bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of mammary gland lactose secretion by colchicine and vincristine
Authors: Guerin M. A. , Loizzi R. F.
Published: December 2017

The possible role of microtubules in milk production was examined in mammary gland slices from lactating guinea pigs. Colchicine, 10(-5)-10(-4) M, depressed lactose secretion within 15 min, maintaining maximal inhibition over 2.5 h, accompanied by retention of lactose within the slices. In colchicine dose-response studies (2 h, 10(-8)-10(-5) M), secretion was depressed 22% by 10(-5) M, whereas tissue lactose increased with dose up to +25% at 10(-5) M. Lactose synthesis was inhibited 3-19% without correlation to colchicine concentration. In another study, incubation with 10(-5) M lumicolchicine yielded one-third less inhibition of secretion than 10(-5) M colchicine with no increase in tissue lactose. Both drugs depressed synthesis 31%. Lactose secretion showed a negative correlation with 10(-7)-10(-4) M vincristine yielding a maximal 66% inhibition at 10(-4) M, whereas tissue retention showed a linear increase with concentration up to 151% of control at 10(-4) M. Effects on synthesis were sporadic. These data suggest that microtubules have a role in facilitating the transport and/or secretion of lactose and perhaps other milk components.
Dependence of energy output on force generation during muscle contraction
Authors: Rall J. A.
Published: December 2017

It has been proposed that the energy (heat + work) output of an isometric twitch is determined by the force that is generated under conditions of invariant activation, irrespective of muscle length. To test the effect of length and force on total energy output, muscles were stretched by increments beyond the muscle length at which twitch force is maximum (LO) and then stimulated; energy output and force then were measured. These data were compared with isovelocity twitches in which stimulated muscles, initially at different lengths, shortened (near maximum velocity) a constant distance and then redeveloped tension at lengths less than LO. If energy liberation was determined by force generation, plots of energy output versus force produced would be parallel with isovelocity twitches liberating extra energy as shortening heat. As predicted, the ratio of the slopes (n = 13) of these relations, 0.98 +/- 0.02, was not different from 1 and the shortening heat coefficient (alphaF/Pot, measured from the difference in intercepts), 0.15 +/- 0.01, was near to the expected value. Therefore, energy liberation in twitches appears to be uniquely determined by force generation and not by muscle length.
Flow and potentials during perfusion of lacrimal gland with electrolyte solutions
Authors: Botelho S. Y. , Fuenmayor N. , Hisada M.
Published: December 2017

Resting membrane potentials, acetylcholine (ACh)-induced change in membrane potentials of lacrimal gland cells, and ACh-induced flow from the excretory duct of the lacrimal gland were recorded during close intra-arterial perfusion of various electrolyte solutions. Resting membrane potentials were unchanged during perfusion with Na+-free, Cl--free, K+-free, and K+-rich solutions. Regardless of the composition of the perfusion solution, ACh induced hyperpolarization of the majority of cells but it also induced depolarization or biphasic potential or no change in potential in a significant number of cells. When the gland was perfused with a Na+-free or a Cl--free solution, the magnitude of the ACh-induced hyperpolarization was not changed but ACh-induced flow was markedly decreased. We conclude that the process(es) responsible for the ACh-induced change in potential may differ from those responsible for the ACh-induced flow and caution should be used when drawing conclusions about mechanisms of secretion solely from analysis of electrophysiological data.
Screening of membrane surface charges by divalent cations: an atomic representation
Authors: D'Arrigo J. S.
Published: December 2017

To arrive at a clear and atomically realistic representation of the process of ionic screening, a model with the following necessary and justifiable constraints was devised. 1) The minimum internuclear distance (IND) between a negative site on the membrane and a cation screening the site is equal to the sum of the site's "equivalent" radius (rs) + the diameter of a water molecule (approximately 2.8 A) + crystal radius of the cation (rc). 2) The average value for the dielectric constant (D) over IND is given by D approximately 80 ((IND - rs - rc)/IND). When this simple atomic model for ionic screening is employed in conjunction with equilibrium ion-selectivity theory, it is possible to predict quantitatively, from coulombic energy calculations, the secondary stereospecific actions of certain alkaline-earth cations as well as the predominant screening effect of these divalent cations at the surfaces of different types of membranes. The model also successfully predicts the transition from a predominantly screening situation to a predominantly binding situation, which was observed experimentally when negative surface charge density was decreased in nerve.
Relationship of intracellular creatine concentration and uptake to muscle mass in vivo
Authors: Hofmann W. W. , Butte J. , Leon H. A.
Published: December 2017

Attempts have been made to evaluate the role of intracellular creatine in conditions leading to increased or decreased amounts of contractile protein in rat skeletal muscles. Resting concentrations of intracellular creatine ([Cr]i) and creatine phosphate ([CrP]i) were compared in gastrocnemius and soleus muscles with those immediately after a 20-s tetanic stimulation. The hydrolysis of creatine phosphate was the same after heavily and lightly loaded contractions, suggesting that hypertrophy of isometric exercise is not mediated by creatine. With atrophy after denervation or interruption of sciatic axoplasmic flow [Cr]i also remained unchanged, though [CrP]i and the rate of Cr uptake fell after denervation. The major change in adult red and white muscle bulk with unaltered [Cr]i suggests that the Cr sensitivity found by others in developing muscle in vitro has been supplemented or replaced by other control mechanisms.
Effect of oxygen at high pressure on spontaneous transmitter release
Authors: Colton J. S. , Colton C. A.
Published: December 2017

The effect of oxygen at high pressure (OHP) on resting membrane properties (effective membrane resistance (Reff) and membrane potential (Vm)) and the spontaneous release of excitatory transmitter were examined at the lobster neuromuscular junction. Pressurization with 100% oxygen to 150 pounds per square inch gauge pressure (psig) or with nitrogen to 150 psig (7,000 mmHg nitrogen and 135 mmHg oxygen) produced a decrease in Reff associated with a hyperpolarization of Vm. These changes, however, returned to control values within 20--30 min after completion of pressurization. Spontaneous release of excitatory transmitter was shown to increase dramatically in the presence of 100% oxygen at 150 psig. The increase in miniature end-plate potential (MEPP) frequency persisted beyond the transient changes seen with Reff and Vm. This effect was selective to oxygen, as pressurization with nitrogen did not produce an increase in MEPP frequency. No change in average MEPP amplitude was seen with either OHP or pressure alone. An OHP-induced increase in MEPP frequency was also seen at the frog neuromuscular junction. The results indicate that both glutamate-mediated and acetylcholine-mediated synaptic transmission are altered by OHP.
Micro-light guides: a new method for measuring tissue fluorescence and reflectance
Authors: Ji S. , Chance B. , Nishiki K. , Smith T. , Rich T.
Published: December 2017

Three-way light guides containing one or more strands of 25-micron or 80-micron diameter optical fibers in each channel have been constructed and used to measure the NADH fluorescence and UV reflectance from mitochondrial suspensions, the perfused, hemoglobin-free rat liver, and the perfused beating interventricular septum of the rabbit. The optical changes measured with these so-called micro-light guides, which have channels containing one or several strands of optical fibers less than 100 micron, are comparable in magnitude with those measured using much larger conventional light guides. The effect of light scattering on the fluorescence channel has been determined and an empirical equation for correcting the fluorescence channel for light scattering has been obtained for mitochondrial suspensions. A mathematical equation characterizing the optical behavior of a two-way micro-light guide has been derived and has been shown to account satisfactorily for reflectance and fluorescence measurements of a mat surface in air.
Interactions of cell volume, membrane potential, and membrane transport parameters
Authors: Jakobsson E.
Published: December 2017

Equations have been written and solved that describe for animal cells the relationships among membrane transport, cell volume, membrane potential, and distribution of permeant solute. The essential system consists of n + 2 equations, where n is the number of permeant solute species. The n of the equations are the n transport equations for the permeant species, one for each species. The other two equations are statements of 1) the condition for bulk electroneutrality inside the cell and 2) the condition for isotonicity between the interior and exterior of the cell. Numerical solutions have been obtained in both the steady-state and time-varying cases for transport equations that are physically and phenomenologically reasonable. In addition to numerical solutions analytic expressions are presented that show the ranges of membrane parameters essential for volume regulation; for values of membrane parameters beyond explicitly defined bounds, the equations do not have real, positive solutions for cell volume.
Stretch-induced growth in chicken wing muscles: myofibrillar proliferation
Authors: Ashmore C. R. , Summers P. J.
Published: December 2017

The patigialis muscle (PAT) in the wing of the chicken can be induced to grow rapidly in length and in diameter by passively stretching the muscle with a spring-loaded aluminum bar (Holly et al., Am. J. Physiol. 238 (Cell Physiol. 7): C62–C71, 1980). Rates of DNA, RNA, and protein synthesis are accelerated. Sarcomere length falls from 3.19 micrometers after 1 day of stretch to only 10% above control values at 7 days of stretch. Myofibrils are wavy and misaligned. Electron microscopy of cross-sectioned muscles shows that the fraction of cell volume occupied by myofibrils remains constant throughout the experimental period, even though cross-sectional area of the muscle fibers increases by 55%. The mean diameter of myofibrils in stretched muscle increases by more than 25%. The number of splitting myofibrils increases from 15% before stretching to 45% after 1 wk of stretch. Splits appear to originate in the center of the I band, and then progress to the A band and the periphery of the myofibril. Elements of the sarcotubular system develop quickly at the origin of the fractures. It is concluded that rapid growth of the myofibril is required for initiation of splitting. Neither neurally mediated active tension nor muscle contraction are required.
Muscle fatigue with prolonged exercise: contractile and biochemical alterations
Authors: Fitts R. H. , Courtright J. B. , Kim D. H. , Witzmann F. A.
Published: December 2017

Alterations in the contractile and biochemical properties of fast and slow skeletal muscle were studied in rats following a prolonged swim to exhaustion. The exercise produced glycogen depletion (less than 1 mg/g tissue) in muscles representative of all three fiber types; the isometric contractile properties were altered in the 84% type I soleus (SOL) and the 60% type IIa extensor digitorium longus (EDL) but not in the 100% type IIb superficial region of the vastus lateralis (SVL). Peak tetanic tension (Po) and the rate of tension development and decline all decreased after prolonged exercise in both the SOL and the EDL. The maximal isotonic shortening velocity was highly correlated with the myofibrillar ATPase activity, and both were relatively resistant to fatigue. Furthermore, the Ca2+ sensitivity of the myofibrils was unaffected by exercise in both fast and slow muscle. The Ca2+ uptake capacity of the sarcoplasmic reticulum (SR) was reduced in both the SOL and the fast-twitch type IIa deep region of the vastus lateralis, whereas the SR ATPase activity was unchanged. Our findings provide evidence that prolonged exercise produces alterations in contractile and biochemical properties of type I and IIa but not type IIb fibers and that muscle fatigue as measured by a decline in Po is not necessarily correlated with glycogen depletion.
Comparison of lipid-mediated blood-brain-barrier penetrability in neonates and adults
Authors: Cornford E. M. , Braun L. D. , Oldendorf W. H. , Hill M. A.
Published: December 2017

The permeability of compounds that penetrate brain capillaries by virtue of their lipoidal solubilities was studied in vivo after a single capillary transit by the intracarotid injection technique. Brain permeabilities of 14C-labeled test isotopes were measured relative to that of tritiated water, a highly diffusible reference substance, with correction for any test isotope remaining in the cerebral vasculature. The brain uptake indices of acetamide, antipyrine, benzyl alcohol, butanol, caffeine, cytosine, diphenyl hydantoin, ethanol, ethylene glycol, heroin, mannitol, methanol, phenobarbital, propylene glycol, thiourea, and urea were measured in ether-anesthetized newborn rabbits. A highly significant correlation (r = 0.86) between brain uptake indices and octanol-saline partition coefficients of these compounds was observed. An almost identical relationship was derived in the adult rat blood-brain barrier where brain uptakes and partition coefficients of some 48 compounds could be correlated (r = 0.86). The similarities in slope-intercept relationships indicate that newborn rabbit and adult rat brain endothelia are functionally similar with respect to lipid-mediated permeability [in contrast to previous studies that have established dramatic differences in selective permeabilities of metabolites transported by saturable, carrier-mediated ("facilitated diffusion") mechanisms]. Permeability-surface area products were also derived; these data confirmed no differences in permeability could be detected between newborn and adult blood-brain-barrier capillaries. A relationship between hydrogen bond number (an alternative indicator of hydrophobic properties( and brain uptake indices derived for the adult rat brain could not be confirmed in the case of the newborn rabbit.
Effect of ionophore A23187 on cytosolic Ca2+ and enzyme secretion
Authors: Stark R. J. , O'Doherty J.
Published: December 2017

As the ionophore A23187 is believed to act by increasing cytosolic Ca2+ ([Ca]i), it offers a mechanism for experimentally controlling [Ca]i. Ca2+-selective microelectrodes were employed to examine the effect of A23187 on [Ca]i and the role of [Ca]i in acinar secretion. The mean [Ca]i in acinar cells of the mouse pancreas was determined to be 0.43 +/- 0.03 microM. When the ionophore was added to the saline bathing the acinar cells, 10(-6) M A23187 depolarized the membrane potential (Em) by 5.2 +/- 0.3 mM and the intracellular Ca-electrode potential (ECs) by 9.8 +/- 0.6 while 10(-5) M A23187 depolarized Em by 7.4 +/- 0.3 mV and ECs by 14.1 +/- 0.8. These changes in potentials reflect an increase in [Ca]i to 0.62 +/- 0.03 microM with 10(-6) M and 0.73 +/- 0.05 microM with 10(-5) M A23187. The increase in [Ca]i observed with 10(-6) M A23187 was similar to that found with concentrations of acetylcholine (Ach) that produced maximal enzyme secretion, whereas the increase in [Ca]i with 10(-5) M was similar in magnitude to that observed with ACh concentration that inhibited or reduced secretion. Measurements of amylase release during 30 min exposure of A23187 produced an 88.4% increase in amylase activity over basal levels with 10(-6) M and little or no change with (10(-5) M, indicating that the ionophore influences secretion through changes in [Ca]i in a manner analogous to the natural secretagogue ACh. This report establishes that acinar secretion occurs only within a narrow range of [Ca]i activities and suggests intracellular increases in both "bound" and "free" calcium may occur during cell activation.
Nonenzymatic separation of myocardial cell nuclei from whole heart tissue
Authors: Liew C. C. , Jackowski G. , Ma T. , Sole M. J.
Published: December 2017

A simple and rapid nonenzymatic method has been developed to isolate myocardial cell nuclei from whole heart tissue. This method consists of a controlled disruption of cells followed by isopycnic gradient centrifugation. We have reviewed and compared our method to others more lengthy and laborious. By using a number of criteria, such as morphometric measurements, chemical composition, functional studies, specific nuclear protein markers, and mathematical analysis, we show that the nonenzymatic digestion method provides a most useful technique for the study of the biochemistry of the myocardial cell nucleus.
Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum
Authors: Fabiato A.
Published: December 2017

The hypothesis of a Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) is supported by experiments done in skinned cardiac cells (sarcolemma removed by microdissection). According to this hypothesis, the transsarcolemmal Ca2+ influx does not activate the myofilaments directly but through the induction of a Ca2+ release from the SR. The stimulus gating CICR is not a small change in free Ca2+ concentration (delta[free Ca2+]) outside the SR but a function of the rate of this change (delta[free Ca2+/delta t]). The initial relatively fast component of the transsarcolemmal Ca2+ current would trigger Ca2+ release; the subsequent slow component, perhaps corresponding to noninactivating Ca2+ channels, would load the SR with an amount of Ca2+ available for release during subsequent beats. Inactivation of CICR is caused by the large increase of [free Ca2+] outside the SR resulting from Ca2+ release, which inhibits further release. This negative feedback helps to explain that CICR is not all or none. During relaxation the Ca2+ reaccumulation in the SR is backed up by the Ca2+ efflux across the sarcolemma through Na+-Ca2+ exchange and the sarcolemmal Ca2+ pump. Computations of the Ca2+ buffering in the mammalian ventricular cell and of the systolic transsarcolemmal Ca2+ influx do not support the alternative hypothesis that this influx of Ca2+ is large enough to activate the myofilaments directly. Yet the hypothesis of a CICR can be challenged because of many problems and uncertainties related to the preparations and methods used for skinned cardiac cell experiments.
Fatigue and phosphocreatine depletion during carbon dioxide-induced acidosis in rat muscle
Authors: Sahlin K. , Edstrom L. , Sjoholm H.
Published: December 2017

Isolated extensor digitorum longus muscles from rat were exposed to atmospheres of 30% CO2 (high-CO2 muscles) or 6.5% CO2 (control muscles) in O2 for 95 min. Muscle contraction characteristics were studied before and after the incubation. Tetanic tension decreased in high-CO2 muscles to 55% of initial value but remained unchanged in control muscles. Relaxation time was prolonged in high-CO2 muscles but not in control muscles. Intracellular pH was 6.67 +/- 0.04 (SD) in high-CO2 muscles and 7.01 +/- 0.04 in control muscles. CO2-induced acidosis had a marked influence on the intermediary energy metabolism as shown by a fourfold increase of glucose 6-phosphate, a 14% increase of ADP, and a decrease of phosphocreatine to 44% of the control value. Lactate and pyruvate contents were unchanged. The observed metabolic changes can be explained by an effect of H+ on the activity of phosphofructokinase and on the creatine kinase equilibrium. It can be concluded that H+ concentration causes muscular fatigue. It is, however, uncertain whether this is an effect of increased H+ per se or by high-energy phosphate depletion induced by acidosis.
Responses of lymphocytes to anisotonic media: volume-regulating behavior
Authors: Grinstein S. , Rothstein A. , Sarkadi B. , Gelfand E. W.
Published: December 2017

The regulatory responses elicited in lymphoid cells suspended in anisotonic media are reviewed. The immediate response approximates osmometric behavior. In addition, in hypotonic media, the initial osmometric swelling is followed by a regulatory volume decrease (RVD), which is associated with KCl loss. The volume-induced effluxes of K+ and Cl- are mediated by two independent conductive pathways. Ca2+-depletion experiments and studies of inhibitor susceptibility suggest that Ca2+ may mediate the activation of the K+ pathway. The responses of the two main lymphocyte subpopulations to hypotonic challenge are different. RVD is much more rapid in T- than in B-cells, regardless of their tissue of origin. Under certain conditions, shrunken lymphocytes will regain their initial volume. This regulatory volume increase (RVI) is due to NaCl uptake, followed by a secondary exchange of Na+ for K+ via the Na+-K+ pump. Na+ is primarily taken up in exchange for H+ through an amiloride-sensitive pathway, whereas Cl- enters in exchange for HCO-3 (or OH-). Anion and cation fluxes responsible for RVI are electroneutral. Some of the volume-sensitive pathways can also be activated in isotonic cells. The conductive K+ pathway is activated by Ca2+ plus ionophore A23187, and the Na+-H+ exchanger can be activated by cytoplasmic acidification. The responses of lymphocytes to anisotonic challenge are compared with those of other cells, and the possible significance of the volume-induced fluxes is discussed.
High-resolution 23Na-NMR studies of human erythrocytes: use of aqueous shift reagents
Authors: Pike M. M. , Fossel E. T. , Smith T. W. , Springer C. S.
Published: December 2017

Aqueous shift reagents were used to clearly distinguish intra-and extracellular 23Na-nuclear magnetic resonance (NMR) signals in samples consisting of whole blood or suspensions of washed human erythrocytes (both fresh and outdated). The lanthanide chelates Dy(PPP)2(7-) and Tm( TTHA )3- were used to shift the extracellular signals upfield, and Dy( TTHA )3- and Tm(PPP)2(7-) were similarly used to shift extracellular resonances downfield. The absolute intensities of the signals were used along with the measured hematocrit to simultaneously determine the intra- and extracellular Na+ concentrations. The results were generally within 5% of the values determined by more time-consuming centrifugation-flame emission photometry measurements on the same samples. Thus the 23Na-NMR signals from both intra- and extracellular cations suffer no NMR invisibility within experimental error. The lower level of intracellular Na+ in fresh erythrocytes (less than 12 mM) is easily distinguished from the higher level (approximately 30 mM) in erythrocytes that have been stored (in the cold) outside the body for some weeks.
Control of potassium transport by turtle colon: role of membrane potential
Authors: Halm D. R. , Dawson D. C.
Published: December 2017

To more clearly define the role of the transepithelial electrical potential difference (V m----s), potassium permeability, and sodium-potassium pump rate in transcellular potassium transport by isolated turtle colon, we measured transmural potassium fluxes under open-circuit conditions in the presence and absence of putative blockers of potassium transport: amiloride and barium. The results were consistent with the notion that V m----s is a major determinant of cellular potassium secretion, whereas active potassium absorption is insensitive to changes in V m----s. These observations suggest that “coupling” between colonic sodium absorption and potassium secretion in vivo could be due primarily to the effect of the lumen negative V m----s on transcellular secretory potassium flow. Amiloride-induced inhibition of potassium secretion appeared to be due to the reductions in V m----s and sodium-potassium pump rate that accompanied the inhibition of active sodium absorption.
Influence of abnormal Cl- impermeability on sweating in cystic fibrosis
Authors: Bijman J. , Quinton P. M.
Published: December 2017

Parameters of electrolyte transport in single sweat glands in normal subjects and cystic fibrosis (CF) patients were monitored and compared. Results indicate that in both normal and CF sweat ducts, Na+ is reabsorbed by an active process in which Cl- follows passively while K+ is secreted. However, while net NaCl reabsorption is markedly lower, the electrical potential associated with sweat emerging from the sweat duct is significantly more negative in CF than in normal subjects. Comparison of the differences in apparent electrochemical potential experienced by ions in the sweat duct during secretion indicates that Na+ is held out of the lumen of both groups of ducts against a large but similar gradient, but that Cl- is held in the CF duct against a much larger gradient than in the normal duct. These results indicate that the mechanism for Na+ reabsorption is not inhibited in the CF duct, but that the decreased NaCl transport in the defective duct is due to an abnormally low permeability to Cl-. Analysis of the electrical potential as a function of the Cl- gradient in the sweat suggests that the normal and defective route of Cl- uptake may be transcellular.
Differential stimulation of PGE2 synthesis in mesangial cells by angiotensin and A23187
Authors: Schlondorff D. , Perez J. , Satriano J. A.
Published: December 2017

The mechanism of arachidonic acid release and prostaglandin E2 (PGE2) synthesis was studied in cultured mesangial cells from rat kidneys. Both the ionophore A23187 and angiotensin II stimulated radioimmunoassayable PGE2 synthesis. The effect of angiotensin occurred within minutes, with half-maximal stimulation around 10(-9) M. In cells prelabeled with [14C]arachidonate, A23187 caused release of [14C]-arachidonate from all phospholipids. In contrast, angiotensin II caused preferential release of [14C]arachidonate from phosphatidylinositol, associated with a significant increase in 14C-labeled phosphatidic acid, mono- and diacylglyceride, and arachidonate. These results indicate that angiotensin preferentially, but not exclusively, stimulates a phosphatidylinositol-specific phospholipase C, whereas A23187 results in unspecific stimulation of phospholipases. The tight coupling between an angiotensin-responsive phospholipid-arachidonate pool and cyclooxygenase may be responsible for the specificity of the response to angiotensin.
Staphylococcal alpha-toxin-induced PGI2 production in endothelial cells: role of calcium
Authors: Suttorp N. , Seeger W. , Dewein E. , Bhakdi S. , Roka L.
Published: December 2017

Studies in erythrocytes indicate that staphylococcal alpha-toxin generates discrete transmembrane channels with an effective diameter of 2-3 nm. In cultured, confluent, pig pulmonary arterial endothelial cells we studied the triggering of the arachidonic acid cascade and its dependence on calcium influx, possibly through toxin-created pores. In endothelial cells alpha-toxin time dependently (5-30 min) and dose dependently (0.1-8 micrograms/ml) stimulated the release of radiolabeled arachidonic acid and prostacyclin (PGI2) production in similar amounts as the calcium ionophore A23187 (10 microM). Preincubation of alpha-toxin with neutralizing antibodies abolished the effect. The toxin response was strictly dose dependent on extracellular calcium but not on magnesium. The toxin effect was accompanied by an up to 10-fold increased passive permeability of pulmonary arterial endothelial cells for 45Ca. Interference with calcium-calmodulin function (trifluoperazine, W7) dose dependently reduced production of PGI2, but blockers of physiological calcium channels (verapamil, nimodipine, nisoldipine, and diltiazem) did not. In contrast to the effect of the ionophore A23187, the toxin effect was accompanied by a release of potassium, but in neither system was there a release of lactate dehydrogenase. In addition, alpha-toxin but not ionophore-exposed endothelial cells showed an increased passive influx of small radiolabeled markers (45Ca and [3H]sucrose) but not of large markers [( 3H]inulin and [3H]dextran). These data are consistent with the concept that alpha-toxin triggers the arachidonic acid cascade in pulmonary arterial endothelial cells by calcium influx and suggest that this calcium influx may proceed through toxin-created transmembrane channels.
Transport properties of the lens
Authors: Mathias R. T. , Rae J. L.
Published: December 2017

Many studies have shown that the lens is a multicellular syncytial tissue whose electrophysiological properties are the integrated result of membrane transport, low-resistance gap junctions interconnecting the cells, and the restricted extracellular space between cells. There are at least three structurally distinct populations of cells within the lens, and the membrane transport properties of each cell type appear to differ. Indeed, there may be subcellular specialization of membrane transport properties in the surface epithelial cells. We review the physical structure of the lens, its electrical structure, and our present knowledge of the membrane transport properties of the different cell types. Our recent work has focused on radially circulating fluxes generated by the spatial localization of membrane transport in surface cell membranes versus inner fiber cell membranes. We review this work and present some simplified models of the results with some discussion of physiological implications.
Selective induction of manganous superoxide dismutase in human monocytes
Authors: Asayama K. , Janco R. L. , Burr I. M.
Published: December 2017

Radioimmunoassays for both human copper-zinc and manganous superoxide dismutases (Cu-Zn SOD and Mn SOD, respectively) have been developed, validated, and utilized to measure the concentrations of these enzymes in cultured monocytes. Monocyte Mn SOD increased 4.7-fold over basal during 3 days of culture, an increase that was markedly enhanced by stimulation with bacterial lipopolysaccharide (LPS). Cu-Zn SOD showed a transient decrease over the culture period but was unaffected by LPS. Stimulation with muramyl dipeptide had minimal effect on Mn SOD and no effect on Cu-Zn SOD during culture, even at a concentration capable of activating the monocytes, as defined by zymosan-induced superoxide production.
Eicosonoid metabolism and beta-adrenergic mechanisms in coronary arterial smooth muscle: potential compartmentation of cAMP
Authors: Rubanyi G. , Galvas P. , DiSalvo J. , Paul R. J.
Published: December 2017

Beta-Adrenergic relaxation in bovine coronary arteries is enhanced by inhibition of eicosonoid metabolism and inhibited by its stimulation. We investigated the interaction between eicosonoid metabolism and beta-adrenergic mechanisms by studying the effect of perturbations of eicosonoid metabolism on vascular adenosine 3',5'-monophosphate (cAMP) content and the cAMP-dependent relaxation of isometric force and activation of glycogen phosphorylase. KCl (35 mM) elicited a contraction, activated phosphorylase, and slightly decreased cAMP content. Isoproterenol (10(-7) M) relaxed the KCl contraction, further increased phosphorylase activity, and increased cAMP. Neither indomethacin (5 X 10(-6) M) nor arachidonic acid (3 X 10(-5) M) affected the KCl contraction, but arachidonic acid increased both cAMP and phosphorylase activity and indomethacin decreased cAMP. Indomethacin potentiated the relaxation induced by isoproterenol but inhibited the activation of phosphorylase and had no effect on the isoproterenol-induced increase in cAMP. Arachidonic acid, on the other hand, inhibited the isoproterenol-induced relaxation but potentiated both the increases of phosphorylase activity and cAMP. Thus neither relaxation nor phosphorylase activity was related in a straightforward manner to the total cAMP content. A direct relation between cAMP, relaxation, and phosphorylase can be reconciled with the antiparallel effects of alterations of eicosonoid metabolism observed in this study by a proposed model in which the effects of cAMP are assumed to be functionally compartmentalized.
The "coat" of kidney intercalated cell tubulovesicles does not contain clathrin
Authors: Brown D. , Orci L.
Published: December 2017

Intercalated cells of kidney collecting ducts contain a population of tubulovesicles in their apical cytoplasm, whose limiting membranes are decorated by arrays of dense, club-shaped projections oriented toward the cytoplasm. These tubulovesicles have been implicated in endo-exocytotic events in these cells. To determine a possible relationship between this “coating” material and clathrin, the coat protein associated with endocytotic coated pits and coated vesicles in other cell types, we applied a monospecific, affinity-purified anti-clathrin antibody to thin sections of rat kidney embedded at low temperature in Lowicryl K4M. We found that no specific labeling was associated with the studlike material of intercalated cell tubulovesicles.
Porous-bottom dishes for culture of polarized cells
Authors: Steele R. E. , Preston A. S. , Johnson J. P. , Handler J. S.
Published: December 2017

Porous-bottom dishes offer several advantages for growing and studying epithelia in culture. Many epithelia differentiate more on porous surfaces than on plastic tissue culture dishes. In addition, separate solutions can be maintained on each side of the epithelium and can be sampled easily for studies of transport and other polarized functions. We describe the fabrication of dishes with a cellulose ester filter, a collagen-coated polycarbonate filter, or a collagen membrane forming the surface for cell attachment at the bottom of the dish.
Interactions of inhibitors on anion transporter of human erythrocyte
Authors: Frohlich O. , Gunn R. B.
Published: December 2017

Chloride tracer efflux was measured from intact human erythrocytes into media containing different chloride concentrations and different concentrations of the inhibitors 4,4'-dinitrostilbene-2-2'-disulfonate (DNDS), N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurine), phloretin, and sulfate. The data were analyzed to test whether these inhibitors were mutually exclusive with each other or whether they could bind at the same time. Under the assumption that mutual exclusiveness is due to steric interference, the data can be used to map out the protein surface near the outward-facing anion binding-transport site. It is concluded that there are separate domains for NAP taurine and phloretin that do not overlap with each other or with the chloride binding site. These two domains do, however, overlap with the binding domain for DNDS that, in addition, excludes the binding of chloride and sulfate.
Mechanism of NaCl transport-stimulated prostaglandin formation in MDCK cells
Authors: Kurtz A. , Pfeilschifter J. , Malmstrom K. , Woodson R. D. , Bauer C.
Published: December 2017

Recently we have found that stimulation of NaCl transport in high-resistance MDCK cells enhances their prostaglandin formation. In the present study, we investigated the mechanisms by which prostaglandin formation could be linked to the ion transport in these cells. We found that stimulation of transport caused a transient stimulation of prostaglandin formation lasting 5–10 min. The rise in prostaglandin formation was paralleled by a rise of free intracellular arachidonic acid. Analysis of membrane lipids revealed that the rise of free arachidonic acid was paralleled by a loss of arachidonic acid from polyphosphoinositides. We failed to obtain indications for the stimulation of calcium-dependent phospholipase A2. However, we did obtain evidence that the incorporation of arachidonic acid into phospholipids was diminished during stimulation of ion transport, indicating a decreased rate of reesterification. Despite the fact that there was no significant fall in total cellular ATP on stimulation of ion transport, we found a high and transient rise of lactate production of the cells on stimulation of the ion transport indicating an alteration of the ADP/ATP ratio. Moreover, prostaglandin formation and lactate formation were linearly correlated in this situation. When glucose utilization was inhibited by mannoheptulose, the rise in lactate formation was abolished, whereas that of PG formation was unaltered, indicating that lactate formation and prostaglandin formation were not causally linked on stimulation of ion transport. Our results suggest that an increase in the rate of sodium chloride transport by MDCK cells stimulates formation by an inhibition of reesterification of free arachidonic acid.(ABSTRACT TRUNCATED AT 250 WORDS)
Complement complex C5b-8 induces PGI2 formation in cultured endothelial cells
Authors: Suttorp N. , Seeger W. , Zinsky S. , Bhakdi S.
Published: December 2017

The effects of the terminal complement sequence on prostacyclin (PGI2) generation in antibody-sensitized pulmonary arterial endothelial cells were examined. Whereas C5b-7 complement complexes induced no PGI2 formation, addition of purified complement component C8 resulted in a time- and dose-dependent burst of PGI2 release in the absence of overt cell damage. Formation of the complete terminal complement complex C5b-9 enhanced PGI2 release but was accompanied by cytolysis. Extracellular Ca2+ was required for C5b-8-dependent PGI2 formation. Three different blockers of physiological calcium channels failed to suppress the observed stimulatory effect. In contrast, W7 [N-(6-amino-hexyl)-5-chloro-1-naphthalene sulfonamide] and trifluoperazine, inhibitors of calmodulin activity, all reduced the C5b-8-dependent PGI2 generation. None of the inhibitors used impaired Ca2+ flux into the cells. One minute after addition of C8 to endothelial cells carrying C5b-7 complexes, a six- to seven-fold enhanced passive influx of 45Ca2+ into the cells was noted. An enhanced passive influx was also observed for 51Cr O4(2-), [3H] aminobutyric acid, and [3H]sucrose, but not for [3H]inulin and [3H]dextran. These data together suggest that complement C5b-8 complexes may serve as Ca2+ bypass gates in endothelial cells, the ensuing influx of Ca2+ leading to subsequent activation of the arachidonic acid pathway.
Angiotensin decreases cyclic GMP accumulation produced by atrial natriuretic factor
Authors: Smith J. B. , Lincoln T. M.
Published: December 2017

Atrial natriuretic factor (ANF) produced rapid increases in cyclic GMP (cGMP) in cultured aortic smooth muscle cells. Angiotensin II (ANG II) markedly decreased the accumulation of cGMP that was evoked by ANF. Arginine vasopressin and ATP, which evoke transient increases in free Ca2+ similarly to ANG II, also inhibited cGMP accumulation. The effect of the calcium mobilizing neurohormones was mimicked by the divalent cation ionophore, A23187. The cyclic nucleotide phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, prevented ANG II from inhibiting ANF-evoked cGMP accumulation. ANG II also inhibited cGMP accumulation induced by nitroprusside, a compound that activates cytosolic guanylate cyclase. These findings support the hypothesis that ANG II decreases cGMP accumulation by stimulating cGMP hydrolysis, apparently via a Ca2+-activated cGMP phosphodiesterase.
Sulfate transport in apical membrane vesicles isolated from tracheal epithelium
Authors: Elgavish A. , DiBona D. R. , Norton P. , Meezan E.
Published: December 2017

Sulfate uptake in apical membrane vesicles isolated from bovine tracheal epithelium is shown to occur into an osmotically sensitive intravesicular space, via a carrier-mediated system. This conclusion is based on three lines of evidence: 1) saturation kinetics; 2) substrate specificity; and 3) inhibition by the anion transport inhibitors SITS and DIDS. The affinity of the transport system is highest in low ionic strength media (apparent Km = 0.13 mM) and decreases in the presence of gluconate (apparent Km = 0.68 mM). Chloride appears to cis-inhibit sulfate uptake and to trans-stimulate sulfate efflux. Cis-inhibition and trans-stimulation studies with a variety of anions indicate that this exchange system may be shared by HCO3-, S2O3(2-), SeO4(2-), and MoO4(2-) but not by H2PO4- or HAsO4(2-). Studies indicate that protons may play two distinct roles in sulfate transport in this system. 1) Their possible modifier role is suggested by the fact that protons affect SO2-4 transport in an uncompetitive manner. 2) The possibility that the proton gradient may act as an energy source for a secondary active transport is indicated by the fact that the imposition of a proton gradient stimulates a transient movement of sulfate in to the tracheal apical membrane vesicle, against its concentration gradient, causing an "overshoot" phenomenon. Our studies show that the carrier-mediated system can function in the absence of chloride. The overshoot observed in the presence of a proton gradient (OH- gradient) indicates that under those conditions the mechanism of transport may be a SO4(2-)-OH- exchange. The fact that chloride cis-inhibits and trans-stimulates SO4(2-) transport indicates that SO2-4 uptake may also occur via a SO4(2-)-Cl- exchange. Studies carried out so far do not enable us to conclude unequivocally whether the tracheal apical membrane system displays two distinct carrier activities (SO4(2-)-Cl-; SO4(2-)-OH-) or one anion exchanger, which like the erythrocyte anion exchanger, may interact with SO4(2-), Cl-, and H+. The fact that the anion transport inhibitors DIDS and SITS inhibit SO4(2-) transport in the presence or absence of chloride suggests that the latter possibility may be the case.
Gentamicin enhanced production of hydrogen peroxide by renal cortical mitochondria
Authors: Walker P. D. , Shah S. V.
Published: December 2017

Agents that affect mitochondrial respiration have been shown to enhance the generation of reactive oxygen metabolites. On the basis of the well-demonstrated ability of gentamicin to alter mitochondrial respiration (stimulation of state 4 and inhibition of state 3), it was postulated that gentamicin may enhance the generation of reactive oxygen metabolites by renal cortical mitochondria. The aim of this study was to examine the effect of gentamicin on the production of hydrogen peroxide (measured as the decrease in scopoletin fluorescence) in rat renal cortical mitochondria. The hydrogen peroxide generation by mitochondria was enhanced from 0.17 +/- 0.02 nmol . mg-1 . min-1 (n = 14) in the absence of gentamicin to 6.21 +/- 0.67 nmol . mg-1 . min-1 (n = 14) in the presence of 4 mM gentamicin. This response was dose dependent with a significant increase observed at even the lowest concentration of gentamicin tested, 0.01 mM. Production of hydrogen peroxide was not increased when gentamicin was added to incubation media in which mitochondria or substrate was omitted or heat-inactivated mitochondria were used. The gentamicin-induced change in fluorescence was completely inhibited by catalase (but not by heat-inactivated catalase), indicating that the decrease in fluorescence was due to hydrogen peroxide. Thus this study demonstrates that gentamicin enhances the production of hydrogen peroxide by mitochondria. Because of their well-documented cytotoxicity, reactive oxygen metabolites may play a critical role in gentamicin nephrotoxicity.
Photobleaching of fura-2 and its effect on determination of calcium concentrations
Authors: Becker P. L. , Fay F. S.
Published: December 2017

This study was performed to determine the effect of photobleaching on the spectral properties of the calcium-sensitive fluorescent dye fura-2. Fura-2, whether in cells or in calibrating solutions, was found to be bleached when exposed to excitation light. In contrast to the widely held belief, photobleaching altered the spectral properties of the dye. Decomposition of the excitation spectra of partially bleached fura-2 solutions revealed an intermediate that is still fluorescent and is not sensitive to calcium over the same range as fura-2, but which can bind calcium in the millimolar range. The presence of this intermediate violates one of the assumptions on which the ratio method of calibration is based; that is, that the only fluorescent species present are the calcium-bound and the free anion forms of fura-2. Thus, if photobleaching occurs, the ratio method will not give accurate calcium concentration values. We calculate that as little as an 8% loss of total fluorescence intensity is sufficient to produce a large error. Photobleaching of fura-2-loaded cells and fura-2 containing calibrating solutions can be minimized by reducing the oxygen concentration and by reducing the excitation light intensity. Strategies are presented to help maintain a high signal-to-noise ratio in fura-2 fluorescence detection systems, despite a lower excitation intensity so that photobleaching, and the resulting inaccuracies in calculated [Ca2+], can be largely avoided.
A microelectrometric titration method for measurement of total intracellular Cl- concentration
Authors: Bomsztyk K. , Calalb M. B. , Smith L. , Stanton T. H.
Published: December 2017

A microelectrometric titration method is described to measure picomole amounts of Cl- present in solutions at micromolar concentrations. This method was used to measure total intracellular chloride concentration ([Cl-]i) in leukocytes. Through the use of submicroliter samples, [Cl-] can be measured in the range of 5-500 microM. There is no measurable interference from other ions normally present in the cell and no intracellular ion is falsely measured as Cl-. [Cl-]i determined by the conventional coulometric titration and the new microelectrometric titration method was the same. Among the commonly used substituting anions, thiocyanate was the only one falsely measured as Cl- and should be avoided in experiments using this method. Because only picomole amounts of Cl- are required, measurements of intracellular pH and total concentrations of other intracellular ions can be done in the same lysate of as few as 5 X 10(5) cells. This feature should make it possible to study the time course of changes in [Cl-]i together with measurements of other intracellular ions following various physiological or experimental maneuvers.
De novo synthesis of adenine nucleotides in different skeletal muscle fiber types
Authors: Tullson P. C. , John-Alder H. B. , Hood D. A. , Terjung R. L.
Published: December 2017

Management of adenine nucleotide catabolism differs among skeletal muscle fiber types. This study evaluated whether there are corresponding differences in the rates of de novo synthesis of adenine nucleotide among fiber type sections of skeletal muscle using an isolated perfused rat hindquarter preparation. Label incorporation into adenine nucleotides from the [1-14C]glycine precursor was determined and used to calculate synthesis rates based on the intracellular glycine specific radioactivity. Results show that intracellular glycine is closely related to the direct precursor pool. Rates of de novo synthesis were highest in fast-twitch red muscle (57.0 +/- 4.0, 58.2 +/- 4.4 nmol.h-1.g-1; deep red gastrocnemius and vastus lateralis), relatively high in slow-twitch red muscle (47.0 +/- 3.1; soleus), and low in fast-twitch white muscle (26.1 +/- 2.0 and 21.6 +/- 2.3; superficial white gastrocnemius and vastus lateralis). Rates for four mixed muscles were intermediate, ranging between 32.3 and 37.3. Specific de novo synthesis rates exhibited a strong correlation (r = 0.986) with muscle section citrate synthase activity. Turnover rates (de novo synthesis rate/adenine nucleotide pool size) were highest in high oxidative muscle (0.82-1.06%/h), lowest in low oxidative muscle (0.30-0.35%/h), and intermediate in mixed muscle (0.44-0.55%/h). Our results demonstrate that differences in adenine nucleotide management among fiber types extends to the process of de novo adenine nucleotide synthesis.