bluuebunny/crossref_metadata_2025 huggingface dataset:
Sphingolipids as mediators of effects of platelet-derived growth factor in vascular smooth muscle cells
Authors: Jacobs L. S. , Kester M.
Published: December 2017

The role of sphingolipids in mediating the action of platelet-derived growth factor (PDGF) has been investigated in the vascular smooth muscle-derived A7r5 cell line. L-Cycloserine (2 mM), an inhibitor of sphingolipid synthesis, caused time-dependent inhibition of [3H]serine incorporation into [3H]sphingomyelin in A7r5 cells. PDGF-AB (10 ng/ml), PDGF-BB (10 ng/ml), or sphingosine (10 microM) independently stimulated [3H]thymidine incorporation into DNA in A7r5 cells. L-Cycloserine (2 mM) inhibited stimulation of DNA synthesis by both PDGF-AB and PDGF-BB. L-Cycloserine (2 mM, 16 h) did not affect the ability of PDGF or sphingosine to increase intracellular free calcium ([Ca2+]i) in A7r5 cells loaded with the fluorescent indicator fura 2. Measurement of adenine nucleotide levels in A7r5 cell extracts by reverse-phase high-performance liquid chromatography indicated that treatment with L-cycloserine did not adversely affect cellular metabolism. To determine directly whether PDGF activates sphingolipid metabolism, A7r5 cells were labeled with [3H]serine for 48 h and then treated with PDGF-AB (10 ng/ml) for 1 h. Sphingolipids were separated by thin-layer chromatography and quantified by liquid scintillation counting. PDGF-AB stimulated an increase in [3H]sphingosine from 25.5 +/- 3.0 to 37.5 +/- 4.1 counts.min-1 (cpm).micrograms protein-1 and a concomitant decrease in [3H]ceramide from 24.3 +/- 3.2 to 18.5 +/- 2.9 cpm/micrograms protein. These data suggest that the PDGF-stimulated increase in [Ca2+]i is not sufficient for induction of DNA synthesis and that mitogenic effects of PDGF in vascular smooth muscle cells are mediated by sphingolipid metabolism.
Fate of antidiuretic hormone water channel proteins after retrieval from apical membrane
Authors: Zeidel M. L. , Hammond T. G. , Wade J. B. , Tucker J. , Harris H. W.
Published: December 2017

In toad bladder granular cells, antidiuretic hormone (ADH) stimulates insertion of vesicles containing water channels (WCV), markedly increasing apical membrane osmotic water permeability (Pf). After withdrawal of ADH stimulation, WCV are removed from the apical membrane and fluid-phase markers endocytosed from the apical solution appear predominantly in endosomes at 10-15 min and multivesicular bodies at 30-60 min. Although the luminal contents of this endocytic pathway have been well characterized, the fate of membrane proteins, including functional ADH water channels in these vesicles remains unclear. Using electron microscopic, flow cytometric, and stopped-flow fluorescence measurements and characterization of labeled vesicle proteins, we examined the fate of membrane proteins contained within WCV. The protein complements of endosomes harvested after 10, 30, and 60 min of ADH withdrawal were similar. Selective covalent labeling of apical proteins during ADH stimulation followed by ADH reversal for 30 or 60 min showed that apical proteins colocalize with fluid-phase marker-labeled endosomes at all times, and most apically labeled protein bands present in the 10-min fraction were also present in the 30- and 60-min endosome fractions. Endosomes at 10 and 30 min but not at 60 min contained functional water channels revealed by high Pf and proton permeability, low activation energy of Pf, and sensitivity of Pf to mercurial reagents. We conclude that a portion of apically exposed membrane proteins, including candidate water channel proteins, travel together with fluid-phase markers from 10-min endosomes into later endosomal compartments. Functional water channels may be inactivated or some essential protein component selectively sorted away between 30 and 60 min after ADH withdrawal.
A putative H(+)-K(+)-ATPase is selectively expressed in surface epithelial cells of rat distal colon
Authors: Jaisser F. , Coutry N. , Farman N. , Binder H. J. , Rossier B. C.
Published: December 2017

Recently, a putative distal colon H(+)-K(+)-ATPase alpha-subunit has been identified and characterized (M. S. Crowson and G. E. Shull. J. Biol. Chem. 267:13740-13748, 1992). In the present study, we report the tissue and cell expression of this putative H(+)-K(+)-ATPase. The results indicate that, first, in the gut, the putative H(+)-K(+)-ATPase alpha-subunit is restricted to the distal part of the colon and is predominantly expressed in surface epithelial cells, in marked contrast to the alpha 1-subunit of Na(+)-K(+)-ATPase that is also expressed in glands. These data suggest that the H(+)-K(+)-ATPase alpha-subunit is a potential marker for terminal differentiation of distal colon. Second, in the uterus, the putative H(+)-K(+)-ATPase is restricted to the region of the myometrium between the inner and midmuscular zone that is very rich in vascular supply and nerve cells. This striking expression suggests that the H(+)-K(+)-ATPase may not be involved in the control of pH and potassium concentration of the uterine fluid but rather in distinct functions of vascular and/or nerve cells. Third, with the use of three independent and different approaches (Northern blot analysis, ribonuclease protection assay, and in situ hybridization), we were unable to detect any significant amount of H(+)-K(+)-ATPase transcripts in kidney tissue. Our data suggest that the putative distal colon H(+)-K(+)-ATPase is probably distinct from the kidney isoform. Finally, we report the sequence of a set of degenerate oligonucleotides that are useful to clone alpha-subunits of the Na(+)-K(+)-/H(+)-K(+)-ATPase gene family in different tissues and different species.
Simultaneous imaging of intracellular [Ca2+] and pH in single MDCK and glomerular epithelial cells
Authors: Wiegmann T. B. , Welling L. W. , Beatty D. M. , Howard D. E. , Vamos S. , Morris S. J.
Published: December 2017

The interrelationships between changes in intracellular calcium concentration ([Ca2+]i) and intracellular pH in Madin-Darby canine kidney cells and kidney glomerular epithelial cells exposed to various stimuli were analyzed simultaneously using a new design of a fluorescence video microscope. Cells were double labeled with indo 1 and SNARF 1 dyes and were excited simultaneously at 350 and 540 nm. Images at four emission wavelengths were captured simultaneously at 405, 475, 575, and 640 nm at 30 frames/s for the two ratio dyes. SNARF sensitivity to pH between 6.5 and 8.0 was unchanged by [Ca2+]i. The SNARF ratio maps were used to correct the pH-dependent changes in the calculation of local cell calcium. NH4Cl loading produced the expected alkalinization and a concurrent rise in [Ca2+]i. When the NH4Cl was removed and the cells became acidic, a second rise in [Ca2+]i was recorded. Both changes in [Ca2+]i were from intracellular stores since they persisted in the absence of extracellular calcium. The findings demonstrate the need for pH correction of indo 1 recordings.
Receptor-activated currents in mouse fibroblasts expressing transfected bombesin receptor subtype cDNAs
Authors: Kusano K. , Gainer H. , Battey J. F. , Fathi Z. , Wada E.
Published: December 2017

BALB/c 3T3 cells do not normally express receptors for bombesin-like peptides [bombesin (Bn), gastrin-releasing peptide (GRP), and neuromedin B (NmB)]. Transfection of BALB/c 3T3 cells with complementary DNA-encoding GRP receptors or NmB receptors leads to stable expression of functional GRP receptors (GRP Rt) or NmB receptors (NmB Rt), respectively, which are coupled to cell membrane ion channels. Whole cell current analysis using patch electrodes shows that the activation of these newly expressed receptors induces cation conductance increases, most frequently a Ca(2+)-activated plasma membrane K+ conductance. The dose-response (peak-current) relations of both transfected receptor subtypes were sigmoidal and exhibited threshold activation concentration in the picomole range and the saturation of responses to higher concentrations than 10(-8) M. The GRP Rt responded about equally to GRP, NmB, and Bn when compared at equimolar levels, despite their known difference in binding affinity for the three peptides (GRP, Bn > NmB). In contrast, for the NmB Rt, the NmB was more potent than GRP or Bn. Among four GRP/Bn-receptor antagonists tested, the [D-Phe6]Bn(6-13) ethyl ester suppressed GRP Rt responses at low concentrations (10(-7) M). N-acetyl-GRP-(20-26) amide, [Leu13-psi(CH2NH)-Leu14]Bn, and [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P also blocked GRP Rt responses but at higher concentrations (10(-5) M). However, at these concentrations, these four antagonists had little effect on NmB Rt responses, thereby showing a specificity of these antagonists for the GRP receptors.
Zn(II) transport and distribution in rat spermatids
Authors: Reyes J. G. , Arrate M. P. , Santander M. , Guzman L. , Benos D. J.
Published: December 2017

Zn(II) is an essential trace element. In spermatozoa, Zn(II) modulates metabolism and chromatin condensation. The mechanisms of uptake and distribution of this ion in sperm cells have not been explored. In rat spermatids, our results indicate that 1) 65Zn(II) binds with fast kinetics to a labile, presumably extracellular, compartment. This binding is temperature insensitive and not modified by metabolic inhibitors. 2) Entry of 65Zn(II) in the absence of externally added proteins occurs through a mediated transport system that allows exchange to reach steady state in approximately 15 min at 34 degrees C. 3) Upon entering the cells, 65Zn(II) binds tightly to cellular organelles. 4) Exchangeable Zn(II) bound to cytoplasmic proteins plus free intracellular Zn(II) appears to be < 20% of total exchangeable Zn(II). 5) The intracellular exchangeable Zn(II) compartment is decreased by metabolic inhibitors, showing a direct or indirect link between energy metabolism and cellular Zn(II) levels. 6) 65Zn(II) efflux from rat spermatids is a process with a rate constant of 0.16 +/- 0.13 min-1 at 34 degrees C. This exit rate of Zn(II) is likely to be affected by Zn(II) release from cytoplasmic binding sites or organelles.
Acidification of rat liver lysosomes: quantitation and comparison with endosomes
Authors: Van Dyke R. W.
Published: December 2017

Both lysosomes and endosomes are acidified by an electrogenic proton pump, although studies in intact cells indicate that the steady-state internal pH (pHi) of lysosomes is more acid than that of endosomes. We undertook the present study to examine in detail the acidification mechanism of purified rat liver secondary lysosomes and to compare it with that of a population of early endosomes. Both endosomes and lysosomes exhibited ATP-dependent acidification, but proton influx rates were 2.4- to 2.7-fold greater for endosomes than for lysosomes because of differences in both buffering capacity and acidification rates, suggesting that endosomes exhibited greater numbers or rates of proton pumps. Lysosomes, however, exhibited a more acidic steady-state pHi due in part to a slower proton leak rate. Changes in medium Cl- increased acidification rates of endosomes more than lysosomes, and the lysosome ATP-dependent interior-positive membrane potential was only partially eliminated by high-Cl- medium. Permeability studies suggested that lysosomes were less permeable to Na+, Li+, and Cl- and more permeable to K+ and PO4(2-) than endosomes. Na-K-adenosine-triphosphatase did not appear to regulate acidification of either vesicle type. Endosome and lysosome acidification displayed similar inhibition profiles to N-ethylmaleimide, dicyclohexyl-carbodiimide, and vanadate, although lysosomes were somewhat more sensitive [concentration producing 50% maximal inhibition (IC50) 1 nM] to bafilomycin A1 than endosomes (IC50 7.6 nM). Oligomycin (1.5-3 microM) stimulated lysosome acidification due to shunting of membrane potential. Overall, acidification of endosomes and lysosomes was qualitatively similar but quantitatively somewhat different, possibly related to differences in the density or rate of proton pumps as well as vesicle permeability to protons, anions, and other cations.
Structural determinants of substrate specificity of the erythrocyte anion transporter
Authors: Galanter W. L. , Hakimian M. , Labotka R. J.
Published: December 2017

The human erythrocyte anion transport protein (AE1) mediates the rapid, tightly coupled, electroneutral transmembrane exchange of bicarbonate for chloride. AE1 transports a wide range of oxyanions, such as phosphate, sulfate and the physiological substrate bicarbonate. In this study, the transport characteristics of the selenium based oxyanions selenite (SeO3(2-)) and selenate (SeO4(2-)) were determined. The pH dependence of selenate influx was consistent with a titratable carrier having a extracellular pK value of 5.67 +2- 0.09. In contrast, the pH dependence of selenite influx had a maximum near pH 7.0, consistent with a hypothesis proposed by Labotka and Omachi (J. Biol. Chem. 263: 1166-1173, 1988) that the pH maximum of the transport of titratable anions is located at the midpoint between the pK of the carrier (5.7) and the pK of the titratable anion (8.3). Analysis of the transport rates and structures of these as well as a variety of other oxyanions reported in the literature suggested that oxyanions bind in a three-oxygen atom binding site, and that the formation of the transition state necessary for transport is sterically restrained by oxyanions that protrude in a direction perpendicular to the three-oxygen binding plane. This hypothesis can be used to predict the relationship between the transport rates of many oxyanions reported in the literature and should prove useful in helping to understand the molecular mechanism of AE1 mediated transport.
Cell-specific regulation of cytosolic aspartate aminotransferase by glucocorticoids in the rat kidney
Authors: Feilleux-Duche S. , Garlatti M. , Aggerbeck M. , Poyard M. , Bouguet J. , Hanoune J. , Barouki R.
Published: December 2017

The basal expression and hormonal regulation of cytosolic aspartate aminotransferase (cAspAT) were investigated in the rat kidney. In adrenalectomized animals, the basal activity was highest in the renal cortex and in the inner stripe of the outer medulla (0.1-0.15 U/mg protein). The glucocorticoid analogue dexamethasone increased cAspAT activity about twofold in the cortex and in the inner stripe of the outer medulla but not in the papilla. A half-maximal increase in the activity was achieved at doses of approximately 5 micrograms/100 g body wt. The mineralocorticoid aldosterone did not modify the cAspAT activity. The cell specificity of the hormonal regulation was analyzed by in situ hybridization. In untreated adrenalectomized rats, a cAspAT cRNA probe labeled mainly the inner stripe of the outer medulla. After dexamethasone or hydrocortisone treatment, labeling was uniformly increased in this part of the medulla and was heterogeneously increased in the renal cortex. The specific increase in labeling within the cortex was shown to be confined to the distal convoluted tubule and the thick ascending limb. We conclude that, in addition to widespread basal expression, cAspAT is regulated by glucocorticoids in a highly cell-specific manner in the renal cortex. The enzyme may thus participate in the increased energy metabolism elicited by these hormones in these cells.
Gamma-linolenic acid suppression of hepatic Ito cell mitogenesis: post-PDGF receptor prostaglandin-independent mechanism
Authors: Beno D. W. , Awad J. A. , Davis B. H.
Published: December 2017

Ito cell mitogenesis occurs during liver injury and fibrogenesis in vivo. Platelet-derived-growth factor (PDGF)-induced mitogenesis was studied in cultured rat hepatic Ito cells, which resemble the myofibroblast associated with liver injury. Pretreatment with gamma-linolenic acid (GLA), an essential fatty acid prostanoid precursor, markedly suppressed the PDGF response in a dose-dependent reversible fashion. Prostaglandins E1 and E2 were found to be the predominant prostanoids formed by cultured Ito cells. GLA depressed endogenous PG production, suggesting that the antimitogenic effect was independent of GLA conversion to a prostanoid metabolite. The PDGF-induced cascade was studied with and without GLA to determine the level of regulation that induced the observed suppression. GLA caused no apparent diminution in the abundance of the surface PDGF-beta receptor nor its subsequent activation and tyrosine phosphorylation after PDGF stimulation. Raf kinase activation and Raf perinuclear translocation were also intact despite the presence of GLA. PDGF induction of nuclear Egr and Fos also occurred with or without GLA. Activation of the serine threonine kinase c-Raf has previously been found to be sufficient to activate egr and fos and to induce mitogenesis. Therefore, the GLA suppressive effect is likely to be operative at a parallel non-Raf pathway or distal to Raf-induced early gene expression.
Role of nonselective cation current in muscarinic responses of canine colonic muscle
Authors: Lee H. K. , Bayguinov O. , Sanders K. M.
Published: December 2017

The mechanism of muscarinic excitation was studied in colonic muscle strips and isolated cells. In whole cell voltage-clamp studies performed at 33 degrees C utilizing the permeabilized patch technique, acetylcholine (ACh) reduced an L-type Ca2+ current. With K+ currents blocked, depolarization to positive potentials in the presence of ACh elicited outward current. Difference currents showed that ACh activated a voltage-dependent current that reversed at about -8 mV; this current (IACh) had properties similar to the nonselective cation conductance found in other smooth muscle cells. The reversal potential of IACh shifted toward negative potentials when external Na+ was reduced, and the inward current elicited at -70 mV decreased when external Na+ was reduced. IACh was facilitated by internal Ca2+. After the current was activated at a holding potential of -70 mV, depolarizations to -30 to 0 mV elicited influx of Ca2+ via voltage-dependent Ca2+ channels. After repolarization to the holding potential, a large inward tail current was observed. IACh was blocked by Ni2+ and Cd2+ at concentrations of 100 microM or less. Quinine (0.5 mM) also blocked IACh. With the use of the sensitivity of IACh to reduced external Na+ and divalent cations, the role of IACh in responses of intact muscles to ACh was examined. When external Na+ was reduced, ACh failed to increase slow-wave duration, and Ni2+ (50 microM) reversed the depolarization caused by ACh. These data suggest an important role for IACh in the electrical responses of colonic muscles. The contribution of IACh appears to prolong slow waves, which would allow greater entry of Ca2+ and increased force development.
RNA subunit of mitochondrial RNA-processing enzyme is induced by contractile activity in striated muscle
Authors: Ordway G. A. , Li K. , Hand G. A. , Williams R. S.
Published: December 2017

A small RNA encoded within the nucleus of yeast and mammalian cells is an essential subunit of a mitochondrial RNA-processing endonuclease (RNase MRP) that generates primers for mitochondrial DNA (mtDNA) replication. We examined expression of MRP-RNA in specialized subtypes of mammalian striated muscles that differ markedly in respiratory activity and in muscles subjected to chronic stimulation via the motor nerve, a potent stimulus to mitochondrial biogenesis. MRP-RNA was more abundant in mitochondria-rich cardiac and slow-twitch skeletal muscles than in glycolytic fast-twitch skeletal muscles. Forced contractile activity resulting from nerve stimulation increased expression of MRP-RNA by 3.5-fold within the first day and by 14-fold within 14 days. Changes in abundance of MRP-RNA preceded but otherwise occurred in parallel to changes in specific activity of citrate synthase, a marker of mitochondrial proliferation shown previously to correlate with mtDNA copy number in this model. Another small RNA (U1) also was induced transiently (1-3 days) by nerve stimulation, but such changes were not sustained and were of less magnitude (< 4-fold) than changes in MRP-RNA. These findings are consistent with the hypothesis that MRP-RNA may have a regulatory function with respect to mtDNA replication and mitochondrial biogenesis.
Phosphocreatine protects transgenic mouse liver expressing creatine kinase from hypoxia and ischemia
Authors: Miller K. , Halow J. , Koretsky A. P.
Published: December 2017

Creatine kinase (CK) is normally found at high levels in muscle and brain and catalyzes the reaction phosphocreatine (PCr) + MgADP + H+<==>creatine (Cr) + MgATP. CK is not normally found at high levels in liver. A line of transgenic mice that express high levels of the BB-dimer of CK (CKB) in liver has allowed us to assess the role of CKB during periods of low oxygen stress. During 40 min of ischemia of normal perfused livers at 25 degrees C, ATP levels are depleted, and pH decreases to 6.6. pH recovers to a preischemic level after 30 min of reperfusion of normal livers; however, P(i) levels are significantly higher and ATP levels significantly lower than preischemic values. In transgenic liver with an initial PCr-to-ATP ratio of 4.5, ATP levels are maintained until PCr is markedly depleted. pH remains at preischemic levels for 16 min of ischemia of transgenic livers. During this length of ischemia in normal livers, pH has dropped to 6.9. pH, P(i), and ATP levels return to preischemic values within 30 min of reperfusion in transgenic livers containing PCr and CK. During 90 min of hypoxia of normal perfused livers at 37 degrees C, ATP is depleted. After 15 min of hypoxia of normal livers, there is a significant increase in the release of lactate dehydrogenase (LDH). In transgenic livers, ATP is maintained, and no increase in LDH release is observed for up to 90 min, depending on the level of PCr before hypoxia. These results demonstrate the role of CKB in buffering ATP levels and regulating intracellular pH during periods of low oxygen stress.(ABSTRACT TRUNCATED AT 250 WORDS)
Full-length and truncated Kv1.3 K+ channels are modulated by 5-HT1c receptor activation and independently by PKC
Authors: Aiyar J. , Grissmer S. , Chandy K. G.
Published: December 2017

In T-cells, the Shaker-related gene, Kv1.3 encodes the type n K+ channel, whereas the type l channel is a product of the Shaw. subfamily gene, Kv3.1. Both these genes are also expressed in the brain. We have used the Xenopus oocyte heterologous expression system to study the modulatory effects of serotonin (5-hydroxytryptamine, 5-HT) on both these cloned channels. In oocytes coexpressing the mouse 5-HT1c receptor and mouse Kv1.3 channel, addition of 100 nM 5-HT causes a complete and sustained suppression of Kv1.3 currents in approximately 20 min. In contrast, 5-HT has no effect on mouse Kv3.1 currents when coexpressed with 5-HT1c receptor. The 5-HT-mediated suppression of Kv1.3 currents proceeds via activation of a pertussis toxin-sensitive G protein and a subsequent rise in intracellular Ca2+, but Ca2+ does not directly block the channel. Protein kinase (PK) C activation is not part of the pathway linking 5-HT1c receptor to Kv1.3 channels. However, phorbol esters independently suppress Kv1.3 currents. Deletion of the first 146 amino acids from the NH2-terminal, containing putative tyrosine kinase and PKA phosphorylation sites, does not alter the time course of 5-HT-mediated suppression of Kv1.3 currents, indicating that these residues are not necessary for modulation. Treatment of oocytes with calmodulin or phosphatase inhibitors does not alter 5-HT-mediated modulation. Collectively, these experiments indicate that the mouse Kv1.3 channel is capable of being modulated by 5-HT via 5-HT1c receptor in a G protein and Ca(2+)-dependent manner, but the subsequent steps in the pathway remain elusive.(ABSTRACT TRUNCATED AT 250 WORDS)
Exercise induces a transient increase in transcription of the GLUT-4 gene in skeletal muscle
Authors: Neufer P. D. , Dohm G. L.
Published: December 2017

Endurance exercise training elicits an increase in mitochondrial density as well as GLUT-4 glucose transporter protein content in skeletal muscle. Corresponding increases in mRNA for respiratory enzymes and GLUT-4 indicate that pretranslational control mechanisms are involved in this adaptive process. To directly test whether transcription of the GLUT-4 gene is activated in response to exercise training, nuclei were isolated from red hindlimb skeletal muscle of rats after 1 wk of exercise training (8% grade, 32 m/min, 40 min, twice/day). Rats were killed either 30 min, 3 h, or 24 h after the last training session. GLUT-4 transcription, determined by nuclear run-on analysis, was unaltered after 30 min, increased by 1.8-fold after 3 h, but was no longer different from controls 24 h after exercise. A similar transient increase in GLUT-4 transcription was evident, but less pronounced (1.4-fold), in untrained rats after a single bout of exercise, suggesting that the postexercise induction in GLUT-4 gene transcription is enhanced by exercise training. GLUT-4 protein content was increased 1.7-fold after 1 wk of training in the absence of any corresponding change in GLUT-4 mRNA, providing evidence that the initial increase in GLUT-4 expression involves translational and/or posttranslational control mechanisms. These findings demonstrate that muscle GLUT-4 expression in response to exercise training is subject to both transcriptional and posttranscriptional regulation. We propose that the increase in GLUT-4 mRNA evident with extended periods of training may result from a shift to pretranslational control and is the cumulative effect of repeated postexercise transient increases in GLUT-4 gene transcription.
Modification of epithelial permeability by cationic polypeptides
Authors: Tzan C. J. , Berg J. R. , Lewis S. A.
Published: December 2017

It has been demonstrated that protamine sulfate (PS; a cationic polypeptide composed of 70% arginine) increases the apical membrane conductance of the mammalian urinary bladder. In this report, synthetic cationic polypeptides (CpP; e.g., polyarginine) were used to determine whether the response of the bladder to PS was due to its cationic nature (i.e., its arginine content). We demonstrate that CpP induce a large increase in the cation and anion conductance of the apical membrane of the rabbit urinary bladder epithelium. The modulation of the membrane conductance by CpP is dependent upon a number of parameters. 1) The magnitude of the conductance change was voltage dependent. 2) An increase in the total charge per molecule increased the rate of conductance change. 3) An increase in the charge density (ratio of charged amino acids to total amino acids) increased the rate of change of conductance. 4) La3+ inhibited the ability of CpP to alter the membrane conductance. 5) The rate of reversal of the CpP-induced conductance was dependent upon the total charge per molecule as well as the charge density. 6) The level of self-inhibition (ability of solution CpP to inhibit the CpP-induced membrane conductance) was inversely correlated with the charge density and was also concentration dependent, with less inhibition occurring at low mucosal CpP concentrations. These data are consistent with a model developed to describe the effect of PS on the conductive properties of the urinary bladder epithelium.
Insulin increases the Na(+)-K(+)-ATPase alpha 2-subunit in the surface of rat skeletal muscle: morphological evidence
Authors: Marette A. , Krischer J. , Lavoie L. , Ackerley C. , Carpentier J. L. , Klip A.
Published: December 2017

The cellular localization of the alpha 2-subunit of the Na(+)-K(+)-ATPase was defined by immunoelectron microscopy, and the effect of insulin on the amount of alpha 2-immunoreactive subunits on the cell surface was quantitated. Two protocols were used for tissue fixation and immunolocalization. Protocol 1 was characterized by fixation with 2% paraformaldehyde, use of a monoclonal antibody, and detection with 3-nm-diameter gold-labeled Fab fragments or 10-nm gold-labeled immunoglobulin G. Protocol 2 was characterized by fixation with 4% paraformaldehyde plus 0.1% glutaraldehyde, use of a polyclonal antibody, and detection with 10-nm gold-labeled protein A. In control muscle, the alpha 2-subunit of the Na(+)-K(+)-ATPase was present at the plasma membrane and in intracellular tubular and vesicular structures located in subsarcolemmal and triadic regions. Acute insulin stimulation increased the number of immunolabeled alpha 2-subunits in the plasma membrane after both fixation protocols. The gain in the plasma membrane ranged from 1.5- to 3.7-fold and was significant at the level of P < 0.005. These results provide morphological quantitative evidence that the alpha 2-subunit of the Na(+)-K(+)-ATPase is present both at the plasma membrane and intracellularly in mammalian skeletal muscle and that insulin acutely increases its abundance in the muscle surface.
Involvement of protein kinase C in macrophage activation by poly(I.C)
Authors: Lake F. R. , Dempsey E. C. , Spahn J. D. , Riches D. W.
Published: December 2017

The expression of cytocidal activity is initiated by the interaction of macrophages with priming [e.g., interferon (IFN)] and triggering stimuli (polyinosinic-polycytidylic acid). We have shown that the triggering step can be initiated in a Ca(2+)-dependent fashion and hypothesized that protein kinase C (PKC) may couple the Ca2+ signal to the expression of a gene product, Bf, that accompanies the expression of macrophage cytocidal activity. Exposure of IFN-primed macrophages to polyinosinic-polycytidylic acid in the presence of the PKC inhibitors H-7 or sphingosine or after downregulation of PKC with phorbol myristate acetate markedly inhibited Bf synthesis. Western blots of macrophage lysates revealed the presence of the alpha-, delta-, and zeta-isozymes of PKC, and all were found to be downregulated by phorbol myristate acetate. Inhibition of PKC also prevented the increase in IFN-beta mRNA levels and partially blocked the response to IFN-beta. These data suggest that the alpha-, delta-, and zeta-isozymes of PKC are involved in signaling leading to Bf expression and that the level of involvement is restricted to the induction and response to IFN-beta.
Hydroperoxide-induced oxidative stress impairs heart muscle cell carbohydrate metabolism
Authors: Janero D. R. , Hreniuk D. , Sharif H. M.
Published: December 2017

Hydrogen peroxide (H2O2) may incite cardiac ischemia-reperfusion injury. We evaluate herein the influence of H2O2-induced oxidative stress on heart muscle hexose metabolism in cultured neonatal rat cardiomyocytes, which have a substrate preference for carbohydrate. Cardiomyocyte exposure to 50 microM-1.0 mM bolus H2O2 transiently activated the pentose phosphate cycle and thereafter inhibited cellular glucose oxidation and glycolysis. These metabolic derangements were nonperoxidative in nature (as assessed in alpha-tocopherol-loaded cells) and occurred without acute change in cardiomyocyte hexose transport or glucose/glycogen reserves. Glycolytic inhibition was supported by the rapid, specific inactivation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The degree of GAPDH inhibition correlated directly with the magnitude of the oxidative insult and was independent of both metal-catalyzed H2O2 reduction to free radicals and lipid peroxidation. Severe GAPDH inhibition was required for a rate-limiting effect on glycolytic flux. Cardiomyocyte pyruvate dehydrogenase was also inhibited by H2O2 overload, but to a lesser degree than GAPDH such that entry of hexose-derived acetyl units into the tricarboxylic acid cycle was not as restrictive as GAPDH inactivation to glycolytic ATP production. An increase in phosphofructokinase activity accompanied GAPDH inactivation, leading to the production and accumulation of glycolytic sugar phosphates at the expense of ATP equivalents. Cardiomyocyte treatment with iodoacetate or 2-deoxyglucose indicated that GAPDH inactivation/glycolytic blockade could account for approximately 50% of the maximal ATP loss following H2O2 overload. Partial restoration of GAPDH activity after a brief H2O2 "pulse" afforded some ATP recovery. These data establish that specific aspects of heart muscle hexose catabolism are H2O2-sensitive injury targets. The biochemical pathology of H2O2 overload on cardiomyocyte carbohydrate metabolism has implications for post-ischemic cardiac bioenergetics and function.
Inhibition of Na(+)-K(+)-ATPase activates Na(+)-K(+)-2Cl- cotransporter activity in cultured ciliary epithelium
Authors: Dong J. , Delamere N. A. , Coca-Prados M.
Published: December 2017

Inhibition of Na(+)-K(+)-ATPase activates Na(+)-K(+)-2Cl- cotransporter activity in cultured ciliary epithelium. Am. J. Physiol. 266 (Cell Physiol. 35): C198-C205, 1994.--86Rb uptake experiments were conducted to measure Na(+)-K(+)-ATPase activity and Na(+)-K(+)-2Cl- cotransporter activity in a cell line derived from rabbit nonpigmented ciliary epithelium. The presence of a Na(+)-K(+)-2Cl- cotransporter was supported by the observation of a bumetanide-sensitive 86Rb uptake component that was dependent on the extracellular concentration of both sodium and chloride. Potassium influx mediated by the Na(+)-K(+)-2Cl- cotransporter and Na(+)-K(+)-ATPase accounted for approximately 46 and 33% of total potassium uptake, respectively, whereas both ouabain- and bumetanide-resistant uptake accounted for 9%. Inhibition of the Na(+)-K(+)-ATPase had a stimulatory effect on Na(+)-K(+)-2Cl- cotransporter activity, which was dependent on the extent and duration of Na(+)-K(+)-ATPase inhibition. Ouabain treatment stimulated the potassium (86Rb) efflux rate and reduced intracellular potassium ([K]i). Potassium channel blockers suppressed the ouabain-activated potassium efflux and inhibited the ouabain-induced activation of the Na(+)-K(+)-2Cl- cotransporter. We conclude that Na(+)-K(+)-ATPase inhibition leads to the opening of potassium channels, which exacerbates the depletion of cellular potassium; Na(+)-K(+)-2Cl- cotransporter stimulation caused by the fall of [K]i overrides the tendency of increased cellular sodium to inhibit the cotransporter.
Polarization-dependent apical membrane CFTR targeting underlies cAMP-stimulated Cl- secretion in epithelial cells
Authors: Morris A. P. , Cunningham S. A. , Tousson A. , Benos D. J. , Frizzell R. A.
Published: December 2017

The relationship between adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion and the cellular location of the cystic fibrosis transmembrane conductance regulator (CFTR) was determined in both polarized (Cl.19A) and unpolarized (parental) HT-29 colonocytes expressing similar levels of CFTR mRNA and protein. CFTR immunolocalized to the apical membrane domain of polarized colonocytes exhibiting cAMP-responsive Cl- secretion. In contrast, CFTR staining was perinuclear in unpolarized colonocytes, which gave little or no cAMP-stimulated Cl- conductance responses. Thus cAMP-stimulated Cl- secretion coincided with an apical localization of CFTR. Brefeldin A (BFA) was used to perturb glycoprotein targeting in these cells. In polarized colonocytes, BFA caused a reversible, time-dependent decrease in the Cl-conductance response to cAMP but not Ca2+. Apical CFTR redistributed into large coalesced intracellular vesicles, located within the same plane as the microtubule organizing center, a marker for the trans-Golgi network (TGN). In preconfluent monolayers or unpolarized HT-29 cells, BFA had no effect on CFTR staining, which remained perinuclear. Mature, Golgi-processed CFTR protein was isolated from both polarized and unpolarized colonocytes. Thus the mechanism for polarization-dependent apical membrane CFTR targeting and the acquisition of cAMP-dependent Cl- secretion lies at or beyond the late Golgi-TGN in epithelial cells.
Effect of Napip on K0 activation of the Na-K pump in adult rat cardiac myocytes
Authors: Kinard T. A. , Liu X. Y. , Liu S. , Stimers J. R.
Published: December 2017

To determine if environmental factors influence the external K (K0) dependence of Na-K pump current (Ip), we systematically varied internal (pipette) Na (Napip) and Na-K pump activity while measuring the K0 dependence in adult rat cardiac myocytes. For each Napip, reactivation of Ip by K0 was dose dependent. The maximal Ip (Ipmax) and apparent affinity for K0 binding to the Na-K pump (K0.5) increased as Napip increased. The results of making an equimolar substitution of tetramethylammonium for K and Cs, and partial Ip inhibition with ouabain, also showed that Ipmax and K0.5 increased as Napip increased. We simulated pump activity as a function of intracellular Na (Nai) and K0 using a cyclic model of the Na-K pump and found that the model predicts K0.5 for K0 binding increases as Na increases, even when the conditions are adjusted by removing pipette K and partial pump inhibition with ouabain.
Human rsk isoforms: cloning and characterization of tissue-specific expression
Authors: Moller D. E. , Xia C. H. , Tang W. , Zhu A. X. , Jakubowski M.
Published: December 2017

Serine-threonine protein kinases in the ribosomal S6 kinase (rsk or p90rsk) family have been implicated as signaling intermediates in the cellular response to several growth factors. To investigate the molecular diversity of human p90rsk isoforms, mixed degenerate oligonucleotide polymerase chain reaction was used to isolate partial rsk cDNAs (1.1 kb). Three closely related human rsk cDNAs were obtained (HU-1, HU-2, HU-3). These cDNAs are encoded by separate genes based on DNA sequence diversity and distinct patterns seen with genomic Southern blots. Northern analysis revealed different sized mRNA transcripts for each isoform. A full-length HU-1 cDNA (3.1 kb) was subsequently isolated from a HeLa cell library. 5'-cDNA clones for HU-2 and HU-3 were isolated using the "rapid amplification of cDNA ends" strategy. Experiments using human x hamster somatic cell hybrids localized the HU-1 gene to human chromosome 3; HU-2 is on chromosome 6; and HU-3 is on the X chromosome. The tissue distribution of human rsk mRNAs was determined using ribonuclease protection assays. HU-3 mRNA was present in multiple RNA samples. HU-2 was expressed in fibroblast > muscle > lymphocyte = placenta > liver. HU-1 was expressed in Epstein-Barr virus lymphocyte > > muscle = liver > fat = placenta. These results indicate that the multiplicity of p90rsk isoforms is increased to at least three for humans and that marked tissue-/cell-specific differences in p90rsk isoform expression are present.
Role of calcium and calmodulin in flow-induced nitric oxide production in endothelial cells
Authors: Kuchan M. J. , Frangos J. A.
Published: December 2017

These experiments demonstrate that exposure of cultured endothelial cells (EC) to well-defined laminar fluid flow results in an elevated rate of NO production. NO production was monitored by release of NOx (NO2- + NO3(2-) and by cellular guanosine 3',5'-cyclic monophosphate (cGMP) concentration. NO synthase (NOS) inhibitor blocked the flow-mediated stimulation of both NOx and cGMP, indicating that both measurements reflect NO production. Exposure to laminar flow increased NO release in a biphasic manner, with an initial rapid production consequent to the onset of flow followed by a less rapid, sustained production. A similar rapid increase in NO production resulted from an increase in flow above a preexisting level. The rapid initial production of NO was not dependent on shear stress within a physiological range (6-25 dyn/cm2) but may be dependent on the rate of change in shear stress. The sustained release of NO was dependent on physiological levels of shear stress. The calcium (Ca2+) or calmodulin (CaM) dependence of the initial and sustained production of NO was compared with bradykinin (BK)-mediated NO production. Both BK and the initial production were inhibited by Ca2+ and CaM antagonists. In contrast, the sustained shear stress-mediated NO production was not affected, despite the continued functional presence of the antagonists. Dexamethasone had no effect on either the initial or the sustained shear stress-mediated NO production. An inducible NOS does not, therefore, explain the apparent Ca2+/CaM independence of the sustained shear stress-mediated NO production.(ABSTRACT TRUNCATED AT 250 WORDS)
Staurosporine, a protein kinase inhibitor, activates K-Cl cotransport in LK sheep erythrocytes
Authors: Bize I. , Dunham P. B.
Published: December 2017

K-Cl cotransport can participate in volume regulation in a number of cell types. Swelling activation of K-Cl cotransport in sheep erythrocytes proceeds by a two-step process, A<-->B<-->C (Dunham et al., J. Gen. Physiol. 101: 733-765, 1993). The A state, with a low flux, predominates at physiological volume. A-->B is rate limiting and can be activated by reducing cell Mg concentration ([Mg]c); complete activation (B-->C) requires cell swelling. Inhibitors of protein kinases and phosphatases were employed in an attempt to identify enzymatic reactions in the activation process. Staurosporine, a kinase inhibitor, activated K-Cl cotransport by approximately sixfold. Swelling of staurosporine-treated cells caused further activation that proceeded without delay. The effects of staurosporine and reducing [Mg]c were not additive. These two results indicate that staurosporine, like reducing [Mg]c, promotes the rate-limiting A-->B conversion. Unlike swelling, staurosporine activated cotransport without delay. Therefore staurosporine activates by promoting the forward reaction in the A<-->B conversions, in contrast to swelling, which activates by inhibiting the reverse reaction. Calyculin A, a phosphatase inhibitor, inhibited K-Cl cotransport but did not inhibit after activation by reducing [Mg]c, confirming earlier proposals that A-->B is promoted by a phosphatase. Calyculin A, added before or after staurosporine, abolished activation by staurosporine, confirming that staurosporine promotes A-->B. It is proposed that the phosphatase promoting this reaction is regulated by an inhibitory kinase, the staurosporine target.
Luminal perfusion of isolated gastric glands
Authors: Waisbren S. J. , Geibel J. , Boron W. F. , Modlin I. M.
Published: December 2017

We have extended to rabbit gastric glands the technique for perfusing single isolated renal tubules. We isolated glands by hand dissection and used concentric glass pipettes to hold them and perfuse their lumina. Parietal cells (PCs), which tended to be located toward the gland opening, were identified by their pyramidal shape, large size, and autofluorescence. Chief cells (CCs) were identified by their round shape and smaller size. In some experiments, we perfused the lumen with hydroxypyrenetrisulfonate, a pH-sensitive fluorophore, at pH 7.4 and used digital image processing to monitor luminal pH (pH1). Solutions were buffered with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid to pH 7.4 at 37 degrees C. With fast perfusion, we found no evidence of decreased pH1, even with stimulation by 10 microM carbachol. With slow perfusion, pH1 often fell below the dye's sensitive range (pH < 5), especially at low perfusate buffering power. In other experiments, we loaded cells with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein and monitored intracellular pH (pHi) in multiple individual PCs and CCs in a single gland. Mean pHi was 7.21 +/- 0.02 (n = 136 cells) for PCs and 7.27 +/- 0.03 (n = 103) for CCs. To examine the response to decreased pH1 and basolateral pH (pHb), we lowered pHb to 6.4 or lowered pH1 to 3.4 or 1.4. Lowering pHb to 6.4 for approximately 1 min caused pHi to fall reversibly by 0.39 +/- 0.05 (n = 53) in PCs and 0.58 +/- 0.03 (n = 50) in CCs. Lowering pH1 to 3.4 or 1.4 caused no significant pHi changes in PCs (n = 38 and 82) or in CCs (n = 44 and 77). Carbachol did not affect the response to changes in pH1 or pHb. We conclude that the apical surfaces of PCs and CCs are unusually resistant to extreme pH gradients.
Cell volume control in vestibular dark cells during and after a hyposmotic challenge
Authors: Wangemann P. , Shiga N.
Published: December 2017

Cell height was measured as an index of volume in a preparation of vestibular dark cells in which the perfusate had access to both sides of the epithelium. In response to a hyposmotic challenge induced by removal of 75 mM NaCl, cell height increased to 107%; however, cell width did not increase. Significantly larger increases in cell height were observed in the absence of Cl- or K+ or in the presence of ouabain, lidocaine, barium, or quinidine, at 7 degrees C, or after fixation with glutaraldehyde. However, no significantly different swelling was observed during a hyposmotic challenge in the absence of Na+ or in the presence of bumetanide or ethoxyzolamide. Subsequent return to control osmolarity caused a regulatory volume increase that was dependent on Na+, Cl-, and K+, inhibited by bumetanide, ouabain, or 7 degrees C, however not inhibited by ethoxyzolamide, barium, quinidine, or lidocaine. The data suggest that cell volume control during the hyposmotic challenge involved a mechanism dependent on cytosolic KCl and the Na(+)-K(+)-ATPase and that the Na(+)-Cl(-)-K+ cotransporter was involved in regulatory volume increase.
Viral gene products and replication of the human immunodeficiency type 1 virus
Authors: Morrow C. D. , Park J. , Wakefield J. K.
Published: December 2017

The acquired immunodeficiency syndrome (AIDS) epidemic represents a modern-day plague that has not only resulted in a tragic loss of people from a wide spectrum of society but has reshaped our viewpoints regarding health care, the treatment of infectious diseases, and social issues regarding sexual behavior. There is little doubt now that the cause of the disease AIDS is a virus known as the human immunodeficiency virus (HIV). The HIV virus is a member of a large family of viruses termed retroviruses, which have as a hallmark the capacity to convert their RNA genome into a DNA form that then undergoes a process of integration into the host cell chromosome, followed by the expression of the viral genome and translation of viral proteins in the infected cell. This review describes the organization of the HIV-1 viral genome, the expression of viral proteins, as well as the functions of the accessory viral proteins in HIV replication. The replication of the viral genome is divided into two phases, the early phase and the late phase. The early phase consists of the interaction of the virus with the cell surface receptor (CD4 molecule in most cases), the uncoating and conversion of the viral RNA genome into a DNA form, and the integration into the host cell chromosome. The late phase consists of the expression of the viral proteins from the integrated viral genome, the translation of viral proteins, and the assembly and release of the virus. Points in the HIV-1 life cycle that are targets for therapeutic intervention are also discussed.
Neuropeptide Y stimulates hypertrophy of adult ventricular cardiomyocytes
Authors: Millar B. C. , Schluter K. D. , Zhou X. J. , McDermott B. J. , Piper H. M.
Published: December 2017

It was investigated whether neuropeptide Y (NPY) could exert a trophic effect on ventricular myocytes isolated from the adult rat heart. Two different culture models were used: day 1 and 7 cultures of cardiomyocytes. In day 1 and 7 cultures, NPY caused an increase in cellular protein mass. In day 1 cultures, NPY (10 nM) increased the protein-to-DNA ratio within 24 h by 10.1 +/- 2.8% (P < 0.01), but did not stimulate the incorporation of [14C]phenylalanine into cell proteins. The degradation of proteins was retarded in presence of NPY, revealed by pulse-chase experiments. In day 7 cultures, NPY (10 nM) increased the protein-to-DNA ratio within 24 h by 33.9 +/- 5.0% (P < 0.01), increased the RNA-to-DNA ratio by 19.2 +/- 6.4%, and stimulated the incorporation of [14C]phenylalanine by 45.5 +/- 4.5% (P < 0.01). As in day 1 cultures, protein degradation was retarded. The specific activities of cytosolic creatine kinase and lactate dehydrogenase were increased in presence of NPY. This study demonstrates for the first time that NPY is a trophic factor for cardiomyocytes. NPY can cause an increase in cellular mass of protein, i.e., hypertrophy, by two mechanisms: 1) reduction of degradation of protein, found in day 1 and 7 cultures, and 2) stimulation of protein synthesis, observed only in day 7 cultures. The responsiveness of protein synthesis to NPY stimulation is induced during prolonged incubation in culture.