Biomedical subjects

C D Hardin

Publications and source records attributed to C D Hardin.

29 records · Page 2Linked to original sources

PubMed1995

Gluconeogenesis during hypoxia in vascular smooth muscle studied by 13C-NMR.

We investigated whether hypoxia altered the utilization of fructose-1,6-bisphosphate as a gluconeogenic or glycolytic intermediate in superfused media from hog carotid artery. Using 13C-NMR, we found that although 3-13C-lactate production from 1-13C-glucose increased compared to that under well-oxygenated conditions, the conversion of exogenously applied 1,6-13C-fructose-1,6-bisphosphate to glucose (gluconeogenesis) or to 3-13C-lactate was not significantly affected by hypoxia. Since hypoxia alters the rate of glucose conversion to lactate but not the conversion of fructose-1,6-bisphosphate to glucose, we conclude that glycolysis and glycogenolysis may continue to be compartmentalized during hypoxia and that a high rate of gluconeogenesis can occur even during hypoxia.

Animals↗

PubMed1994

Simultaneous and separable flux of pathways for glucose and glycogen utilization studied by 13C-NMR.

Glucose utilization and glycogen turnover was studied by 13C-NMR in segments of hog carotid artery smooth muscle. After superfusion of carotid segments for 8-16 h at 37 degrees C with C-1 labeled glucose. 13C-NMR spectra revealed substantial incorporation into glycogen, lactate and into resonances tentatively identified as glutamate at the C-2 and C-4 positions. The rate of net glycogen incorporation was approximately linear over 8 h in unstimulated muscle. After washing out the initial labeled glucose, carotids were contracted by 80 mM KCl in the presence of C-2 labeled glucose. During the contraction simultaneous flux of glycogenolysis and glycolysis was observed with production of lactate labeled at the C-2 position (from glucose labeled at the second carbon) and with the magnitude of the glycogen resonance decreasing. However, no lactate labeled at the C-3 position (derived from C-3 glycogen) was observed at the end of a prolonged contraction. If complete mixing of the intermediates of the two pathways occurred, lactate derived from both glucose and glycogen would have been observed. When similar experiments are performed in the presence of 5 mM NaCN to block oxidative metabolism, then lactate derived from glucose and from glycogen was clearly observed. Therefore, when oxidative metabolism was intact, the intermediates of glycogenolysis and glycolysis did not normally appear to fully mix despite simultaneous flux of the two pathways during contraction. These separable cytosolic pathways for carbohydrate utilization are proposed to be governed by a reaction-diffusion class of mechanism. Such an organization of cytosolic enzymes may represent a general feature of cell metabolism.

Animals↗

PubMed1994

Metabolism of exogenously applied fructose 1,6-bisphosphate in hypoxic vascular smooth muscle.

Exogenously administered fructose 1,6-bisphosphate reportedly protects ischemic or hypoxic tissue and facilitates metabolic recovery. The mechanism of action of exogenous fructose 1,6-bisphosphate has been an issue of considerable debate, since there is a lack of direct evidence that fructose 1,6-bisphosphate can cross the cell membrane and act as an intermediate in glycolysis. We synthesized [1,6-13C]fructose 1,6-bisphosphate and directly examined its cellular metabolism in hog carotid artery segments using 13C-nuclear magnetic resonance (NMR) spectroscopy. [1,6-13C]fructose 1,6-bisphosphate (2.1 mM) was metabolized by hog carotid artery during normoxia and hypoxia with a major metabolic product being [3-13C]lactate. The production of [3-13C]lactate was greater during hypoxia than during normoxia, indicating that fructose 1,6-bisphosphate metabolism responded to the energetic state of the tissue. We found that exogenously added fructose 1,6-bisphosphate at 2.1 mM did not significantly improve the ability of hypoxic hog carotid artery to maintain isometric force, whereas 20 mM fructose 1,6-bisphosphate did significantly, although modestly, improve isometric force maintenance. These results indicate that exogenously added fructose 1,6-bisphosphate is capable of entering cells and serving as a glycolytic intermediate.

Adenosine Triphosphate↗

PubMed1993

Regulation of glycolytically fueled Ca2+ uptake in smooth muscle plasmalemmal vesicles by phosphorylation.

A highly purified plasma membrane vesicular preparation from porcine antrum had an endogenous protein kinase activity with substrates of molecular weights of 11, 15, 20.5, 25, 35, 44, 155, and 230 x 10(3). Phosphorylation of the plasma membranes by the endogenous protein kinase activity resulted in a stimulation of initial rates of Ca2+ uptake into inside-out vesicles, which was associated with an increase in the maximum velocity of the Ca2+ pump with no apparent changes in the half-maximal effective concentration for calcium. Because we have previously reported that a membrane-associated glycolytic system may preferentially provide ATP to fuel the Ca2+ pump (9), we examined the effects of phosphorylation on Ca2+ uptake when glycolysis was the sole source of ATP for the pump. We found that the stimulation of Ca2+ uptake by phosphorylation was more pronounced when Ca2+ uptake was supported by glycolysis rather than 2 mM ATP. When ATP was added at a level similar to that produced by endogenous glycolysis, the stimulation of Ca2+ uptake by phosphorylation was comparable to when glycolysis supported the Ca2+ pump. Our observations suggest that the dynamic range (up to threefold) for regulation of the plasmalemmal Ca2+ pump by phosphorylation is considerably larger than previously reported and thus likely to be of physiological significance.

Adenosine Triphosphate↗

PubMed1992

Localization of two glycolytic enzymes in guinea-pig taenia coli.

The bound fractions of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and of fructose 1,6-diphosphate aldolase (ALD) were measured in intact Taenia coli. ALD was approximately 60% bound and GAPDH was approximately 41% bound. Bound ALD activity remaining in chemically demembranated Taenia coli was similar to that in intact tissue indicating a localization to the contractile apparatus. ALD was found to be specifically bound in the demembranated preparation. Chemical demembranation resulted in almost complete loss of all GAPDH activity indicating a localization of bound GAPDH to cellular membranes.

Animals↗

PubMed1992

Vascular oxidative metabolism under different metabolic conditions.

Control of respiration in vascular smooth muscle was examined while the metabolic state of the tissue was manipulated. During KCl-induced contractures in the presence of 5 mM glucose, oxygen consumption increased by 10 nmol/per min g without any decrease in phosphocreatine (PCr) or ATP as determined by 31P-NMR indicating a control of respiration which does not involve changes in high-energy phosphates (e.g., ADP, phosphorylation potential). However, when aortae with resting tone in the absence of substrate were then provided with 5 mM 2-deoxyglucose as the sole substrate, oxygen consumption increased 7.4 nmol/min per g while PCr decreased by more than 50% (resulting in a 2-fold increase in the calculated free ADP) with no change in tension from resting tone. During a subsequent KCl induced contracture in the presence of 2-deoxyglucose, oxygen consumption increased an additional 7.2 nmol/min per g while PCr continued to decline. Therefore, at least two mechanisms of respiratory control may exist in sheep aorta, one dependent and the other independent of changes in high-energy phosphates.

Adenosine Diphosphate↗

PubMed1992

Comparison of endogenous and exogenous sources of ATP in fueling Ca2+ uptake in smooth muscle plasma membrane vesicles.

A smooth muscle plasma membrane vesicular fraction (PMV) purified for the (Ca2+/Mg2+)-ATPase has endogenous glycolytic enzyme activity. In the presence of glycolytic substrate (fructose 1,6-diphosphate) and cofactors, PMV produced ATP and lactate and supported calcium uptake. The endogenous glycolytic cascade supports calcium uptake independent of bath [ATP]. A 10-fold dilution of PMV, with the resultant 10-fold dilution of glycolytically produced bath [ATP] did not change glycolytically fueled calcium uptake (nanomoles per milligram protein). Furthermore, the calcium uptake fueled by the endogenous glycolytic cascade persisted in the presence of a hexokinase-based ATP trap which eliminated calcium uptake fueled by exogenously added ATP. Thus, it appears that the endogenous glycolytic cascade fuels calcium uptake in PMV via a membrane-associated pool of ATP and not via an exchange of ATP with the bulk solution. To determine whether ATP produced endogenously was utilized preferentially by the calcium pump, the ATP production rates of the endogenous creatine kinase and pyruvate kinase were matched to that of glycolysis and the calcium uptake fueled by the endogenous sources was compared with that fueled by exogenous ATP added at the same rate. The rate of calcium uptake fueled by endogenous sources of ATP was approximately twice that supported by exogenously added ATP, indicating that the calcium pump preferentially utilizes ATP produced by membrane-bound enzymes.

Adenosine Triphosphate↗

PubMed1992

Tension responses of sheep aorta to simultaneous decreases in phosphocreatine, inorganic phosphate and ATP.

1. Tension responses of sheep aortae were investigated when different substrates were included in the superfusing medium. The magnitude of tension development was similar whether or not 5 mM glucose was present in the medium. However, the rate of tension development was greater in the absence of glucose. 2. When 5 mM 2-deoxyglucose (2DG) was present in the medium, the magnitude of tension generation was 1.6 times that in the absence of exogenous substrate. A second sequential contraction with 2DG generated tension 1.25 times that in the absence of exogenous substrate. The rate of tension development during the first contraction in the presence of 2DG was similar to that in the absence of substrate. However, the second contraction in the presence of 2DG had a substantially increased rate of tension development. 3. 31P nuclear magnetic resonance (NMR) spectroscopy revealed that, at resting tone, in the presence of 2DG, inorganic phosphate (P(i)) and phosphocreatine (PCr) simultaneously decrease while 2-deoxyglucose-6-phosphate accumulates. During contraction-relaxation cycles, in the presence of 2DG, P(i) and PCr become undetectable while ATP declines to approximately 50% of control values as determined by NMR. During the second contraction in the presence of 2DG, the area of the ADP resonance was similar to that of the alpha-ATP resonance. 4. The increase in the magnitude of tension generation, during 2DG administration, correlated with a decrease in P(i) levels. The rate of relaxation from a contraction, in the presence of 2DG, was slower than in the presence of glucose or in the absence of exogenous substrate. These results are consistent with the role of P(i) in the release of the proposed 'latch-bridge' state of maintained contraction at low energy demand. 5. The increase in isometric tension generation during contraction in the presence of 2DG appears to be related to the decreased levels of P(i). In the presence of 2DG, the reduction of PCr and of ATP occur to a similar extent to that during hypoxia, yet no inhibition of force takes place. The low levels of ATP and PCr reported with 2DG administration in these studies do not energetically limit the contractile apparatus.

Adenosine Triphosphate↗

PubMed1989

Preferential support of Ca2+ uptake in smooth muscle plasma membrane vesicles by an endogenous glycolytic cascade.

Studies of intact smooth muscle have suggested that its anomalous aerobic lactate production may reflect an intracellular compartmentation of glycolytic enzyme cascades designed to support specific exergonic processes. In particular, we have postulated a membrane-associated glycolytic cascade that preferentially supports the ATP requirements of membrane functions. We tested this hypothesis by using a smooth muscle plasma membrane fraction (PMV) purified for calcium pump activity. We show that glycolytic enzymes are endogenous in PMV and can produce NADH, ATP, and lactate from fructose 1,6-diphosphate in the presence of glycolytic cofactors. This glycolytic cascade can fuel the calcium pump despite the presence of an ATP trap that eliminated calcium uptake fueled by exogenously added ATP. This plasma membrane glycolytic cascade is coupled to calcium pump function in a tissue with both oxidative and glycolytic metabolism. Thus coupling of metabolic cascades with the specific processes they subserve may be a more general feature of cellular organization than was previously thought.

Adenosine Triphosphate↗

PubMedDate not supplied

Oleate oxidation and mitochondrial substrate selection in vascular smooth muscle.

The purpose of this study was to determine the contribution of long-chain fatty acids relative to other mitochondrial substrates in active vascular smooth muscle (VSM). Hog carotid arteries were isometrically contracted in physiological saline solution containing 0.71 mM U-(13)C-oleic acid (bound to albumin at a ratio of 6.8:1), 5 mM 1-(13)C-glucose and 1 mM acetate in the presence or absence of 5 mM carnitine for 6 h at 37 degrees C. Substrate oxidation was determined using (13)C-isotopomer analysis of glutamate. Although oxidation of oleic acid could not be measured at physiological concentrations [0.5 mM (1:1)], oleic acid oxidation was approximately 5% of the total substrates oxidized at the higher concentration examined. Although insignificant, carnitine increased oleic acid oxidation to approximately 8%, and resulted in a decrease in endogenous lipid oxidation, which was 2-12% of the total substrates oxidized. Oxidation of glucose and acetate did not significantly change due to the inclusion of oleic acid in the incubation solutions. Therefore, we conclude that exogenous long-chain fatty acids are minor contributors to substrate oxidation (approximately 5%) in VSM compared to other mitochondrial substrates, such as glucose and acetate, which account for approximately 80% of the substrates oxidized by VSM.

Acetates↗

PubMedDate not supplied

Vascular smooth muscle glycogen metabolism studied by 13C-NMR.

Vascular smooth muscle glycogen stores are traditionally thought to be small compared to other glycogen-containing tissues such as striated muscle or liver. However, glycogen has been thought to be an important carbon substrate for oxidative metabolism in support of contraction in vascular smooth muscle. We examined the synthesis and degradation of glycogen in isometrically mounted hog carotid artery using 13C-NMR spectroscopy. The rate of net glycogen synthesis from 1-13C-glucose was found to be constant during the first 8 h of incubation of carotid arteries with 10 mM glucose at 37 degrees C and then decreased towards a rate of zero by 14 h of incubation. During 8 h of incubation in the presence of 5 mM glucose, the content of glycogen increased from 1.5 to 8.1 mumol/g blot weight in the absence of insulin and to 11.4 mumol/g blot weight in the presence of 0.5 U/ml insulin. During prolonged glycogen loading, there was a simultaneous degradation of previously synthesized 6-13C-glycogen during synthesis of 1-13C-glycogen from 1-13C-glucose indicating substrate cycling of glycogen metabolism. This substrate cycling results in a pattern of glycogen utilization in which the most recently synthesized glucosyl units of glycogen are utilized only slightly more readily than the previously synthesized glucosyl units of glycogen. We conclude that glycogen stores are larger and more dynamic than previously thought in vascular smooth muscle consistent with an important role for glycogen as a carbon source for smooth muscle energy metabolism.

Animals↗