bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of vesicular transport in ADH-stimulated aggregate delivery
Authors: Ding G. , Franki N. , Bourguet J. , Hays R. M.
Published: December 2017

It has been assumed from studies in toad bladder that antidiuretic hormone (ADH)-stimulated particle delivery to the luminal membrane is mediated by particle-carrying tubular structures (aggrephores). We report studies in frog and toad urinary bladder showing that vesicles, rather than aggrephores, appear to play the major role in particle delivery in the frog and that vesicle and aggrephore delivery proceed in parallel in the toad. Our principal evidence for this view is that in the frog, transmission electron microscopy shows virtually no fused aggrephores. Supporting evidence includes the following. 1) Freeze-fracture studies show that the diameters of fusion events delivering particles can be quite small, indicating that they are formed by fused vesicles rather than fused aggrephores. 2) A significant population of small fusion events is also seen in the toad, along with larger fusion events related to both aggrephores and large vesicles. 3) Surface aggregate areas in both species are small, consistent with vesicular delivery. 4) Freeze-fracture replicas indicate delivery from shallow pits. We propose a system of transport of particles in which aggrephores act largely as intermediate storage organelles in the frog and as storage and fusion organelles in the toad.
Coordination of electromechanical and metabolic properties of cat soleus motor units
Authors: Martin T. P. , Bodine-Fowler S. , Edgerton V. R.
Published: December 2017

Motor units were studied in the soleus muscle of normal adult cats and adult cats that had undergone complete spinal cord transection approximately 4 mo earlier. Intracellular recording and stimulation techniques were used to study selected electrical properties of the motoneuron and isometric contractile properties of the muscle unit. Motor unit fibers were depleted of their glycogen through repetitive stimulation of the motoneuron and identified by a quantitative histochemical determination of glycogen. A sample of muscle fibers from the glycogen-depleted unit and from fibers not depleted of glycogen were analyzed for cross-sectional area, succinate dehydrogenase (SDH), alpha-glycerolphosphate dehydrogenase (GPD), and alkaline myofibrillar adenosine triphosphatase. It was observed that the fiber-to-fiber variability in cross-sectional area and SDH and GPD activity within units of normal and transected cats was significantly larger than that measured in repeated samples from a single fiber. Additionally, for each of these properties, the range found among fibers within a unit was similar to that found among nondepleted fibers of the same myosin type. The influence of spinal cord transection on some muscle fibers seemed to result in a metabolic shift from the generalized category of slow-oxidative toward fast-oxidative glycolytic. This shift in metabolic properties appeared to be coupled with a similar shift in the physiological properties of the muscle unit and motoneuron from slow to fast.
pH regulation in single glomerular mesangial cells. II. Na+-dependent and -independent Cl(-)-HCO3- exchangers
Authors: Boyarsky G. , Ganz M. B. , Sterzel R. B. , Boron W. F.
Published: December 2017

We used the pH-sensitive dye 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) to further characterize the mechanisms of intracellular pH (pHi) regulation in renal mesangial cells. In the accompanying paper [Am. J. Physiol. 255 (Cell Physiol. 24): C844-C856, 1988], we showed that acid extrusion from mesangial cells is mediated by both an ethylisopropylamiloride (EIPA)-sensitive Na+-H+ exchanger and a 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS)-sensitive-HCO3(-)-dependent mechanism. In this study, we examined the ionic dependencies of pHi-regulatory mechanisms in the presence of CO2-HCO3-. We found that in CO2-HCO3-, approximately 90% of the net acid extrusion occurring during recovery from an acid load is blocked by removing external Na+. Short-term (less than 15 min) removal of external Cl- has little effect on the rate of recovery in CO2-HCO3-. In contrast longer periods of external Cl- removal (1-2 h) blocks 40-60% of the rate of recovery, which is consistent with the hypothesis that a large fraction of the SITS-sensitive-HCO3(-)-dependent recovery mechanism described in the preceding paper is also Na+- and Cl(-)-dependent. Therefore, this Cl(-)-dependent component is probably mediated by a Na+-dependent Cl(-)-HCO3- exchanger. As much as 16% of total acid extrusion is insensitive to EIPA and long-term Cl- removal but is blocked by SITS. Thus either 1-2 h of Cl- removal is insufficient to wash out all internal Cl-, or a small component of acid extrusion is mediated by a Cl(-)-independent mechanism, such as the electrogenic Na+/HCO3- cotransporter. We also studied the effect on pHi of the removal and readdition of external Cl-, observing pHi changes consistent with the existence of a Na+-independent Cl(-)-HCO3- exchanger, which would presumably function as an acid loader. In contrast to the Na+-H+ exchanger and Na+-dependent Cl(-)-HCO3- exchanger, which are stimulated at low pHi, the Cl(-)-HCO3- exchanger is stimulated at high pHi. Thus the acid-extruding and acid-loading mechanisms have opposite pHi dependencies.
Metabolic alterations induced in cultured skeletal muscle by stretch-relaxation activity
Authors: Hatfaludy S. , Shansky J. , Vandenburgh H. H.
Published: December 2017

Avian pectoralis muscle cells differentiated in vitro are mechanically stimulated by repetitive stretch-relaxations of the cell's substratum. Their metabolic response to mechanical activity is determined by measuring rates of [3H]deoxy-D-glucose uptake and lactate efflux. These two metabolic parameters show a similar biphasic response to mechanical stimulation. During the first 4-6 h of activity, [3H]deoxy-D-glucose uptake and lactate efflux increase 34 and 26%, respectively; at 8 and 24 h of activity, [3H]deoxy-D-glucose uptake and lactate efflux are no longer elevated relative to control values. With continued activity beyond 24 h, their rates are again significantly elevated (150 and 93% by 48 h, respectively). The long-term increases in [3H]deoxy-D-glucose uptake and lactate efflux occur independently of medium growth factors. Protein synthesis is required for the short-term increase in [3H]deoxy-D-glucose uptake, but muscle electrical activity is not required for either short- or long-term increases in [3H]deoxy-D-glucose uptake because both occur in the presence of tetrodotoxin. This new model system allows for the analysis of mechanically induced metabolic alterations in aneural skeletal muscle cells under the defined conditions of tissue culture.
A human airway smooth muscle cell line that retains physiological responsiveness
Authors: Panettieri R. A. , Murray R. K. , DePalo L. R. , Yadvish P. A. , Kotlikoff M. I.
Published: December 2017

We report the development of a nontransformed line of human airway smooth muscle cells retaining smooth muscle-specific contractile protein expression and physiological responsiveness to agonists implicated in inflammatory airway diseases. Specific responses to histamine, leukotrienes, bradykinin, platelet-activating factor, substance P, and thromboxane analogues are demonstrated as well as functional coupling to beta-adrenergic receptors. The cell line was characterized using indirect immunofluorescence, as well as electrophoretic separation and immunoblot analysis of smooth muscle-specific actin. Functional responses were assessed by measurements of cytosolic calcium and stimulation of adenosine 3',5'-cyclic monophosphate production. The cells retain their responsiveness over many population doublings and should be a useful model to examine specific receptor-effector mechanisms, as well as the effects of neurohumoral agents on the regulation of airway smooth muscle growth and differentiation.
Carbohydrate metabolism by primary cultures of rabbit proximal tubules
Authors: Tang M. J. , Suresh K. R. , Tannen R. L.
Published: December 2017

Renal proximal tubular epithelia were used to assess the factors responsible for the induction of glycolysis in cultured cells. Primary cultures of rabbit proximal tubules, which achieved confluency at 6 days, exhibited hormonal responsiveness and brush-border characteristics typical of proximal tubular cells. Beginning at day 4, these cultured cells exhibited increased glycolytic metabolism reflected by enhanced glucose uptake and lactate production, along with parallel increases in activity of the glycolytic enzymes, pyruvate kinase and lactate dehydrogenase. The gluconeogenic enzymes, phosphoenolpyruvate carboxykinase (PEPCK) and fructose-1,6-bisphosphatase (FDP), were downregulated, and the cultured cells exhibited lower oxygen consumption rates than fresh tubules. Cells grown on a rocker, to mitigate hypoxia, exhibited a metabolic and enzymatic profile similar to cells grown under still conditions. ATP levels in cultured cells were higher than in fresh tubules. Furthermore, pyruvate kinase activity was higher in cells grown in media containing 0.5 as contrasted with 25 mM glucose. The enhanced glycolytic metabolism exhibited by cultured proximal tubular cells appears to be a characteristic of proliferation and is not a response to hypoxia, the Pasteur effect, or environmental glucose.
Effects of growth factors on myogenic differentiation
Authors: Florini J. R. , Magri K. A.
Published: December 2017

It has now been well established that the terminal differentiation of muscle cells in culture is subject to control by hormones and growth factors in the incubation medium. Thus far the most potent and most extensively studied agents are fibroblast growth factor (FGF), the insulinlike growth factors (IGFs), and transforming growth factor-beta (TGF-beta). Independent reports from several laboratories have established that both FGF and TGF-beta are potent inhibitors of differentiation and both appear to act at early stages of commitment to differentiation. Stimulation of differentiation by the IGFs (and by insulin at concentrations in the microgram/ml range) has also been observed and confirmed repeatedly. FGF and IGF are mitogenic for muscle cells, and TGF-beta either has no effect or suppresses cell proliferation, so previous generalizations that mitogens inhibit myogenic differentiation are clearly not valid when results with purified agents in well-defined media are considered. Work with oncogenes and specific toxins is beginning to reveal the mechanisms by which these agents might affect differentiation, and there is reason for optimism that an understanding of the molecular events that control terminal differentiation may be attained in the near future.
Cytosolic free magnesium concentration in cultured chick heart cells
Authors: Rotevatn S. , Murphy E. , Levy L. A. , Raju B. , Lieberman M. , London R. E.
Published: December 2017

Cytosolic free magnesium (Mgi) was measured in embryonic chick heart cells loaded with one of two newly developed 19F nuclear magnetic reasonance (NMR)-sensitive magnesium chelators, 4-methyl,5-fluoro-2-aminophenol-N,N,O-triacetate (MF-APTRA) and 5-fluoro-2-aminophenol-N,N,O-triacetate (5F-APTRA). The cells, embedded in strands of collagen, were superfused at a rate that allowed for solution changes in 2 min. In this preparation 19F- and 31P-NMR spectra were stable for at least 3.5 h. Because Na-coupled Mg countertransport may be a possible mechanism of Mg transport, in some experiments extracellular Na was reduced to 1 mM (choline substituted). This manipulation caused a 2.5-fold increase in Mgi from the basal level of 0.56 mM. A significant proportion of this increase in Mgi could be secondary to an increase in Cai that occurs with low extracellular Na (Nao) perfusion (Nai-Cao exchange). Perfusing cells with nominally Ca-free, 1 mM Na salt solution substantially attenuated the increase in Mgi that occurred with Ca present (1.25 mM) in the low Na (1 mM) solution. Furthermore, perfusion with 1 mM Na, Mg-free salt solution caused a 1.5-fold increase in Mgi, which cannot be attributable to Nai-Mgo exchange. Therefore attempts to describe the regulation of Mgi in heart cells must differentiate between the effects of Nai-Mgo exchange and competition for binding sites that are secondary to stimulation of ion gradient-coupled mechanisms.
Binding of adenylosuccinate synthetase to contractile proteins of muscle
Authors: Manfredi J. P. , Marquetant R. , Magid A. D. , Holmes E. W.
Published: December 2017

The muscle isozyme of adenylosuccinate synthetase (AdSS), an enzyme of the purine nucleotide cycle, has previously been shown to bind to purified F-actin in buffers of low ionic strength and pH (Ogawa et al. Eur. J. Biochem. 85: 331-338, 1978). We have extended these observations by measuring the association of both crude and purified AdSS with the contractile proteins of muscle in buffers of physiological ionic strength and pH. Under these conditions, the enzyme binds to F-actin, actin-tropomyosin complexes, reconstructed thin filaments, and myofibrils but not to myosin. The apparent dissociation constant of 1.2 microM and binding maximum of 2.6 nmol enzyme/mg myofibrils indicate that binding of AdSS to myofibrils can be physiologically significant. The results suggest that AdSS in muscle may be associated with the thin filament of myofibrils.
Swelling, reductive stress, and cell death during chemical hypoxia in hepatocytes
Authors: Gores G. J. , Flarsheim C. E. , Dawson T. L. , Nieminen A. L. , Herman B. , Lemasters J. J.
Published: December 2017

In rat hepatocytes, we examined the relationship between cell volume, bleb formation, and loss of cell viability during chemical hypoxia with KCN plus iodoacetic acid. In hypotonic media (150-200 mosmol/kgH2O), cells swelled to a greater extent during chemical hypoxia than in isotonic media, but rates of cell killing were identical. Sucrose (300 mM) added to isotonic media prevented early cell swelling but actually accelerated cell killing. In contrast, mannitol (300 mM) improved cell survival but did not prevent cell swelling. Bleb formation occurred regardless of buffer tonicity. The antioxidants desferrioxamine and cyanidanol but not superoxide dismutase +/- catalase delayed lethal cell injury. Cell killing was greater during aerobic compared with anaerobic chemical hypoxia. Hydroperoxide formation was measured using a dichlorofluorescin assay and was accelerated during aerobic but not anaerobic chemical hypoxia. The results indicate that cell swelling is not the driving force for bleb formation or lethal cell injury. We conclude that “reductive stress” caused by respiratory inhibition favors formation of toxic oxygen species and may contribute to lethal cell injury during intermittent or incomplete oxygen deprivation.
Ca2+-activated K channels of canine colonic myocytes
Authors: Carl A. , Sanders K. M.
Published: December 2017

K channels in enzymatically dispersed circular smooth muscle cells from the canine proximal colon were studied with the patch-clamp technique. The most prominent channel in cell-attached and excised, inside-out patches was a K channel, which had slope conductances of approximately 100 pS at a holding potential of 0 mV in a physiological K+ gradient and approximately 200 pS in symmetrical 140 mM K+ solutions. The relative permeabilities of the channel for monovalent cations were 1.0 K+:0.5 Rb+: less than 0.07 Li+:less than 0.07 Na+. The channels were activated by potential and intracellular Ca2+. At Ca2+ concentrations less than 10(-7) M, channel openings were rare except at very positive potentials. At Ca2+ concentrations between 10(-7) and 10(-6) M the probability of channel opening increased steeply, and the voltage for channel activation shifted to a negative potential range, which cells experience during electrical slow wave events in situ. The effect of Ca2+ on the open-state probability of single channels was mainly due to a decrease in mean close time. Channels were blocked by 1 mM tetraethylammonium applied to the outside of the patch but up to 10 mM tetraethylammonium applied to the inside of the patch, and 4-aminopyridine applied to either side did not block the channel. The data suggest that this channel mediates a current important in the termination of electrical slow waves, which are the primary excitable event in colonic circular muscles.
ATP receptor-induced Ca2+ transients in cardiac myocytes: sources of mobilized Ca2+
Authors: De Young M. B. , Scarpa A.
Published: December 2017

Addition of micromolar concentrations of extracellular ATP to adult rat cardiac ventricular myocytes increases cytosolic Ca2+ concentration ([Ca2+]). Experiments were performed on fura-2-loaded myocytes to determine whether the [Ca2+] rise was due to Ca2+ influx, release of Ca2+ from the sarcoplasmic reticulum (SR), or a combination of both. BAY K 8644 and nifedipine affected ATP-induced [Ca2+] transients, indicating involvement of voltage-sensitive Ca2+ channels. Addition of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or Ca2+ channel blockers significantly reduced cytosolic [Ca2+] changes due to addition of ATP or KCl without depleting Ca2+ stores (shown by ionomycin treatment in a Ca2+-free medium), demonstrating that these responses require Ca2+ influx. Depletion of intracellular Ca2+ stores by caffeine or ryanodine also diminished cytosolic [Ca2+] responses, indicating that a portion of the increased cytosolic [Ca2+] is due to Ca2+ release from SR. Norepinephrine potentiates the ATP-Ca2+ response, and this effect was not inhibited by depletion of intracellular stores. Although the data show that there are two Ca2+ sources in the cytosolic Ca2+ response to ATP, the pattern is also consistent with the hypothesis of Ca2+-induced Ca2+ release from cardiac SR.
Mechanisms by which mitochondria transport calcium
Authors: Gunter T. E. , Pfeiffer D. R.
Published: December 2017

It has been firmly established that the rapid uptake of Ca2+ by mitochondria from a wide range of sources is mediated by a uniporter which permits transport of the ion down its electrochemical gradient. Several mechanisms of Ca2+ efflux from mitochondria have also been extensively discussed in the literature. Energized mitochondria must expend a significant amount of energy to transport Ca2+ against its electrochemical gradient from the matrix space to the external space. Two separate mechanisms have been found to mediate this outward transport: a Ca2+/nNa+ exchanger and a Na(+)-independent efflux mechanism. These efflux mechanisms are considered from the perspective of available energy. In addition, a reversible Ca2(+)-induced increase in inner membrane permeability can also occur. The induction of this permeability transition is characterized by swelling of the mitochondria, leakiness to small ions such as K+, Mg2+, and Ca2+, and loss of the mitochondrial membrane potential. It has been suggested that the permeability transition and its reversal may also function as a mitochondrial Ca2+ efflux mechanism under some conditions. The characteristics of each of these mechanisms are discussed, as well as their possible physiological functions.
Dual effects of carbachol on cytosolic Ca2+ and contraction in intestinal smooth muscle
Authors: Mitsui M. , Karaki H.
Published: December 2017

The effects of carbachol on muscle tension and cytosolic Ca2+ concentrations ([Ca2+]cyt), measured with fura-2, were examined in the guinea pig intestinal smooth muscle. Carbachol induced an initial transient increase followed by a sustained increase in [Ca2+]cyt and muscle tension. Higher concentrations of carbachol induced larger transient changes and smaller sustained changes. In the presence of carbachol, application of Ca2+ to a Ca2(+)-depleted muscle induced a contraction that was smaller in the presence of higher concentrations of carbachol. High concentrations of carbachol inhibited the high-K(+)-stimulated muscle tension and [Ca2+]cyt. Contractile and inhibitory effects of carbachol were inhibited by a muscarinic M2 antagonist. Increase in the external Ca2+ concentration or addition of BAY K 8644, a Ca2(+)-channel activator, antagonized the inhibitory effect. There was a linear correlation between log [Ca2+]cyt and muscle tension under the conditions employed in the present experiments (r = 0.949). These results suggest that lower concentrations of carbachol increase [Ca2+]cyt and induce contraction, whereas high concentrations of carbachol have an additional effect to decrease [Ca2+]cyt and inhibit contraction by a Ca2(+)-channel blocker-like action.
Sodium affinity of brain Na(+)-K(+)-ATPase is dependent on isozyme and environment of the pump
Authors: Brodsky J. L. , Guidotti G.
Published: December 2017

The sodium affinities for the two forms of the Na(+)-K(+)-ATPase in brain were characterized. To mimic physiological conditions, synaptosomes, which are pinched off presynaptic nerve termini, were used. Examination of the pump in vitro was performed by preparing synaptic plasma membranes (SPMs). It was first shown that synaptosomes contain the two forms of the Na(+)-K(+)-ATPase, alpha 1 and alpha 2, and that these forms have markedly different affinities for the inhibitory cardiac glycoside ouabain. The apparent dissociation constant (K0.5) of alpha 1 for sodium changed from 12 to 9 mM when going from synaptosomes to membranes. For alpha 2, however, a shift from 36 to 12.5 mM was evident. The conclusion is that in vivo alpha 2 exists as a low sodium affinity species but can be altered to a high-affinity form simply by vesicle disruption. By comparison, the Na(+)-K(+)-ATPase from the mouse fibroblast cell line, 3T3-F442A cells, expressed only the alpha 1-isozyme, as shown by immunoblotting and by measurement of its ouabain and sodium affinities. The physiological relevance of these observations is also presented.
Acetylcholine activates nonselective cation channels in guinea pig ileum through a G protein
Authors: Inoue R. , Isenberg G.
Published: December 2017

Acetylcholine (ACh) depolarizes the membrane of mammalian intestinal myocytes by activating a nonselective cation channel (G. D. Benham, T. B. Bolton, and R. J. Lang. Nature Lond. 316: 345-347, 1985; R. Inoue, K. Kitamura, and H. Kuriyama. Pfluegers Arch. 410: 69-74, 1987). Here, we present evidence that occupation of the muscarinic receptor by ACh couples to channel activation via a G protein; the coupling can be blocked by pertussis toxin or by intracellular guanosine 5'-O-(2-thio-diphosphate) (GDP beta S), whereas intracellular guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) activates the channel in the absence of ACh. The currents, activated by either ACh or GTP gamma S, are nonadditive, conduct sodium ions, and are similar in their voltage dependence and facilitation by submicromolar calcium ions in the cytosol.
Effects of changes of pH on the contractile function of cardiac muscle
Authors: Orchard C. H. , Kentish J. C.
Published: December 2017

It has been known for over 100 years that acidosis decreases the contractility of cardiac muscle. However, the mechanisms underlying this decrease are complicated because acidosis affects every step in the excitation-contraction coupling pathway, including both the delivery of Ca2+ to the myofilaments and the response of the myofilaments to Ca2+. Acidosis has diverse effects on Ca2+ delivery. Actions that may diminish Ca2+ delivery include 1) inhibition of the Ca2+ current, 2) reduction of Ca2+ release from the sarcoplasmic reticulum, and 3) shortening of the action potential, when such shortening occurs. Conversely, Ca2+ delivery may be increased by the prolongation of the action potential that is sometimes observed and by the rise of diastolic Ca2+ that occurs during acidosis. This rise, which will increase the uptake and subsequent release of Ca2+ by the sarcoplasmic reticulum, may be due to 1) stimulation of Na+ entry via Na(+)-Ca2+ exchange; 2) direct inhibition of Na(+)-Ca2+ exchange; 3) mitochondrial release of Ca2+; and 4) displacement of Ca2+ from cytoplasmic buffer sites by H+. Acidosis inhibits myofibrillar responsiveness to Ca2+ by decreasing the sensitivity of the contractile proteins to Ca2+, probably by decreasing the binding of Ca2+ to troponin C, and by decreasing maximum force, possibly by a direct action on the cross bridges. Thus the final amount of force developed by heart muscle during acidosis is the complex sum of these changes.
Differential localization of two glucose transporter isoforms in rat kidney
Authors: Thorens B. , Lodish H. F. , Brown D.
Published: December 2017

The localization of two glucose transporter isoforms was mapped in the rat kidney: the high-Michaelis constant (Km; 15-20 mM) low-affinity "liver" transporter and the low-Km (1-2 mM) high-affinity "erythroid/brain" transporter. Both are basolateral membrane proteins, but the liver transporter was present exclusively in the S1 part of the proximal tubule, whereas the erythroid/brain transporter was expressed at variable levels in different nephron segments. Staining intensity was low in the straight proximal tubule (S3), intermediate in the medullary thin and thick ascending limbs, and highest in connecting segments and collecting ducts. In the collecting duct, the erythroid/brain glucose transporter was expressed at the highest level in intercalated cells; less was present in principal cells. In the papilla, only intercalated cells expressed this transporter isoform. These results suggest specific involvements of each transporter isoform in transepithelial glucose reabsorption by different segments of the proximal tubule. They also indicate that while the liver glucose transporter is present in gluconeogenic cells, there is a good correlation between the level of expression of the erythroid/brain glucose transporter and the glycolytic activity of the different nephron segments.
Protein content and myosin light chain phosphorylation in uterine arteries during pregnancy
Authors: Annibale D. J. , Rosenfeld C. R. , Stull J. T. , Kamm K. E.
Published: December 2017

During pregnancy, the ovine uterine artery changes from a low- to a high-stress artery. We investigated the hypotheses that the increased stress reflects alterations in vessel wall cellularity, smooth muscle cell contractile protein contents, or activation properties. Uterine artery diameter increased during pregnancy, whereas the fractional cellular composition and thickness of the muscularis were unchanged. Results of morphometry suggest that vessel growth is associated with cell elongation. Uterine arteries from pregnant ewes had greater protein contents than those from nonpregnant ewes (104 vs. 69 mg/g, respectively); there were corresponding increases in the absolute cellular contents of actin and myosin. While the fraction of light chain phosphorylated in response to phenylephrine was unaltered, the total amount of myosin light chain phosphorylated per gram wet weight increased significantly during pregnancy. In addition, the distribution of myosin heavy chain isoforms was also altered during pregnancy. The increased stress observed in the uterine artery during ovine pregnancy reflects, in part, increases in cellular contractile protein concentrations associated with hypertrophy.
Glycolytic component of rat spermatid energy and acid-base metabolism
Authors: Reyes J. G. , Velarde M. V. , Ugarte R. , Benos D. J.
Published: December 2017

The impact of glycolysis on rat spermatid energy metabolism is made apparent by the simultaneous occurrence of the following three events upon glucose addition to the extracellular medium of a rat spermatid cell suspension: decrease in ATP content, exit of acid equivalents, and increased lactate production and efflux. In this work, we have studied the interrelations between these three phenomena. By measuring ATP content, net acid transport, lactate exit, oxygen consumption, intracellular pH, CO2 production, and glycolytic intermediates in the presence of glucose and glucose analogues, we conclude that 1) lactate production, decrease in ATP content, and acid equivalent exit are dependent on the metabolism of glucose up to different stages in glycolysis. 2) The decrease in ATP content is not directly related to the exit of acid equivalents from rat spermatids. 3) Glucose metabolism is a net ATP-consuming process at high intracellular ATP content but is a net ATP-producing process at low intracellular ATP concentration in rat spermatids. 4) Acid equivalent production arises from the metabolism of glucose beyond glyceraldehyde 3-phosphate dehydrogenase. 5) Lactic acid diffusion and/or lactate transport and CO2 production and exit could account for the glucose-dependent acid equivalent efflux in rat spermatids.
Xenopus oocyte K+ current. III. Phorbol esters and pH regulate current at gap junctions
Authors: Greenfield L. J. , Hackett J. T. , Linden J.
Published: December 2017

Xenopus follicles consist of a single large oocyte surrounded by a monolayer of follicle cells attached to the oocyte by gap junctions. Adenosine 3',5'-cyclic monophosphate (cAMP) activates an outward K+ current which is completely abolished if follicle cells are removed or if phorbol esters (which have been reported to reduce gap junctional conductance) are added. In this study we show that phorbol esters do not reduce cAMP levels in follicles and that acid pH, another known stimulus for reducing gap junctional conductance, mimics the action of phorbol esters to inhibit the cAMP-stimulated K+ current. We also examined electrical coupling between oocytes of pairs of follicles placed in physical contact (across 2 oocyte-follicle cell and 1 follicle cell-follicle cell gap junction). Phorbol esters and acid pH (5.5-6.5) decreased electrical coupling without eliciting a shunt current, since slope conductance of current-voltage curves recorded during voltage clamp was simultaneously decreased. Increasing cAMP, which has been reported to enhance gap junctional conductance in mammalian cells, increased slope conductance without decreasing electrical coupling between pairs of follicles. The data suggest that cAMP increases and phorbol esters and acid pH decrease K+ currents at least in part by effects on gap junctions. The effects of phorbol esters and acid pH to reduce electrical coupling between oocytes cannot be due to blockade of K+ channels, since such an action would increase electrical coupling (as verified by computer simulations). These findings are consistent with the idea that cAMP-activated K+ currents originate in follicle cells and are communicated to the oocyte via gap junctions.
Cyclosporin A and vehicle toxicity in primary cultures of rabbit renal proximal tubule cells
Authors: Sokol P. P. , Capodagli L. C. , Dixon M. , Holohan P. D. , Ross C. R. , Coulson R. , Jones D. B.
Published: December 2017

The capability of cyclosporin to produce direct injury to primary proximal tubular renal cells was studied. These cells, when grown on Millicell inserts, retain the functional polarity of the proximal tubule, i.e., generate a transepithelial pH gradient (apical compartment acidic) that is reversibly blocked by amiloride addition only if it is added to the apical compartment. Administration of ouabain to the basal compartment also blocks the generation of the transepithelial pH gradient. Additionally, the cells were more responsive to parathyroid hormone (PTH), a proximal tubule characteristic, than to arginine vasopressin (AVP), a distal tubule characteristic. The following substances were tested for their effect on the capacity of these cells to generate a pH gradient: Sandimmune, the commercial form of cyclosporin A; the free form of the drug; Cremophor EL, the vehicle used in the commercial preparation; and ethanol, the vehicle used to dissolve the free form. Sandimmune, at 25-50 microM, inhibited the generation of the pH gradient within 24 h. Surprisingly, Cremophor also blocked the development of a pH gradient, although somewhat less effectively. In contrast, 10 microM cyclosporin, regardless of the form tested, had no effect for up to 96 h. These findings show that cyclosporin, in the form of Sandimmune, has a direct toxic effect on these cells; they also suggest that the vehicle, Cremophor, may contribute to the well-established nephrotoxicity of cyclosporin A.
Vasopressin depolymerizes F-actin in toad bladder epithelial cells
Authors: Ding G. H. , Franki N. , Condeelis J. , Hays R. M.
Published: December 2017

Vasopressin (AVP) induces the rapid fusion of water channel-containing vesicles with the luminal membrane of its target cell. We have carried out a quantitative study of the F-actin content of toad bladder epithelial cells, using the rhodamine phalloidin binding assay. As early as 1 min after AVP stimulation, there is a significant 15% reduction of cellular F-actin, which remains reduced by 20-30% for the duration of action of AVP. Comparable reductions were seen following 8-bromoadenosine 3',5'-cyclic monophosphate, 1-desamino-8-D-arginine vasopressin, and forskolin. F-actin content rose to and then exceeded that of control bladders after AVP washout. Inhibition of prostaglandin synthesis enhanced both water flow and the decrease of F-actin. In the living cell, stabilization of F-actin with NBD-phallacidin selectively inhibited water flow. In view of the rapidity of the response, we conclude that AVP shifts the equilibrium between F-actin and G-actin monomers, and this depolymerization may be required for vesicle fusion.
Satellite cell activation in the stretch-enlarged anterior latissimus dorsi muscle of the adult quail
Authors: Winchester P. K. , Davis M. E. , Alway S. E. , Gonyea W. J.
Published: December 2017

Satellite cell activity was examined in the stretch-enlarge anterior latissimus dorsi muscle (ALD) of the adult quail. Thirty-seven birds had a weight equal to 10% of their body mass attached to one wing while the contralateral wing served as an intra-animal control. At various time intervals after application of the wing weight (from 1 to 30 days), the birds were injected with tritiated thymidine and killed 1 h later. Stretched muscle length was greater by day 1 and mass by day 3 when compared with the contralateral muscle. Satellite cells actively synthesizing DNA were quantitated in fiber segments of the control and stretched ALD. A minimum of 1,500 muscle nuclei (satellite cell nuclei and myonuclei) were counted in each muscle. Labeling in stretched muscle was expressed by the percent labeled nuclei per total nuclei counted. Satellite cell labeling was initiated by day 1, peaked between days 3 and 7, and was not statistically different from control values at day 30. These results demonstrate that satellite cells are induced to enter the cell cycle in the stretch-enlarged ALD muscle from the adult quail, and the peak of proliferative activity is within the first week of stretch.
Perpetuation of muscle fibers after removal of stretch in the Japanese quail
Authors: Alway S. E.
Published: December 2017

Stretch-overload has been shown to increase muscle mass in the anterior latissimus dorsi (ALD) of the adult quail by increasing both fiber size and number, but it is not known whether the new muscle fibers or fiber size is maintained after removal of the stretch stimulus. A weight was added to the right wing of 40 adult quail while the left wing in each bird served as an intra-animal control. The weight was removed after 30 days of stretch, and terminal experiments were conducted 0, 30, 60, or 90 days thereafter. Average slow beta-fiber area increased by 53.4 +/- 17.5% (SE) after 30 days of stretch, but it was not different from control area by 30 days poststretch removal. Total fiber number was determined after nitric acid digestion of connective tissue. It increased by 41.3 +/- 2.3% after 30 days of stretch, but it was 28.5 +/- 5.2% greater than control after unweighting for 30- days. Thus, once the overload was removed, fiber number returned to control levels more slowly than fiber mass or volume. The data suggest that mechanisms that downregulate fiber number and fiber size may differ in the ALD of the Japanese quail.
Insulin and IGF-I induce pronounced hypertrophy of skeletal myofibers in tissue culture
Authors: Vandenburgh H. H. , Karlisch P. , Shansky J. , Feldstein R.
Published: December 2017

Skeletal myofibers differentiated from primary avian myoblasts in tissue culture can be maintained in positive nitrogen balance in a defined serum-free medium for at least 6-7 days when embedded in a three-dimensional collagen gel matrix. Incubation of established myofiber cultures for 3-7 days with insulin (1 microM) or insulin-like growth factor I (IGF-I, 32 nM) stimulates both cell hyperplasia and myofiber hypertrophy. Mean myofiber diameter increases 71-98%. Insulin-like growth factor II stimulates cell hyperplasia but not myofiber hypertrophy. Cell growth results from a 42-62% increase in total protein synthesis and a 28-38% decrease in protein degradation. Myosin heavy-chain content increases 183-258% because of a 55% stimulation of myosin synthesis and 33-61% inhibition of degradation. Associated with myofiber hypertrophy is a 87-148% increase in the number of myofiber nuclei per unit myofiber length. The results indicate that insulin and IGF-I, but not IGF-II, can induce rapid myofiber hypertrophy in vitro, most likely by stimulating myoblast proliferation and/or fusion to established myofibers.
Kinetics of DIDS inhibition of HL-60 cell anion exchange rules out ping-pong model with slippage
Authors: Restrepo D. , Cronise B. L. , Snyder R. B. , Spinelli L. J. , Knauf P. A.
Published: December 2017

According to the ping-pong model of band 3-mediated anion exchange, the transport protein has a single transport site, which can exist in either an inward-facing or an outward-facing conformation. Anions bind to these unloaded forms of the carrier, and translocation takes place only when a suitable anion is bound to the transport site. In a previous paper [Am. J. Physiol. 257 (Cell Physiol. 26): C520-C527, 1989], we had shown that the substrate kinetics of Cl-Cl exchange in the promyelocytic HL-60 cell cannot be explained by this simple ping-pong model of anion exchange but is consistent with a simultaneous model according to which both extracellular and intracellular anions must bind before simultaneous translocation can take place. In the present paper we show that external 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibits anion exchange in HL-60 cells by competing with Cl- for binding to the outward-facing transport site. Furthermore, there is a linear dependence of the slope of the Dixon plot for inhibition by DIDS on the reciprocal of the intracellular Cl- concentration. This result clearly rules out a simple ping-pong scheme. In addition, the data also rule out a ping-pong model in which some translocation of the unloaded carrier is allowed (ping-pong model with slippage). The observed inhibition kinetics can be modeled by a simultaneous model of Cl-Cl exchange with competitive inhibition by DIDS.
Ethylisopropylamiloride-sensitive pH control mechanisms modulate vascular smooth muscle cell growth
Authors: Bobik A. , Grooms A. , Little P. J. , Cragoe E. J. , Grinpukel S.
Published: December 2017

The reported effects of alterations in Na-H exchange activity on mitogenesis are variable and appear dependent on the cell type examined. We examined the effects of reductions in ethylisopropylamiloride (EIPA)-sensitive pH-regulating mechanisms including Na-H exchange and alterations in intracellular pH (pHi) on the growth characteristics of rat aortic smooth muscle cells (RASM) cultured in serum-containing bicarbonate-buffered medium. Exposure of RASM replicating in bicarbonate-containing medium to the Na-H exchange inhibitors EIPA, dimethylamiloride (DMA), or amiloride (A) attenuated their replication rate. The order of potency of the inhibitors (EIPA greater than DMA much greater than A) was similar to their documented effects on Na-H exchange activity and to their order of potency for inhibiting recovery from CO2-induced acidosis in these cells. Reductions in pHi induced by lowering extracellular pH also attenuated the incorporation of [3H]-thymidine into DNA, while increases in pHi were associated with an acceleration in the rate of incorporation of [3H]thymidine into DNA. The effects of the Na-H exchange inhibitors on RASM replication were due to a reduction in the ability of the smooth muscle cells to enter the S phase of the mitotic cell cycle. This appeared predominantly the consequence of effects late within the G1 phase of the cell cycle. Concentrations of EIPA that markedly reduced the ability of RASM to enter S phase and to replicate also attenuated the increase in protein synthesis occurring 6-8 h after exposure to serum.(ABSTRACT TRUNCATED AT 250 WORDS)
Alpha-adrenergic regulation of phosphoinositide metabolism and protein kinase C in isolated cardiac myocytes
Authors: Kaku T. , Lakatta E. , Filburn C.
Published: December 2017

alpha 1-Adrenergic regulation of phosphoinositide metabolism and protein kinase C translocation was studied in isolated rat cardiac myocytes. Exposure of [3H]inositol-labeled myocytes to norepinephrine in the presence of propranolol caused a dose-dependent increase in [3H]inositol phosphates. Norepinephrine also increased the level of membrane-associated protein kinase C from approximately 10% of total activity to 18%, with a dose response similar to that for generation of inositol phosphates. Depolarization of myocytes with 30 mM KCl had no effect on inositol phosphates or membrane-associated protein kinase C but potentiated the effect of submaximal norepinephrine on both parameters. The potentiation of protein kinase C translocation was amplified when extracellular Ca2+ was increased to 4 mM, resulting in membrane association of one-third of the total cellular activity. These data show that activation of protein kinase C occurs during alpha 1-adrenergic stimulation of cardiac myocytes and that elevation of intracellular Ca2+ amplifies this effect at least in part through increased phosphoinositide metabolism.
Antibody-detectable changes in fibrinogen adsorption affecting platelet activation on polymer surfaces
Authors: Shiba E. , Lindon J. N. , Kushner L. , Matsueda G. R. , Hawiger J. , Kloczewiak M. , Kudryk B. , Salzman E. W.
Published: December 2017

Reactivity of platelets with an artificial surface exposed to whole blood is correlated with the concentration of adsorbed fibrinogen detectable by antifibrinogen antibodies. To examine the effect on platelets of the organization (distribution, orientation, conformation) of fibrinogen adsorbed on a hydrophobic surface, we studied the binding of polyclonal and monoclonal antifibrinogen antibodies to polyalkyl methacrylate polymers previously exposed to purified fibrinogen solution or diluted plasma and compared the results with platelet retention in methacrylate bead columns. There was an increase in platelet retention following diluted plasma pretreatment, which was eliminated by a polyclonal antibody against fibrinogen or against a gamma-(395-411) peptide from fibrinogen and was reduced by monoclonal antibodies (4A5, 4-2) against other COOH-terminal gamma-chain epitopes. Monoclonal antibody 10E5 against the fibrinogen receptor GpIIb/IIIa totally inhibited platelet retention in the bead columns. Our data suggest that different methacrylate polymers induce different changes in adsorbed fibrinogen, which may interfere with its interaction with platelets, and that platelet retention in a methacrylate bead column involves interaction of the COOH-terminal end of the gamma-chain of adsorbed fibrinogen with platelet GpIIb/IIIa receptors.
IgG from amyotrophic lateral sclerosis affects tubular calcium channels of skeletal muscle
Authors: Delbono O. , Garcia J. , Appel S. H. , Stefani E.
Published: December 2017

Amyotrophic lateral sclerosis (ALS) is a devastating human disease of upper and lower motoneurons. We studied the action of the immunoglobulin G (IgG) from ALS and disease control patients on dihydropyridine (DHP)-sensitive Ca2+ channels in single mammalian skeletal muscle fibers with the double Vaseline gap technique. The peak of the Ca2+ current (ICa) and the charge movement were reduced when the fibers were incubated in ALS IgG. These effects were lost when the IgG was boiled or adsorbed with skeletal tubular membranes. ALS IgG reduced skeletal muscle ICa in a similar fashion as nifedipine; the ICa blockade was voltage dependent, and the associated charge movement was reduced. These observations suggest that IgG from ALS patients reacts with the skeletal muscle DHP-sensitive Ca2+ channels or some associated regulatory moiety.
Differential regulation of Na-K-ATPase isoform gene expression by T3 during rat brain development
Authors: Corthesy-Theulaz I. , Merillat A. M. , Honegger P. , Rossier B. C.
Published: December 2017

A fetal rat telencephalon organotypic cell culture system was found to reproduce the developmental pattern of Na-K-adenosinetriphosphatase (ATPase) gene expression observed in vivo [Am. J. Physiol. 258 (Cell Physiol. 27): C1062-C1069, 1990]. We have used this culture system to study the effects of triiodothyronine (T3; 0.003-30 nM) on mRNA abundance and basal transcription rates of Na-K-ATPase isoforms. Steady-state mRNA levels were low at culture day 6 (corresponding to the day of birth) but distinct for each isoform alpha 3 much greater than beta 1 = beta 2 greater than alpha 2 greater than alpha 1. At culture day 6, T3 did not modify mRNA abundance of any isoform. At culture day 12 (corresponding to day 7 postnatal), T3 increased the mRNA level of alpha 2 (4- to 7-fold), beta 2 (4- to 5-fold), alpha 1 (3- to 6-fold), and beta 1 (1.5-fold), whereas alpha 3 mRNA levels remained unchanged. Interestingly, the basal transcription rate for each isoform differed strikingly (alpha 2 greater than alpha 1 much greater than beta 1 = beta 2 greater than alpha 3) but remained stable throughout 12 days of culture and was not regulated by T3. Thus we observed an inverse relationship between rate of transcription and rate of mRNA accumulation for each alpha-isoform, suggesting that alpha 1- and alpha 2-mRNA are turning over rapidly whereas alpha 3-mRNA is turning over slowly. Our data indicate that one of the mechanisms by which T3 selectively controls Na-K-ATPase gene expression during brain development in vitro occurs at the posttranscriptional level.
Effect of flow on prostaglandin E2 and inositol trisphosphate levels in osteoblasts
Authors: Reich K. M. , Frangos J. A.
Published: December 2017

Osteoblasts in culture respond to mechanical strains. Fluid flow has been shown to increase intracellular adenosine 3',5'-cyclic monophosphate levels in cultured osteoblasts, and this response is mediated by prostaglandin synthesis. The signal transduction pathway of these cells exposed to fluid flow is still unknown. In the present study, we have demonstrated a 9- and 20-fold increase in the rate of prostaglandin E2 (PGE2) production in osteoblasts exposed to low (6 dyn/cm2) and high (24 dyn/cm2) steady shear, respectively. We further observed that fluid flow induced increases in the intracellular levels of inositol trisphosphate (IP3), another important second messenger. A shear stress of 24 dyn/cm2 increased IP3 levels dramatically for up to 2 h. Removal of flow resulted in a gradual return of IP3 to basal levels. The stimulation of IP3 levels was partially inhibited by 20 microM ibuprofen and 14 microM indomethacin, indicating that the IP3 response was partly dependent on flow-induced prostaglandin synthesis. The IP3 response was unaffected by daltroban, a specific thromboxane antagonist. These results show that fluid flow induced prostaglandin E2 production and increased intracellular levels of IP3 in osteoblasts. This suggests that flow may be the external signal produced by loading and that these messengers may be involved in the transduction of mechanical strain into a biochemical response.