bluuebunny/crossref_metadata_2025 huggingface dataset:
Osmoregulation of Na(+)-inositol cotransporter activity and mRNA levels in brain glial cells
Authors: Paredes A. , McManus M. , Kwon H. M. , Strange K.
Published: December 2017

During plasma hypertonicity brain volume is regulated acutely by electrolyte uptake and chronically by accumulation of organic solutes such as inositol. Cultured rat C6 glioma cells, an astrocyte-like cell line, show a similar pattern of volume control. Volume regulatory accumulation of inositol requires external inositol, indicating that membrane transport plays a central role in this process. The inositol uptake pathway is Na+ dependent and exhibits Michaelis-Menten kinetics. Chronic hypertonic acclimation results in a twofold increase in the maximum velocity of the transporter without changing the Km. Hypertonic stress also results in a 17-fold increase in transporter mRNA. Elevation of mRNA levels precedes activation of the transporter by 4-6 h, suggesting that increased inositol uptake is mediated by synthesis and membrane insertion of new transport proteins. Reacclimation of hypertonic cells to isotonicity causes a rapid reduction of transporter mRNA levels to control levels within 4 h. In contrast, downregulation of transport activity does not begin until between 10 and 24 h after reexposure to isotonicity.
Fish antifreeze proteins block Ca entry into rabbit parietal cells
Authors: Negulescu P. A. , Rubinsky B. , Fletcher G. L. , Machen T. E.
Published: December 2017

Many fish and insects have adapted to life at subfreezing temperatures by evolving so-called antifreeze proteins (AFP) that noncolligatively depress the freezing temperatures of aqueous solutions without affecting the melting temperature. AFP have been thought to function solely as antifreezes. Recently, however, we discovered that AFP also protect mammalian cells and organs from damage caused by exposure to hypothermic (above freezing) temperatures. It has been proposed that hypothermic damage is caused by changes in intracellular ionic content due to a reduction of active transport that is required to balance passive ion transport across cell membranes. Given this possibility, we tested whether AFP isolated from the Newfoundland ocean pout might reduce the Ca ion permeability of a mammalian cell, the rabbit gastric parietal cell, which has been particularly well studied in terms of Ca transport and signaling. Digital image processing of the Ca-sensitive fluorescent indicator fura-2 was used to measure intracellular free Ca in these cells. During stimulation with the cholinergic agonist carbachol, AFP inhibited passive Ca entry across the cell membrane without interfering with either the release of Ca from internal stores (indicating that the carbachol receptor and other signaling events were operational) or the normal active rates of Ca efflux from the cell (indicating that Ca pumping was also still intact). These results suggest that, in addition to their actual antifreeze properties, AFP may also help to confer cold tolerance in animals by preventing passive Ca entry into epithelial cells.
Force-length relation of isometric sarcomeres in fixed-end tetani
Authors: Horowitz A. , Pollack G. H.
Published: December 2017

The higher force observed in fixed-end tetani relative to sarcomere-isometric tetani is commonly attributed to sarcomere length inhomogeneity; sarcomeres in the end regions of the fiber shorten extensively at the expense of the central sarcomeres. By shortening, these sarcomeres supposedly attain higher force production capacity and can thus account for the extra force. However, the fibers could also contain sarcomeres that stay isometric throughout most of the tetanic force plateau. If such sarcomeres undergo slight shortening before their isometric phase, their force-length relation should be elevated (A. Horowitz, H. P. M Wussling, and G. H. Pollack. Biophys. J. 63: 3-17, 1992). These sarcomeres may therefore account for the higher force in fixed-end tetani. To test this possibility, single frog semitendinosus fibers were tetanized under fixed-end conditions. Sarcomere length change during the tetanus was measured at different locations along the fiber by optical diffraction. Fibers stretched to average sarcomere lengths between 2.2 and 3.2 microns contained sarcomeres that, except for some initial shortening during the early part of the tetanus, remained isometric. These sarcomeres were located between the ends and the central region of the fibers. Their force-length relation was higher than the linear force-length relation based on sarcomere length clamps by an average of 14% between sarcomere lengths of 2.4-3.2 microns. Thus slight (1-5%) shortening may explain the relatively higher fixed-end force-length relation.
Tyrosine kinase regulates epithelial sodium transport in A6 cells
Authors: Matsumoto P. S. , Ohara A. , Duchatelle P. , Eaton D. C.
Published: December 2017

Insulin increases epithelial Na+ reabsorption, and many of its actions involve tyrosine kinase. We used tyrosine kinase inhibitors to examine the role of tyrosine kinase in the action of insulin. Pretreatment of Na+ transporting cells with tyrosine kinase inhibitors attenuates the subsequent action of insulin, suggesting that the action of insulin on epithelial Na+ transport involves tyrosine kinase activity. In addition to their effect on insulin-induced Na+ transport, the tyrosine kinase inhibitors also significantly reduce Na+ transport in Na(+)-transporting epithelial cells, suggesting that there is a significant tonic tyrosine kinase activity that modulates epithelial Na+ transport. Using patch-clamp methods, we found that one inhibitor, genistein, reduces the number of active Na+ channels in cell-attached patches without significantly affecting the open probability of any remaining channels. The effects of the tyrosine kinase inhibitors are not due to inhibition of protein kinase A (PKA), since H89, a PKA inhibitor, does not affect Na+ transport of control cells (as the tyrosine kinase inhibitors do), and the tyrosine kinase inhibitor, genistein or tyrphostin 23, does not alter the stimulation of ion transport by 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, a membrane-permeable adenosine 3',5'-cyclic monophosphate analogue (as H89 does).
Brefeldin A inhibits phosphate transport in opossum kidney cells
Authors: Capparelli A. W. , Heng M. C. , Li L. , Jo O. D. , Yanagawa N.
Published: December 2017

Brefeldin A (BFA) is a fungal metabolite that blocks the transport processes between the endoplasmic reticulum and the Golgi apparatus. In the present study, we have tested the effect of BFA on phosphate transport in a kidney epithelial cell line, opossum kidney (OK) cells. Electron microscopy showed that exposure of OK cells to BFA caused a rapid and reversible disorganization of Golgi apparatus. Addition of BFA also caused a time (2-8 h)- and dose (1-10 micrograms/ml)-dependent inhibition of Na(+)-dependent cell phosphate uptake. The inhibition of cell phosphate uptake by BFA was reversible and was associated with a decrease in the maximum velocity of phosphate transport. Both the inhibition and the stimulation of cell phosphate uptake by parathyroid hormone and insulin, respectively, were not affected by BFA. BFA at 1 microgram/ml concentration did not affect protein synthesis as determined by [3H]leucine incorporation but diminished the adaptive increase in cell phosphate uptake in response to 2 or 8 h of incubation in nominally phosphate-free medium. On the other hand, inhibition of protein synthesis by cycloheximide (5 microM) abolished the adaptive increase in cell phosphate uptake in response to 8 but not 2 h of incubation in nominally phosphate-free medium, indicating the existence of an early response to phosphate deprivation, which does not require new protein synthesis but is sensitive to the effect of BFA. In summary, results of these studies show that, in OK cells, BFA inhibits phosphate uptake and curtails the adaptive response to phosphate deprivation.(ABSTRACT TRUNCATED AT 250 WORDS)
Desensitization and reactivation of ACh-regulated exocrine secretion in hen tracheal epithelium
Authors: Winding B. , Bindslev N.
Published: December 2017

We have studied acetylcholine (ACh) desensitization of Cl- secretion and its reactivation by atropine in the hen tracheal epithelium employing voltage-clamp technique. Adding 4 microM ACh to the serosal side induces a maximal bumetanide-sensitive Cl- secretion. The ACh-induced Cl- secretion is desensitized nearly 100% at 512 microM ACh. The desensitization of Cl- secretion may be reduced 30-40% by 0.32 microM atropine (reactivation). From a kinetic model for desensitization and reactivation with two muscarinic receptors (mAChR), we have estimated the maximal secretory response, the apparent dissociation coefficients, and the apparent Hill coefficients for ACh and atropine at the two receptors. Based on the kinetic model we can predict an optimal fixed concentration ratio (Ofcor) between ACh and atropine that will maintain Cl- secretion above 40% of maximum over a wide concentration range, ACh 1-4,100 microM, and we have confirmed the predicted Ofcor experimentally. A cellular model is proposed with two mAChR, one stimulatory and another inhibitory, that accounts for an observed dissociation between desensitization in short-circuit current and conductance.
cAMP-dependent phosphorylation modulates voltage gating in an endothelial Cl- channel
Authors: Vaca L. , Kunze D. L.
Published: December 2017

Using the patch clamp technique in the cell-attached, inside-out, and outside-out configurations, we have identified a voltage-gated outwardly rectifying, large conductance (400 pS) Cl- channel in patches from the surface membrane of a cultured monolayer of bovine aortic endothelial cells. The channel is activated in cell-attached patches with 1 microM isoproterenol or 1 mM dibutyryladenosine 3',5'-cyclic monophosphate. In excised inside-out patches the voltage dependence of this channel could be fitted by a Boltzmann distribution with a half-activation voltage (V1/2) at 0 mV. Adenosine 3',5'-cyclic monophosphate (cAMP)-dependent phosphorylation induces a shift of -50 mV in V1/2. Alkaline phosphatases restores the voltage dependence of the channel to control values. The channel is reversibly blocked by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. The half-inhibitory concentration was approximately 70 microM. Our results suggest that beta-adrenergic stimulation (which increases intracellular cAMP levels in this endothelium) may increase Cl- permeability at the cell resting potential by shifting the voltage dependence of this channel.
Enhanced keratinocyte prostaglandin synthesis after UV injury is due to increased phospholipase activity
Authors: Kang-Rotondo C. H. , Miller C. C. , Morrison A. R. , Pentland A. P.
Published: December 2017

The possibility that increased eicosanoid synthesis in skin after ultraviolet light irradiation is due to enhanced phospholipase activity was examined. [3H]arachidonic acid-labeled human keratinocyte cultures exposed to 30 mJ/cm2 ultraviolet (UV) B were studied 6 h after injury. Bradykinin-stimulated release of [3H]arachidonic acid was increased 1.8-fold over release from control cultures by prior irradiation. In unlabeled cultures, prior irradiation produced a threefold increase in bradykinin-stimulated prostaglandin (PG) E2 synthesis as measured by immunoassay. The relative contribution of increased phospholipase vs. cyclooxygenase activity was therefore examined using stable isotope mass measurements of PGE2. By this method, prior irradiation increased bradykinin-stimulated phospholipase activity 3.5-fold, while no change in total cellular cyclooxygenase activity was observed. The effects of irradiation on phospholipase activity were then assessed in more detail. The activities of phospholipase A2, arachidonoyl-CoA synthetase, and arachidonoyl-CoA lysophosphatide acyltransferase in cell homogenates were determined. No effect of UV exposure on the activity of these enzymes was observed. These results suggest that the increase in prostaglandin synthesis produced after UV irradiation is due to increased phospholipase activity, thus enhancing arachidonate release.
High sensitivity of hypertensive aortic myocytes to norepinephrine and angiotensin
Authors: Bodin P. , Travo C. , Stoclet J. C. , Travo P.
Published: December 2017

Single-mass primary cultures were used for as long as 5 wk as the source of subcultured vascular smooth muscle cells for the study of their change of shape on the addition of agonists. We have compared the responses to angiotensin II, vasopressin, norepinephrine, and serotonin of myocytes derived from three different areas or the thoracic aorta (aortic arch and midthoracic and diaphragm areas) of adult Wistar-Kyoto normotensive rats (WKY) and spontaneously hypertensive rats (SHR). Once in secondary culture, vascular myocytes displayed different mean sizes according to their origin along the organ, as previously described in freshly dispersed aortic myocyte suspensions. Responses of the cells from the area of the aortic arch of both strains had the maximal amplitude to all agonists. Angiotensin and norepinephrine were more potent on myocytes derived from the three areas in SHR than in WKY. As this hypersensitivity persisted even after 5 wk in culture, it is believed to be pressure independent and thus might have a genetic rather than an adaptive origin.
Na(+)-K(+)-ATPase alpha 1- and beta 1-subunit degradation: evidence for multiple subunit specific rates
Authors: Lescale-Matys L. , Putnam D. S. , McDonough A. A.
Published: December 2017

Na(+)-K(+)-ATPase is a heterodimeric plasma membrane protein consisting of an alpha-catalytic and a beta-glycoprotein subunit. Because these two subunits are derived from two separate genes, they may not be synthesized with stoichiometric equivalence. The aim of this study was to estimate relative rates of synthesis and degradation of nascent and mature Na(+)-K(+)-ATPase alpha- and beta-subunits to determine whether either of the nascent subunits accumulates in excess and, if so, the fate of the excess subunits. We studied a pig kidney cell line (LLC-PK1/Cl4) that expresses only alpha 1- and beta 1-subunits. Relative synthesis and degradation rates of nascent subunits were first estimated by pulsing cells for 10 min with [35S]methionine followed by chase periods of up to 120 min and by immunoprecipitation. We found that directly after labeling, beta-subunits were present in threefold excess over alpha-subunits and that nearly 50% of this beta-subunit pool was degraded by 60 min. Nascent alpha-subunits were not degraded during the chase period. In a second strategy to examine relative rates of nascent alpha- vs. beta-subunit accumulation, cells were pulsed for 5-60 min and immunoprecipitated directly (without chase). The rate of accumulation of labeled alpha was greater than that of beta between 5 and 60 min, consistent with the results of the pulse-chase strategy, demonstrating a significant component of degradation of beta during this period. Despite the very different degradation rates of newly synthesized alpha- vs. beta-subunits, the degradation rates of alpha- and beta-subunits beyond 4 h after synthesis were indistinguishable (t0.5 = 10-12 h).(ABSTRACT TRUNCATED AT 250 WORDS)
Stimulation of phospholipase D activity and phosphatidic acid production by norepinephrine in rat aorta
Authors: Jones A. W. , Shukla S. D. , Geisbuhler B. B.
Published: December 2017

We sought to relate norepinephrine (NE) stimulation of phosphatidic acid (PA) production to functional responses of rat aorta and pathways for PA production. The time course for changes in PA was closely related to Ca-dependent tonic responses in 42K efflux and contraction. NE (30 microM for 1 min) increased PA and reduced phosphatidylcholine (PC) and phosphatidylinositol (PI) based on Pi analyses and 32P labeling of phospholipids. The 32P-to-Pi ratio in PA (0.8 +/- 0.2, n = 13) was similar to PC (0.8 +/- 0.1, n = 14) but was significantly lower (P < 0.001) than PI (4.6 +/- 0.5, n = 14). The 32P-to-Pi ratio in PA was also lower (P < 0.02) than phosphatidylinositol phosphate and phosphatidylinositol bisphosphate. NE also increased [3H]PA twofold (P < 0.05) when PC was selectively labeled with [3H]myristic acid. These observations are more consistent with PA being formed from the hydrolysis of PC by phospholipase D (PLD) than by the phosphorylation of diacylglycerol produced by the action of phospholipase C. PLD was assayed by the formation of phosphatidylethanol (PEt) via a transphosphatidylation reaction with ethanol (half-maximal stimulation at 0.4-0.5% vol/vol). The time course for PLD stimulation by NE was similar to PA, with significant increases (P < 0.002) during 10 s to 30 min exposure. Once formed, PEt was degraded slowly, with a half time > 3 h. It is concluded that NE stimulates PLD in rat aorta, which forms a significant amount of PA from the hydrolysis of PC.(ABSTRACT TRUNCATED AT 250 WORDS)
Arachidonic acid and its metabolites increase Cai in cultured rat oligodendrocytes
Authors: Soliven B. , Takeda M. , Shandy T. , Nelson D. J.
Published: December 2017

Fluorescence measurements of intracellular calcium (Cai) were made on cultured rat spinal cord oligodendrocytes (OLGs) using the dye fura-2. Exposure of OLGs to arachidonic acid (AA) (5-50 microM) elicited a concentration-dependent increase in Cai that was derived mainly from extracellular Ca2+. AA at 50 microM also released Ca2+ from intracellular stores. The response to AA was not decreased by nifedipine or by inhibition of Na(+)-Ca2+ exchange. AA-induced Ca2+ influx pathway was permeable to Mn2+ and Co2+ but not to Ba2+ and was not markedly influenced by depolarization, suggesting that AA activates a voltage-independent, not strictly selective, Ca2+ channel. The Cai response to AA was partially attenuated in the presence of indomethacin, indicating that the Cai response was mediated in part by cyclooxygenase products of AA. However, the AA-induced Cai response far exceeded that induced by prostaglandins and was mimicked by linoleic acid. We conclude that AA modulates Cai of OLGs via two mechanisms: 1) indirectly via cyclooxygenase pathway and 2) directly via membrane lipid-protein interaction.
Acid and base effects on avian osteoclast activity
Authors: Carano A. , Schlesinger P. H. , Athanasou N. A. , Teitelbaum S. L. , Blair H. C.
Published: December 2017

Osteoclasts generate a massive acid flux to mobilize bone calcium. Local extracellular acidification by polarized vacuolar-type H(+)-ATPase, balanced by contralateral HCO3-(-)Cl- exchange to maintain physiological intracellular pH, is theorized to drive this process. It follows that extracellular pH, PCO2, or HCO3- concentration ([HCO3-]) should impact bone matrix dissolution. However, the effects on bone resorption of the concentrations of these ions or their transmembrane gradients are unknown. Furthermore, because bone management is a vital process, regulatory feedback may minimize such effects. Thus a complex relationship between bone resorption and pH, PCO2, and [HCO3-] is expected but requires experimental determination. We measured bone resorption by isolated avian osteoclasts while varying these parameters across the physiological range. Bone degradation increased 50% from pH 7.3 to 6.7, whether achieved by changing [HCO3-] (2.3-38 mM) at constant HCO3- or PCO2 (15-190 mmHg) at constant [HCO3-]. However, at constant pH, changing PCO2 and [HCO3-] within physiological limits did not affect bone resorption. In contrast, total HCO3- removal at pH 7.4 reduced bone degradation by rat or avian osteoclasts substantially, confirming that normal acid secretion requires HCO3-. These observations support a model coupling osteoclastic bone resorption to proton and HCO3- transport but indicate that [HCO3-] is not rate limiting under physiological conditions. Extracellular pH changes affect osteoclastic bone resorption measurably, but not dramatically, at physiological [HCO3-].
Effects of extracellular ATP on ICa, [Ca2+]i, and contraction in isolated ferret ventricular myocytes
Authors: Qu Y. , Himmel H. M. , Campbell D. L. , Strauss H. C.
Published: December 2017

The effects of extracellular ATP on the voltage-activated "L-type" Ca current (ICa), action potential, resting and transient intracellular Ca2+ levels, and cell contraction were examined in enzymatically isolated myocytes from the right ventricles of ferrets. With the use of the whole cell patch-clamp technique, extracellular ATP (10(-7) to 10(-3) M) inhibited ICa in a time- and concentration-dependent manner. ATP decreased the peak amplitude of ICa without altering the residual current at the end of 500-ms clamp steps. The concentration-response relationship for ATP inhibition of ICa was well described by a conventional Michaelis-Menten relationship with a half-maximal inhibitory concentration of 1 microM and a maximal effect of 50%. Consistent with its inhibitory effect on ICa, ATP hyperpolarized the plateau phase and shortened the action potential duration. In fura-2-loaded myocytes, extracellular ATP did not change the resting myoplasmic Ca2+ levels; however, when current was elicited under voltage-clamp conditions, ATP both decreased the myoplasmic intracellular Ca2+ transient and inhibited the degree of cell shortening. Our results suggest that ATP could be a genuine and potent extracellular modulator of cardiac function in ferret ventricular myocardium.
Distribution of calcium/calmodulin-dependent protein kinase II in rat ileal enterocytes
Authors: Matovcik L. M. , Haimowitz B. , Goldenring J. R. , Czernik A. J. , Gorelick F. S.
Published: December 2017

Ca2+/calmodulin (CaM)-dependent protein kinase II is a major effector of the Ca2+ signaling pathway. It has a wide tissue distribution and phosphorylates multiple substrates. Villus enterocytes from rat ileum contain a Ca2+/CaM-dependent kinase activity that phosphorylates the exogenous neural substrate synapsin I. This phosphorylation is blocked by a specific peptide inhibitor. Antibodies made to rat brain Ca2+/CaM-dependent protein kinase II label a single band with a relative molecular mass of approximately 50 kDa in isolated rat enterocytes by immunoblot. Almost one-half of this immunoreactive protein is preferentially found in a particulate compared with a soluble subcellular fraction of the enterocytes. Virtually all of the 50-kDa band in the particulate fraction is insoluble in nonionic detergent, suggesting that the kinase is associated with the enterocyte cytoskeleton. Antibodies to Ca2+/CaM-dependent protein kinase II immunocytochemically detect fibrillar structures concentrated in the terminal web region of intestinal epithelial cells that colocalized with myosin II. This enzyme may have a role in regulating the intestinal epithelial cytoskeleton.
Supernatant of endothelial cells exposed to laminar flow inhibits mesangial cell proliferation
Authors: Morigi M. , Zoja C. , Figliuzzi M. , Remuzzi G. , Remuzzi A.
Published: December 2017

We investigated the effects of culture medium conditioned with endothelial cells exposed to hemodynamic shear forces on modulation of mesangial cell (MC) growth. Confluent monolayers of bovine aortic endothelial cells, grown in medium containing 10% fetal calf serum, were exposed to static or to laminar flow conditions for 24 h using a cone-and-plate device. Endothelial cell-conditioned medium was used to study the growth of bovine MC by [3H]thymidine uptake. The proliferative response of MC to fresh medium (containing 10% fetal calf serum) and to culture medium from endothelial cells under static flow [66.7 +/- 34.1 vs. 73.9 +/- 30.0 counts/min (cpm) x 10(-3)] was comparable. In contrast, medium conditioned with endothelial cells exposed to laminar shear stress of 8 dyn/cm2 almost completely abolished MC proliferation (5.8 +/- 6.9 cpm x 10(-3), P < 0.01). To establish whether this effect is due to endothelial cell production of a substance that inhibits MC proliferation or simply to metabolization of serum growth factors in the culture medium, we performed shear stress experiments using serum free medium and we added 10% fetal calf serum after shear exposure just before the proliferation assay. In this condition a significant antiproliferative effect of endothelial cell supernatant under laminar flow was obtained (27.7 +/- 23.4 vs. 68.8 +/- 45.8 cpm x 10(-3), laminar vs. static, P < 0.05), suggesting that endothelial cells under shear stress effectively produce a factor that inhibits MC proliferation. These results would suggest that local glomerular capillary blood flow could play a role in the regulation of MC mitogenesis.
Endocytosis in proximal tubule cells involves a two-phase membrane-recycling pathway
Authors: Nielsen S.
Published: December 2017

The aims of the present study were to examine the initial events of endocytosis with respect to migration, internalization, and recycling of protein binding sites on the luminal plasma membrane of genuine renal proximal tubule cells (not cultured cells), and in particular the sequential involvement of dense apical tubules (DAT). Isolated rabbit proximal tubules were first perfused at low temperature (9 degrees C) with insulin-gold to label the binding sites and then with disuccinimidyl suberate (DSS) for 20 min to covalently cross-link insulin-gold and binding sites. The tubules were subsequently perfused for 4-30 min at 25 or 37 degrees C before fixation to follow the pathways of the binding sites. The present experimental approach allowed a temporal separation of the subcellular processes involved in endocytosis. The identity and organization of the different organelles were certified by serial sectioning of the experimental tubules. In tubules fixed directly at low temperature after cross-linking the label was located on the microvilli membranes and in plasma membrane invaginations. The labeling of microvilli decreased with time of perfusion at 25 degrees C in parallel with a simultaneous increase of labeling of invaginations, suggesting lateral migration of the binding sites. The invaginations pinched off from the surface to form endocytic vesicles. The binding sites were subsequently trafficked either to 1) tubular elongations from endocytic vesicles or small vacuoles, which again form DAT, or 2) in a later sequence trafficked to larger vacuoles, from which tubular elongations and DAT also form. The elongations formed the DAT that transported binding sites back to the luminal plasma membrane. Lysosomes and Golgi apparatus were not involved in membrane recycling. Thus the present study provides evidence for a two-phase recycling pathway of membrane proteins involved in endocytosis.
Eosin-5-maleimide inhibits red cell Cl- exchange at a noncompetitive site that senses band 3 conformation
Authors: Knauf P. A. , Strong N. M. , Penikas J. , Wheeler R. B. , Liu S. Q.
Published: December 2017

Eosin-5-maleimide (EM) has been used as a fluorescent probe for the external-facing transport site of the human erythrocyte band 3 protein. Changes in chloride concentration at both sides of the membrane have no significant effect on the inhibitory potency of EM as a reversible inhibitor of Cl- exchange at 0 degrees C, however, demonstrating that it is not a competitive inhibitor. The affinity of EM for the form of band 3 with the transport site facing outward is approximately five times greater than for the form with the transport site facing the cytoplasm; binding of iodide to the external transport site causes no statistically significant decrease in affinity for EM. Eosin, without the maleimide moiety, is a slightly more potent inhibitor than is EM. Erythrosin, an analogue with four iodide atoms replacing the four bromide atoms in eosin, is a much more potent inhibitor, with a half-inhibitory concentration of only 3.1 microM, > 30 times lower than that of EM. Neither eosin nor erythrosin inhibition is affected by changes in chloride concentration as would be expected for a competitive inhibitor. Thus EM and the other eosin derivatives bind to a site separate from the external transport site, but one that is affected by the changes of transport site conformation from the inward-facing to the outward-facing state.
Mechanisms of regulatory volume decrease in UC-11MG human astrocytoma cells
Authors: Medrano S. , Gruenstein E.
Published: December 2017

Swelling of astrocytes commonly occurs after cerebral ischemia and other brain injuries. Because these cells constitute 20-25% of human brain volume, their swelling is a major factor in the morbidity and mortality associated with cerebral edema. Many cells, including astrocytes, resist or reverse the tendency to swell by activating transport pathways that lead to a regulatory volume decrease. Here we report the results of studies designed to elucidate the mechanisms of the regulatory volume decrease that occurs after astrocytes are swollen by exposure to hypotonic medium. Using UC-11MG cells, a well-characterized, human, astrocytoma-derived line, we observed an increase in membrane permeability to both K+ and Cl- during regulatory volume decrease, consistent with a net loss of these ions. Neither the increase in K+ exit nor the regulatory volume decrease was affected by bumetanide, an inhibitor of anion-cation cotransport. On the other hand, the increased K+ efflux, as well as the regulatory volume decrease, was blocked by Gd3+, suggesting a putative role of stretch-activated cationic channels in the process of volume regulation. Although increases in intracellular free Ca2+ were also observed during hypotonic treatment, they occurred well after the onset of the regulatory volume decrease. Furthermore, the regulatory volume decrease was not affected by blocking the intracellular free Ca2+ increase with dimethyl 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or by removal of extracellular Ca2+. These results indicate that the regulatory volume decrease in UC-11MG cells may involve stretch-activated channels that operate independently of changes in intracellular free Ca2+.
Mg2+ buffering in cultured chick ventricular myocytes: quantitation and modulation by Ca2+
Authors: Koss K. L. , Putnam R. W. , Grubbs R. D.
Published: December 2017

To characterize the Mg2+ buffering of cultured chick ventricular myocytes, cytosolic Mg2+ was increased by liberating Mg2+ normally chelated by ATP upon total depletion of ATP content. Because the total Mg content and cell volume remained constant during this time, the difference between the amount of Mg2+ liberated (2.7 mM) and the 0.9 mM increase in cytosolic Mg2+ activity measured fluorometrically with mag-fura-2 indicates a sizable Mg2+ buffering. A new term, the Mg2+ buffer coefficient (BMg), was derived to quantify this buffering. We also determined that cytosolic Mg2+ activity increased by only 0.6 mM in cells acutely exposed to zero external Ca2+ during ATP depletion. In the absence of extracellular Ca2+, the basal cytosolic Ca2+ activity (alpha Ca2+i) was reduced by 72%, whereas the increase in alpha Ca2+i induced by ATP depletion was substantially blunted; no difference in either the time course of adenine nucleotide changes or the Ca and Mg content was observed. The BMg value calculated for these cells indicates that Mg2+ buffering is substantially greater in the absence of extracellular Ca2+ (2.5) than when extracellular Ca2+ is present (1.4), indicating that alpha Ca2+i affects cytosolic Mg2+ activity in ventricular myocytes. Therefore the Mg2+ buffering of ventricular myocytes appears to be comprised of at least two components: 1) a Ca(2+)-insensitive adenine nucleotide pool and 2) a Ca(2+)-sensitive nonadenine nucleotide pool.
A dramatic pH-dependent alteration in ANP receptor density: a note for using cultured cells
Authors: Katafuchi T. , Hagiwara H. , Ito T. , Hirose S.
Published: December 2017

Culture media tend to become acidic when rapidly growing cells are cultured under batch conditions using a CO2/HCO3- buffer system. The effects of this inherent lowering of pH on cellular makeup of cultured cells, which have long been ignored, were examined by monitoring the pH and number of the atrial natriuretic peptide (ANP) receptors expressed on the cultured bovine endothelial cells. The Eagle's minimum essential medium was adjusted to three different pH values of 7.0, 7.4, and 7.7 and used for 48-h batch cultures. After this 48-h incubation, the pH values of the media were found to be 7.0, 7.1, and 7.4, respectively. These pH shifts had unexpectedly strong influences on the ANP receptor levels without affecting the affinity. Cells maintained in the slightly higher pH medium had a trace amount of the receptor (< 10 sites/cell), while those in the lower pH environment exhibited a large number of binding sites (40,000 sites/cell). Similar situations might occur in other cellular components and in other types of cells, and therefore, such possibilities should be kept in mind when cultured cell systems are used.
Masking of A-type K+ channel in guinea pig cardiac cells by extracellular Ca2+
Authors: Inoue M. , Imanaga I.
Published: December 2017

Removal of extracellular Ca2+ induced transient outward currents (Io) at membrane potentials more positive than 0 mV in the guinea pig cardiac cell. This current reached a peak within a few milliseconds of stimulation, then decreased exponentially. External Cd2+ (0.1 mM) mimicked the inhibitory effect of Ca2+ on Io. Addition of D 600 (1 microM) or quinidine (0.1 mM) in the perfusate produced a reversible suppression, and replacement of internal K+ with tetraethylammonium induced a complete inhibition of Io. The steady-state inactivation of the transient component of Io was expressed by a Boltzmann relation with a half-inactivation voltage of -33.5 mV and a slope factor of 7.5 mV. This transient component was completely or almost completely inhibited by substitution of 4-aminopyridine for external cations. We conclude that in guinea pig cardiac cells, extracellular Ca2+ at physiological concentrations is masking the activity of an A-type K+ channel. This finding implies that even should a channel gene or transcript be identified using molecular biological techniques, the channel may not necessarily function under physiological conditions.
Angiotensin II-aldosterone interactions on protein synthesis in vascular smooth muscle cells
Authors: Ullian M. E. , Hutchison F. N. , Hazen-Martin D. J. , Morinelli T. A.
Published: December 2017

We examined the effects of mineralocorticoid-mediated increases in angiotensin II receptors on angiotensin II-stimulated protein synthesis in cultured rat aortic vascular smooth muscle cells. Incubation of quiescent (serum-deprived) cells for 24 h with angiotensin II alone resulted in concentration-dependent increases in leucine incorporation (protein synthesis), e.g., 57% over control after 1 microM angiotensin II, whereas incubation for 24 h with aldosterone alone resulted in concentration-dependent decreases in leucine incorporation, e.g., 40% less than control after 1 microM aldosterone. Incubation of serum-replete cells with 10 nM aldosterone for 24 h followed by serum deprivation and incubation with 100 nM angiotensin II and 1 nM aldosterone for an additional 48 h (experimental conditions in which angiotensin II receptor number was increased but the direct negative effects of aldosterone on leucine incorporation were minimized) resulted in increases in angiotensin II-stimulated protein synthesis by 53%, and this augmentation was inhibited by the aldosterone receptor antagonist spironolactone. The aldosterone effect was not universal, as aldosterone did not upregulate binding of or potentiate leucine incorporation stimulated by thromboxane A2 mimetics; nor was the aldosterone effect mediated by inhibition of angiotensin II metabolism, because angiotensin II concentrations were not increased by incubation with aldosterone. In summary, aldosterone-mediated increases in angiotensin II receptor number are associated with enhanced angiotensin II-stimulated protein synthesis.
Effects of angiotensin-converting enzyme inhibition on mitochondrial number in the aging mouse
Authors: Ferder L. , Inserra F. , Romano L. , Ercole L. , Pszenny V.
Published: December 2017

To investigate the effects of angiotensin-converting enzyme (ACE) inhibitors on the cellular function and structure in a variety of organs during the aging process, one hundred CF1 mice were divided into four groups of 25 animals each. Groups A, B, and C received enalapril in drinking water from weaning until the age of 24 mo. Doses administered were (in mg/l): group A, 20; group B, 10; group C, 5. Group D is the control. Animals were killed, and morphometric studies were performed in heart, kidney, liver, and spleen. As a result, there was a decrease of renal and myocardial sclerosis and an increase in the number of mitochondria in heart and liver cells, which is associated with a significant increase in survival of animals receiving ACE inhibitors. These findings lead us to think that natural aging mechanisms have been altered in those animals.
Microprobe analysis of Tc-MIBI in heart cells: calculation of mitochondrial membrane potential
Authors: Backus M. , Piwnica-Worms D. , Hockett D. , Kronauge J. , Lieberman M. , Ingram P. , LeFurgey A.
Published: December 2017

Hexakis (2-methoxyisobutylisonitrile) technetium-99m (99mTc-MIBI) is a gamma-emitting radiopharmaceutical probe currently in clinical use to evaluate myocardial perfusion. Biochemical and cellular pharmacological studies have suggested that Tc-MIBI, a lipophilic cation, is sequestered in mitochondria in response to transmembrane potentials. To assess directly the subcellular distribution of the probe in heart tissue, cultured chick heart cells were analyzed by electron-probe X-ray microanalysis (EPXMA) following equilibration in micromolar concentrations of carrier-added 99Tc-MIBI, the ground-state radiopharmaceutical. Quantitation of the physiological elements Na, Ca, Mg, K, S, P, and Cl was correlated with exposure to increasing concentrations of 99Tc-MIBI. EPXMA signals indicated that 99Tc-MIBI was concentrated up to 1,000 times into mitochondria in a dose-dependent fashion based on measured Tc content in the mitochondria. Inner membrane potential (delta psi) of individual mitochondria was calculated as -117 mV using the Nernst equation. Concentrations of 99Tc-MIBI > 36 microM caused a significant efflux of K and Mg from the cell, as well as an increase in Cl in the mitochondria. Comparison of cell ultrastructure with conventional electron microscopy at extracellular 99Tc-MIBI concentrations of 36-72 microM showed no changes compared with control. 99Tc-MIBI allows valuable in situ investigation of cellular bioenergetics with EPXMA by quantitation of delta psi.
Mechanism and regulation of swelling-activated inositol efflux in brain glial cells
Authors: Strange K. , Morrison R. , Shrode L. , Putnam R.
Published: December 2017

Rat C6 glioma cells chronically acclimated to hypertonic media accumulate large quantities of inositol. When returned to isotonic conditions, the cells swell and lose inositol slowly via a four- to fivefold increase in the rate of passive inositol efflux. The inositol efflux pathway is a Na(+)-independent transport mechanism with low affinity for inositol and is inhibited by quinidine, quinine, various anion transport blockers, and cis-unsaturated fatty acids. Ionomycin-induced elevation of intracellular Ca2+ (Ca2+i) had no effect on basal or swelling-induced inositol efflux. Inositol efflux was not inhibited by chelation of Ca2+i with 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid. In addition, Ca2+i measured with fura 2 did not change during cell swelling, indicating that increases in Ca2+i do not regulate inositol efflux. Exposure of C6 cells to 20 nM phorbol 12-myristate 13-acetate, 0.5 mM adenosine 3',5'-cyclic monophosphate (cAMP), or 50 microM forskolin had no effect on basal inositol efflux but stimulated swelling-induced inositol loss by 2.6-, 2.2-, and 3.4-fold, respectively. Exposure to the protein kinase inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine or staurosporine or downregulation of protein kinase C (PKC) activity, however, had no inhibitory effect on inositol efflux, and cellular cAMP levels were not altered by cell swelling. Taken together, these results indicate that stimulation of PKC and protein kinase A modulates the activity of the efflux pathway but is not required for swelling-induced activation. Ketoconazole, cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate, and gossypol, inhibitors of lipoxygenase enzymes, blocked both basal and swelling-induced inositol efflux, suggesting indirectly that lipoxygenase metabolites may be responsible for swelling-induced activation of the efflux mechanism. The characteristics of inositol efflux in C6 cells are similar to those described for volume regulatory sorbitol and taurine efflux in a number of cell types, suggesting the existence of a common transport mechanism.
A model of stress relaxation in cross-bridge systems: effect of a series elastic element
Authors: Luo Y. , Cooke R. , Pate E.
Published: December 2017

Many experimental protocols employed in the study of muscle mechanics use tension transients as a probe of the magnitudes of the kinetic rates in the underlying cross-bridge dynamics. These transients could potentially be modified by the elastic elements that exist both within the fiber and at the points of attachment to the experimental apparatus. To better understand the magnitude of such modifications, we have used computer simulation to investigate the transients that would be expected for cross bridges acting on an actin filament attached to an elastic element. The original model of cross-bridge mechanics by A.F. Huxley was used (Prog. Biophys. 7: 255-318, 1957). After an isometric equilibrium is achieved, a tension transient is produced by changing the dissociation rate constant, g1, while holding the attachment rate constant, f1, fixed. This decreases the number of attached, force-producing cross bridges. We find that the tension transients are markedly slowed by the presence of even a few (> or = 2) nanometers of series elastic strain per half-sarcomere. Thus some rate constants inferred from mechanical transients (e.g., those induced by caged ligands) may underestimate the actual kinetic rates of the cross-bridge processes.
Disopyramide blocks pancreatic ATP-sensitive K+ channels and enhances insulin release
Authors: Hayashi S. , Horie M. , Tsuura Y. , Ishida H. , Okada Y. , Seino Y. , Sasayama S.
Published: December 2017

An antiarrhythmic agent, disopyramide, was found to enhance the insulin secretory capacity of Wistar rat pancreatic islets with a half-maximal concentration of 23.3 microM. Employing a patch-clamp technique, disopyramide was found to inhibit ATP-sensitive K+ (KATP) channel activity in rat pancreatic beta-cells in primary culture without altering the unitary conductance. Half-maximal inhibition was achieved by the addition of 3.6 microM disopyramide to the intracellular bathing solution in the inside-out mode, 11.0 microM to the extracellular bathing solution in the outside-out mode, and 87.4 microM in the cell-attached mode. The binding of [3H]glibenclamide to pancreatic islets was inhibited by unlabeled glibenclamide but not by unlabeled disopyramide. Based on these observations, it is concluded that disopyramide blocks pancreatic KATP channels via binding to a site(s) distinct from the sulfonylurea receptor. This effect may be causatively involved in disopyramide-induced hypoglycemia.
Mechanism of diltiazem's immunomodulatory effects after hemorrhage and resuscitation
Authors: Meldrum D. R. , Ayala A. , Chaudry I. H.
Published: December 2017

Although diltiazem improves immune responses after hemorrhage and resuscitation, its mechanism remains unknown. To study this, C3H/HeN mice were bled to a mean blood pressure of 35 mmHg, maintained at that level for 1 h, and then adequately resuscitated and treated with diltiazem (400 micrograms/kg body wt) or saline (vehicle). One hour after hemorrhage and resuscitation, splenic microvascular blood flow was determined by laser Doppler flowmetry. Splenocytes were also harvested and ATP levels and cytoplasmic free Ca2+ concentration ([Ca2+]i) were determined by 31P nuclear magnetic resonance and fluo 3 flow cytometry, respectively. Splenocyte functions were determined by measuring interleukin (IL)-1, IL-2, IL-3, IL-6, and tumor necrosis factor concentrations in concanavalin A-stimulated supernatant with cytokine-specific cellular assays. Hemorrhage and resuscitation caused a significant decrease in the splenocyte's ability to release cytokines, which was correlated with significant reductions in splenocyte ATP levels and splenic microvascular blood flow as well as a significant increase in splenocyte [Ca2+]i. Diltiazem significantly decreased splenocyte [Ca2+]i while improving splenocyte ATP levels, cytokine production, and splenic microvascular blood flow. Nitroglycerin (71 micrograms/kg body wt) administration improved splenic microvascular blood flow to diltiazem-treated levels but failed to concomitantly improve splenocyte ATP levels or cytokine production. Thus diltiazem's immunoprotective effects appear to be the result of decreased Ca(2+)-induced splenocyte injury.
Twitch-dependent SR Ca accumulation and release in rabbit ventricular myocytes
Authors: Bassani J. W. , Bassani R. A. , Bers D. M.
Published: December 2017

Using caffeine-induced contractures (Ccaf) and thapsigargin (TG), we estimated the fraction of sarcoplasmic reticulum (SR) Ca released at one twitch and also the number of twitches required to reload a Ca-depleted SR. Similar results were obtained for twitches or intracellular Ca (Cai) transient with the fluorescent indicator, indo 1. Sustained exposure to 10 mM caffeine completely depletes the SR of Ca in < 5 s (as assessed by a second Ccaf). After such Ca depletion, four to five twitches are necessary to reload the SR to the steady-state level (with a twitch constant, tau = 1.6 twitches). We also determined the time required for complete inhibition of the SR Ca-adenosinetriphosphatase (ATPase) by TG. After SR Ca depletion, 5 microM TG was applied for different periods of time before a train of "reloading" twitches. A TG exposure period of 90 s was sufficient to completely prevent Ccaf after these reloading twitches. When SR is Ca depleted, the twitch is larger in the presence of TG, indicating that the SR Ca-ATPase can limit the ability of Ca influx to activate contraction. To assess SR Ca released at one twitch in cells with normally Ca-loaded SR, 5 microM TG was applied for 90 s to prevent SR Ca reuptake. Then one or several twitches were activated (causing SR Ca release, but with reuptake completely blocked). After the twitch (or train), a Ccaf was used to assess remaining SR Ca.(ABSTRACT TRUNCATED AT 250 WORDS)