Metabolism In Vascular Smooth Muscle Research Articles

Abstract 80: Abnormal Water Balance in Salt-Sensitivity of Blood Pressure

We studied body weight (BW), serum Na, osmolality (Osm), urine sodium excretion (UNaV) and sodium balance (NaBal) responses to a 24-hr 460 mEq sodium load in 53 subjects (32 hypertensive HTN, 21 normotensive NTN) classified as salt-resistant (SR) or salt-sensitive (SS) with an in-patient protocol. Cutoff for SS was a fall in mean daytime systolic BP (Spacelabs 90207) of ≥10 mmHg from high- to low-salt (10 mEq Na diet plus furosemide 40 mg x 3). UNaV (SR 362±22 vs SS 353±21 mEq/day) and NaBal (SR 98±22 vs SS 107±21 mEq/day) did not differ between groups. In contrast, SR had non-significant BW loss (-0.2±0.2 kg) whereas SS gained BW (0.8±0.2, p<0.001), suggesting different water balances; supported by a differential trend of changes in Na between SR (0.6±0.4 mEq) and SS (-0.3±0.5, p~0.07) and lack of change of Osm in SR (0.04±0.86 mOsm) versus a significant reduction in SS (-1.70±0.87, p<0.03). These observations were similar in HTN and NTN. We also measured hemodynamics (echocardiogram) in NTN during the high- and low-salt days. On low-salt, cardiac output (CO, 3.7±0.2 vs 4.0±0.5 L/min), mean arterial pressure (MAP, 85±2 vs 83±3 mmHg), and total peripheral resistance (TPR, 1896±92 vs 1767±163 dyn*cm -5 *sec -1 ), were not different between SR and SS. On high-salt, SR significantly reduced TPR (-347±84 dyn*cm -5 *sec -1 ; p<0.001) and maintained MAP (Δ -1±3 mmHg), whereas SS failed to vasodilate (ΔTPR +24.3±97.4, ns) and increased MAP (Δ 14±3; p<0.001). Atrial filling pressures increased equally with high-salt in both groups. In contrast, left ventricular (LV) filling pressures (E/e’ ratios) were higher in SS than in SR on both days (low-salt 9.0±0.9 vs 5.4±0.4, p<0.005, and high-salt 9.8±0.7 vs 7.7±0.3, p<0.03). Our data suggest that: 1. SR are able to store Na without concomitant water in an undefined compartment whereas this storage is impaired in SS; 2. water retention by SS is not intravascular because despite small effects on Na and Osm, their cardiac output response to salt was no greater than that in SR; and 3. inability of SS to vasodilate in response to salt and increased LV filling pressures regardless of salt-balance suggest a role for abnormal water metabolism in vascular smooth muscle and cardiomyocyte dysfunction, the former probably mediating pressure natriuresis.

Matrix Gla Protein Metabolism in Vascular Smooth Muscle and Role in Uremic Vascular Calcification

Matrix Gla protein (MGP) is an inhibitor of vascular calcification but its mechanism of action and pathogenic role are unclear. This was examined in cultured rat aortas and in a model of vascular calcification in rats with renal failure. Both carboxylated (GlaMGP) and uncarboxylated (GluMGP) forms were present in aorta and disappeared during culture with warfarin. MGP was also released into the medium and removed by ultracentrifugation, and similarly affected by warfarin. In a high-phosphate medium, warfarin increased aortic calcification but only in the absence of pyrophosphate, another endogenous inhibitor of vascular calcification. Although GlaMGP binds and inactivates bone morphogenic protein (BMP)-2, a proposed mediator of vascular calcification through up-regulation of the osteogenic transcription factor runx2, neither warfarin, BMP-2, nor the BMP-2 antagonist noggin altered runx2 mRNA content in aortas, and noggin did not prevent warfarin-induced calcification. Aortic content of MGP mRNA was increased 5-fold in renal failure but did not differ between calcified and noncalcified aortas. Immunoblots showed increased GlaMGP in noncalcified (5-fold) and calcified (20-fold) aortas from rats with renal failure, with similar increases in GluMGP. We conclude that rat aortic smooth muscle produces both GlaMGP and GluMGP in tissue-bound and soluble, presumably vesicular, forms. MGP inhibits calcification independent of BMP-2-driven osteogenesis and only in the absence of pyrophosphate, consistent with direct inhibition of hydroxyapatite formation. Synthesis of MGP is increased in renal failure and deficiency of GlaMGP is not a primary cause of medial calcification in this condition.

Overexpression of caveolin‐1 results in increased plasma membrane targeting of glycolytic enzymes: The structural basis for a membrane associated metabolic compartment

Although membrane-associated glycolysis has been observed in a variety of cell types, the mechanism of localization of glycolytic enzymes to the plasma membrane is not known. We hypothesized that caveolin-1 (CAV-1) serves as a scaffolding protein for glycolytic enzymes and may play a role in the organization of cell metabolism. To test this hypothesis, we over-expressed CAV-1 in cultured A7r5 (rat aorta vascular smooth muscle; VSM) cells. Confocal immunofluorescence microscopy was used to study the distribution of phosphofructokinase (PFK) and CAV-1 in the transfected cells. Areas of interest (AOI) were analyzed in a central Z-plane across the cell transversing the perinuclear region. To quantify any shift in PFK localization resulting from CAV-1 over-expression, we calculated a periphery to center (PC) index by taking the average of the two outer AOIs from each membrane region and dividing by the central one or two AOIs. We found the PC index to be 1.92 +/- 0.57 (mean +/- SEM, N = 8) for transfected cells and 0.59 +/- 0.05 (mean +/- SEM, N = 11) for control cells. Colocalization analysis demonstrated that the percentage of PFK associated with CAV-1 increased in transfected cells compared to control cells. The localization of aldolase (ALD) was also shifted towards the plasma membrane (and colocalized with PFK) in CAV-1 over-expressing cells. These results demonstrate that CAV-1 creates binding sites for PFK and ALD that may be of higher affinity than those binding sites localized in the cytoplasm. We conclude that CAV-1 functions as a scaffolding protein for PFK, ALD and perhaps other glycolytic enzymes, either through direct interaction or accessory proteins, thus contributing to compartmented metabolism in vascular smooth muscle.

Bilirubin Production and Oxidation in CSF of Patients with Cerebral Vasospasm after Subarachnoid Hemorrhage

Delayed cerebral vasospasm after subarachnoid hemorrhage (SAH) remains a significant cause of mortality and morbidity; however, the etiology is, as yet, unknown, despite intensive research efforts. Research in this laboratory indicates that bilirubin and oxidative stress may be responsible by leading to formation of bilirubin oxidation products (BOXes), so we investigated changes in bilirubin concentration and oxidative stress in vitro, and in cerebral spinal fluid (CSF) from SAH patients. Non-SAH CSF, a source of heme oxygenase I (HO-1), and blood were incubated, and in vitro bilirubin production measured. Cerebrospinal fluid from SAH patients was collected, categorized using stimulation of vascular smooth muscle metabolism in vitro, and information obtained regarding occurrence of vasospasm in the patients. Cerebral spinal fluid was analyzed for hemoglobin, total protein and bilirubin, BOXes, malonyldialdehyde and peroxidized lipids (indicators of an oxidizing environment), and HO-1 concentration. The formation of bilirubin in vitro requires that CSF is present, as well as whole, non-anti-coagulated blood. Bilirubin, BOXes, HO-1, and peroxidized lipid content were significantly higher in CSF from SAH patients with vasospasm, compared with nonvasospasm SAH CSF, and correlated with occurrence of vasospasm. We conclude that vasospasm may be more likely in patients with elevated BOXes. The conditions necessary for the formation of BOXes are indeed present in CSF from SAH patients with vasospasm, but not CSF from SAH patients without vasospasm.

Endothelial- and nitric oxide-dependent effects on oxidative metabolism of intact artery

Oxidative metabolism and its possible modulation by nitric oxide (NO) was examined in endothelial-intact and endothelial-denuded segments of porcine carotid arteries. Endothelial-intact arteries displayed appropriate NO-mediated vasorelaxation to acetylcholine (ACh). Endothelial-denuded arteries demonstrated absent vasorelaxation to ACh stimulation and depressed contractile responsiveness to K + depolarization, which was normalized by inhibition of NO synthesis by N ω-nitro- l-arginine methylester ( l-NAME). Confirmation that carotid arteries continued to produce NO despite removal of the endothelium was indicated by detection of NO metabolites in the incubation medium bathing the arteries. O 2 consumption and the oxidation of glucose and fatty acid were depressed in endothelial-denuded arteries. Depression of O 2 consumption and glucose oxidation was completely reversed by treatment with l-NAME. We conclude that endogenous NO produced by non-endothelial vascular cells depresses contractility, O 2 consumption, and oxidation of energy substrates in vascular smooth muscle. The endothelium may play a role in oxidative metabolism of vascular smooth muscle possibly by modulating the effects of NO produced by other cells of the vessel wall, or by other factors.

Caveolae and the organization of carbohydrate metabolism in vascular smooth muscle

We have previously found that glycolysis and gluconeogenesis occur in separate "compartments" of the VSM cell. These compartments may result from spatial separation of glycolytic and gluconeogenic enzymes (Lloyd and Hardin [1999] Am J Physiol Cell Physiol. 277:C1250-C1262). We have also found that an intact plasma membrane is essential for compartmentation to exist (Lloyd and Hardin [2000] Am J Physiol Cell Physiol. 278:C803-C811), suggesting that glycolysis and gluconeogenesis may be associated with distinct plasma membrane microdomains. Caveolae are one such microdomain, in which proteins of related function colocalize. Thus, we hypothesized that membrane-associated glycolysis occurs in association with caveolae, while gluconeogenesis is localized to non-caveolae domains. To test this hypothesis, we disrupted caveolae in vascular smooth muscle (VSM) of pig cerebral microvessels (PCMV) with beta methyl-cyclodextrin (CD) and examined the metabolism of [2-(13)C]glucose (a glycolytic substrate) and [1-(13)C]fructose 1,6-bisphosphate (FBP, a gluconeogenic substrate in PCMV) using (13)C nuclear magnetic resonance spectroscopy. Caveolar disruption reduced flux of [2-(13)C]glucose to [2-(13)C]lactate, suggesting that caveolar disruption partially disrupted the glycolytic pathway. Caveolae disruption may also have resulted in a breakdown of compartmentation, since conversion of [1-(13)C]FBP to [3-(13)C]lactate was increased by CD treatment. Alternatively, the increased [3-(13)C]lactate production may reflect changes in FBP uptake, since conversion of [1-(13)C]FBP to [3-(13)C]glucose was also elevated in CD-treated cells. Thus, a link between caveolar organization and metabolic organization may exist.

In Vitro Therapy with Dobutamine, Isoprenaline and Sodium Nitroprusside Protects Vascular Smooth Muscle Metabolism from Subarachnoid Haemorrhage Induced Cerebral Vasospasm

The cerebrospinal fluid (CSF) from subarachnoid haemorrhage (SAH) patients with cerebral vasospasm stimulates vasoconstriction and oxygen consumption in the porcine carotid artery in vitro. Stimulation of oxygen consumption has been used as an in vitro model of vasospasm to assess the relative benefits of nimodipine, isoprenaline, dobutamine, and sodium nitroprusside (SNP). Samples of human CSF were obtained from SAH patients and applied to de-endothelialised porcine carotid artery. Stimulation of oxygen consumption (as an in vitro marker for a stimulation of the vessels) was monitored and the effects of SNP, isoprenaline, dobutamine or nimodipine were measured. The CSF from SAH patients with evidence of vasospasm stimulated oxygen consumption to 0.91 +/- 0.17 (microM O2/min/g dry wt, +/- SD p < or = 0.01) and CSF from SAH patients without vasospasm did not significantly stimulate oxygen consumption 0.27 +/- 0.02, with 0.23 +/- 0.03 (microM O2/min/g dry wt) being an unstimulated rate of respiration for the porcine carotid artery. SNP, isoprenaline or dobutamine significantly (p < or = 0.01) decreased the stimulation of oxygen consumption of the porcine carotid artery whereas nimodipine did not. In a cohort of 41 SAH patients who received nimodipine alone or nimodipine and dobutamine, the in hospital mortality rate of the patients who received only nimodipine was 42% as compared to an in hospital mortality rate of 17% in the nimodipine plus dobutamine group P < or = 0.076). The in vivo data on the 41 patients is not statistically significant, so further studies are required to determine if the differences are important. SNP, isoprenaline and dobutamine significantly decreased oxygen consumption of the porcine carotid arteries exposed to CSF from SAH patients who had vasospasm whereas nimodipine did not. Our in vitro results suggest that these compounds require further study in patients with SAH who are at risk for vasospasm because they may have a direct benefit for the vasospastic arteries.

Effect of serum albumin on vascular smooth muscle metabolism

In studies on metabolism of vascular smooth muscle, it was observed that incubation of intact porcine carotid artery strips with 3% bovine or porcine serum albumin had profound effects on the oxidation of substrates and O 2 consumption. Arteries incubated over 180 min with charcoal-treated and dialyzed albumin demonstrated time-dependent stimulation of glucose oxidation (145%; P<0.0001, n=6) and O 2 consumption (116%; P<0.001, n=6). These results were not mimicked by incubation with 3% solutions of ovalbumin or porcine skin gelatin. However, the oxidation of the medium chain fatty acid octanoate was inhibited in the presence of albumin over a broad range of octanoate concentrations (0.5–5.0 mM). Short chain fatty acid oxidation (acetate, 5 mM), in contrast, was not inhibited by albumin. Wash-out of albumin only partially reversed the stimulation of O 2 consumption and incubation of arteries with a polyanionic compound, polyethylene sulfonate (5 mg/ml), blunted the stimulatory effect of albumin on O 2 consumption. Albumin also produced anaplerosis of the Krebs cycle, and an increase in the content of glutamate and alanine ( P<0.005, n=8). The metabolic effects of albumin were associated with time-dependent uptake of albumin (30.9±1.5 nmol/g per 210 min; P<0.01, n=15). ATP-dependent proteolysis of the albumin taken up was also observed. These results demonstrate novel and important intracellular effects of serum albumin on energy metabolism of vascular smooth muscle.

Influence of glycogen storage on vascular smooth muscle metabolism.

The role of glycogen as an oxidative substrate for vascular smooth muscle (VSM) remains controversial. To elucidate the importance of glycogen as an oxidative substrate and the influence of glycogen flux on VSM substrate selection, we systematically altered glycogen levels and measured metabolism of glucose, acetate, and glycogen. Hog carotid arteries with glycogen contents ranging from 1 to 11 micromol/g were isometrically contracted in physiological salt solution containing 5 mM [1-(13)C]glucose and 1 mM [1, 2-(13)C]acetate at 37 degrees C for 6 h. [1-(13)C]glucose, [1, 2-(13)C]acetate, and glycogen oxidation were simultaneously measured with the use of a (13)C-labeled isotopomer analysis of glutamate. Although oxidation of glycogen increased with the glycogen content of the tissue, glycogen oxidation contributed only approximately 10% of the substrate oxidized by VSM. Whereas [1-(13)C]glucose flux, [3-(13)C]lactate production from [1-(13)C]glucose, and [1, 2-(13)C]acetate oxidation were not regulated by glycogen content, [1-(13)C]glucose oxidation was significantly affected by the glycogen content of VSM. However, [1-(13)C]glucose remained the primary ( approximately 40-50%) contributor to substrate oxidation. Therefore, we conclude that glucose is the predominate substrate oxidized by VSM, and glycogen oxidation contributes minimally to substrate oxidation.

Role of microtubules in the regulation of metabolism in isolated cerebral microvessels.

We used (13)C-labeled substrates and nuclear magnetic resonance spectroscopy to examine carbohydrate metabolism in vascular smooth muscle of freshly isolated pig cerebral microvessels (PCMV). PCMV utilized [2-(13)C]glucose mainly for glycolysis, producing [2-(13)C]lactate. Simultaneously, PCMV utilized the glycolytic intermediate [1-(13)C]fructose 1,6-bisphosphate (FBP) mainly for gluconeogenesis, producing [1-(13)C]glucose with only minor [3-(13)C]lactate production. The dissimilarity in metabolism of [2-(13)C]FBP derived from [2-(13)C]glucose breakdown and metabolism of exogenous [1-(13)C]FBP demonstrates that carbohydrate metabolism is compartmented in PCMV. Because glycolytic enzymes interact with microtubules, we disrupted microtubules with vinblastine. Vinblastine treatment significantly decreased [2-(13)C]lactate peak intensity (87.8 +/- 3.7% of control). The microtubule-stabilizing agent taxol also reduced [2-(13)C]lactate peak intensity (90.0 +/- 2. 4% of control). Treatment with both agents further decreased [2-(13)C]lactate production (73.3 +/- 4.0% of control). Neither vinblastine, taxol, or the combined drugs affected [1-(13)C]glucose peak intensity (gluconeogenesis) or disrupted the compartmentation of carbohydrate metabolism. The similar effects of taxol and vinblastine, drugs that have opposite effects on microtubule assembly, suggest that they produce their effects on glycolytic rate by competing with glycolytic enzymes for binding, not by affecting the overall assembly state of the microtubule network. Glycolysis, but not gluconeogenesis, may be regulated in part by glycolytic enzyme-microtubule interactions.

Effects of insulin and metformin on glucose metabolism in rat vascular smooth muscle.

Glucose metabolism in vascular smooth muscle cells (VSMCs) is characterized by substantial lactate production even in fully oxygenated conditions. Insulin and metformin, an insulin-sensitizing agent, have direct effects on the vascular tissue metabolism. We investigated whether insulin or metformin can induce a switch in VSMC glucose metabolism from lactate production to pyruvate oxidation, by measuring lactate oxidation as determined by the conversion of [1-14C]-D,L-lactate to [1-14C]-pyruvate and subsequent oxidation to acetyl coenzyme A and 14CO2 by pyruvate dehydrogenase (PDH). Lactate oxidation was measured in control rat aortic cultured VSMCs incubated for 30 minutes in media with and without additional glucose compared with VSMCs cultured in the presence of insulin or metformin. The addition of glucose to VSMCs decreased lactate oxidation (4.6+/-1.7 v 9.6+/-2.4 pmol/cell/min, P < .001). In the absence of additional glucose, metformin decreased lactate oxidation in VSMCs compared with controls (4.9+/-1.4 v 9.6+/-2.4 pmol/cell/min, P < .01). Metformin in the presence of glucose caused the greatest decline in lactate oxidation (2.5+/-0.4 pmol/cell/min, P < .001). In contrast to the effects of metformin, insulin increased lactate oxidation both with (12.9+/-1.5 pmol/cell/min, P < .001) and without (17.9+/-4.4, P < .01) additional glucose. This suggests that insulin facilitates VSMC utilization of lactate as a source of pyruvate and energy production even during noncontractile periods.

Pattern of substrate utilization in vascular smooth muscle using 13C isotopomer analysis of glutamate.

Although vascular smooth muscle (VSM) derives the majority of its energy from oxidative phosphorylation, controversy exists concerning which substrates are utilized by the tricarboxylic acid (TCA) cycle. We used 13C isotopomer analysis of glutamate to directly measure the entry of exogenous [13C]glucose and acetate and unlabeled endogenous sources into the TCA cycle via acetyl-CoA. Hog carotid artery segments denuded of endothelium were superfused with 5 mM [1-13C]glucose and 0-5 mM [1,2-13C]acetate at 37 degreesC for 3-12 h. We found that both resting and contracting VSM preferentially utilize [1,2-13C]acetate compared with [1-13C]glucose and unlabeled substrates. The entry of glucose into the TCA cycle (30-60% of total entry via acetyl-CoA) exhibited little change despite alterations in contractile state or acetate concentrations ranging from 0 to 5 mM. We conclude that glucose and nonglucose substrates are important oxidative substrates for resting and contracting VSM. These are the first direct measurements of relative substrate entry into the TCA cycle of VSM during activation and may provide a useful method to measure alterations in VSM metabolism under physiological and pathophysiological conditions.

Differential Regulation of Glucose and Glycogen Metabolism in Vascular Smooth Muscle by Exogenous Substrates

The aim of this study was to determine whether the pathways of glycolysis and glycogenolysis can be independently modulated by the provision of acetate or pyruvate as exogenous substrates. Hog carotid artery segments were allowed to replete glycogen stores to over 6μmol/g of new13C-labeled glycogen by incubation at 37°C with 5 mm[1-13C]glucose for 6–16 h and then were isometrically contracted for 3 h with 80 mmKCl in the presence of 5 mm[2-13C]glucose and either 2 mmsodium acetate or 5 mmsodium pyruvate. Measurements were made of total lactate production, glucose utilization, glycogen utilization, isometric force, [2-13C]lactate and [3-13C]lactate production. Compared to experiments with glucose as the sole exogenous substrate, provision of pyruvate significantly decreased glucose utilization (by 28%) but insignificantly decreased glycogen utilization. In contrast, provision of acetate resulted in a statistically insignificant decrease in glucose utilization (by 23%) and an increase in glycogen utilization (by 20%). The fraction of [3-13C]pyruvate derived from glycogen that was converted to [3-13C]lactate was significantly decreased in the presence of acetate despite the enhanced glycogen utilization. Despite these alterations in cellular energy balance, isometric force generation and maintenance was similar for all experimental groups. This differential regulation of glycolysis and glycogenolysis may either reflect the compartmentation of these pathways or suggest a novel regulation of carbohydrate metabolismin vivo.

Substrate-dependent alteration in O2 consumption and energy metabolism in vascular smooth muscle.

Energy metabolism and the substrate utilization pattern of intact porcine carotid artery were investigated in the presence or absence of glucose and/or octanoate during the phases of isometric contraction induced by K+ depolarization. During the early phase of contraction, there was a rapid increase in the rate of O2 uptake that was independent of the rate of force generation but dependent on the availability of intracellular pyruvate, the source of which was glucose and not glycogen. Lactate production increased linearly from the onset of contractile stimulation and was not suppressed by octanoate oxidation. There was no alteration from the basal resting state in the concentrations of the metabolites of the tricarboxylic acid cycle in the presence or absence of octanoate. During the phase of steady-state force maintenance, O2 consumption was increased compared with the basal unstimulated rate but was not increased when glucose and octanoate were present, which is consistent with the Crabtree effect. This was associated with increased aerobic lactic acid production and inhibition of the tricarboxylic acid cycle at the citrate synthase step. Alteration of the high-energy phosphate content could not account for the pattern of O2 consumption during contraction under different substrate conditions. In the absence of glucose, the energy from octanoate oxidation could substitute for the energy ordinarily derived from aerobic glycogen and lactic acid production. It is concluded that energy metabolism of vascular smooth muscle is coordinated during contraction by integration of the pathways of aerobic glycolysis and oxidative phosphorylation.

EFFECT OF INSULIN ON GLUCOSE OXIDATION IN VASCULAR SMOOTH MUSCLE CELLS.† 556

Glucose metabolism in vascular smooth muscle (VSM) is characterized by production of lactic acid rather than pyruvate oxidation (Am J Physiol 1995). An increase in aerobic lactate production by VSM may result in lactic acidosis in certain vascular diseases such as hypertension and atherosclerosis. Activation of pyruvate dehydrogenase (PDH) by insulin in adipocytes and fibroblasts increases pyruvate oxidation and decreases lactate oxidation in the presence of glucose. Experiments were designed to determine if insulin would divert glucose metabolism in VSM cells from lactate production to pyruvate oxidation. Lactate oxidation was determined in control VSM cells cultured from rat aortic tissue and incubated in media without insulin or glucose (9.6 ± 2.4 μmol/1×106 cells/min) by measuring the conversion of [1-14C]-D,L-lactate to [1-14C]-pyruvate and the subsequent oxidation to acetyl CoA and 14CO2 by PDH. Lactate oxidation measured in VSM cells with glucose alone (4.6 ± 1.7μmol/1×106 cells/min) was decreased compared to control(p<0.001). Insulin alone (100 μU/ml) induced [1-14C]-D,L-lactate oxidation in VSM cells (17.9 ± 4.4 μmol/1×106 cells/min) and was greater than both control (p< 0.01) and glucose alone(p< 0.001). However, unlike in adipocytes and fibroblasts, lactate oxidation did not decrease in VSM when insulin and glucose were both added to the media (12.9 ± 1.56 μmol/1×106 cells/min). This suggests that insulin does not divert glucose from lactate production to generate pyruvate in VSM but instead may utilize lactate as a source of pyruvate and energy production even during noncontractile periods.

Metabolite utilization and compartmentation in porcine carotid artery: a study using beta-guanidinopropionic acid

The relationship between substrate and metabolism in vascular smooth muscle has been investigated by studying the acute energetic effects caused by the creatine analogue beta-guanidinopropionic acid (beta-GPA) on porcine carotid arteries using 31P-nuclear magnetic resonance (NMR). Porcine carotid arteries were superfused for 12 h with Krebs-Henseleit buffer at 22 degrees C, containing 50 mM beta-GPA, and either 11 mM glucose or 5 mM pyruvate as substrate. beta-GPA enters the cells and becomes phosphorylated by creatine kinase to produce beta-GPA-P. Perfusion with beta-GPA leads to the formation of NMR observable beta-GPA-P (after 2.5 h). The appearance of beta-GPA-P with time was significantly greater when glucose was used as substrate. To differentiate between oxidative and glycolytic metabolism in the phosphorylation of beta-GPA, 1 mM cyanide was included in the perfusion buffer containing 50 mM beta-GPA and 11 mM glucose. No phosphocreatine (PCr) was observed with these conditions, and there was a small but significant decrease in ATP concentration ([ATP]) compared with glucose perfusion without cyanide (0.56 +/- 0.02 to 0.47 +/- 0.02 mumol/g wet wt), that was greater than the concentration with pyruvate as substrate (0.25 +/- 0.03 mumol/g wet wt). Thus the [ATP] during cyanide treatment is maintained with glycolytic metabolism. Despite the relatively high [ATP], accumulation of beta-GPA-P only occurred over a much slower time course ( > 10 h) than without cyanide.(ABSTRACT TRUNCATED AT 250 WORDS)

1014-96 Dichloroacetate Impairs Energy Metabolism and Produces a Negative Inotropic Effect in Vascular Smooth Muscle

Dichloroacetate (DCA) is a promising agent for treatment of congestive heart failure. By stimulation of pyruvate dehydrogenase, it facilitates the oxidation of glucose (and its metabolites pyruvate and lactate), decreases O 2 consumption, and optimizes efficiency of energy production by heart muscle. Its effect on the energy metabolism of vascular smooth muscle, however, is unknown. The effect of DCA on vascular smooth muscle was investigated in segments of porcine carotid arteries mounted in an organ bath and incubated in physiological salt solution (PSS) containing glucose and presence or absence of 1 mM DCA. DCA suppressed the production of lactic acid (0.03 ± 0.006 vs. 0.11 ± 0.01 μ mol/g/min.; p &lt; 0.001, n = 8) and the level of pyruvate was reduced by 56% (p &lt; 0.01) consistent with enhanced oxidation of glucose. However, O 2 consumption was unaffected. The levels of the Krebs cycle metabolites citrate and oxaloacetate were unaltered by DCA but aspartate and oxoglutarate were decreased by 66% (p &lt; 0.005) and 30% (p &lt; 0.02) respectively, suggesting increased Krebs cycle activity. Despite this, the level of ATP + Phosphocreatine was decreased by DCA (1.00 ± 0.09 vs. 1.40 ± 0.09 μ mol/g, p &lt; 0.02, n = 8). DCA had no effect on high energy phosphate levels in control carotid arteries incubated in glucose-free PSS containing fatty acid (0.5 mM octanoate). Carotid arteries incubated in DCA and then stimulated to contract by K + -depolarization displayed a reduced rate of tension generation when compared with controls (p &lt; 0.05, n = 17). It is concluded that, in contrast to cardiac muscle, DCA impairs energy metabolism and exerts a negative inotropic effect in vascular smooth muscle in vitro. The impairment in energy metabolism is related to altered activity of the Krebs cycle as a result of excessive oxidation of glucose. Whether this effect on glucose metabolism and contractility of vascular smooth muscle occurs in vivo requires further investigation. The effects of DCA on the peripheral vasculature may play a role in its beneficial effects in congestive heart failure.

Production of lactic acid and energy metabolism in vascular smooth muscle: effect of dichloroacetate.

Vascular smooth muscle metabolism is characterized by substantial production of lactic acid even under fully oxygenated conditions. The role the aerobic production of lactate plays in the energetics of smooth muscle is obscure and was investigated in this study. Helical strips of porcine carotid arteries were incubated in medium containing 1 mM dichloroacetate (DCA), an agent that stimulates pyruvate dehydrogenase and promotes the oxidation of glucose. Lactate production in resting muscle was decreased in the presence of DCA (0.033 +/- 0.006 vs. 0.111 +/- 0.014 mumol.g-1.min-1, P < 0.02), indicating diversion of glucose metabolism from lactate production to enhanced glucose oxidation. This was associated with reduction in the level of ATP+phosphocreatine (PCr) (0.99 +/- 0.01 vs. 1.40 +/- 0.09 mumol/g, P < 0.05) and cataplerosis of the tricarboxylic acid (TCA) cycle. Contraction by KCl was also associated with reduced lactate production in the presence of DCA (0.086 +/- 0.017 vs. 0.20 +/- 0.002 mumol.g-1.min-1, P < 0.01), but ATP+PCr normalized, and there was anaplerosis of the TCA cycle. Glycogen in control arteries declined by approximately 1.3 mumol/g over 30 min K+ contraction but was unchanged in the presence of DCA. By calculation, the glycogen spared could be accounted for by the quantity of glucose diverted from lactate production to glucose oxidation during contraction. It is concluded that the aerobic production of lactate is a mechanism affording optimal coordination and modulation of glucose supply and oxidative energy production with energy demand.

Compartmentation of glucose and fructose 1,6-bisphosphate metabolism in vascular smooth muscle.

We examined the metabolism of exogenously added 13C-labeled fructose 1,6-bisphosphate (either labeled at the first and sixth carbons or labeled at the first carbon only) and of [2-13C]glucose in well-oxygenated and well-superfused hog carotid artery segments. Exogenously added fructose 1,6-bisphosphate was utilized by hog carotid artery and primarily participated in gluconeogenesis while the production of [3-13C]lactate was not significantly different from zero. When [1,6-13C]fructose 1,6-bisphosphate or [1-13C]fructose 1,6-bisphosphate was utilized individually, gluconeogenic flux occurred without metabolism through aldolase and triosephosphate isomerase resulting in formation of [1,6-13C]-glucose and [1-13C]glucose respectively. When [2-13C]glucose was the sole exogenous substrate, it was utilized and exclusively participated in glycolytic flux with production of [3-13C]lactate and no gluconeogenic flux from the trioses to [5-13C]glucose. When both glucose and fructose 1,6-bisphosphate were provided together as exogenous substrates, glucose still participated exclusively in glycolytic flux with no trioses participating in gluconeogenesis while fructose 1,6-bisphosphate participated in glycolytic flux with [3-13C]lactate production approximately being approximately half of the [1,6-13C]glucose production from [1,6-13C]fructose 1,6-bisphosphate. In the presence of glucose, [1-13C]fructose 1,6-bisphosphate also participated in glycolytic flux and gluconeogenic flux simultaneously. However in the presence of [2-13C]glucose, [1-13C]fructose 1,6-bisphosphate underwent isomerization through the trioses prior to gluconeogenesis since [6-13C]glucose was produced.(ABSTRACT TRUNCATED AT 250 WORDS)

Fatty acid, tricarboxylic acid cycle metabolites, and energy metabolism in vascular smooth muscle.

The influence of octanoate on O2 consumption, tricarboxylic acid (TCA) cycle intermediates, and high-energy phosphates was examined in intact resting porcine carotid artery to investigate the role of fatty acid in energy metabolism and its integration with glucose metabolism in vascular smooth muscle. Incubation of resting arteries with octanoate (0.5 mM), which was previously shown to inhibit aerobic glycolysis (6), inhibited lactate production by 64% and increased O2 consumption by 30%. The increase in O2 consumption with octanoate was approximately equal to that calculated to account for the ATP production lost by inhibition of aerobic lactate production by octanoate. In glucose-free medium, the level of high-energy phosphate was reduced but was restored when octanoate was included in the incubation medium. This was associated with an increase in O2 consumption. These results suggest that the energy requirements of resting carotid artery can be largely met by the oxidative metabolism of fatty acid. Octanoate induced anaplerosis of the TCA cycle, as indicated by a 70% increase in the level of citrate. Extracellular glucose was necessary for octanoate-induced anaplerosis, probably by providing the extra carbon via pyruvate carboxylation, whereas a coupled transamination involving aspartate was a less important anaplerotic mechanism.