glucose and [1-¹³C]acetate (n = 3) for 12-20 h at 37°C. After the incubation period, the tissues were briefly rinsed in fresh PSS without substrate, rapidly weighed, and frozen in liquid nitrogen. Frozen tissues were pulverized in liquid nitrogen with a mortar and pestle and then extracted in 10 or 20 ml of an 8% perchloric acid (PCA)-40% methanol solution and placed in the freezer for at least 1 h. The powdered tissue was then homogenized with a Polytron tissue homogenizer (5 × 30 s; Brinkmann), and 9 or 19 ml of the superfusate was centrifuged at 10,000 g for 30 min at 4°C. The supernatant was neutralized with 5 M K₂CO₃, placed in the freezer (20°C) until cooled, and centrifuged at 2,800 g for 15 min. The supernatant was then transferred to a new centrifuge tube and lyophilized in a Speed Vac (Savant Instruments) for ¹³C NMR analysis as described in NMR Spectroscopy. ¹³C NMR was conducted to examine the ¹³C-labeling patterns of glutamate. We examined the relative substrate utilization using either [1-¹³C]glucose and [1,2-¹³C]acetate or [1,2-¹³C]glucose and [1-¹³C]acetate as substrates to determine whether the labeling patterns of the substrates influenced the calculated relative substrate utilization.

¹³C isotopomer analysis of glutamate. Analysis of ¹³C-glutamate isotopomers from ¹³C NMR spectra was performed as described previously by Malloy et al. (23). Briefly, the two ¹³C-labeled substrates supplied in the superfusate and the endogenous unlabeled substrates will generate three possible labeling patterns of acetyl-CoA ([1,2-¹³C]acetyl-CoA, [2-¹³C]acetyl-CoA, and unlabeled acetyl-CoA). The contribution of [1,2-¹³C]acetyl-CoA, [2-¹³C]acetyl-CoA, and unlabeled acetyl-CoA to the labeling pattern of glutamate represents the entry of substrate to the TCA cycle via acetyl-CoA and thus via citrate synthase. The calculation involves the analysis of peak intensities of the third (C-3) (~32.5 ppm) and fourth (C-4) (~37 ppm) carbon resonance peak regions of glutamate. ¹³C enrichment in glutamate at only C-4 would appear as a singlet, whereas ¹³C enrichment at both C-3 and C-4 would result in a doublet with a specific coupling constant. The utilization of [1-¹³C]glucose is determined by the intensity of the doublet at C-4 caused by [1-¹³C]glucose (D_G) divided by total C-4 resonance and multiplied by the ratio of total C-4 resonance to the total C-3 resonance. The peak intensities of the glucose resonances were doubled to account for the fact that [1-¹³C]glucose can provide a maximum of only 50% fractional enrichment of ¹³C to acetyl-CoA. Similarly, substrate utilization of [1,2-¹³C]acetate was determined by dividing the C-4 quartet caused by [1,2-¹³C]acetate (Q_A) by the total C-4 resonance and multiplying by the ratio of the total C-4 resonance to the total C-3 resonance. The unlabeled substrate input can then be calculated because the total input via citrate synthase equals the sum of the inputs from [1,2-¹³C]acetate, [1-¹³C]glucose, and unlabeled substrates. Therefore, because all relative substrate input via citrate synthase must equal 100%, the unlabeled input can be quantified as equal to 100% minus the sum of the percent input from glucose and the percent input from acetate.

Dependence of acetate concentration on pattern of substrate utilization. To determine whether the concentration of exogenous acetate altered the pattern of substrate utilization in resting or contracting VSM, we incubated isometrically mounted hog carotid artery segments (~140-370 mg) in 5 mM [1-¹³C]glucose with varying concentrations of [1,2-¹³C]acetate. For resting VSM, [1,2-¹³C]acetate was provided at 1 (n = 4), 2 (n = 3), or 5 mM (n = 3) for 9 h at 37°C. For contracting VSM, [1,2-¹³C]acetate was provided at 0 (n = 3), 0.1 (n = 3), 0.5 (n = 3), 1 (n = 2), or 5 (n = 3) mM for 6 h of isometric contraction (80 mM added KCl) at 37°C. The tissues were extracted in PCA-methanol for ¹³C NMR analysis of the glutamate peaks for determination of substrate utilization as described in ¹³C isotopomer analysis of glutamate.

Time course of substrate utilization. Hog carotid artery segments were cut into four sections (~100 mg each) and isometrically mounted on glass rods. The arteries were then incubated in PSS containing 5 mM [1-¹³C]glucose and 5 mM [1,2-¹³C]acetate at 37°C. At 3-h intervals, one section of each artery was removed, rinsed in PSS, rapidly weighed, and frozen in liquid nitrogen. The four carotid segments, each from different arteries, from each time period were pooled. The frozen tissues from the 3-, 6-, 9-, and 12-h incubations were extracted, and ¹³C NMR was conducted to examine the evolution of the ¹³C-labeling patterns of glutamate and substrate utilization as described in ¹³C isotopomer analysis of glutamate. Each extract was considered as n = 1. The same procedure was followed but with the addition of 80 mM KCl to PSS to determine whether the pattern of substrate utilization changed with isometric contraction of VSM.

Force measurements. Isometric force measurements (n = 24) were made using a 7-ml water-jacketed organ chamber (Radnoti Glass Technology) equipped with isometric force transducers sensitive to the 0- to 20-g range (Radnoti Glass Technology). Hog carotid arteries were cut into rings ranging from 1 to 3 mm in width and from 1 to 2.5 mm in diameter. After the arteries were mounted on the tissue mounts, the rings were stretched 140% of their resting lengths. The superfusate was alternated twice from PSS containing 5 mM glucose to PSS with 5 mM glucose and 80 mM added KCl for 20-min intervals and finally back to PSS with 5 mM glucose. After the final 20-min incubation in PSS with 5 mM glucose, the superfusate was switched to PSS containing 5 mM glucose and 80 mM added KCl for a prolonged (>6 h) contraction with the superfusate maintained at 37°C and continuously equilibrated with 95% O₂-5% CO₂. After 4 h of contraction, the superfusate was drained and replaced with fresh PSS also containing 5 mM glucose and 80 mM added KCl.

NMR spectroscopy. Frozen PCA-methanol extracts were lyophilized in a Speed Vac. After resuspension of the powder in 800 µl of 99% ²H₂O with 25 mM 3-(trimethylsilyl)-1-propane-sulfonic acid (TMSPS), 650 µl were transferred to a 5-mm NMR tube. A Bruker DRX 500 spectrometer was used to perform ¹³C NMR with the following acquisition parameters: 5,120 scans with 64 dummy scans, sweep width 33,333 Hz, 30° pulse angle at 125.77 MHz, and a 1-s predelay with broadband proton decoupling; 32,768 points were acquired and then processed with 1-Hz line broadening Fourier transform. The TMSPS peak at 0 ppm was used as a chemical shift and peak intensity reference. Processing of the data was done using Bruker software for peak intensity determination.

Statistical analysis. Statistics were calculated with Microsoft Excel version 7.0 for the PC using single-factor ANOVA and a two-tailed Student's t-test for two samples assuming unequal variance. Significance was considered at a P value of 0.05.

	RESULTS

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Figure 1 represents the C-4 glutamate resonance region of a typical ¹³C NMR spectrum of VSM harvested after 6 h of contraction in PSS containing (5 mM each) [1-¹³C]glucose and [1,2-¹³C]acetate at 37°C. The third (Fig. 1, inset) and fourth carbon resonance peak regions of glutamate are the peaks pertinent for isotopomer analysis of substrate utilization via citrate synthase and anaplerotic reactions. Therefore, by 6 h of incubation we have an adequate signal-to-noise ratio for measurement of ¹³C incorporation into glutamate.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Representative 13C NMR spectrum from an extract of hog carotid arteries contracted in presence of 5 mM [1-13C]glucose and 5 mM [1,2-13C]acetate at 37°C for 6 h. Region of spectrum where C-4 carbon of glutamate resonates is shown. Respective peaks are labeled and referenced to 3-(trimethylsilyl)-1-propane-sulfonic acid peak at 0 ppm. Inset, C-3 carbon of glutamate consisting of a doublet and a triplet. Triplet exists because coupling constants between 3rd and 4th carbon are virtually identical to those between 3rd and 2nd carbon. Ratio of peak areas of the C-3/C-4 regions is central to calculation of anaplerotic flux (see text for details). SG, singlet from [1-13C]glucose; DG, doublet from [1-13C]glucose; DA, doublet from [1,2-13C]acetate; QA, quartet from [1,2-13C]acetate.

To determine the pattern of substrate utilization in unmounted, resting VSM, we incubated hog carotid arteries with 5 mM [1-¹³C]glucose and 5 mM [1,2-¹³C]acetate for 12-20 h. ¹³C NMR analysis revealed that the input (mean percentage of entry via acetyl-CoA ± SE) into the TCA cycle from [1-¹³C]glucose, [1,2-¹³C]acetate, and unlabeled substrates was 30.0 ± 2.5, 71.3 ± 1.4, and 1.39 ± 3.4, respectively. However, it is possible that this technique for determining the pattern of substrate entry into the TCA cycle via acetyl-CoA may be biased by the labeling pattern of the substrates provided. For example, if nuclear Overhauser effects or line widths of the singlet (S_G) and doublet (D_G) in the C-4 resonance of glutamate, resulting from the oxidation of [1-¹³C]glucose via the TCA cycle, are substantially different from those of D_A and Q_A derived from [1,2-¹³C]acetate, then the peak magnitude determination could pose a bias in the intensity determination for the glutamate resonances. By performing the same experiments with reciprocally labeled substrates, we were able to determine whether the choice of labeling patterns of the substrates influenced the measured pattern of substrate utilization. When hog carotid artery segments were incubated in the presence of [2-¹³C]acetate and [1,2-¹³C]glucose, the input (mean percentage of entry via acetyl-CoA ± SE) into the TCA cycle from [1,2-¹³C]glucose, [2-¹³C]acetate, and unlabeled sources was 37.0 ± 2.0, 71.5 ± 4.7, and 8.5 ± 5.1, respectively. Shown in Fig. 2 is a summary of the data from these two series of experiments. No significant difference in substrate utilization was seen between the two reciprocally labeled substrate selections. Therefore, the choice of labeling patterns for exogenous metabolic substrates did not affect the measurement of substrate utilization. For all other studies, the more economical substrate combination of [1,2-¹³C]acetate and [1-¹³C]glucose was used for measurements of substrate utilization.

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Mean (±SE, n = 3) percentage of entry into tricarboxylic acid (TCA) cycle of 5 mM [1,2-13C]glucose and 5 mM [2-13C]acetate (filled bars) or [1-13C]glucose and [1,2-13C]acetate (open bars) after 12-20 h of incubation. Measured pattern of substrate utilization did not significantly (P < 0.05) depend on 13C-labeling pattern of substrates provided.

Although the ¹³C-labeling pattern in acetate and glucose did not influence the pattern of substrate utilization in resting VSM, alterations in the concentrations of acetate might be expected to influence the pattern of substrate utilization. Isometrically mounted hog carotid arteries were incubated with 5 mM [1-¹³C]glucose and 1, 2, or 5 mM [1,2-¹³C]acetate for 9 h to determine the changes, if any, in the pattern of substrate utilization (Fig. 3). Alterations in the [1,2-¹³C]acetate concentration from 1 to 5 mM had no significant effect on the extent of utilization of [1-¹³C]glucose, [1,2-¹³C]acetate, or unlabeled carbon substrates. Therefore, the utilization of glucose remains constant despite a fivefold change in acetate concentration.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Mean (±SE) percentage of entry into TCA cycle of [1-13C]glucose, [1,2-13C]acetate, and unlabeled sources with varying concentrations of [1,2-13C]acetate in resting hog carotid artery. Superfusates contained 5 mM [1-13C]glucose and either 1 (gray bars, n = 4), 2 (open bars, n = 3), or 5 (filled bars, n = 3) mM [1,2-13C]acetate. Increasing acetate concentrations did not significantly change utilization of glucose, acetate, or unlabeled substrates.

Because resting VSM has a lower overall energy utilization than contracting VSM, we investigated whether the concentration of exogenous acetate influenced the utilization of glucose or endogenous stores in contracting VSM. Shown in Fig. 4 is the substrate utilization (entry to the TCA cycle via citrate synthase) with 5 mM [1-¹³C]glucose and 0, 0.1, 0.5, 1, or 5 mM [1,2-¹³C]acetate. [1-¹³C]glucose utilization was not significantly altered as [1,2-¹³C]acetate concentration was varied from 0.1 to 5 mM compared with when no acetate was provided. However, there was a significant difference in glucose utilization when 0.1 mM acetate was provided compared with when 5 mM acetate was provided. Therefore, glucose utilization remains remarkably constant despite acetate concentrations varying over the physiological and pathophysiological range. The plasma acetate concentration is ~0.1 mM in normal human subjects (8) and can approach 1 mM in diabetic subjects (1). The utilization of [1,2-¹³C]acetate was significantly higher when the concentration of [1,2-¹³C]acetate was 5 mM compared with when [1,2-¹³C]acetate was provided at 0.1 and 0.5 mM. When acetate was absent, the lack of rise in glucose utilization occurred because there was a significant and measurable contribution from unlabeled (endogenous) substrates.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Substrate utilization (via citrate synthase) of [1-13C]glucose, [1,2-13C]acetate, and unlabeled substrates in hog carotid artery segments that were isometrically contracted for 6 h in presence of 5 mM [1-13C]glucose and 0 (hatched bars, n = 3), 0.1 (open bars, n = 3), 0.5 (light gray bars, n = 3), 1 (dark gray bars, n = 2), or 5 (filled bars, n = 3) mM [1,2-13C]acetate. [1-13C]glucose utilization was not significantly altered as [1,2-13C]acetate concentration was varied from 0.1 to 5 mM compared with when no acetate was provided. However, there was a significant difference in glucose utilization when 0.1 mM acetate was provided compared with when 5 mM acetate was provided. The utilization of [1,2-13C]acetate at 5 mM was significantly higher than that at 0.1 and 0.5 mM [1,2-13C]acetate. The utilization of unlabeled substrates was significantly higher at 0 mM [1,2-13C]acetate compared with that at any other concentration of [1,2-13C]acetate. Significance was determined by single-factor one-way ANOVA.

Although the utilization of [1-¹³C]glucose was not significantly altered by large changes in the concentration of exogenous [1,2-¹³C]acetate in either resting or contracting VSM, the anaplerotic flux (all input to the TCA cycle not through citrate synthase) might be expected to change in response to alterations in [1,2-¹³C]acetate concentration. Shown in Fig. 5 is the anaplerotic flux (scaled to the flux via citrate synthase) in resting and contracting hog carotid artery segments incubated with 5 mM [1-¹³C]glucose and varying concentrations of [1,2-¹³C]acetate. Anaplerotic flux did not significantly change when the [1,2-¹³C]acetate concentration was varied from 0.1 to 5 mM. However, when no exogenous [1,2-¹³C]acetate was provided, the anaplerotic flux was significantly lower than when [1,2-¹³C]acetate was provided.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Input to TCA cycle by anaplerotic reactions in resting (solid line) and contracting (dashed line) hog carotid artery segments incubated with 5 mM [1-13C]glucose and varying concentrations of [1,2-13C]acetate. Anaplerotic flux is scaled to input to TCA cycle via citrate synthase. Data are taken from experiments depicted in Figs. 3 and 4; values are expressed as means ± SE. Anaplerotic flux does not significantly change when acetate concentrations varied from 0.1 to 5 mM. At 0 mM acetate, anaplerotic flux is significantly lower compared with anaplerotic flux at any concentration of acetate 0.1 mM.

An increase in ATP demand associated with contraction might be expected to alter the pattern of carbon substrate utilization in VSM. To determine whether VSM contraction results in an altered pattern of substrate utilization, we incubated hog carotid artery segments for 3, 6, 9, and 12 h in PSS containing 5 mM [1,2-¹³C]acetate and 5 mM [1-¹³C]glucose in the presence (contracted) or absence (relaxed) of 80 mM KCl. The time course was examined to determine whether the pattern of substrate utilization changed during a prolonged contraction.

Shown in Fig. 6 is the time course for the pattern of substrate utilization in resting and contracting hog carotid artery. The mean [1-¹³C]glucose, [1,2-¹³C]acetate, and unlabeled utilization values of resting and contracting hog carotid artery for all time points were not significantly (ANOVA, P < 0.05) different from each other indicating that substrate utilization was at steady state throughout the time course of this study. However, the signal-to-noise ratio of the ¹³C-glutamate resonance progressively increased from 3 to 12 h of incubation (data not shown). Because the signal-to-noise ratio is highest at longer incubation times, it would be expected that differences in substrate utilization between contracting and relaxed VSM would become more apparent at longer incubation times. [1-¹³C]glucose utilization was not significantly different compared with resting and contracting tissues. However, contraction resulted in a significant decrease in acetate utilization at the 9- and 12-h time points. The larger SE of the measurements associated with earlier time points was likely responsible for the lack of significance at 3 and 6 h of incubation.

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Substrate utilization (via citrate synthase) of [1-13C]glucose (open bars, relaxed; hatched bars, contracted), [1,2-13C]acetate (light gray bars, relaxed; crosshatched bars, contracted), and unlabeled substrates (dark gray bars, relaxed; solid bars, contracted) in hog carotid artery segments that were either relaxed or isometrically contracted for 3, 6, 9, or 12 h in the presence of 5 mM [1-13C]glucose and 5 mM [1,2-13C]acetate. Values are expressed as means ± SE. Noncontracting artery segments had a sample size (n) at 3, 6, 9, and 12 h of incubation of 3, 4, 5, and 5, respectively. Contracting artery segments had n at 3, 6, 9, and 12 h of incubation of 4, 3, 4, and 4, respectively.

One concern with the studies depicted in Fig. 6 is the prolonged nature of the contractions. To determine whether isometric force is adequately maintained during such prolonged contractions, we measured isometric force of hog carotid artery rings during 8 h of KCl-induced contraction (Fig. 7). By 6 h of contraction, hog carotid artery segments maintained ~60% of the isometric force developed by 20 min of exposure to 80 mM KCl. Therefore, our measurements of substrate utilization occurred over a period of time in which the hog carotid artery was capable of maintaining substantial levels of isometric force. It is important to note that although there is a decline in isometric force maintenance over 8 h of contraction (Fig. 7), the pattern of substrate utilization did not change over 12 h of contraction (Fig. 6).

View larger version (13K): [in this window] [in a new window]   Fig. 7.   Normalized isometric force (± SE, n = 24) of hog carotid artery rings. Six hours after initiation of contraction rings maintained ~60% of initial force.

	DISCUSSION

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VSM produces most of its energy from the oxidation of acetyl-CoA derived from various endogenous and exogenous sources; however, the relative contributions of these substrates have not been well characterized in VSM. In addition, it has been proposed that carbohydrate metabolism is compartmented in VSM (13, 25, 26) with separate functional compartments for glycolysis, glycogenolysis, and gluconeogenesis (11, 14-16). Such a compartmentation of metabolism might be expected to influence the pattern of substrate utilization and the regulation of the pattern of substrate utilization in VSM. Therefore, the purpose of this study was to determine the relative inputs of exogenous ¹³C-labeled glucose and acetate, and endogenous unlabeled substrates to the TCA cycle via acetyl-CoA and anaplerotic reactions, in VSM under resting and contracting conditions. These studies represent the first simultaneous determination of the entry of multiple substrates into the TCA cycle via acetyl-CoA in VSM and demonstrate for the first time the applicability of ¹³C isotopomer analysis of glutamate to the study of VSM metabolism.

Advantages and disadvantages of ¹³C isotopomer analysis. Both radioisotope techniques and stable isotope techniques (such as NMR) have distinct advantages and disadvantages. The primary advantage of radioisotope methods is that they provide more sensitivity than ¹³C NMR. However, ¹³C NMR has some clear advantages compared with radioisotope techniques. Within the limits of the signal-to-noise ratio, ¹³C NMR can measure all nonvolatile metabolic products of a ¹³C-labeled substrate in a single sample with no need for the error and assumptions used with small molecule separation. In addition, much more information is provided by stable isotope techniques. For ¹³C NMR experiments the ¹³C-labeling position within a molecule can be assessed, thereby enabling different substrates to carry ¹³C labels at different positions. The products of metabolism can be traced to the original substrate by using the information of labeling position within the molecules. Finally, the technique does not rely on a constant pool size for glutamate because the technique simply examines the proportion of the glutamate pool (regardless of pool size) that carries specific ¹³C labeling patterns.

Anaplerotic input to TCA cycle. The anaplerotic flux is taken to be the input to the TCA cycle from all reactions other than citrate synthase. Total input to the TCA cycle is the sum of the input via citrate synthase and the anaplerotic pathways. In the heart, anaplerotic flux was <10% of the total substrate entry into the TCA cycle (23, 29). However, in VSM we report that anaplerotic flux is substantially higher (Fig. 5). In the absence of exogenous acetate, anaplerotic flux is ~20% of the flux via citrate synthase (or ~17% of total input to the TCA cycle). However, under conditions of physiological levels of exogenous acetate (~0.1 mM), anaplerotic flux significantly increases to ~50-60% of input via citrate synthase (or ~33-38% of total TCA cycle input). Although the physiological significance of such a high anaplerotic flux is not known, it may be related to the relatively low oxidative ATP production of VSM compared with other muscles (13, 25, 26). It has been proposed that glucose is required for anaplerosis in VSM and that the carboxylation of pyruvate to oxaloacetate may be the primary anaplerotic pathway (5). Although the fraction of total cellular CO₂ that is produced by the mitochondria is likely to be small, the ¹⁴CO₂ is produced by oxidative phosphorylation in the mitochondria and pyruvate carboxylase uses the CO₂ pool in the mitochondria. Therefore, this close juxtaposition of ¹⁴CO₂ production and CO₂ consumption may make difficulties in interpretation of ¹⁴C measurements of substrate utilization. If pyruvate carboxylase is the primary anaplerotic pathway, then difficulties with interpretation of ¹⁴CO₂ measurements of substrate oxidation may be especially pronounced in VSM, with a possible underestimation of ¹⁴CO₂ production. With ¹³C isotopomer analysis of TCA cycle inputs, anaplerosis is distinctly measured from input via citrate synthase, thereby avoiding such ambiguities.

Glucose utilization by TCA cycle. A key finding of the current study is that approximately one-half of the substrate for the TCA cycle via citrate synthase is derived from glucose (see Figs. 2-4 and 6). VSM has a high rate of glucose uptake and lactate production (see Refs. 13, 24, and 25). In addition, the respiratory quotient of this tissue has been reported to be near 1.0, indicating exclusive oxidation of glucose (18, 19). We report that glucose entry into the TCA cycle is ~30-60% of total substrate input via acetyl-CoA. This fraction is remarkably constant despite changes in activation state and provision of exogenous acetate over a range of 0-5 mM. These results suggest that glucose oxidation is modest in this tissue and that the extent of glucose oxidation is regulated separately from lipid oxidation. Contrary to studies showing a high respiratory quotient for VSM (18, 19), it has been suggested that the high rate of lactate production from glucose corresponds to a low entry of pyruvate derived from glucose into the TCA cycle (see Refs. 11 and 21).

Utilization of exogenous acetate. Previous investigations have examined the O₂ consumption resulting from the metabolism of a variety of exogenous and endogenous sources (2, 3, 6, 24). It was reported that in resting rabbit aorta exogenous glucose utilization accounted for only ~5% of total O₂ consumption (measured as ¹⁴CO₂ production), whereas exogenous amino acids and ketones each contributed <8% of total O₂ consumption (6). However, endogenous fatty acids were shown to account for 76% of total O₂ consumption (24). Those studies were carried out in the absence of exogenous fatty acids and thus may overestimate the physiological use of endogenous lipid stores. In resting or contracting hog carotid artery, exogenous octanoate oxidation accounted for 80% of the oxygen consumption, suggesting that when both glucose and exogenous fatty acids are provided glucose oxidation is minor (2). In our studies, O₂ consumption was not measured by either ¹⁴C-labeling techniques or polarographic methods; rather, we directly assessed entry of substrates into the TCA cycle via acetyl-CoA. With the simple substrate regimes in this study, we found that exogenous acetate provided ~55-65% of acetyl-CoA to the TCA cycle in resting and contracting VSM despite variations in [1,2-¹³C]acetate concentration from 1 to 5 mM. The total fatty acid levels range from 0.5 to 1 mM (27) and the plasma free acetate concentration is ~0.1 mM in normal human subjects (8) and can approach 1 mM in diabetic subjects (1). In our studies, even when acetate concentration was 0.1 mM, acetate utilization was still ~40% of the input to the TCA cycle. When acetate was absent, the entry of unlabeled substrates into the TCA cycle became significant (>40% of the total substrate entry via acetyl-CoA; Fig. 4). Therefore, at acetate concentrations ranging from physiological to pathophysiological levels and above, acetate utilization is a major input to the TCA cycle and glucose input to the TCA cycle remains at ~30-60%.

Nature and utilization of endogenous substrates. In our current study any substrate that was not ¹³C labeled could contribute to the acetyl-CoA as an unlabeled substrate. Because no other exogenous carbon substrates were present, the unlabeled substrates were endogenous. Possible endogenous sources include fatty acids of various lengths, amino acids, and glycogen. In resting VSM with exogenous glucose present, net glycogen synthesis, rather than net glycogen degradation, occurs (4, 12). Therefore, unlabeled glycogen will not be a significant endogenous substrate in resting VSM and will not contribute to TCA cycle input. However, in resting VSM we previously observed a simultaneous synthesis and degradation of glycogen (12). Therefore, in resting VSM it is possible that some glycogen derived from [1-¹³C]glucose may contribute to the TCA cycle and be measured as part of the [1-¹³C]glucose contribution. In contracting VSM, our initial glycogen stores were likely to be ~1-2 µmol/g (16) because glycogen stores were not repleted before contraction. Therefore, in contracting VSM the contribution of glycogen to the unlabeled substrate utilization signal would be small and not observable in most of our measurements. In our study, typically no contribution from endogenous stores was observed when acetate was provided. Only when acetate was absent did we observe a substantial (>40%) contribution from unlabeled substrate stores. Our findings are in good agreement with the findings of Odessey and Chace (24), who suggested that as much as 77% of oxygen consumption was from endogenous triglyceride fatty acids when only glucose was provided as an exogenous substrate.

Contribution of exogenous substrates to endogenous stores. It is possible that either exogenous glucose or acetate could contribute to endogenous pools of substrate (such as triglycerides), and the labeled carbon inputs that have been designated as derived from glucose or acetate utilization may actually be more directly derived from endogenous triglyceride stores. Although we have not directly assessed this possibility in the current study, previous work by ourselves and others suggests this is unlikely. The contribution of glucose to endogenous metabolite stores other than glycogen is likely to be small, because we previously reported that when resting hog carotid artery is superfused with 5 mM [1-¹³C]glucose for up to 16 h, the intracellular resonances observed corresponded to [1-¹³C]glycogen and [¹³C] glutamate (11). No other intracellular resonances derived from [1-¹³C]glucose were observed. Therefore, if incorporation of glucose into endogenous lipids did occur, such incorporation was relatively small. This is further supported by early work by Hashimoto and Dayton (17), who showed in rat aorta that the incorporation rate of glucose into lipid was ~2-3% of the rate of glucose oxidation. Whether this small pool of lipid derived from glucose is readily utilized remains unclear. Therefore, our estimates of the relative input of glucose to the TCA cycle via citrate synthase are at most overestimated by 1/50th. Less is known about the incorporation of exogenous acetate into endogenous lipid stores in VSM tissue. Although it was not directly measured in the current study, several pieces of evidence suggest that the contribution of acetate to endogenous stores that are subsequently utilized is unlikely. Unless acetate incorporation into endogenous stores was rapid, we would have expected to have measurable entry of unlabeled endogenous stores early in the incubations (3 h) and a decrease in that contribution over the subsequent 9 h of incubation. However, the contribution of endogenous stores was low and relatively constant throughout the incubations. We cannot exclude the possibility of a rapid incorporation of labeled acetate into endogenous lipid pools in our experiments.

Substrate utilization in contracting carotid arteries. An increase in ATP demand associated with contraction might be expected to alter the pattern of carbon substrate utilization in VSM. Overall, we observed (Fig. 6) a significantly decreased utilization of exogenous acetate in contracting VSM. Interestingly, the relative entry of glucose into the TCA cycle was not different in contracting compared with resting VSM. Other investigators have found that the absolute rate of oxidation of acetate and octanoate significantly increased during contraction (2). Because oxygen consumption also increased, it is likely that the fractional contribution of acetate or octanoate to total oxidation also remained relatively constant, consistent with our direct assessment. We showed previously (15) that 2 mM acetate did not alter the development or maintenance of isometric force induced by KCl in hog carotid arteries. Therefore, any alterations in substrate utilization caused by changes in acetate concentration are not the result of changes in isometric force and hence ATP demand.

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