bluuebunny/crossref_metadata_2025 huggingface dataset:
Preservation of structural and functional polarity in isolated epithelial cells
Authors: Torres R. J. , Altenberg G. A. , Copello J. A. , Zampighi G. , Reuss L.
Published: December 2017

Pages C1864-C1874: R. J. Torres, G. A. Altenberg, J. A. Copello, G. Zampighi, and L. Reuss. “Preservation of structural and functional polarity in isolated epithelial cells.” Pages Cl866 (Fig. 2), Cl869 (Fig. 8, and Cl873 (Fig. 9): due to a printer processing error at the final publication stage, Figs. 2, 5, and 9 were poorly reproduced.
Osmotic regulation of Na-myo-inositol cotransporter mRNA level and activity in endothelial and neural cells
Authors: Wiese T. J. , Dunlap J. A. , Conner C. E. , Grzybowski J. A. , Lowe W. L. , Yorek M. A.
Published: December 2017

Pages C990-C997: T. J. Wiese, J. A. Dunlap, C. E. Conner, J. A. Grzybowski, W. L. Lowe, Jr., and M. A. Yorek. “Osmotic regulation of Na- myo-inositol cotransporter mRNA level and activity in endothelial and neural cells.” Page C994, right-hand column, line 4: the statement that reads “BAE cells were not examined because a cDNA probe for the bovine SMIT gene was not available” is erroneous, because a paper was published in 1994 by C. Zhou, N. Agarwal, and P. R. Cammarata (Invest. Ophthalmol. Visual Sci. 35: 1236-1242, 1994) entitled “Osmoregulatory alterations in myo-inositol uptake by bovine lens epithelial cells. Part 2: cloning of a 626 bp cDNA portion of a Na+/myo-inositol cotransporter, an osmotic shock protein.” The GenBank accession number of that bovine Na-myo-inositol cotransporter cDNA is S72877.
Role of ion channels in aqueous humor formation
Authors: Jacob T. J. , Civan M. M.
Published: December 2017

The aqueous humor is secreted by the ciliary epithelium, a bilayered syncytial epithelium comprising a pigmented (PE) cell layer abutting the stroma and a nonpigmented (NPE) cell layer facing the aqueous phase. As in other epithelia, secretion depends on the transfer of solute, with water passively following. Na+, K+, and Cl- enter the syncytium principally through a Na(+)-K(+)-2Cl- symport, diffusing to the aqueous surface of the NPE cells. The Na+, K+, and Cl- are secreted into the aqueous humor through the Na+/K+ exchange pump, K+ channels, and Cl- channels, respectively. Na+ is also secreted between the cells in response to a small transepithelial potential. The K+ channels are critical not only for K+ release but also for hyperpolarizing the membrane, providing an electrical driving force for Cl- secretion. Some of the K+ channels are Ca2+ sensitive and can be activated by Ca2+ entry through T- and L-type Ca2+ channels. The roles of the ciliary epithelial nonselective and Na+ channels are less clear. This review describes the ion channels thus far identified in the ciliary epithelium in terms of the activation and inactivation of their macroscopic currents, the open probabilities and conductances of the single channels, and their locations and regulation. The review relates each class of channel to known families of channels and indicates how those channels can contribute to the secretion of the aqueous humor.
Modulation of Ca2+ current in canine colonic myocytes by cyclic nucleotide-dependent mechanisms
Authors: Koh S. D. , Sanders K. M.
Published: December 2017

Regulation of Ca2+ currents by cyclic nucleotide-dependent mechanisms was studied in circular muscle cells isolated from canine proximal colon. Whole cell Ca2+ currents were recorded at 32 degrees C with the use of amphotericin B-perforated patches. The effects of several agents known to increase levels of adenosine 3',5'-cyclic monophosphate (cAMP) were tested. Vasoactive intestinal peptide (VIP) and isoproterenol (10(-7) to 10(-5) M) increased Ca2+ current in a concentration-dependent manner. Forskolin (10(-7) M) and dibutyryl cAMP (10(-6) to 10(-5) M) also increased Ca2+ current. Higher concentrations of forskolin (10(-6) to 10(-5) M) caused inhibition of Ca2+ current. Low concentrations (10(-5) to 10(-7) M) of dibutyryl cAMP or 8-bromo-cAMP caused concentration-dependent enhancement in Ca2+ current, and these effects were reversible on washout of the cAMP analogues. When the concentration of cAMP analogues was increased (10(-3) to 10(-4) M), we observed inhibition of Ca2+ current similar to the effects of forskolin. Membrane-permeable analogues of guanosine 3',5'-cyclic monophosphate produced exclusively inhibitory effects. The nonspecific protein kinase inhibitor H-7 (up to 60 microM) failed to block the effects of VIP, isoproterenol, and forskolin, and it produced inhibitory effects on Ca2+ current, independent of agonist stimulation. The data suggest that low levels of cAMP may, via phosphorylation by protein kinase A, enhance L-type Ca2+ current, but higher concentrations of cAMP may “cross over” and activate protein kinase G. Phosphorylation by protein kinase G appears to produce a dominant inhibition of Ca2+ current.
Impairment of insulin secretion in pancreatic islets isolated from Walker 256 tumor-bearing rats
Authors: el Razi Neto S. , Zorn T. M. , Curi R. , Carpinelli A. R.
Published: December 2017

Previous study has shown that insulin secretion in response to a glucose stimulus (16.7 mM) is reduced in islets isolated from Walker 256 tumor-bearing rats compared with controls. The ultrastructure, 45Ca2+ and 86Rb+ fractional outflow rate, phosphoinositide hydrolysis, and [U-14C]glucose decarboxylation were examined in islets isolated from tumor-bearing and control rats. The general morphological features of the islets from the control and experimental groups were very similar. The 86Rb+ fractional outflow rate was not changed, whereas the 45Ca2+ fractional outflow rate, [U-14C]glucose decarboxylation, and phosphoinositide metabolism were markedly reduced in islets from tumor-bearing rats. The changes in 45Ca2+ fractional outflow rate in islets from tumor-bearing rats were not due to impaired functioning of voltage-dependent calcium channels. By perifusing the islets in the presence of high potassium concentration, evidence was obtained that phospholipase C from islets from tumor-bearing rats reduced response to calcium. To further examine the mechanism involved in the impairment of insulin secretion by islets from tumor-bearing rats, islets isolated from normal rats were perifused after preincubation in the presence of serum from tumor-bearing rats. The results suggest that a thermolabile circulating factor is partially responsible for the changes described in islets isolated from Walker 256 tumor-bearing rats.
Amino-terminal processing of the catalytic subunit from Na(+)-K(+)-ATPase
Authors: Pressley T. A. , Allen J. C. , Clarke C. H. , Odebunmi T. , Higham S. C.
Published: December 2017

The first five amino acids of the catalytic alpha 1-subunit predicted from its cDNA are not found in purified mammalian Na(+)-K(+)-ATPase, suggesting co- or posttranslational cleavage. To facilitate evaluation of amino-terminal structure and the cleavage process, we developed a site-directed antibody (anti-VGR) specific for the first nine residues of nascent alpha 1 from rat. In immunoblots of polypeptides generated by in vitro translation, anti-VGR detected a prominent band with a mobility appropriate for the alpha 1-subunit (100 kDa). Immunoblots of total protein from various rat organs, however, revealed no significant binding, implying that virtually all the alpha 1-subunit expressed in vivo was modified. We also assessed amino-terminal structure in various heterologous expression systems. Binding of anti-VGR was observed in Escherichia coli transformed with a vector containing an alpha 1/troponin fusion protein and in insect cells infected with baculovirus containing full-length alpha 1 or alpha 1T. This suggests that modification of the introduced alpha 1 in these expression systems was absent or different from that in mammals. In contrast, green monkey kidney cells (COS-1) transfected with alpha 1 did not reveal significant binding of the antibody, indicating that the introduced isoform was processed appropriately. These results demonstrate that the structure of the alpha 1-subunit's amino terminus differs among various expression systems. The results further imply that efficient co- or posttranslational processing of nascent alpha 1 is conserved among various organs within the rat, yet the required modification enzymes are not present in distant phyla.
Buffering of plasmalemmal Ca2+ current by sarcoplasmic reticulum of guinea pig urinary bladder myocytes
Authors: Yoshikawa A. , van Breemen C. , Isenberg G.
Published: December 2017

The effects of cyclopiazonic acid (CPA), an inhibitor of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), on cytosolic Ca2+ concentration ([Ca2+]c) and membrane currents were studied in isolated urinary bladder myocytes to test the hypothesis that the sarcoplasmic reticulum (SR) buffers Ca2+, which enters the myocyte at a slow to moderate rate. Inhibition of SERCA by CPA was demonstrated by the following modifications of the caffeine-induced [Ca2+]c transients: 1) CPA prolonged the 90% decay time from peak to resting [Ca2+]c from 2.2 +/- 0.3 to 8.3 +/- 0.92 s (n = 5), 2) CPA abolished the “undershoot” of the [Ca2+]c transient that follows the washout of caffeine, and 3) CPA prevented caffeine from inducing a second [Ca2+]c transient. CPA reversibly increased resting [Ca2+]c. Starting from a control [Ca2+]c of 137 +/- 10 nM, 19 of 24 cells responded with a monotonic increase in [Ca2+]c to a steady [Ca2+]c of 238 +/- 10 nM, whereas 5 of 24 cells responded with a transient rise of [Ca2+]c to 472 nM (within 2.8 +/- 0.5 s) followed by a decay to a steady [Ca2+]c of 161 +/- 10 nM. The CPA-mediated rise in [Ca2+]c was augmented by increasing extracellular Ca2+ concentration ([Ca2+]o), suggesting a “leakage pathway” for Ca2+ influx that is unmasked by SERCA blockade. CPA reduced [Ca2+]c transients and Ca(2+)-activated K+ currents (IK,Ca), induced by depolarizing clamp steps from -60 to 0 mV, compatible with suppression of SR Ca2+ release on depletion of SR Ca2+. To reduce the contribution due to Ca(2+)-induced Ca2+ release, the cells were depolarized with a slow ramplike command (-60 to 0 mV, 15 mV/s). In 12 of these 40 cells, CPA increased [Ca2+]c and IK,Ca signals. If spontaneous transient outward currents were present, they were suppressed by CPA. CPA reduced the peak L-type Ca2+ channel current apparently through increased Ca2+ inactivation of voltage-gated Ca2+ channels. The current could be restored to control by elevating [Ca2+]o from 2.5 to 5 mM. Under these conditions, CPA increased the ramp-induced [Ca2+]c transients from 105.1 +/- 22 to 162.0 +/- 31 nM (n = 9, P < 0.05). These results suggest that Ca2+ sequestration by the SR can buffer part of the Ca2+ influx during slow depolarizations.
Interleukin-1-induced nitric oxide production modulates glutathione synthesis in cultured rat hepatocytes
Authors: Kuo P. C. , Abe K. Y. , Schroeder R. A.
Published: December 2017

In cultured rat hepatocytes, we have previously demonstrated that inhibition of interleukin-1 (IL-1)-mediated nitric oxide (NO) synthesis is associated with depletion of intracellular reduced glutathione (GSH) in toxin-mediated oxidative injury. To further examine NO's effects on GSH metabolism in rat hepatocytes, IL-1-mediated NO synthesis was examined in the context of 1) cysteine, cystine, and methionine uptake; 2) gene transcription and enzyme activities for gamma-glutamylcysteine synthetase, the rate-limiting enzyme in GSH synthesis, glutathione reductase, and glutathione peroxidase; and 3) GSH and oxidized glutathione (GSSG) levels. Inhibition of NO synthesis decreased the GSH content and GSH/GSSG ratio in a guanylyl cyclase-independent fashion. Enzyme activity and steady-state levels of mRNA for gamma-glutamylcysteine synthetase were also depressed. Nuclear run-on analysis demonstrated ablation of gamma-glutamylcysteine synthetase gene transcription. Hepatocellular uptake of cysteine, cystine, and methionine was not altered. Activity and steady-state mRNA levels for glutathione reductase and glutathione peroxidase were not affected. These results indicate that IL-1-mediated NO synthesis regulates hepatocyte GSH synthesis through a mechanism that is dependent on transcriptional regulation of the rate-limiting enzyme in GSH synthesis. In the setting of oxidative stress and IL-1 exposure, hepatocyte synthesis of NO may be protective through regulation of GSH synthesis.
Detergent-resistant membrane microdomains from Caco-2 cells do not contain caveolin
Authors: Mirre C. , Monlauzeur L. , Garcia M. , Delgrossi M. H. , Le Bivic A.
Published: December 2017

In this study we analyzed the relationship between detergent-resistant microdomains and caveolae in Caco-2 cells. Caveolin was not detected on Western blots or Northern blots or by immunoprecipitation in these cells, in contrast to A 431 cells. Triton X-100-resistant membranes from Caco-2 and A 431 cells showed the same morphological aspect by electron microscopy and peaked at the same isopycnic density on sucrose gradients. Detergent-resistant microdomains from Caco-2 cells were enriched in glycosyl phosphatidylinositol (GPI)-anchored proteins, in sucrase-isomaltase, an apical marker, and in most of the proteins found in caveolin-rich membranes such as src-like proteins, fimbrin, ezrin, and Gi alpha. Caveolae-like structures were present in A 431 but absent from Caco-2 cells at the electron microscopic level. Detergent-resistant microdomains from Caco-2 cells resemble caveolin-rich microdomains in their molecular composition but do not seem to derive from morphologically identified caveolae. Our results also indicate that caveolin is not necessary for sorting of GPI-linked proteins to the apical membrane of Caco-2 cells.
Modulation of phospholipase C pathway and level of Gq alpha/G11 alpha in rat myometrium during gestation
Authors: Lajat S. , Tanfin Z. , Guillon G. , Harbon S.
Published: December 2017

The regulation of the receptor-G protein-phospholipase C (PLC) cascade was investigated in rat myometrium at midgestation (day 12) and at term (day 21) comparatively to the estrogen-treated tissue (day 0). Carbachol-mediated generation of [3H]inositol phosphates was insensitive to pertussis toxin and was enhanced at days 12 and 21 two- and threefold, respectively, with no alteration of muscarinic sites (M3 subtype). A similar increase could be detected in the production of inositol 1,4,5-trisphosphate, indicating the stimulation of a PLC degrading phosphatidylinositol 4,5-bisphosphate. AlF4- also enhanced PLC activation during gestation, suggesting pregnancy-related regulations that bypass receptor activation. Immunoreactive G proteins, Gq alpha and G11 alpha, and PLC-beta 3 were detected in all myometrial preparations. The amount of PLC-beta 3 was similar in day 0 and day 21 myometrium, although decreasing by 75% at midgestation. Of significance was the increased amount of Gq alpha in day 12 and day 21 myometrium (3- and 2-fold, respectively) which coincided with the enhanced phosphoinositide breakdown. The upregulation of Gq alpha may contribute to the enhanced PLC activity during pregnancy and at term.
Prolactin enables normal development of ACh-stimulated current in cultured larval bullfrog skin
Authors: Takada M. , Yai H. , Takayama-Arita K. , Komazaki S.
Published: December 2017

The response to acetylcholine (ACh) can be used as a marker for larval-type bullfrog skin because apically applied ACh induces an increase in short-circuit current (SCC) in larval-type but not adult-type skin. EDTA-treated larval skin, which contains only basal cells and does not respond to ACh, was used as the starting material for our culture. ACh, carbamylcholine, and choline stimulated SCC in skin that had been cultured with aldosterone (5 x 10(-7) M) supplemented with prolactin (PRL; 2 micrograms/ml). Atropine and d-tubocurarine each inhibited the ACh-induced stimulation of SCC in skin so cultured. Eserine, an inhibitor of acetylcholinesterase, also inhibited the ACh response. Amiloride stimulated SCC itself, but it reduced the ACh response. All of these results are quite similar to those seen in intact larval skin, suggesting that a larval-skin had differentiated from the basal cells used as the starting point for our culture. This is the first physiological report that PRL induces differentiation in vitro into a true larval-type bullfrog skin.
Role of PLA2, PLC, and PLD in bradykinin-induced release of arachidonic acid in MDCK cells
Authors: Kennedy C. R. , Proulx P. R. , Hebert R. L.
Published: December 2017

The role of cytosolic phospholipase A2 (cPLA2), phosphatidylcholine-specific phospholipase C (PC-PLC) and phospholipase D (PLD) in the bradykinin (BK)-stimulated release of arachidonic acid (AA) was examined in Madin-Darby canine kidney (MDCK) cells. Release of AA, phosphorylcholine, choline, and phosphatidic acid (PA) or the transphosphatidylation product, phosphatidylethanol, was detected after 1 min of BK stimulation. A role for PC-PLC was confirmed with D609, which reduced BK-stimulated AA by 70%. Ethanol (EtOH), which blunts PA formation, diminished BK-stimulated AA release by 50%. Together, D609 and EtOH inhibited this release almost completely. Evidence indicated that diacylglycerol and PA can enhance PLA2 activity when added to cytosol extracts. The enzyme responsible for AA release was characterized as cPLA2, since PLA2 activity assayed in cell extracts was largely inhibited by an antibody to this enzyme. The membrane fraction PLA2 activity increased significantly in BK-stimulated cells. We conclude that BK signaling in MDCK cells is mediated by the lipid products of PC-PLC and PLD, increasing cPLA2 activity, possibly by causing perturbations in the bilayer structure of its substrate, by a direct effect on the enzyme or by activation of protein kinases such as protein kinase C.
ET-1 induces IL-6 gene expression in human umbilical vein endothelial cells: synergistic effect of IL-1
Authors: Stankova J. , D'Orleans-Juste P. , Rola-Pleszczynski M.
Published: December 2017

Endothelins are a family of potent vasoactive peptides that have also been implicated in inflammation. To examine the consequence of endothelin stimulation on cytokine production, we explored the effect of endothelin-1 (ET-1) on interleukin-6 (IL-6) gene expression. ET-1 augmented the production of IL-6 in human umbilical vein endothelial cells (HUVEC) by 2- to 5-fold and synergized with IL-1 to induce a 150-fold increase in IL-6 secretion, compared with spontaneous production. The peak of IL-6 production was in the presence of 100 pM ET-1 after 12 h of stimulation. ET-1 augmented IL-6 mRNA expression at the optimal concentration of 100 pM, in the presence of 1 ng/ml of IL-1. ET-1-induced IL-6 mRNA expression was significant within 4 h of stimulation and persisted for 18 h. Similarly, the ETB receptor-specific agonist, IRL-1620, also augmented IL-6 mRNA expression and IL-6 protein production in HUVEC. Our results suggest that endothelin may play a role at the vascular inflammatory site by modulating cytokine production through ETB receptor activation in endothelial cells of human origin.
Inadequacy of high K+/nigericin for calibrating BCECF. II. Intracellular pH dependence of the correction
Authors: Boyarsky G. , Hanssen C. , Clyne L. A.
Published: December 2017

In the accompanying study [G. Boyarsky, C. Hanssen, and L.A. Clyne. Am. J. Physiol. 271 (Cell Physiol. 40): C1131-C1145, 1996], it was demonstrated that steady-state intracellular pH (pHi) determined using high K+/nigericin calibrations was systematically in error in vascular smooth muscle (VSM) cells by approximately 0.2 pH units. In this paper the possibility is explored that this correction (pHcor) to the nigericin-calibrated pHi (pHnig) might not be a constant but could vary as pHi varies. The range of pHi during exposures to "null solutions" designed to bracket pHi was extended to acidic and alkaline levels relative to the starting pHi in VSM cells. The pHcor necessary to correct pHnig was linearly dependent on pHnig, increasing from near zero at approximately 6.0 to approximately 0.2 at steady-state pHi, to approximately 0.3 at alkaline pHnig. It is shown how to retrieve previously acquired (tabulated) data using the linear relationship between pHcor and pHnig. Also examined were what corrections must be made to high K+/nigericin calibration curves to correct for this pHi-dependent pHcor. The following changes in the calibration parameters were found: the maximal fluorescence ratio increased from 16.75 to 17.28; the minimal fluorescence ratio decreased from 2.15 to 1.57; and the pK of 2',7'-bis (carboxy-ethyl)-5 (6)-carboxyfluorescein decreased from 7.13 to 6.93. Three potential explanations for these changes are discussed: external [K+] in the nigericin solutions could have been too low; internal [K+] changes during the calibration because of the finite buffering power of cells; and other acid-base transport/generation could have been contributing during the nigericin calibrations (i.e., nigericin does not overwhelm to insignificance other processes generating/consuming H+). The nonconstancy of pHcor is shown to have profound implications for measuring changes in pHi.
Electrophysiological and functional effects of adenosine on ventricular myocytes of various mammalian species
Authors: Song Y. , Belardinelli L.
Published: December 2017

The goal of this study was to determine the electrophysiological and functional effects of adenosine on ventricular myocytes of guinea pig, rabbit, rat, and ferret hearts. Adenosine (100 microM) shortened the action potential durations of rat and ferret myocytes by 14 +/- 1 and 57 +/- 7%, reduced the amplitudes of cell twitch shortening by 13 +/- 1 and 54 +/- 5%, and increased outward currents by 15 +/- 4 and 55 +/- 5%, respectively, but had no effect on guinea pig and rabbit myocytes. The properties of adenosine-activated outward current in rat and ferret ventricular myocytes indicated that this current is the adenosine-sensitive K+ current [IK(Ado)]. Adenosine had no significant effect on basal Ca2+ current but specifically inhibited isoproterenol-stimulated L-type Ca2+ current in myocytes of all species studied. Binding studies revealed that the density of A1 adenosine receptors (A1AdoR) was highest in ferret and lowest in rabbit myocytes, but the differential effects of adenosine among species could not be solely explained by differences in A1AdoR density. In summary, adenosine shortened the action potential and reduced the twitch shortening of rat and ferret but not of guinea pig and rabbit ventricular myocytes. Shortening of the action potential was associated with the activation of IK(Ado). The anti-beta-adrenergic action of adenosine appeared to be independent of species.
Glucose and pyruvate regulate cytokine-induced nitric oxide production by cardiac myocytes
Authors: Oddis C. V. , Finkel M. S.
Published: December 2017

Metabolic requirements for the production of nitric oxide (NO) by cytokine-stimulated neonatal rat cardiac myocytes (CM) were studied. CM were cultured for 48 h in media containing interleukin-1 beta (IL-1 beta) and free fatty acids. Removal of glucose from the media partially inhibited IL-1 beta-stimulated nitrite (NO2-) production [8.1 +/- 0.3 vs. 4.4 +/- 0.6 nmol.(1.25 X 10(5) cells)-1.48 h-1; P < 0.01; n = 12]. The glycolytic inhibitor 2-deoxy-D-glucose (2-DG) completely inhibited IL-1 beta-stimulated NO2- production [0.7 +/- 0.5 nmol.(1.25 X 10(5) cells)-1.48 h-1; P < 0.01; n = 12]. The addition of the glycolytic end product, pyruvate, completely blocked the 2-DG inhibition of IL-1 beta-stimulated NO2- production [7.4 +/- 0.4 nmol.(1.25 X 10(5) cells)-1.48 h-1; P < 0.01; n = 12]. Pyruvate alone did not significantly enhance NO2- production in the presence or absence of glucose (n = 12). The inactive analogue 3-O-methylglucose had no effect on NO2- production (n = 12). Reverse transcription-polymerase chain reaction revealed that pyruvate blocked 2-DG inhibition of inducible NO synthase mRNA expression. Neither 2-DG nor pyruvate had any effect on GTP-cyclohydrolase I mRNA expression in CM. We report for the first time that optimal IL-1 beta-stimulated NO production by CM requires both glucose and the glycolytic end product pyruvate.
Cell-specific expression of amiloride-sensitive, Na(+)-conducting ion channels in the kidney
Authors: Ciampolillo F. , McCoy D. E. , Green R. B. , Karlson K. H. , Dagenais A. , Molday R. S. , Stanton B. A.
Published: December 2017

Amiloride-sensitive, electrogenic Na+ absorption across the distal nephron plays a vital role in regulating extracellular fluid volume and blood pressure. Recently, two amiloride-sensitive, Na(+)-conducting ion channel cDNAs were cloned. One, an epithelial Na(+)-selective channel (ENaC), is responsible for Na+ absorption throughout the distal nephron. The second, a guanosine 3',5'-cyclic monophosphate (cGMP)-inhibitable cation channel, is conductive to Na+ and Ca2+ and contributes to Na+ absorption across the inner medullary collecting duct (IMCD). As a first step toward understanding the segment-specific contributions(s) of cGMP-gated cation channels and ENaC to Na+ and Ca2+ uptake along the nephron, we used in situ reverse transcription-polymerase chain reaction (RT-PCR) hybridization, solution-phase RT-PCR, and Western blot analysis to examine the nephron and cell-specific expression of these channels in mouse kidney cell lines and/or dissected nephron segments. cGMP-gated cation channel mRNA was detected in proximal tubule, medullary thick ascending limb (mTAL), distal convoluted tubule (DCT), cortical collecting duct (CCD), outer medullary collecting duct (OMCD), and IMCD. cGMP-gated cation channel protein was detected in DCT, CCD, and IMCD cell lines. These observations suggest that hormones that modulate intracellular cGMP levels may regulate Na+, and perhaps Ca2+, uptake throughout the nephron. mRNA for alpha-mENaC, a subunit of the mouse ENaC, was detected in mTAL, DCT, CCD, OMCD, and IMCD. Coexpression of alpha-mENaC and cGMP-gated cation channel mRNAs in mTAL, DCT, CCD, OMCD, and IMCD suggests that both channels may contribute to Na+ absorption in these nephron segments.
Endotoxin induces cardiac HSP70 and resistance to endotoxemic myocardial depression in rats
Authors: Meng X. , Brown J. M. , Ao L. , Nordeen S. K. , Franklin W. , Harken A. H. , Banerjee A.
Published: December 2017

Endotoxin (bacterial lipopolysaccharide, LPS) depresses myocardial function. However, heat shock and sublethal LPS can confer cardiac resistance to postischemic dysfunction. We hypothesized that a prior exposure to LPS stress induces the expression of cardiac heat shock protein 70 (HSP70) and resistance to endotoxemic myocardial depression. Moreover, induction of HSP70 by hyperthermia should also increase cardiac resistance to LPS toxicity. LPS (500 micrograms/kg ip) depressed rat left ventricular developed pressure (LVDP) maximally at 6 h (58.4 +/- 3.72 vs. 101 +/- 1.46 mmHg in saline control, P < 0.01), and myocardial contractile function recovered at 24 h. In rats pretreated with LPS 24 h earlier, subsequent LPS exposure did not depress LVDP (97.0 +/- 3.53 mmHg at 6 h, P < 0.01 vs. single exposure). Both LPS and hyperthermia (42 degrees C, 15 min) induced HSP72 mainly in the cardiac interstitial cells, including macrophages at 24 h after treatment. When hyperthermia-pretreated animals were similarly challenged with LPS, myocardial depression at 6 h was partially abrogated (LVDP 80.1 +/- 5.67 vs. 62.2 +/- 4.91 mmHg in sham+LPS group, P < 0.01). We conclude that LPS induces HSP70 in rat heart and that an exposure to LPS or heat stress confers cardiac resistance to endotoxemic myocardial depression.
Modeling spiral Ca2+ waves in single cardiac cells: role of the spatial heterogeneity created by the nucleus
Authors: Dupont G. , Pontes J. , Goldbeter A.
Published: December 2017

Excitation-contraction coupling in cardiomyocytes is known to rely on the Ca(2+)-induced Ca2+ release mechanism. This autoamplification process is also very apparent when voltage-clamped or Ca(2+)-overloaded myocytes exhibit fast-propagating Ca2+ waves. Although most of the fronts are planar, some adopt a spiral shape, revealing additional characteristics about the excitability and structure of the cardiac cell (P. Lipp and E. Niggli, Biophys. J. 65: 2272-2276, 1993: J. Engel, M. Fechner, A. Sowerby, S. Finch, and A. Stier, Biophys. J. 66: 1756-1762, 1994). Using a previously developed model for Ca2+ oscillations and waves (A. Goldbeter, G. Dupont, and M.J. Berridge, Proc. Natl. Acad. Sci. USA 87: 1461-1465, 1990; G. Dupont and A. Goldbeter, Biophys. J. 67: 2191-2204, 1994), we study by numerical simulations different conditions in which spiral Ca2+ waves can occur as a result of the spatial heterogeneity created by the nucleus in a system with geometry resembling that of a myocyte. A region of the cell lacking Ca2+ pools, acting as an obstacle able to break the propagation of planar waves, suffices to initiate a spiral wave; however, this region must be properly placed with respect to the pacemaker. An obstacle behaving as a barrier to diffusion is also able to create the initial bending that can lead to the spiral wave. We study how the occurrence of spiral Ca2+ waves in single cardiomyocytes is influenced by factors such as the stimulus location and the position, shape, and dimensions of the obstacle to planar wave propagation.
Activity-dependent induction of slow myosin gene expression in isolated fast-twitch mouse muscle
Authors: Barton-Davis E. R. , LaFramboise W. A. , Kushmerick M. J.
Published: December 2017

We demonstrate that direct electrical stimulation of isolated fast-twitch muscle in an organ culture system can induce expression of the slow myosin heavy chain (beta-MHC) gene, indicative of a phenotype transformation. Pairs of extensor digitorum longus (EDL) muscles were isolated from adult mice, incubated at resting length in separate chambers, and superfused with the same recirculated media One muscle was subjected to twitch stimulation (5-s trains of 5-Hz pulses at supramaximal voltage every minute), and force was recorded to assess function. The contralateral muscle was incubated without stimulation, to control for effects of the experimental preparation. Both muscle were rapidly frozen for RNA purification and oligo(dT)-primed reverse transcription; serial studies were carried out to 36 h. Polymerase chain reaction was performed utilizing primers specific for cytoplasmic beta-actin (beta-actin), a constitutive marker, and beta-MHC, a gene that is either inactive or expressed at very low levels in control EDL. After 30 h of stimulation, beta-MHC was consistently detected at a level severalfold higher in stimulated EDL than in incubated control EDL when band intensities were normalized to those of beta-actin. These results show that signals or fiber-specific transformations reside within the muscle and that this shift begins rapidly after induction of continuous stimulation.
Interpretation of extraordinary kinetics of Na(+)-K(+)-ATPase by a phase change
Authors: Post R. L. , Klodos I.
Published: December 2017

We interpret at a molecular level an extraordinary response in the transient kinetics of the phosphointermediate of Na(+)-K(+)-ATPase (I. Klodos, R. L. Post, and B. Forbush III. J. Biol. Chem. 269: 1734-1743, 1994). The phosphointermediate comprises two principal states. The partition between these states varies with salt concentration. A jump in salt concentration changes the partition of some of the molecules more rapidly than they interconvert in a steady state at constant salt concentration. We propose that interconversion is limited by free volume in the lipid of the surrounding membrane. This lipid is partitioned into phases that vary with salt concentration. Free volume is larger at the interface between these phases than within the phases themselves. Na(+)-K(+)-ATPase molecules are distributed at random in the membrane. When the phase boundary moves in response to a jump in salt concentration, it crosses some Na+ -K+ -ATPase molecules, which transiently experience an increase in free volume of the surrounding lipid. Thus their phosphointermediate states equilibrate more rapidly than at a constant salt concentration. Functional and structural heterogeneity of Na(+)-K(+)-ATPase molecules is discussed.
Contraction-induced injury to single fiber segments from fast and slow muscles of rats by single stretches
Authors: Macpherson P. C. , Schork M. A. , Faulkner J. A.
Published: December 2017

Susceptibility to contraction-induced injury was investigated in single permeabilized muscle fiber segments from fast extensor digitorum longus and slow soleus muscles of rats. We tested the hypotheses that, after single stretches of varying strains and under three conditions of Ca2+ activation (none, submaximum, and maximum), 1) the magnitude of the deficit in maximum isometric force is dependent on the work done to stretch the fiber, and 2) for each condition of activation and strain, fast fibers incur greater force deficits than slow fibers. When all data on force deficits were analyzed together, the best predictors of the overall force deficits for both fast and slow muscle fibers were linear regression models that introduced the simultaneous but independent effects of strain and average force (r2 = 0.52 and 0.63, respectively). Under comparable conditions, greater force deficits were produced in fast than slows fibers. Despite differences in the strain required to produce injury in fast and slow muscle fibers, for a given force deficit, the ultrastructural damage was strikingly similar.
Inhibition of amiloride-sensitive Na+ channel by isothiouronium derivatives
Authors: Avigdor A. , Asher C. , Tal D. M. , Karlish S. J. , Garty H.
Published: December 2017

The effects on the amiloride-blockable Na+ channel of a family of recently synthesized isothiouronium derivatives were measured in plasma membrane vesicles from rat distal colon. Some of these derivatives act as high-affinity Na(+)-like antagonists on the Na(+)-K(+)-adenosinetriphosphatase. One of the reagents tested, 1-bromo-2,4,6-tris(isothiouronium methyl)-benzene tribromide (Br-TITU), was found to be a potent blocker of the Na+ channel. At neutral pH, Br-TITU rapidly inhibits the channel mediated 22Na+ uptake, with an inhibition constant of 94 +/- 39 nM. The inhibition observed is specific and reversible. 1,3-Dibromo-2,4,6-tris(isothiouronium methyl)benzene tribromide and Br-TITU derivatives with methyl and phenyl substitutions on the isothiouronium moiety were much less effective blockers. Incubation of cells with Br-TITU at alkaline (but not neutral) pH produces irreversible inactivation of channels, possibly due ot covalent modification of a lysine residue. This inactivation can be attenuated by amiloride but not by Na+. Thus Br-TITU may be a useful reagent in identifying essential residues of the channel protein.
Creatine depletion elicits structural, biochemical, and physiological adaptations in rat costal diaphragm
Authors: Levine S. , Tikunov B. , Henson D. , LaManca J. , Sweeney H. L.
Published: December 2017

To elucidate adaptations elicited by creatine (Cr) depletion in the costal diaphragm (Dia), 16 12-wk-old male Fisher 344 rats had 2% beta-guanidinopropionic acid (beta-GPA), a competitive inhibitor of Cr transport into muscle, added to their food; a control group (Con) of 16 rats ate normal rat chow. After 18 wk, beta-GPA and Con Dia did not differ histochemically with respect to fiber-type distribution; however, the cross-sectional area of type II(b + x) fibers was 33% less in beta-GPA than Con Dia. Biochemically, the proportion of myosin heavy chain IIb in beta-GPA Dia was decreased 42% from Con Dia, whereas the proportions of myosin heavy chains I and IIa were increased. Physiologically, both peak twitch tension and tetanic tension in beta-GPA Dia were decreased 40% from Con. To assess fatigability, we used the protocol of Kelsen and Nochomovitz (J. Appl. Physiol. 53; 440-447, 1982) for 2-6 min duration; the percentage of initial force exhibited by beta-GPA Dia was approximately twice that of Con Dia. We conclude that these structural, biochemical, and physiological adaptations elicited by Cr depletion can all be explained by selective atrophy of IIb muscle fibers in the Dia.
Presence of a metabotropic and an ionotropic purinergic receptor on rat submandibular ductal cells
Authors: Amsallem H. , Metioui M. , VandenAbeele A. , Elyamani A. , Moran A. , Dehaye J. P.
Published: December 2017

ATP (1 microM-1 mM) increased the intracellular Ca2+ concentration ([Ca2+]i) in rat submandibular ductal cells. The dose-response curve was biphasic with a first plateau approximately 10 microM and a second increase at concentrations higher than 100 microM. This second increase was abolished in the absence of extracellular calcium or in the presence of Coomassie blue and could be mimicked by benzoyl-ATP. The activation of this response increased the uptake of extracellular manganese and the release of kallikrein, a ductal cell marker. The response at low concentrations of ATP was mimicked by ADP, 2-methylthioadenosine 5'-triphosphate (2-MeS-ATP; which was the most potent agonist), adenosine 5'-O-(2-thiodiphosphate), and UTP. Removal of extracellular calcium did not abolish this response, but ADP or 2-MeS-ATP had no effect after a preincubation with thapsigargin. A preincubation with 2-MeS-ATP desensitized the receptor involved in the response to both ADP and UTP. Similarly, a pretreatment with UTP prevented a further response to ADP. ADP increased the intracellular concentration of inositol 1,4,5-trisphosphate but did not affect the secretion of kallikrein. In conclusion, rat submandibular ductal cells possess two types of purinergic receptors: a metabotropic (P2y1) receptor and a P2x ionotropic purinergic receptor coupled to a manganese-permeant calcium channel and to kallikrein secretion.
Regulation of human erythrocyte Na+/H+ exchange by soluble and particulate guanylate cyclase
Authors: Petrov V. , Lijnen P.
Published: December 2017

Guanylate cyclase activity in human erythrocytes is investigated by evaluating the intracellular guanosine 3',5'-cyclic monophosphate (cGMP) content in the presence of various agents that exert specific effects on soluble or particulate guanylate cyclase. The increase in the intraerythrocyte cGMP content by the soluble guanylate cyclase activators nitroprusside and NaNO2 suggests the presence of this enzyme in human erythrocytes. The effects of four different atrial natriuretic peptide (ANP) fragments on the intraerythrocyte cGMP content is also studied. ANP II and ANP III increase the intraerythrocyte cGMP content, whereas ANP I and des-Ser5,des-Ser6-ANP III are ineffective. Thus our data show that human erythrocytes possess particulate guanylate cyclase together with the soluble enzyme. The ANP fragments ANP II and ANP III also activate the erythrocyte Na+/H+ exchange. Nitroprusside, M & B 22948 (an inhibitor of cGMP phosphodiesterase), and the cGMP analogues dibutyryl cGMP and 8-bromoguanosine 3',5'-cyclic monophosphate also increase the erythrocyte Na+/H+ exchange rate. The latter data also suggest that the erythrocyte Na+/H+ exchange is regulated by cGMP.
Na(+)-K(+)-2Cl- cotransport, Na+/H+ exchange, and cell volume in ferret erythrocytes
Authors: Mairbaurl H. , Herth C.
Published: December 2017

Ferrets have high-Na+ and low-K+ erythrocytes (113 and 5.4 mmol/l cell water) due to the lack of Na(+)-K+ pumps. Because ferret erythrocytes have a high capacity for Na(+)-K(+)-2Cl- cotransport, the present study was undertaken to evaluate cell volume-related changes in cotransport activity and its role in volume regulation. With cell shrinkage, Na(+)-K(+)-2Cl- cotransport is activated about twofold. A large bumetanide-insensitive Na+ uptake component that has not yet been described is found in shrunken erythrocytes. Its inhibition by amiloride (concn inhibiting 50% of maximal response = 12 microM) and the Na+ dependence of amiloride-sensitive extracellular pH changes measured in cells suspended in hypertonic unbuffered medium indicate that this flux represents Na+/H+ exchange. Shrinkage activation of both transporters follows a time lag of approximately 3 min and also requires normal levels of ATP. ATP depletion inhibits Na(+)-K(+)-2Cl- cotransport even at normal cell volume. Both transporters are partially inhibited by the protein kinase inhibitors staurosporine and K252a, and activators of protein kinases A and C do not affect transport. Okadaic acid inhibition of protein phosphatases activates Na(+)-K(+)-2Cl- cotransport to its maximal activity (same after shrinkage), but shrinkage and okadaic acid activation are not additive. In contrast, okadaic acid activates Na+/H+ exchange even in shrunken cells. These results indicate that cell shrinkage activates Na(+)-K(+)-2Cl- cotransport and Na+/H+ exchange probably by phosphorylation processes.
Theoretical and experimental discrimination between Cl(-)-H+ symporters and Cl-/OH- antiporters
Authors: Alvarado F. , Vasseur M.
Published: December 2017

A Cl(-)-H+ symport and a Cl-/OH- antiport cannot be readily distinguished physicochemically, but a kinetic distinction is theoretically possible, because a Cl(-)-H+ symporter involves a two-site carrier whereas a Cl-/OH- antiporter involves a single-site carrier. Accordingly, we have developed kinetic models and equations that we have tested by studying Cl- uptake by isolated guinea pig ileal brush-border membrane vesicles as a function of Cl- or H+ concentration. We conclude that a two-site Cl(-)-H+ symporter with a 1:1 stoichiometry explains the pH-dependent Cl- uptake and Cl-/Cl- exchange activities of the brush-border membrane in terms of a single random nonobligatory mobile carrier where exchange occurs by counterflow. This symport, probably involving an anion exchanger (AE 2) protein, differs, therefore, functionally from the erythrocyte's band 3 AE1, which involves an antiport. The question is whether members of the AE gene family can be functionally diverse, even when their primary structures exhibit up to 50% overall homologies.
Characterization of the rat Na+/H+ exchanger isoform NHE4 and localization in rat hippocampus
Authors: Bookstein C. , Musch M. W. , DePaoli A. , Xie Y. , Rabenau K. , Villereal M. , Rao M. C. , Chang E. B.
Published: December 2017

The kinetics of the Na+/H+ exchanger (NHE) isoform NHE4 were studied by measuring 22Na+ fluxes in stably transfected NHE-deficient fibroblasts. Unlike NHE1, NHE2, and NHE3, activation of this isoform is dependent on hyperosmolarity-induced cell shrinkage. It is virtually inactive at isosmolarity and most active at 490 mosM. When induced by cell shrinkage, NHE4 exhibits a sigmoidal response to increasing extracellular Na+ concentrations, suggesting allosteric or cooperative binding kinetics. In comparison, NHE1 and -3 exhibit hyperbolic velocity vs. extracellular Na+ concentration responses at both iso- and hyperosmolar conditions. Unlike NHE1 and NHE4, hyperosmolarity-induced cell shrinkage inhibits NHE3 activity in transfected fibroblasts, reducing maximum velocity by 40%, with no effect on binding affinity to extracellular Na+.NHE4 is relatively insensitive to inhibition by amiloride analogues in the order 5-(N,N-dimethyl)amiloride > 5-(N,N-hexamethylene)amiloride ride > amiloride > 5-(N-ethyl-N-isopropyl)amiloride. Time-dependent inhibition of activity by cytochalasin D suggests a relationship between the actin cytoskeleton and regulation by cell shrinkage. By in situ hybridization of fixed tissues, NHE4 mRNA was found to be highly expressed in the cavi amnoni fields of rat hippocampus. The kinetics of this exchanger, when considered with its unusual tissue distribution in renal inner medullary collecting tubules and hippocampus, are-consistent with NHE4 having a specialized role in cell functions.
Na-K-ATPase in lacrimal gland acinar cell endosomal system: correcting a case of mistaken identity
Authors: Gierow J. P. , Yang T. , Bekmezian A. , Liu N. , Norian J. M. , Kim S. A. , Rafisolyman S. , Zeng H. , Okamoto C. T. , Wood R. L. , Mircheff A. K.
Published: December 2017

Na-K-ATPase is associated with a variety of membrane populations in lacrimal acinar cells. Acinus-like structures formed by rabbit acinar cells in primary culture were incubated with horseradish peroxidase (HRP) to label basolateral and endosomal membranes and then analyzed by electron microscopy cytochemistry with the 3-3'-diaminobenzidine reaction or by fractionation and measurement of marker catalytic activities or immunoreactivities. HRP adsorbed to basolateral membranes at 4 degrees C. Fractionation showed it associated with low-density membranes enriched in acid phosphatase and TGN38 but containing only minor amounts of Na-K-ATPase. Cells internalized HRP to cytoplasmic vesicles, Golgi structures, and lysosomes at 37 degrees C. The major endosomal compartment revealed by fractionation coincided with major peaks of Na-K-ATPase and Rab6 and secondary peaks of galactosyltransferase and gamma-adaptin. Carbachol (10 microM) increased lysosomal and Golgi labeling. Thus most of the Na-K-ATPase is located in the basolateral membrane-oriented endosomal system, concentrated in a compartment possibly related to the trans-Golgi network. Constitutive and stimulation-accelerated traffic to and from this compartment may serve several exocrine cell functions.
Functional identification of a sarcolemmal chloride channel from bovine tracheal smooth muscle
Authors: Salvail D. , Alioua A. , Rousseau E.
Published: December 2017

The biophysical and pharmacological characteristics of unitary Cl- currents from bovine tracheal smooth muscle cells were studied after reconstitution of microsomal vesicles into planar lipid bilayers. Two types of currents were recorded simultaneously in KCl buffer: the well-defined Ca(2+)-dependent K+ conductance [GK(Ca)] and a much smaller Cl- current, indicating that the Cl- channels under scrutiny originate from the same membrane as the GK(Ca)-type channels, the plasma membrane of airway smooth muscle (ASM) cells. The GK(Ca) activities were eliminated by the use of CsCl buffer. The average unitary Cl- conductance measured in 50 mM trans-250 mM cis CsCl was 77 +/- 6 pS (n = 21), and the reversal potential measured in various CsCl gradients followed the Cl- equilibrium potential as determined from the Nernst equation. In contrast with the previous reports describing the Ca2+ sensitivity of macroscopic ASM Cl- currents, this channel was found to be insensitive to cytoplasmic and extracellular Ca2+ levels. Phosphorylation cocktails, including protein kinases A, G, or C, did not alter the activity of the channel nor did changes in pH. Among a series of Cl- channel inhibitors, 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid [50% effective concentration (EC50) = 30 microM] and 5-nitro-2-(3-phenylpropylamino) benzoic acid (EC50 = 130 microM) were the most potent blockers of the current examined. The exact role of this surface Cl- conductance remains unclear, and its involvement in cellular activity needs further investigation.