bluuebunny/crossref_metadata_2025 huggingface dataset:
Depression of force by phosphate in skinned skeletal muscle fibers of the frog
Authors: Stienen G. J. , Roosemalen M. C. , Wilson M. G. , Elzinga G.
Published: December 2017

The relation between isometric force and phosphate concentration in skinned skeletal muscle fibers of the frog is found to depend on fiber size. Force decreased with increasing phosphate concentration, but depression of force in thick fibers was smaller than in thin segments. When the external phosphate concentration was abruptly altered during a sustained contracture, force changed. The half-time of the force change was proportional to the cross-sectional area of the preparation. From this relation, a value for the diffusion constant of phosphate in skinned fibers of 0.9 x 10(-10) m2/s was derived. The rate of phosphate production was determined photometrically via the enzymatic coupling of the resynthesis of ATP to the oxidation of nicotinamide adenine dinucleotide. The average value (+/- SE) of the rate of ATP hydrolysis (at 4 degrees C) was 2.7 +/- 0.3 mumol.s-1.g dry wt-1, which corresponds to 0.34 mmol.l-1.s-1. From a calculation based on the diffusion constant and the rate of phosphate production determined, it follows that the dependency of the force-phosphate relation on fiber diameter is due to phosphate accumulation inside the fiber.
H+/base transport in principal cells characterized by confocal fluorescence imaging
Authors: Wang X. , Kurtz I.
Published: December 2017

A dual-excitation inverted confocal laser-scanning microscope has been developed for measuring intracellular pH (pHi) using 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) in individual cells in the isolated perfused cortical collecting tubule (CCT). This new microscope has superior depth discrimination, which eliminates the contribution of fluorescence information from cells outside the plane of focus. pHi was monitored in real time from a spot 0.55 microns in diameter within a single cell. Experiments were performed to examine the apical and basolateral membrane H+/base transport properties of single principal cells. The results indicate that principal cells possess a basolateral membrane Na(+)-independent Cl-/base exchanger, a Na(+)-H+ antiporter, and a Na+/base cotransporter. No evidence was found for an apical membrane Na(+)-independent Cl-/base exchanger. The data provide evidence for base efflux pathways in the principal cell and are compatible with the hypothesis that principal cells contribute importantly to H+/base transport in the CCT. The new methodology described in this report can be applied to other epithelia that are optically heterogeneous in the depth dimension.
Neurons from neonatal hypertensive rats exhibit abnormal membrane properties in vitro
Authors: Jubelin B. C. , Kannan M. S.
Published: December 2017

The in vitro membrane properties of neurons from superior cervical ganglia (SCG) of neonatal spontaneously hypertensive (SH), Wistar-Kyoto (WKY), and Sprague-Dawley (SD) rats were studied with microelectrodes. Neurons were obtained by enzymatic dissociation, plated, irradiated, and studied after 2-5 wk. Most SH neurons showed multiple action potentials in response to an intracellular long-duration depolarizing pulse (multiple firing), whereas most neurons from WKY or SD rats generated only one or two action potentials. Multiple firing was inhibited by low concentrations of cobalt (10(-5) M) but not by tetrodotoxin (TTX) (3 x 10(-6) M). Neither high calcium (5-10 x 10(-3) M) nor the Ca2+(-)channel opener BAY K 8644 (10(-6) M) could induce multiple firing in SD or WKY neurons. However, multiple firing was readily induced by apamin (10(-6) M) or tetraethylammonium chloride (5 x 10(-3) M) (Ca2+(-)activated K+(-)channels blockers), with cobalt and TTX sensitivities similar to native multiple-firing neurons. We conclude that 1) multiple firing is characteristic of neonate SH rats SCG neurons in vitro and depends on regenerative Ca2+ currents; 2) multiple firing in SH neurons results from a lack of activation of a Ca2+(-)activated K+ conductance and not from a lack of internal Ca2+ availability; and 3) multiple firing in SCG neurons mirrors a default in K+ conductance common to all cells in genetically hypertensive individuals.
Uptake of biotin by native Xenopus laevis oocytes
Authors: Said H. M. , Polenzani L. , Khorchid S. , Hollander D. , Miledi R.
Published: December 2017

The present study examined biotin uptake by Xenopus laevis oocytes in vitro. Uptake of low (0.03 microM) and high (10 microM) concentrations of biotin was linear with time for up to 4 h of incubation and occurred with little initial binding to oocytes. Uptake of biotin was dependent on extracellular Na+ concentration [Na+]o and was severely inhibited when Na+ was replaced by other monovalent cations [choline, tetraethylammonia, Li+, and tris(hydroxymethyl)aminomethane]. The initial rate of biotin uptake was saturable as a function of concentration with an apparent Michaelis constant of 3.9 +/- 0.5 microM and maximum velocity of 1,559 +/- 70 fmol.oocyte-1.h-1. Addition to the incubation medium of biotin structural analogues desthiobiotin and thioctic acid caused significant and concentration-dependent inhibition in the uptake of [3H]biotin. This inhibition was found to be competitive in nature with inhibition constant values of 9 and 17.5 microM. In contrast, neither the structural analogue biocytin nor biotin methyl ester (compounds in which the carboxyl group of the valeric acid moiety is blocked) showed any effect on the uptake of [3H]biotin. Biotin uptake was significantly blocked by the metabolic inhibitors dinitrophenol, cyanide, and azide and by incubation at 4 degrees C. Also, the sulfhydryl group blocker p-(chloromercuri)phenylsulfonate caused significant inhibition in biotin uptake. These results demonstrate that Xenopus oocytes possess an uptake system for biotin in its cell membrane that is Na+, energy, and temperature dependent. These characteristics of biotin uptake are similar to those reported in mammalian cells. It is suggested that Xenopus oocytes might be a useful in vitro model system to study the details of the mechanisms and regulation of biotin movement across biological membranes.(ABSTRACT TRUNCATED AT 250 WORDS)
Influence of ATP turnover and metabolite changes on IMP formation and glycolysis in rat skeletal muscle
Authors: Sahlin K. , Gorski J. , Edstrom L.
Published: December 2017

Deamination of AMP to inosine monophosphate (IMP) and NH3 is thought to be regulated by the observed increases in ADP, AMP, and H+. We have examined this hypothesis by comparing the rate of IMP accumulation in contracting and noncontracting rat skeletal muscle. The rate of IMP formation was high during ischemic contraction, and consistent with previous studies, formation of IMP was associated with high levels of muscle lactate, depletion of phosphocreatine (PCr), and increased levels of ADP and AMP. When the contraction period was followed by 5-min anoxic recovery, the metabolic changes were maintained, but no further IMP or lactate was formed. During long-term (2-4 h) anoxia, the rate of IMP formation was less than 4% of that during contraction, despite similar changes in PCr, lactate, ADP, and AMP. It is concluded that the observed changes in the intracellular chemical environment are not sufficient to explain the high rate of IMP formation during contraction but that a combination of metabolic stress and a high ATP turnover rate is required. It is suggested that a high ATP turnover rate during conditions of metabolic stress results in transient increases in ADP and AMP at the site of ATP hydrolysis and that these activate AMP deaminase and glycolysis. An alternative hypothesis is that these processes are regulated by the increase in cytosolic Ca2+ in a contracting muscle.
pH regulation and response to AVP in A10 cells differ markedly in the presence vs. absence of CO2-HCO3-
Authors: Kikeri D. , Zeidel M. L. , Ballermann B. J. , Brenner B. M. , Hebert S. C.
Published: December 2017

The fluorescent pH-sensitive dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) was used to determine the effect of ambient CO2-HCO3- on the regulation of intracellular pH (pHi) and the pHi response to arginine vasopressin (AVP) in A10 vascular smooth muscle (VSM) cells. Steady-state pHi averaged 7.04 +/- 0.02 in the absence and 7.25 +/- 0.01 in the presence of CO2-HCO3-. In the absence of CO2-HCO3-, virtually all (greater than 96%) of the acid extrusion from acidification occurred by amiloride-sensitive Na(+)-H+ exchange. However, in the presence of CO2-HCO3-, acid extrusion after acidification occurred by both Na(+)-H+ exchange and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive Na(+)-dependent Cl(-)-HCO3- exchange. In CO2-HCO3(-)-containing media, amiloride-sensitive Na(+)-H+ exchange mediated 85% of acid extrusion at a pHi of 6.48, but the DIDS-sensitive acid extrusion mechanism (NA(+)-dependent Cl(-)-HCO3- exchange) was the dominant acid extrusion mechanism at a pHi of 6.94. Base exited A10 cells by a DIDS-sensitive process consistent with Na(+)-independent Cl(-)-HCO3- exchange. Both amiloride- and DIDS-sensitive processes regulated steady-state pHi in CO2-HCO3-. AVP (10(-7) M) alkalinized steady-state pHi in the absence of CO2-HCO3- (delta pHi = 0.08 +/- 0.01 pH units) by stimulating Na(+)-H+ exchange; however, AVP did not alter pHi of untreated cells in CO2-HCO3- (delta pHi = -0.01 +/- 0.01 pH units) because of concomitant stimulation of Na(+)-independent Cl(-)-HCO3-exchange. We conclude that the steady-state pHi, the mechanisms of pHi regulation, and the pHi response to AVP in A10 cells are critically influenced by the presence of extracellular CO2-HCO3-. Thus the potential contribution of pHi changes to VSM cell responses to vasoactive agents should be evaluated in the presence of CO2-HCO3-.
Enzymatic plasticity of medial gastrocnemius fibers in the adult chronic spinal cat
Authors: Jiang B. , Roy R. R. , Edgerton V. R.
Published: December 2017

The metabolic plasticity of single fibers in adult cat medial gastrocnemius (MG) 6 mo after complete spinal cord transection (Sp) at T12-T13 was studied. Some Sp cats were trained to weight support (Sp-WS) 30 min/day beginning 1 mo posttransection. Cross-sectional area, succinate dehydrogenase (SDH), alpha-glycerophosphate dehydrogenase (GPD), and myofibrillar adenosinetriphosphatase (ATPase) activities were determined in fibers identified in frozen serial sections. Fibers were categorized as light or dark based on myosin ATPase staining, alkaline preincubation. The percentage of dark ATPase fibers was higher in Sp and Sp-WS (approximately 85%) than in control (approximately 60%). All dark ATPase fibers reacted positively to a fast myosin heavy chain monoclonal antibody. In both spinal groups, a higher percentage of dark ATPase fibers reacted to both fast and slow myosin heavy chain antibodies than in controls. Neither Sp nor Sp-WS cats showed fiber atrophy. Compared with control, SDH activity was decreased in both fiber types of Sp cats. Daily weight-support training ameliorated this adaptation. There were no differences among the three groups in mean GPD and ATPase activities for either fiber type. There was a slight tendency, however, for spinal cats to have higher GPD and ATPase activities (independent of type) than control, probably reflecting the larger proportion of dark ATPase fibers in these cats. These observations indicate that 6 mo after spinalization in adult cats, some of the fibers of a fast muscle became "faster" and developed oxidative and glycolytic enzyme profiles that normally are exhibited in fast fatigable motor units.(ABSTRACT TRUNCATED AT 250 WORDS)
Presence of a novel influx pathway for Mg2+ in MDCK cells
Authors: Quamme G. A. , Dai L. J.
Published: December 2017

Basal free Mg2+ concentration was 0.49 +/- 0.03 mM in normal single Madin-Darby canine kidney (MDCK) cells as measured by fluorescence with the aid of mag-fura-2. Accordingly, Mg2+ may enter the cell down a transmembrane electrical gradient. The present study describes some aspects of Mg2+ entry into the established MDCK cell line. MDCK cells were Mg2(+)-depleted (0.26 +/- 0.01 mM) by culturing in Mg2(+)-free media for 16-20 h. Cells were subsequently exposed to 5 mM MgCl2, and intracellular Mg2+ concentration ([Mg2+]i) was monitored with fluorescence. [Mg2+]i returned to normal basal levels, 0.56 +/- 0.05 mM, with a refill rate of 272 +/- 39 nM/s, n = 4. Mg2+ entry was not changed by 5.0 mM external Ca2+ but was completely inhibited with 5.0 mM La3+. Intracellular Ca2+ concentration was not altered by Mg2+ depletion or during Mg2+ repletion. Mg2+ uptake was inhibited by verapamil (0 +/- 27 nM/s, n = 3), was inhibited less so by diltiazem (141 +/- 34 nM/s, n = 3), and was not affected by nifedipine (300 +/- 53 nM/s, n = 6). These inhibitors were fully reversible on removal, and [Mg2+]i returned to normal levels. These data indicate the presence of a unique Mg2+ entry pathway in MDCK cells that may be important in Mg2+ homeostasis. The model of Mg2+ refill into Mg2(+)-depleted cells may be useful in other cell types.
Osmotic activation of Na(+)-H+ exchange in human endothelial cells
Authors: Escobales N. , Longo E. , Cragoe E. J. , Danthuluri N. R. , Brock T. A.
Published: December 2017

Regulation of intracellular pH (pHi) via a Na(+)-H+ exchange-dependent mechanism was studied in cultured human umbilical vein endothelial cells (HEC) using the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein, as well as measuring 22Na influx. Basal pHi of HEC incubated in a bicarbonate-free Na+ medium was 6.99 +/- 0.03. In HEC that had been acid-loaded using nigericin or a NH4Cl prepulse, pHi recovery occurred via a Na(+)-dependent mechanism that was inhibited by 5-(N-ethyl-N-isopropyl)amiloride (EIPA). The potency of amiloride derivatives to inhibit 22Na influx was EIPA greater than 5-(N,N-dimethyl)amiloride greater than amiloride [Ki (extracellular Na = 30 mM) = 17 nM, 150 nM, and 8.8 microM, respectively]. EIPA-sensitive 22Na influx in acid-loaded HEC was a saturable function of the external Na+ concentration (0-130 mM), exhibiting an approximate Km and Vmax of 19.70 +/- 0.14 mM and 34.01 +/- 2.2 nmol.10(6) cells-1.min-1, respectively. H+ efflux was also dependent on external Na+ and blocked by EIPA. At resting pHi, HEC Na(+)-H+ exchange was slightly stimulated by increases in medium osmolality. However, when HEC were acid-loaded in the presence of hypertonic (sucrose) medium, Na(+)-H+ exchange activity (22Na influx or pHi recovery) increased markedly. Overall, these data indicate that pHi in cultured HEC can be regulated by a Na(+)-H+ exchanger and that its activity can be markedly influenced by osmolality at acidic pHi.
Hepatic cytochrome P-450 in rats submitted to chronic hypobaric hypoxia
Authors: Costa L. E.
Published: December 2017

Hepatic cytochrome P-450 content in adaptation to hypobaric hypoxia was studied in three groups of rats and in their respective controls at sea level atmospheric pressure. The experimental groups were as follows: 1) young male and female rats submitted to 4,400 m (simulated altitude) for 6-8 mo, 2) the same animal model of group 1 submitted to 5,500 m (simulated altitude) for a subsequent period of 2-3 mo, and 3) adult males exposed to 5,500 m for 35 days. Hypoxia caused a marked polycythemia in all three groups, body weight loss in males of the three groups and at 5,500 m also in females, whereas liver weight was normal in groups 1 and 2 and slightly decreased in group 3. Cytochrome P-450 content measured in microsomal suspensions of groups 1 and 2 was unchanged. In liver homogenates, cytochrome P-450 content was normal at 4,400 m (group 1) and decreased at 5,500 m (groups 2 and 3). Therefore, endoplasmic reticulum mass, calculated as the ratio of cytochrome P-450 in the homogenates and in the isolated microsomes, was unchanged in group 1 (4,400 m) and decreased in group 2 (5,500 m). The content of antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase, measured in liver homogenates of group 3, were markedly decreased (by 40, 30, and 35%, respectively). Results do not support the hypothesis that an increase in cytochrome P-450 content plays a role in adaptation to hypoxia.
Spatial dynamics of intracellular calcium in agonist-stimulated vascular smooth muscle cells
Authors: Neylon C. B. , Hoyland J. , Mason W. T. , Irvine R. F.
Published: December 2017

Vasoconstrictor agonists stimulate smooth muscle contraction by inducing a rise in intracellular free Ca2+. Digital-imaging microscopy of fura-2 fluorescence from single vascular smooth muscle cells cultured from the human internal mammary artery has allowed us to record the subcellular alterations in Ca2+ that occur immediately after stimulation by receptor agonists. The thrombin-induced rise in cytoplasmic free Ca2+ begins in a discrete region typically located close to the end of the cell. Subsequently, this region of elevated Ca2+ expands until Ca2+ is elevated throughout the cell cytoplasm. The rate of spreading in the region of elevated Ca2+ in a linear direction averaged 10.1 microns/s, enabling it to traverse the length of most cells within approximately 5 s, and involved rises in Ca2+ of between 200 and 500 nM. In some cells, the Ca2+ rise began at both ends and collided midway. Similar dynamic changes in the spatial distribution of Ca2+ were recorded in cells stimulated by acetylcholine. The novel observation that vasoconstrictor agonists induce an elevation of Ca2+ in a localized region which subsequently expands throughout the cytoplasm of single smooth muscle cells may provide new insight into the nature of Ca2+ signaling in vascular tissue.
AVP stimulates adenylyl cyclase and phospholipase C in reciprocal fashion in cultured RIMCT cells
Authors: Teitelbaum I. , Strasheim A.
Published: December 2017

In cultured rat inner medullary collecting tubule (RIMCT) cells, arginine vasopressin (AVP) stimulates adenylyl cyclase (AC) activity in dose-dependent fashion, with no response at concentrations of 10(-10) M or below and with peak activity at 10(-7) M AVP. In contrast, AVP-stimulated phospholipase (PLC) activity is greatest at concentrations at which there is no effect on AC and decreases at higher concentrations of AVP, becoming undetectable at 10(-7) M. Increasing cellular adenosine 3',5'-cyclic monophosphate (cAMP) content with either exogenous ClPheScAMP or forskolin eliminates inositol trisphosphate production in response to 10(-13) M AVP. Conversely, inhibition of AC by 2',5'-dideoxyadenosine (DDA) unmasks PLC activity in response to 10(-7) M AVP that is not observed in the absence of DDA. Similarly, DDA prevents inhibition of epidermal growth factor-stimulated PLC by AVP. These findings demonstrate the reciprocal relationship between AVP-stimulated AC and PLC activities in cultured RIMCT cells, which may explain previous divergent results regarding the ability of AVP to stimulate PLC in this tissue.
Reduced prostacyclin formation after reoxygenation of anoxic endothelium
Authors: Hempel S. L. , Haycraft D. L. , Hoak J. C. , Spector A. A.
Published: December 2017

Human umbilical vein endothelial cells subjected to 24 h of anoxia followed by reoxygenation released less prostacyclin (PGI2) in response to thrombin, calcium ionophore A23187, or arachidonic acid. This was associated with a substantial increase in stimulated platelet adherence. Increased lactate dehydrogenase and 51Cr release occurred after 1 h of reoxygenation, but the high rate of release did not persist during the subsequent 23 h of reoxygenation. The changes in platelet adherence and PGI2 release partially resolved over 24 h. PGI2 formation from prostaglandin H2 was not reduced, suggesting that cyclooxygenase activity, but not prostacyclin synthase, is affected by reoxygenation. A decrease in arachidonic acid release from cellular lipids also occurred. The reduction in cyclooxygenase activity, but not arachidonic acid release, was prevented by the presence of ibuprofen during reoxygenation. Addition of catalase or superoxide dismutase during reoxygenation increased PGI2 release but did not completely overcome the reduction relative to control cultures. These findings suggest that the increase in platelet adherence during reoxygenation may be mediated in part by a change in cyclooxygenase activity. This is only partly overcome by extracellular oxygen species scavengers but is prevented by the presence of a reversible cyclooxygenase inhibitor during reoxygenation.
Genetic complementation in cystic fibrosis pancreatic cells by somatic cell fusion
Authors: Jilling T. , Cunningham S. , Barker P. E. , Green M. W. , Frizzell R. A. , Kirk K. L.
Published: December 2017

The biochemical defect that underlies the genetic disorder cystic fibrosis (CF) has been proposed to involve an altered regulation of epithelial Cl- permeability by agents such as adenosine 3',5'-cyclic monophosphate (cAMP). We report here the successful complementation of this functional defect achieved by using the technique of somatic cell fusion to introduce the normal CF allele into mutant cells. CF epithelial cells were fused with transfectant mouse fibroblasts that contain the normal human gene. The resulting heterokaryons were examined for restoration of cAMP-activated Cl- transport using an optical assay of Cl- permeability. Our results provide direct evidence for the involvement of the protein product of the normal CF allele in modulating epithelial Cl- permeability.
15-Lipoxygenase products of arachidonate play a role in proliferation of transformed erythroid cells
Authors: Postoak D. , Nystuen L. , King L. , Ueno M. , Beckman B. S.
Published: December 2017

The role of specific products of the lipoxygenase pathway of arachidonic acid metabolism has been investigated in the Friend erythroleukemia cell line, a model system for erythroid cell differentiation. When triggered with agents such as hexamethylene-bis-acetamide, these cells mature as normal erythroid cells. 15-Hydroxyeicosatetraenoic acid (15-HETE) was identified by reverse-phase high-performance liquid chromatography and by radioimmunoassay as the principal lipoxygenase metabolite produced by Friend cells. Its production was significantly lower (903 +/- 73 pg/ml) in stationary-phase cells compared with logarithmic-phase cells (1,496 +/- 24 pg/ml). In addition, inhibitors of both the cyclooxygenase and lipoxygenase pathways (phenidone, BW 755C, caffeic acid, nordihydroguaiaretic acid and BW 4AC) significantly blocked DNA synthesis (P less than 0.05), whereas neither specific inhibitor of the cyclooxygenase pathway (aspirin or sodium meclofenate) blocked DNA synthesis. The addition of 15-hydroperoxyeicosatetraenoic acid as well as 15-HETE to Friend cells produced an increase in DNA synthesis as assessed by [3H]thymidine incorporation in differentiating cells but not in proliferating cells. These data support a role for 15-lipoxygenase products of arachidonic acid metabolism in maintaining DNA synthesis.
Somatostatin increases voltage-dependent potassium currents in rat somatotrophs
Authors: Chen C. , Zhang J. , Vincent J. D. , Israel J. M.
Published: December 2017

To study the modulatory effects of somatostatin on membrane K+ currents, whole cell voltage-clamp recordings were performed on identified rat somatotrophs in primary culture. In the presence of Co2+ (2 mM) and tetrodotoxin (1 microM) in the bath solution to block Ca2+ and Na+ inward currents, two types of voltage-activated K+ currents were identified on the basis of their kinetics and pharmacology. First, a delayed rectifier K+ current (IK) had a threshold of -20 mV, did not decay during voltage steps lasting 300 ms, and was markedly attenuated by extracellular application of tetraethylammonium (TEA, 10 mM). Second, a transient outward K+ current (IA) was activated at -40 mV (from a holding potential of -80 mV) and persisted despite the presence of TEA. This IA was blocked by 4-aminopyridine (2 mM). Somatostatin (10 nM) increased IK by 75% and IA by 45% without obvious effects on steady-state voltage dependency of activation or inactivation, and these effects were reversible. This increase in K+ currents may contribute in part to the inhibitory effect of somatostatin on growth hormone release.
Na(+)-K(+)-ATPase in adipocyte differentiation in culture
Authors: Russo J. J. , Manuli M. A. , Ismail-Beigi F. , Sweadner K. J. , Edelman I. S.
Published: December 2017

Differentiation of 3T3-L1 cells from a fibroblast to an adipocyte phenotype results in an approximately 50% decline in Na(+)-K(+)-ATPase activity and ouabain-sensitive 86Rb uptake. Kinetic analysis revealed a K 1/2 for Na+ of approximately 14 mM, a Km for ATP of approximately 0.4 mM, and maximal activation by sodium dodecyl sulfate at a 0.05 (wt/wt) detergent/protein ratio in both mature fibroblasts and adipocytes. Both fibroblasts and adipocytes exhibited Na(+)-K(+)-ATPase activity with an inhibition constant (Ki) for ouabain of approximately 10(-4) M. In addition, adipocytes exhibited a second component representing 30% of total activity with a Ki of approximately 5 x 10(-7) M. The emergence of biphasic ouabain inhibition kinetics in adipocytes raised the possibility of a change in alpha-subunit isoform composition with cytodifferentiation. This inference was evaluated by isoform-specific mRNA analysis (Northern blots) and by alpha-isoform-specific immunoassays (Western blots). Northern blots revealed a modest decrease in mRNA alpha 1, a striking increase in mRNA alpha 2, and a significant loss of mRNA beta content with differentiation of fibroblasts to adipocytes. By immunoassay, fibroblasts exhibited the alpha 1-isoform. Adipocytes exhibited an admixture of alpha 1- and alpha 2-isoforms, with alpha 2 being the more abundant isoform. There was no one-to-one correspondence either between the mRNA isoform and alpha-subunit abundances or between alpha-subunit abundances and enzymatic activity, suggesting that regulation occurs at multiple levels in this system. Findings indicate, however, that a shift in alpha-isoform composition accompanied by a change in ouabain inhibition kinetics occurs with cytodifferentiation.
Synthesis, transfer, and phosphorylation of phosphoinositides in cardiac membranes
Authors: Wolf R. A.
Published: December 2017

Compartmentation of phosphoinositide synthesis and transfer of endogenous phosphatidylinositol (PI) were characterized in membrane fractions prepared from rabbit myocardium. De novo synthesis of PI was highly enriched in free sarcoplasmic reticulum (551 pmol.mg-1. min-1) compared with that in sarcolemma (26.8 pmol.mg-1. min-1) and junctional sarcoplasmic reticulum (178 pmol.mg-1. min-1). In contrast, PI phosphorylation was highly enriched in sarcolemma (2.69 nmol.mg-1.min-1) compared with that in free sarcoplasmic reticulum (0.22 nmol.mg-1.min-1) and junctional sarcoplasmic reticulum (0.38 nmol.mg-1.min-1). Phosphorylation of endogenous phosphatidylinositol 4-phosphate to phosphatidylinositol 4,5-bisphosphate was also enriched in sarcolemma (38.5 pmol.mg-1.min-1) compared with that in free sarcoplasmic reticulum (less than 5.0 pmol.mg-1.min-1) and junctional sarcoplasmic reticulum (6.5 pmol.mg-1.min-1). Transfer of endogenous PI was characterized as a mechanism to overcome compartmentation of PI synthesis in cardiac membranes. A 29-kDa PI transfer protein was purified 1,500-fold from cytosol of rabbit myocardium. Both cytosol and purified PI transfer protein catalyzed the transfer of endogenous PI from microsomal sites of synthesis to sarcolemma. In conclusion, synthesis of PI is highly enriched in free sarcoplasmic reticulum, whereas phosphorylation of phosphoinositides is highly enriched in sarcolemma. A 29-kDa PI transfer protein in myocardial cytosol can mediate in vitro transfer of de novo-synthesized PI to the sarcolemma.
[Ca2+]i modulation of Cl- content controls cell volume in single salivary acinar cells during fluid secretion
Authors: Foskett J. K.
Published: December 2017

Differential interference contrast microscopy and low-light-level digital imaging of the fluorescent chloride indicator dye 6-methyl-1-(3-sulfonatopropyl)quinolinium (SPQ) were performed simultaneously in single mammalian salivary gland acinar cells to examine the relationship between cytoplasmic chloride concentration [( Cl-]i) and cell volume during stimulus-secretion coupling. Agonist stimulation of Cl(-)-driven fluid secretion is associated with rapid, Ca2(+)-dependent changes of cell volume, which are temporally coupled to changes of [Cl-]i. The agonist-induced changes in [Cl-]i, if accompanied by cations and water, quantitatively account for the cell volume changes, demonstrating in a single cell that cell volume is determined by cell solute content. Agonist-induced modulation of cell volume appears to be a consequence of the requirement to develop appropriate ion gradients necessary for vectorial salt (and fluid) transport.
Internal alkalinization by reversal of anion exchange in human neutrophils: regulation of transport by pH
Authors: Simchowitz L. , Davis A. O.
Published: December 2017

When steady-state human neutrophils bathed in 148 mM Cl- are transferred to a Cl(-)-free medium containing 0.5 mM HCO3- and 148 mM glucuronate or aspartate as nominally inert replacement ions, there is a rapid efflux of 36Cl- from the cells. The accelerated loss of Cl- is accompanied by an intracellular alkalinization of 0.7-0.9 pH units. Both the Cl- efflux and intracellular pH (pHi) transient are dependent on extracellular HCO3- and are sensitive to inhibition by SITS and alpha-cyano-4-hydroxycinnamate, which block anion exchange, thereby indicating that these processes are due to the countertransport of internal Cl- for external HCO3-. Rate of anion exchange is strongly influenced by pH, which functions to regulate carrier activity; alkalinization stimulates the transport velocity, whereas acidification inhibits it. The relationship to pHi follows a Hill equation with pK' approximately 7.40 and Hill coefficient of 3.3, thereby suggesting that approximately 3 HCO3- may be required to bind to the modifier site. Neutrophils placed in glucuronate medium progressively shrink during the first 7.5 min of incubation due to the net loss of osmotically active particles through Cl(-)-HCO3- exchange. However, between 7.5 and 30 min, cells regain their normal cell size. This volume recovery phase correlates with the time course of 22Na+ and [14C]glucuronate influxes, whose kinetics can be dissociated from that of anion exchange. Uptake of glucuronate is largely Na+ dependent (whereas Cl(-)-HCO3- exchange is not), is resistant to amiloride, and can be blocked by furosemide, which suggests that glucuronate probably enters via a volume-activated pathway such as Na+ + glucuronate cotransport.
Effects of adenosine on cAMP production and cytosolic Ca2+ in cultured rabbit medullary thick limb cells
Authors: Burnatowska-Hledin M. A. , Spielman W. S.
Published: December 2017

The cellular signaling mechanism of adenosine action has been studied in highly purified populations of cultured cells from the rabbit medullary thick ascending limb of Henle's loop (MTAL). The effects of specific adenosine-receptor agonists 5'(N-ethylcarboxamido)adenosine (NECA; A2) and N6-cyclohexyladenosine (CHA; A1) on basal and hormone-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) production, cytosolic free calcium concentration ([Ca2+]f), and formation of inositol phosphates were examined. Production of cAMP was stimulated by high doses of NECA and was inhibited by low doses of CHA. The inhibitory effect of CHA was observed in cells in which cAMP production was first stimulated with vasopressin, isoproterenol, prostaglandin E2 (10(-6) M), or calcitonin (100 ng/ml) and was abolished by pretreating the cells with pertussis toxin (PT) for 12-20 h. A highly selective adenosine A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine (CPX), also abolished the inhibitory effect of CHA. Both NECA and CHA induced a rapid (10 s) and transient increase in [Ca2+]f, and this was associated with an increased inositol trisphosphate (IP3) production. Single-cell [Ca2+]f measurements indicated that all MTAL cells responded to CHA. The removal of extracellular Ca2+ failed to inhibit these responses. Pretreatment with PT or administration of CPX abolished both the increase in [Ca2+]f and the formation of IP3 occurring in response to CHA and NECA. Our results suggest that both adenylate cyclase-coupled inhibitory (A1) and stimulatory (A2) adenosine receptors are present in pure populations of cultured MTAL cells. Moreover, activation of an adenosine receptor coupled to a PT substrate results in the increased production of inositol phosphate and elevation of [Ca2+]f.
Effect of diabetes on Na(+)-H+ exchange by single isolated hepatocytes
Authors: Boyarsky G. , Rosenthal N. , Barrett E. , Boron W. F.
Published: December 2017

We used the fluorescent dye 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) to examine intracellular pH (pHi) regulation in single hepatocytes isolated from control rats and rats with either spontaneous or drug-induced diabetes mellitus (DM). In the absence of CO2-HCO3-, both control and DM cells recovered from cellular acid loads applied by the NH4+ prepulse technique. Because the pHi recovery was blocked by either Na+ withdrawal or ethylisopropylamiloride in both control and DM cells, it was presumably mediated by Na(+)-H+ exchange. In the control cells, the pHi threshold above which the rate of change of pHi (dpHi/dt) was zero was 7.06, and the slope of the dpHi/dt-pHi relationship was -0.030 s-1. In the DM cells, the pHi threshold was 7.22 and the slope was -0.017 s-1. Thus, at pHi values below approximately 6.9, the pHi recovery was slower in the DM cells. Inasmuch as we observed no difference in the cellular buffering power between control and DM cells, diabetes inhibits Na(+)-H+ exchange within this low pHi range. At pHi values above approximately 6.9, however, Na(+)-H+ exchange was apparently stimulated by diabetes. Thus diabetes induces two distinct alterations of Na(+)-H+ exchange, an alkaline shift in pHi threshold and decrease in slope. Treatment of diabetic rats with insulin for 48 h restored both Na(+)-H+ exchange parameters to normal. On the other hand, insulin added in vitro to DM cells for 2-5 h shifted the threshold toward the control value without affecting the slope, thus leading to a further inhibition of Na(+)-H+ exchange over the entire pHi range.(ABSTRACT TRUNCATED AT 250 WORDS)
Role of protein phosphatase in activation of KCl cotransport in human erythrocytes
Authors: Kaji D. M. , Tsukitani Y.
Published: December 2017

We investigated the effects of okadaic acid, a novel highly specific inhibitor of protein phosphatases on swelling-activated transport in human erythrocytes. Nanomolar concentrations of this compound inhibited swelling-activated K transport. Complete inhibition of this transport was observed at 1 microM. Analysis of the time course of activation of K transport upon swelling revealed that okadaic acid not only decreased the final steady-state flux but also markedly increased the time lag for activation. These results suggest that okadaic acid decreases the rate constant for the conversion of KCl transporters from the resting to the active form. Okadaic acid also inhibited N-ethylmaleimide (NEM)-stimulated K transport, and this inhibition was also observed when cells were first treated with NEM before exposure to okadaic acid. The latter finding suggests that NEM activation of KCl transport is reversible and that a later step for this activation may involve the net dephosphorylation of the KCl transport protein. These results provide the first evidence that activation of KCl cotransport in human erythrocytes is regulated by phosphoprotein phosphatase.
Effect of low-density lipoprotein on endothelial cell membrane fluidity and mononuclear cell attachment
Authors: Pritchard K. A. , Schwarz S. M. , Medow M. S. , Stemerman M. B.
Published: December 2017

To determine the effects of prolonged low-density lipoprotein (LDL) exposure in vitro on cultured endothelial cell (EC) lipid dynamics and cellular function, human umbilical vein ECs were incubated in LDL concentrations [cholesterol (Chol) = 240 mg/dl] associated with the premature development of atherosclerosis. After 4 days of incubation, cells were examined for changes in cellular lipid composition and for membrane fluidity. Results indicate that LDL-EC have increased Chol content (control EC vs. LDL-EC = 22.4 +/- 5.26 vs. 38.9 +/- 0.24 nmol/10(6) cells, P less than 0.05) and cellular Chol-to-phospholipid ratio (0.61 +/- 0.10 vs. 1.21 +/- 0.10 mol/mol, P less than 0.05). Augmentation of EC Chol content was accompanied by a marked decrease in EC cellular membrane fluidity as assessed by fluorescence polarization (anisotropy, r values, 0.172 +/- 0.019 vs. 0.226 +/- 0.014, P less than 0.0001). LDL-induced changes in EC lipid dynamics were associated with enhanced EC binding of monocytes (P less than 0.05) and U937 cells (P less than 0.01). Both LDL-induced decreases in membrane fluidity and enhanced attachment of mononuclear cells were reversed to control levels following a 2-min incubation of LDL-EC with the membrane mobility agent, A2C. These data therefore suggest that LDL-induced modulations in lipid dynamics play an important role in perturbation of EC function.
Sepsis does not impair tricarboxylic acid cycle in the heart
Authors: Hotchkiss R. S. , Song S. K. , Neil J. J. , Chen R. D. , Manchester J. K. , Karl I. E. , Lowry O. H. , Ackerman J. J.
Published: December 2017

Sepsis has been reported to cause mitochondrial dysfunction and inhibition of key enzymes that regulate the tricarboxylic acid (TCA) cycle. We investigated the effect of sepsis on high-energy phosphates, glycolytic and TCA cycle intermediates, and specific amino acids that are involved in regulating the size of the TCA cycle pool during changes in metabolic state of the heart. Sepsis was induced in 12 female rats by the cecal ligation and perforation technique under halothane anesthesia; seven control rats underwent cecal manipulation without ligation. At 36-42 h postsurgery, the rats were reanesthetized, the chest was opened, and the hearts were freeze-clamped. Perchloric acid extracts of the hearts were analyzed with fluorometric enzymatic methods and 31P nuclear magnetic resonance spectroscopy. There were no significant differences in the levels of the TCA cycle intermediates or high-energy phosphates between the septic and control rats. The major metabolic changes were the 28% decrease in alanine and the 31% decrease in glutamate in the septic hearts compared with control (P less than 0.05 and P less than 0.005, respectively). Phosphocholine, a component of membrane phospholipids, was increased by 91% in the septic hearts (P less than 0.01). We conclude that sepsis does not impair the TCA cycle or induce significant cellular ischemia in the heart. The increase in phosphocholine may represent significant cellular membrane disruption during sepsis.
Release of intact endothelium-derived relaxing factor depends on endothelial superoxide dismutase activity
Authors: Mugge A. , Elwell J. H. , Peterson T. E. , Harrison D. G.
Published: December 2017

Endothelium-derived relaxing factor (EDRF) is rapidly inactivated by radicals. Endothelial cells possess several antioxidant defense mechanisms. It is not clear which intrinsic antioxidant defense systems are important to preserve the release of biologically active EDRF. We impaired antioxidant defense in normal vascular tissue by inhibiting catalase activity with 3-amino-1,2,4-triazole (AT), superoxide dismutase with diethyldithiocarbamate (DETC), and by reducing glutathione content via inhibiting glutathione synthesis with L-buthionine-(S,R)-sulfoximine (BSO). Pretreatment of rabbit aorta in vitro with DETC markedly reduced endothelium-dependent relaxation in response to acetylcholine and calcium ionophore A23187 and, to a lesser extent, reduced endothelium-independent relaxation in response to nitroprusside. Pretreatment of cultured bovine aortic endothelial cells (BAEC) with DETC did not alter release of nitrogen oxides (measured by chemiluminescence), but, the effluent of pretreated cells showed marked depression in vasodilator activity (measured by bioassay). Pretreatment of rabbit aorta in vitro with AT did not alter endothelium-dependent and -independent relaxations. Pretreatment of BAEC with BSO did not alter the release of nitrogen oxides or the vasodilator activity. These results suggest that endothelial superoxide dismutase activity, but not catalase or glutathione, is necessary for the release of biologically active EDRF. An imbalance of the intrinsic superoxide dismutase and the production of superoxide anions may therefore predispose to impaired endothelium-dependent relaxations and alter vascular reactivity.
Stimulation by cGMP of apical Na channels and cation channels in toad urinary bladder
Authors: Das S. , Garepapaghi M. , Palmer L. G.
Published: December 2017

The effects of 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) on apical membrane cation conductances in the toad urinary bladder were investigated. 8-BrcGMP (1 mM) added to the serosal solution increased the amiloride-sensitive short-circuit current (INa) after a delay of 5 min to a steady-state value 1.8 times that of controls achieved after 30 min. Similar effects were seen when the bladders were bathed on the serosal side with a normal NaCl Ringer solution and with a high-K sucrose solution to depolarize the basolateral membrane. Under the latter conditions, the amiloride-sensitive transepithelial conductance increased in parallel with the short-circuit current, indicating stimulation of apical membrane Na channels. The threshold concentration for observing the stimulation of INa was 100 microM, 10-100 times larger than the concentration of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) required to elicit an increase in INa. Currents through an outwardly rectifying Ca-sensitive cation conductance (Iout) were also increased by 1.8-fold relative to controls. This stimulatory effect occurred after a delay of 15 min and reached maximal levels 90-120 min after addition of the nucleotide. The effects of cGMP on INa were not additive with those of 8-BrcAMP or with antidiuretic hormone, an agent known to act by increasing cAMP within the cell. Addition of 1 mM 3-isobutyl-1-methylxanthine to the serosal side of the bladders stimulated INa by 1.3-fold and Iout by 2.4-fold. In both cases, subsequent addition of cGMP produced no further activation of either conductance.(ABSTRACT TRUNCATED AT 250 WORDS)
Cadmium block of isometric contractions of isolated bullfrog atrial cells
Authors: Shepherd N. , Kavaler F. , Spielman W.
Published: December 2017

We studied the effect of cadmium, verapamil, and quinacrine on the force of contraction (Fp) of isolated, single, field-stimulated bullfrog atrial cells. All agents were applied or removed rapidly (t1/2 approximately 15 ms) to minimize intracellular concentration changes other than intracellular calcium concentration. Two components of twitch force were observed, one blocked by micromolar Cd2+ and the other by millimolar Cd2+. The two contributed about equally to the activation of the twitch. The "cadmium-sensitive" portion of force (that affected by [Cd] less than or equal to 100 microM) had a K1/2 approximately 1 microM, was identical in magnitude to, and not additive with, a "verapamil-sensitive" (10 microM) component of force, was most strongly affected by 50-ms pulses of Cd2+ when they were applied in the mechanical latent period, and was potentiated by catecholamines. The cadmium-insensitive portion of force was abolished by the removal of extracellular calcium and was greatly potentiated by quinacrine (3 or 10 microM), a blocker of Na-Ca exchange. The results are consistent with the idea that activating calcium enters the cell via both an inactivating cadmium-sensitive L-type channel and a noninactivating cadmium-insensitive mechanism that is not Na-Ca exchange and leaves the cell via Na-Ca exchange.
Modulation of Gs activity by phorbol myristate acetate in rat hepatocytes
Authors: Hernandez-Sotomayor S. M. , Macias-Silva M. , Malbon C. C. , Garcia-Sainz J. A.
Published: December 2017

Activation of protein kinase C promotes heterologous desensitization of hepatic adenylate cyclase. The basis for this desensitization was explored by use of a strategy with several independent approaches. Although not influencing the amount of forskolin-stimulated adenylate cyclase activity (catalyst), treatment with phorbol 12-myristate 13-acetate (PMA) decreased adenylate cyclase activation in response to either sodium fluoride or guanylyl imidodiphosphate [Gpp(NH)p]. Adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in cholera toxin-treated hepatocytes and both the basal and GTP-stimulated adenylate cyclase activity of membranes from toxin-treated cells displayed a marked reduction in response to PMA. The ability of cholate extracts of hepatocyte membranes to reconstitute beta-adrenergic-stimulated adenylate cyclase activity of membrane of S49 mouse lymphoma cyc- cells was reduced by treatment with PMA. Cholera toxin-catalyzed labeling of Gs alpha-subunits was likewise diminished by phorbol ester treatment. Immunoblots of membranes from control or PMA-treated hepatocytes showed no difference in the amount of Gs alpha. Immunoprecipitation studies failed to detect phosphorylation of this G protein alpha-subunit. The data demonstrate that PMA induces an alteration in the functional status of Gs without altering the amount of this transmembrane signaling element. The alteration in Gs function may play a significant role in heterologous desensitization.
Myofilament overlap in swimming carp. I. Myofilament lengths of red and white muscle
Authors: Sosnicki A. A. , Loesser K. E. , Rome L. C.
Published: December 2017

To assess myofilament overlap during locomotion, we estimated the length of myosin and actin filaments in axial red and white muscle of carp. Myosin filament lengths were 1.52 +/- 0.009 and 1.50 +/- 0.037 micron (means +/- SD) in the red and white muscle, respectively, as measured from thin sections. After correction for shrinkage (using the troponin-based 385-A axial periodicity), thin filaments were 0.96 +/- 0.009 and 0.97 +/- 0.023 micron in the red and white muscles, respectively. Filaments were also isolated from the white muscle and negatively stained. Myosin filaments were 1.56 +/- 0.025 microns, and actin filaments were 0.99 +/- 0.024 micron in length. The data from thin sections and isolated filaments agreed within 2% for actin and 4% for myosin filaments. The number of actin filament periods (24 for the red and white muscle) and the length of the filaments are the same as in frog. This suggests that the classic sarcomere length-tension curve of frog muscle may be used to estimate the functional properties of carp red and white muscle.