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.     

Compartmentation of ATP synthesis and utilization in smooth muscle: roles of aerobic glycolysis and creatine kinase

The phosphocreatine content of smooth muscle is of similar magnitude to ATP. Thus the function of the creatine kinase system in this tissue cannot simply be regarded as an energy buffer. Thus an understanding of its role in smooth muscle behavior can point to CK function in other systems. From our perspective CK function in smooth muscle is one example of a more general phenomenon, that of the co-localization of ATP synthesis and utilization. In an interesting and analogous fashion distinct glycolytic cascades are also localized in regions of the cell with specialized energy requirements. Similar to CK, glycolytic enzymes are known to be localized on thin filaments, sarcoplasmic reticulum and plasma membrane. In this chapter we will describe the relations between glycolysis and smooth muscle function and compare and contrast to that of the CK system. Our goal is to more fully understand the significance of the compartmentation of distinct pathways for ATP synthesis with specific functions in smooth muscle. This organization of metabolism and function seen most clearly in smooth muscle is likely representative of many other cell types.     

Motility, heat, and lactate production in ejaculated bovine sperm

Effects of various inhibitors on motility, heat, and lactate production of ejaculated bovine sperm were determined in the presence of antimycin A and rotenone. erythro-9-[3-(2-Hydroxynonyl)]adenine (EHNA) and polyvinylpyrrolidone (PVP-360) stopped motility and reduced heat or lactate production by 30-50%. Carbodiimides resulted in loss of motility and a reduction of metabolism by 60-75%. Quercetin treatment, which enhanced rather than inhibited motility, depressed heat and lactate production by 50-60%. Since mechanical immobilization reduced heat production by only 30%, the question arises as to what other cellular processes are major contributors to the energy budget. Inhibitors of ion flux had little-to-no effect on heat or lactate production, suggesting that neither mitochondrial nor Na+/K+ ATPases were major ATP-requiring processes. Calcium flux at the plasma membrane also was minimal and previous reports eliminated glycolytic substrate cycling as major consuming processes for ATP. Although quercetin inhibited lactate production in intact cells, no effect of quercetin on cell-free glycolysis and the ATPase activities of isolated dynein was detected. Quercetin did, however, inhibit ATPase activity of plasma membrane, suggesting that this unidentified ATPase may contribute to the formation of ADP and Pi required for lactate production by the intact cell. We propose (a) that the bioenergetic costs of motility are divided between regulatory events and dynein-microtubule interaction (dynein ATPase), (b) that some of the membrane-related processes may be "inefficient," and (c) that quercetin may render these steps more "efficient," in a manner analogous to its action on the Na+/K+ pump of Ehrlich ascites tumor cells.     

Protection of ROC-1 Hybrid Glial Cells by Polyethylene Glycol Following ATP Depletion

Abstract: Oligodendroglia-glioma hybrid cells (ROC-1) subjected to inhibition of glycolytic and oxidative ATP synthesis undergo a sequence of changes, including ATP depletion, parallel processes of cell swelling and blebbing, and finally plasma membrane disruption and cell death. The morphological and biochemical changes that follow ATP depletion were studied in the presence and absence of polyethylene glycol (M, 8,000), a nonpermeant oncotic agent. Polyethylene glycol prevented cell swelling and membrane blebbing. It significantly delayed, but did not prevent, the release of lactate dehydrogenase into the medium; it did not affect the fall in [ATP]. These results suggest that osmotic cell swelling may be a contributing factor in the loss of cell viability when ROC-1 cells are depleted of ATP.     

Kinetics and Energetics of Mg2+,ATP-Dependent Ca2+ Transport in the Plasma Membrane of Smooth Muscle Cells

Calcium ions play a crucial role in the excitation/contraction coupling in smooth muscles. I would like to interpret the biochemical mechanisms underlying Ca²⁺ exchange and dynamics of such an exchange in the smooth muscles. Particular emphasis is laid on the examination of kinetic, energetic, and catalytic properties of the membrane-linked energy-dependent Ca²⁺-transporting systems involved in regulation of the intracellular Ca²⁺ concentration in smooth muscle cells (SMC). It was suggested that the Mg²⁺,ATP-dependent plasma membrane calcium pump (Ca²⁺,Mg²⁺-ATPase) plays a key role in regulation of the Ca²⁺ concentration in SMC. The purpose of this review is to analyze some of our own results concerning kinetic, energetic, and catalytic properties of the calcium pump of the SMC plasma membrane. In our experiments, we used different biochemical models (namely, fractions of the membrane subcellular structures, highly purified Ca²⁺,Mg²⁺-ATPase of the SMC plasma membrane solubilized and reconstituted in the lyposomes, and suspension of digitonin-treated SMC) and a number of methods (including preparative biochemistry, enzymology, membranology, tracer ⁴⁵Ca²⁺ flux analysis, and chemical and enzymological kinetics). We have shown that sodium azide-insensitive Mg²⁺,ATP-dependent Ca²⁺ accumulation in ureter smooth muscle microsomes is determined by two components. One component represents the Mg²⁺,ATP-dependent calcium pump of the sarcoplasmic reticulum functionally potentiated by Ca²⁺-precipitating permeating anions, oxalate or phosphate and inhibited by thapsigargin or cyclopiazonic acid, the highly selective inhibitors of the calcium pump of sarco(endo)plasmic rerticulum. Another component represents the Mg²⁺,ATP-dependent calcium pump of the plasma membrane functionally potentiated by phosphate. This pump is not inhibited by thapsigargin and cyclopiazonic acid. The effects of temperature, dielectric permeability (D), and ionic strength on the activity of purified Ca²⁺,Mg²⁺-ATPase solubilized from the myometrial sarcolemma were studied. The results suggest that changes in the polarity of the incubation medium markedly affect the activity of transport Ca²⁺,Mg²⁺-ATPase, and electrostatic interactions between the enzyme activity center and specific ligands (Mg·ADP^-, in particular) significantly contribute to the energetics of ATP hydrolysis. Therefore, our data show that changes in the incubation medium polarity significantly affects the ATP-hydrolase activity of Ca²⁺,Mg²⁺-ATPase solubilized from the SMC plasma membranes, and electrostatic interactions between the enzyme active sites and reactants (in particular, Mg·ADP^-) contribute to a significant extent to the energetics of ATP hydrolysis. We cannot rule out that under physiological conditions the local D values of the myoplasm may differ from that of water, and, moreover, may change (especially near the membrane surface) depending on the metabolic level of SMC. We suppose that local changes in the cytoplasmic D value will affect the plasma membrane calcium pump and, consequently, the efficiency of control of intracellular Ca²⁺ homeostasis in smooth muscle. So, our biochemical models are suitable experimental objects for studying the kinetic, energetic, and catalytic properties of the Mg²⁺,ATP-dependent calcium pump of the SMC plasma membrane. In addition, our data might be useful for screening of the mechanisms underlying the action of different physico-chemical factors involved in modulation of the contraction/relaxation cycle.     

Cell Swelling, Blebbing, and Death Are Dependent on ATP Depletion and Independent of Calcium During Chemical Hypoxia in a Glial Cell Line (ROC-1)

The morphological and biochemical changes that occur during chemical hypoxic injury in a neural cell line were studied in the presence and absence of calcium. Oligodendroglial-glioma hybrid cells (ROC-1) were subjected to inhibitors of glycolytic and oxidative ATP synthesis (chemical hypoxia). Complete respiratory inhibition depleted [ATP] to less than 5% of control by 4 min. Blebs appeared on the cell surfaces and cells began to swell within a few minutes of ATP depletion. A 200% increase in cell volume and bleb coalescence preceded irreversible cell injury (lactate dehydrogenase release) which began at approximately 20 min with 50% cell death by 40 min. In energized cells an equivalent degree of osmotic swelling induced by ouabain inhibition of the Na+, K(+)-ATPase pump did not produce blebbing or cell death. Partial inhibition of respiration decreased [ATP] to approximately 10% of control by 40 min. Blebbing and swelling began at 40 min and bleb coalescence preceded plasma membrane disruption which began at approximately 55 min. ATP depletion, blebbing, swelling, and death followed similar time courses in the presence or absence of extracellular calcium ([Ca2+]e). Intracellular calcium ([Ca2+]i) was measured using fura-2. In calcium-containing medium metabolic inhibition caused a transient increase in resting [Ca2+]i (100 +/- 17 nM) followed by a low steady-state level preceding plasma membrane disruption. Following deenergization in calcium-free medium, [Ca2+]i remained below 60 nM throughout injury and death. These data suggest that decreased ATP initiates a sequence of events including bleb formation and cell swelling that lead to irreversible cell injury in the absence of large increases in [Ca2+]i.     

The muscle-specific calmodulin-dependent protein kinase assembles with the glycolytic enzyme complex at the sarcoplasmic reticulum and modulates the activity of glyceraldehyde-3-phosphate dehydrogenase in a Ca2+/calmodulin-dependent manner

The skeletal muscle specific Ca(2)+/calmodulin-dependent protein kinase (CaMKIIbeta(M)) is localized to the sarcoplasmic reticulum (SR) by an anchoring protein, alphaKAP, but its function remains to be defined. Protein interactions of CaMKIIbeta(M) indicated that it exists in complex with enzymes involved in glycolysis at the SR membrane. The kinase was found to complex with glycogen phosphorylase, glycogen debranching enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and creatine kinase in the SR membrane. CaMKIIbeta(M) was also found to assemble with aldolase A, GAPDH, enolase, lactate dehydrogenase, creatine kinase, pyruvate kinase, and phosphorylase b kinase from the cytosolic fraction. The interacting proteins were substrates of CaMKIIbeta(M), and their phosphorylation was enhanced in a Ca(2+)- and calmodulin (CaM)-dependent manner. The CaMKIIbeta(M) could directly phosphorylate GAPDH and markedly increase ( approximately 3.4-fold) its activity in a Ca(2+)/CaM-dependent manner. These data suggest that the muscle CaMKIIbeta(M) isoform may serve to assemble the glycogen-mobilizing and glycolytic enzymes at the SR membrane and specifically modulate the activity of GAPDH in response to calcium signaling. Thus, the activation of CaMKIIbeta(M) in response to calcium signaling would serve to modulate GAPDH and thereby ATP and NADH levels at the SR membrane, which in turn will regulate calcium transport processes.     

Calcium transporters and signalling in smooth muscles

Two P-type Ca transporters, the plasma membrane Ca-ATPase (PMCA) and the sarcoplasmic reticulum (SR) Ca-ATPase (SERCA), play a crucial role in maintaining Ca homeostasis, controlling contractility and contributing to excitably and cell signalling in smooth muscle cells. There is considerable structural homology between the two Ca-ATPases; they both have transmembrane spanning regions, have similar ATP-phosphorylated intermediaries, counter transport protons and are regulated by several second messengers. They both also exist in several isoforms and have many splice variants, which presumably impart some of their tissue specific functions. We describe the relative contribution of PMCA and the Na-Ca exchanger to Ca efflux in relaxation to smooth muscle, including recent data from transgenic mice, which has begun to elucidate the specific contributions of individual isoforms to Ca signalling. We then consider Ca release and uptake into the SR in smooth muscle. Experiments investigating the distribution of SERCA in smooth muscle cells have provided new insights into control of SR luminal Ca, and the effects of SR Ca load on signalling, is discussed. This is followed by a detailed consideration of the interactions between the surface membrane and SR membrane pumps, exchangers and ion channels in smooth muscle, along with their distribution to caveolae and cholesterol-rich membrane domains. Where relevant the importance of these functions to health and disease are noted. We conclude that the dynamic changes in splice variants expressed, constituents of membrane microdomains and environment of the sub-sarcolemmal space, close to the SR, need to be the focus of future research, so that the full importance of Ca transporters to smooth muscle signalling cascades can be better understood.     

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.     

Cortical alveoli of Paramecium: a vast submembranous calcium storage compartment

The plasma membrane of Paramecium is underlain by a continuous layer of membrane vesicles known as cortical alveoli, whose function was unknown but whose organization had suggested some resemblance with muscle sarcoplasmic reticulum. The occurrence of antimonate precipitates within the alveoli first indicated to us that they may indeed correspond to a vast calcium storage site. To analyze the possible involvement of this compartment in calcium sequestration more directly, we have developed a new fractionation method, involving a Percoll gradient, that allows rapid purification of the surface layer (cortex) of Paramecium in good yield and purity and in which the alveoli retain their in vivo topological orientation. This fraction pumped calcium very actively in a closed membrane compartment, with strict dependence on ATP and Mg2+. The pumping activity was affected by anti-calmodulin drugs but no Triton-soluble calmodulin binding protein could be identified, using gel overlay procedures. The high affinity of the pump for calcium (Km = 0.5 microM) suggests that it plays an important role in the normal physiological environment of the cytosol. This may be related to at least three calcium-regulated processes that take place in the immediate vicinity of alveoli: trichocyst exocytosis, ciliary beating and cytoskeletal elements dynamics during division.     

Cover Picture: J. Biophotonics 8/2014

Optical induction of smooth muscle contraction using a femtosecond‐pulsed laser: When femtosecond laser pulses (yellow beam) are focused on the cytosol of the smooth muscle cell (upper left), free electrons (blue particle) are generated in the focal area. Laser‐induced free electrons subsequently induce intracellular production of reactive oxygen species (ROS, red particles), which are amplified via inter‐mitochondrial networks. Amplified ROS signals can stimulate the sarcoplasmic reticulum to release calcium ion (green particles) into the cytosol. Locally increased calcium ion can induce global calcium wave in whole cytosolic area through intrinsic calcium‐induced calcium release process. This calcium wave propagates to adjacent cells via a gap‐junction, which is located at the plasma membrane. Thus, femtosecond‐pulsed laser stimulation into a single muscle cell can induce contraction of whole tissue (lower right) via intrinsic cascades, which are composed of low‐density plasma, ROS, calcium ion, and calcium‐induced calcium propagation. (Picture: J. Yoon et al., pp. 597–606 in this issue)     

Energy-dependent calcium uptake activity of microsomes from the aorta of normal and hypertensive rats

Energy-dependent calcium uptake activity of microsomes isolated from the rat aorta has been characterized. The microsomes consist of smooth membrane vesicles which in the presence of Mg · ATP as an energy source continuously sequester calcium over a 60-min period. This calcium uptake is greatly stimulated by oxalate anion which serves as a calcium trapping agent. Unlike the calcium uptake of miltochondria this uptake is not inhibited by sodium azide. Sucrose density gradient analysis of the microsomal calcium uptake suggests that the system is associated with the sarcoplasmic reticulum. In presence of 5 mM Mg · ATP and 20μM calcium approximately 38 nmol of calcium per mg of microsormal protein are taken up in 20 min. In the absence of ATP, less than 2 nmol of calcium per mg of protein are taken up in the first 2 min. with no further uptake of calcium in subsequent time periods. When calcium uptake activity is plotted against calcium or ATP concentration of the medium, half maximal activity is calculated for 24.3 μM calcium and for 1.6 mM ATP. The calcium uptake characteristics of the rat aorta microsomes are compatible with a postulated role in the relaxation of the vascular smooth muscle and the provision of an intracellular calcium store for muscle contraction.Aorta microsomes from SHR rats (a genetic strain that is spontaneously hypertensive) have a significantly reduced calcium uptake when compared with the corresponding nonhypertensive control strain. The level of calcium and ATP for half maximal activity of the rat aorta microsomal calcium uptake system is approximately the same in the SHR and the control strain. The rate of release of calcium from rat aorta microsomes is apparently identical in SHR strain and control. The calcium uptake activity of kidney and liver microsomes isolated from the SHR rat appears to be identical to that found in the control strain.Rats were treated with the steroid deoxycorticosterone acetate for ten and thirty days to induce hypertension. After ten days of deoxycorticosterone acetate although hypertension is present, there is no change in calcium uptake activity of aorta microsomes, renal microsomes or renal plasma membranes. After 30 days of deoxycorticosterone acetate treatment calcium uptake activity of renal microsomes is reduced. A variable decrease in calcium uptake activity is observed with aorta microsomes. Renal plasma membrane calcium uptake remains unchanged.     

Identification and biochemical characterization of two novel UDP-2,3-diacetamido-2,3-dideoxy-alpha-D-glucuronic acid 2-epimerases from respiratory pathogens

For a long period lactate was considered as a dead-end product of glycolysis in many cells and its accumulation correlated with acidosis and cellular and tissue damage. At present, the role of lactate in several physiological processes has been investigated based on its properties as an energy source, a signalling molecule and as essential for tissue repair. It is noteworthy that lactate accumulation alters glycolytic flux independently from medium acidification, thereby this compound can regulate glucose metabolism within cells. PFK (6-phosphofructo-1-kinase) is the key regulatory glycolytic enzyme which is regulated by diverse molecules and signals. PFK activity is directly correlated with cellular glucose consumption. The present study shows the property of lactate to down-regulate PFK activity in a specific manner which is not dependent on acidification of the medium. Lactate reduces the affinity of the enzyme for its substrates, ATP and fructose 6-phosphate, as well as reducing the affinity for ATP at its allosteric inhibitory site at the enzyme. Moreover, we demonstrated that lactate inhibits PFK favouring the dissociation of enzyme active tetramers into less active dimers. This effect can be prevented by tetramer-stabilizing conditions such as the presence of fructose 2,6-bisphosphate, the binding of PFK to f-actin and phosphorylation of the enzyme by protein kinase A. In conclusion, our results support evidence that lactate regulates the glycolytic flux through modulating PFK due to its effects on the enzyme quaternary structure.     

Characterization of membrane calcium pumps by simultaneous immunoblotting and 32P radiography

Calcium pumps of various plasma membrane, endoplasmic reticulum and sarcoplasmic reticulum preparations were visualized by simultaneous immunoblotting and autoradiography of the 32P-labelled phosphoenzymes. The pump proteins and their fragments produced by a proteolytic pretreatment of the membranes were selectively phosphorylated by [gamma-32P]ATP, separated on an acidic SDS-polyacrylamide gel, blotted onto nitrocellulose and reacted with polyclonal antibodies raised against the purified human erythrocyte and rat skeletal muscle sarcoplasmic reticulum calcium pumps, respectively. The immuno-reaction was detected by peroxidase staining, while the phosphoproteins were shown by autoradiography of the same blot. An antibody against the erythrocyte calcium pump, reacting on the blot with the 140 kDa erythrocyte calcium pump and its 80 kDa proteolytic fragment, did not show a cross-reaction with the calcium pump of similar molecular mass in rat synaptosome membranes or with any of the endoplasmic- or sarcoplasmic-type calcium pumps. An anti-sarcoplasmic reticulum calcium pump antibody cross reacted with several sarcoplasmic and endoplasmic calcium pump proteins and their proteolytic fragments but with none of the plasma membrane pumps. This sensitive double-labelling method can be applied to study structural relationships and molecular alterations in various ion pump proteins.     

Dense-cored membranous structures in smooth muscle cells of the vas deferens of the rat

Summary Smooth muscle cells from rat vas deferens were studied by electron microscopy. Vesicular and tubular membranous structures containing an electron-opaque material were found in the smooth muscle cells. Similar structures were also found in a subfraction (F3) of microsomes of vas deferens smooth muscle which was shown to be rich in both plasma membrane and putative endoplasmic reticulum markers. Treatment of the tissues with calcium-free Krebs solution containing EGTA prior to fixation eliminated almost completely the presence of these dense-cored membranous structures (DMS), whereas incubation of the subcellular membrane fraction with EGTA solution had no effect on the appearance of the DMS. Plasma membrane infoldings were found in the smooth muscle cells extending well into their interior. Horseradish peroxidase penetrates vesicles in a location similar to that of DMS in smooth muscle cells, suggesting that some of the DMS may be connected to the extracellular space. We conclude that the dense-core material within the DMS is calcium dependent. We also suggest that some of the DMS represent infoldings of the plasma membrane extending into the cell's interior.     

Caveolae and calcium handling, a review and a hypothesis

Caveolae are associated with molecules crucial for calcium handling. This review considers the roles of caveolae in calcium handling for smooth muscle and interstitial cells of Cajal (ICC). Structural studies showed that the plasma membrane calcium pump (PMCA), a sodium-calcium exchanger (NCX1), and a myogenic nNOS appear to be colocalized with caveolin 1, the main constituent of these caveolae. Voltage dependent calcium channels (VDCC) are associated but not co-localized with caveolin 1, as are proteins of the peripheral sarcoplasmic reticulum (SR) such as calreticulin. Only the nNOS is absent from caveolin 1 knockout animals. Functional studies in calcium free media suggest that a source of calcium in tonic smooth muscles exists, partly sequestered from extracellular EGTA. This source supported sustained contractions to carbachol using VDCC and dependent on activity of the SERCA pump. This source is postulated to be caveolae, near peripheral SR. New evidence, presented here, suggests that a similar source exists in phasic smooth muscle of the intestine and its ICC. These results suggest that caveolae and peripheral SR are a functional unit recycling calcium through VDCC and controlling its local concentration. Calcium handling molecules associated with caveolae in smooth muscle and ICC were identified and their possible functions also reviewed.     

Role of skeletal muscle Na+-K+ ATPase activity in increased lactate production in sub-acute sepsis

Bacterial sepsis is frequently accompanied by increased blood concentration of lactic acid, which traditionally is attributed to poor tissue perfusion, hypoxia and anaerobic glycolysis. Therapy aimed at improving oxygen delivery to tissues often does not correct the hyperlactatemia, suggesting that high blood lactate in sepsis is not due to hypoxia. Various tissues, including skeletal muscle, demonstrate increased lactate production under well-oxygenated conditions when the activity of the Na+-K+ ATPase is stimulated. Although both muscle Na+-K+ ATPase activity and muscle plasma membrane content of Na+, K+-ATPase subunits are increased in sepsis, no studies in vivo have demonstrated correlation between lactate production and changes in intracellular Na+ and K+ resulting from increased Na+-K+ pump activity in sepsis. Plasma concentrations of lactate and epinephrine, a known stimulator of the Na+-K+ pump, were increased in rats made septic by E. coli injection. Muscle lactate content was significantly increased in septic rats, although muscle ATP and phosphocreatine remained normal, suggesting oxygen delivery remained adequate for mitochondrial energy metabolism. In septic rats, muscle intracellular ratio of Na+:K+ was significantly reduced, indicating increased Na+-K+ pump activity. These data thus demonstrate that increased muscle lactate during sepsis correlates with evidence of elevated muscle Na+-K+ ATPase activity, but not with evidence of impaired oxidative metabolism. This study also further supports a role for epinephrine in this process.     

Mg2+- or Ca2+-activated ATPase activities of plasma membranes isolated from vascular smooth muscle

Studies of ATP hydrolysis by various subcellular fractions isolated from rat mesenteric arteries and veins indicate that an apparent ATPase activity, which can be activated by Mg2+ or Ca2+, is primarily associated with the plasma membranes. Although both Mg2+-activated and Ca2+-activated ATPase activities under the optimal condition are substantially lower in venous than in arterial plasma membrane fraction, their dependence on the concentration of Mg2+ and Ca2+ are quite similar in arterial as well as venous plasma membrane fractions. No synergistic effect on ATP hydrolysis was observed in the presence of both Mg2+ and Ca2+. In addition, Mg2+-activated and Ca2+-activated ATPase activities show similar pH dependence, inhibition by deoxycholate, stability toward heat inactivation and substrate specificity. Furthermore, Mg2+-activated and Ca2+-activated ATPase activities were similarly reduced in vascular smooth muscles of spontaneously hypertensive rats. These results suggest that the activation of ATP hydrolysis by Mg2+ or Ca2+ may represent a single enzyme moiety in the plasma membrane of vascular smooth muscle. The possible involvement of such ATPase in the Ca2+ transport function of vascular smooth muscle is discussed.     

Ecto-ATPases and 5′-nucleotidases in the caveolae of smooth muscle. Enzyme-histochemical evidence may indicate a role for caveolae in neurotransmission.

We have demonstrated the localization of ecto-Ca-ATPase and 5′-nucleotidase activity in the caveolae of smooth muscle cells of guinea pig vas deferens and the ileum longitudinal muscle strips with a cerium-precipitation enzyme-cytochemical method. The activities seemed to be strongest in the caveolae. Since the simultaneous presence of the 5′-nucleotidase activity supports the hypothesis that this ecto-Ca-ATPase activity does not have a pump function, but, together with 5′-nucleotidase, may play a role in neurotransmission, these specific membrane invaginations, the caveolae, have a functional relationship with transverse tubules of striated muscle.     

Effects of dinitrophenol and oligomycin on the coupling between anaerobic metabolism and anaerobic sodium transport by the isolated turtle bladder

Dinitrophenol (1 x 10^-5 M) has been found to inhibit anaerobic sodium transport by the isolated urinary bladder of the fresh water turtle. Concurrently, anaerobic glycolysis was stimulated markedly. However, tissue ATP levels diminished only modestly, remaining at approximately 75% of values observed under anaerobic conditions without DNP. The utilization of glucose (from endogenous glycogen) corresponded closely to that predicted from the molar quantities of lactate formed. Thus the glycolytic pathway was completed in the presence of DNP and if ATP were synthesized normally during glycolysis, synthesis should have been increased. On the other hand, the decrease in Na transport should have decreased ATP utilization. Oligomycin did not block sodium transport either aerobically or anaerobically, but ATP concentrations did decrease. When anaerobic glycolysis was blocked by iodoacetate, pyruvate did not sustain sodium transport thus suggesting that no electron acceptors were available in the system. Two explanations are entertained for the anaerobic effect of DNP: (a) Stimulation by DNP of plasma membrane as well as mitochondrial ATPase activity; (b) inhibition of a high energy intermediate derived from glycolytic ATP or from glycolysis per se. The arguments relevant to each possibility are presented in the text. Although definitive resolution is not possible, we believe that the data favor the hypothesis that there was a high energy intermediate in the anaerobic system and that this intermediate, rather than ATP, served as the immediate source of energy for the sodium pump.