bluuebunny/crossref_metadata_2025 huggingface dataset:
Aquaporin 2 is a vasopressin-independent, constitutive apical membrane protein in rat vas deferens
Authors: Stevens Anna L. , Breton Sylvie , Gustafson Corinne E. , Bouley Richard , Nelson Raoul D. , Kohan Donald E. , Brown Dennis
Published: December 2017

Aquaporin 2 (AQP2), the vasopressin-regulated water channel, was originally identified in renal collecting duct principal cells. However, our recent description of AQP2 in the vas deferens indicated that this water channel may have extra-renal functions, possibly related to sperm concentration in the male reproductive tract. In this study, we have examined the regulation and membrane insertion pathway of AQP2 in the vas deferens. The amino acid sequence of vas deferens AQP2 showed 100% identity to the renal protein. AQP2 was highly expressed in the distal portion (ampulla) of the vas deferens, but not in the proximal portion nearest the epididymis. It was concentrated on the apical plasma membrane of vas deferens principal cells, and very little was detected on intracellular vesicles. Protein expression levels and cellular localization patterns were similar in normal rats and vasopressin-deficient Brattleboro homozygous rats, and were not changed after 36 h of dehydration, or after 3 days of vasopressin infusion into Brattleboro rats. AQP2 was not found in apical endosomes (labeled with Texas Red-dextran) in vas deferens principal cells, indicating that it is not rapidly recycling in this tissue. Finally, vasopressin receptors were not detectable on vas deferens epithelial cell membranes using a [3H]vasopressin binding assay. These data indicate that AQP2 is a constitutive apical membrane protein in the vas deferens, and that it is not vasopressin-regulated in this tissue. Thus AQP2 contains targeting information that can be interpreted in a cell-type-specific fashion in vivo.
Monocarboxylic acid transporters, MCT1 and MCT2, in cortical astrocytes in vitro and in vivo
Authors: Hanu Rekha , McKenna Mary , O'Neill Andrea , Resneck Wendy G. , Bloch Robert J.
Published: December 2017

We used sequence-specific antibodies to characterize two monocarboxylic acid transporters, MCT1 and MCT2, in astrocytes. Both proteins are expressed in primary cultures of cortical astrocytes, as indicated by immunoblotting and immunofluorescence. Both MCT1 and MCT2 are present in small, punctate structures in the cytoplasm and at the cell membrane. Cells showing very low levels of labeling for glial fibrillary acidic protein (GFAP) also label more dimly for MCT2, but not for MCT1. In vivo, double-label immunofluorescence studies coupled with confocal microscopy indicate that MCT1 and MCT2 are rare in astrocytes in the cortex. However, they are specifically labeled in astrocytes of the glial limiting membrane and in white matter tracts. Both transporters are also present in the microvasculature. Comparison of labeling for MCT1 and MCT2 with markers of the blood-brain barrier shows that the transporters are not always limited to the astrocytic endfeet in vivo. Our results suggest that the level of expression of monocarboxylic acid transporters MCT1 and MCT2 by cortical astrocytes in vivo is significantly lower than in vitro but that astrocytes in some other regions of the brain can express one or both proteins in significant amounts.
Pyrrolidine dithiocarbamate-induced apoptosis depends on cell type, density, and the presence of Cu2+ and Zn2+
Authors: Erl Wolfgang , Weber Christian , Hansson Göran K.
Published: December 2017

Pyrrolidine dithiocarbamate (PDTC) has been found to induce or inhibit apoptosis in different cell types. Here we show that PDTC dose-dependently reduced the viability of rat smooth muscle cells (rSMC), human fibroblasts, and endothelial cells at low but not at high cell density. Endothelial cells were least sensitive, fibroblasts showed a medium sensitivity, and rSMC showed a high sensitivity to PDTC-mediated cell death. An early reduction in the mitochondrial membrane potential indicated a rapid onset of apoptosis in rSMC. Apoptosis was further confirmed by annexin V staining and DNA fragmentation analysis. Gel shift analysis demonstrated increased nuclear factor (NF)-κB activity in high-density rSMC compared with low-density cells. NF-κB has recently been shown to regulate the induction of anti-apoptotic proteins. Although PDTC is widely used as an inhibitor for NF-κB and a radical scavenger, our data show that PDTC rather enhanced NF-κB activity and, alone or in combination with menadione, induced oxygen radical generation. Notably, PDTC failed to reduce rSMC viability in medium without Cu2+ or Zn2+, and addition of Cu2+ or Zn2+resulted in a dose-dependent increase in PDTC-induced cell death. Addition of both Cu2+ and Zn2+ showed synergistic effects. Our results indicate that the induction of apoptosis by PDTC requires Cu2+ and Zn2+ and is dependent on cell type and density. Such differential effects may have implications for studies of PDTC as an anti-atherosclerotic or immunomodulatory drug.
Bioactive products of arginine in sepsis: tissue and plasma composition after LPS and iNOS blockade
Authors: Lortie Mark J. , Ishizuka Shunji , Schwartz Doron , Blantz Roland C.
Published: December 2017

Blockade or gene deletion of inducible nitric oxide synthase (iNOS) fails to fully abrogate all the sequelae leading to the high morbidity of septicemia. An increase in substrate uptake may be necessary for the increased production of nitric oxide (NO), but arginine is also a precursor for other bioactive products. Herein, we demonstrate an increase in alternate arginine products via arginine and ornithine decarboxylase in rats given lipopolysaccharide (LPS). The expression of iNOS mRNA in renal tissue was evident 60 but not 30 min post-LPS, yet a rapid decrease in blood pressure was obtained within 30 min that was completely inhibited by selective iNOS blockade. Plasma levels of arginine and ornithine decreased by at least 30% within 60 min of LPS administration, an effect not inhibited by the iNOS blockerl- N 6(1-iminoethyl)lysine (l-NIL). Significant increases in plasma nitrates and citrulline occurred only 3–4 h post-LPS, an effect blocked byl-NIL pretreatment. The intracellular composition of organs harvested 6 h post-LPS reflected tissue-specific profiles of arginine and related metabolites. Tissue arginine concentration, normally an order of magnitude higher than in plasma, did not decrease after LPS. Pretreatment with l-NIL had a significant impact on the disposition of tissue arginine that was organ specific. These data demonstrate changes in arginine metabolism before and after de novo iNOS activity. Selective blockade of iNOS did not prevent uptake and can deregulate the production of other bioactive arginine metabolites.
PKA and arachidonic acid activation of human recombinant ClC-2 chloride channels
Authors: Tewari Kirti P. , Malinowska Danuta H. , Sherry Ann M. , Cuppoletti John
Published: December 2017

An HEK-293 cell line stably expressing the human recombinant ClC-2 Cl− channel was used in patch-clamp studies to study its regulation. The relative permeability P x/ P Cl calculated from reversal potentials was I− > Cl− = NO3 − = SCN−≥Br−. The absolute permeability calculated from conductance ratios was Cl− = Br− = NO3 −≥ SCN− > I−. The channel was activated by cAMP-dependent protein kinase (PKA), reduced extracellular pH, oleic acid (C:18 cisΔ9), elaidic acid (C:18 transΔ9), arachidonic acid (AA; C:20 cisΔ5,8,11,14), and by inhibitors of AA metabolism, 5,8,11,14-eicosatetraynoic acid (ETYA; C:20 transΔ5,8,11,14), α-methyl-4-(2-methylpropyl)benzeneacetic acid (ibuprofen), and 2-phenyl-1,2-benzisoselenazol-3-[2H]-one (PZ51, ebselen). ClC-2 Cl− channels were activated by a combination of forskolin plus IBMX and were inhibited by the cell-permeant myristoylated PKA inhibitor (mPKI). Channel activation by reduction of bath pH was increased by PKA and prevented by mPKI. AA activation of the ClC-2 Cl− channel was not inhibited by mPKI or staurosporine and was therefore independent of PKA or protein kinase C activation.
Reversible regulation of P2Y2 nucleotide receptor expression in the duct-ligated rat submandibular gland
Authors: Ahn Jae Suk , Camden Jean M. , Schrader Ann M. , Redman Robert S. , Turner John T.
Published: December 2017

Ligation of the main excretory duct of the rat submandibular gland (SMG) produces a pronounced atrophy that is reversed upon ligature removal. Based on previous studies by our group and others suggesting that P2Y2 nucleotide receptors are upregulated in response to tissue damage, we hypothesized that P2Y2 receptor activity and mRNA levels would increase after duct ligation and return to control levels after ligature removal. Our results support this hypothesis. Intracellular Ca2+ mobilization in response to the P2Y2 receptor agonist UTP in SMG cells was increased significantly after ligation periods of 1.5 to 7 days, whereas no significant response was observed in the contralateral, nonligated gland. P2Y2 receptor mRNA, as measured by semiquantitative RT-PCR, increased about 15-fold after 3 days of ligation. These increases reverted to control levels by 14 days after ligature removal. In situ hybridization revealed that the changes in P2Y2 receptor mRNA abundance occurred mostly in acinar cells, which also were more adversely affected by ligation, including an increase in the appearance of apoptotic bodies. These findings support the idea that P2Y2 receptor upregulation may be an important component of the response to injury in SMG and that recovery of normal physiological function may signal a decreased requirement for P2Y2 receptors.
Low extracellular Ca2+ activates a transient Cl− current in chicken ovarian granulosa cells
Authors: Qin Wuxuan , Rane Stanley G. , Asem Elikplimi K.
Published: December 2017

The effects of low Ca2+ on ion currents in hen ovarian granulosa cells were examined. A fast activating and inactivating transient outward current (TOC) and a slowly activating outward current (SOC) could be observed. In the presence of normal Ca2+concentration (2.5 mM) and with a holding potential of −80 mV, SOC was activated in all cells with command pulses more positive than −20 mV. In 2.5 mM Ca2+, TOC appeared in 10% of cells at the command pulse of +80 mV and in 60–85% of cells at +100 to +120 mV. In low-Ca2+ solution and command potential of +80 mV (holding potential of −80 mV), the amplitude of TOC was enhanced in cells that expressed it in normal Ca2+, and TOC appeared in 43% of the cells that did not express it initially in normal Ca2+. At both normal and low Ca2+ levels, TOC decreased as the holding potential became more positive. TOC was reduced in Cl−-deficient solution and in the presence of 5-nitro-2-(3-phenylpropylamino)benzoic acid, a Cl− channel blocker. These findings suggest that chicken granulosa cells express a Ca2+-inactivated TOC carried by Cl−. This current may serve as a signal for some of the reduced metabolic functions of granulosa cells associated with Ca2+deficiency.
Forskolin-induced apical membrane insertion of virally expressed, epitope-tagged CFTR in polarized MDCK cells
Authors: Howard Marybeth , Jiang Xiaosui , Stolz Donna Beer , Hill Warren G. , Johnson Jennifer A. , Watkins Simon C. , Frizzell Raymond A. , Bruton Christina M. , Robbins Paul D. , Weisz Ora A.
Published: December 2017

Channel gating of the cystic fibrosis transmembrane conductance regulator (CFTR) is activated in response to cAMP stimulation. In addition, CFTR activation may also involve rapid insertion of a subapical pool of CFTR into the plasma membrane (PM). However, this issue has been controversial, in part because of the difficulty in distinguishing cell surface vs. intracellular CFTR. Recently, a fully functional, epitope-tagged form of CFTR (M2–901/CFTR) that can be detected immunologically in nonpermeabilized cells was characterized (Howard M, Duvall MD, Devor DC, Dong J-Y, Henze K, and Frizzell RA. Am J Physiol Cell Physiol 269: C1565–C1576, 1995; and Schultz BD, Takahashi A, Liu C, Frizzell RA, and Howard M. Am J Physiol Cell Physiol 273: C2080–C2089, 1997). We have developed replication-defective recombinant adenoviruses that express M2–901/CFTR and used them to probe cell surface CFTR in forskolin (FSK)-stimulated polarized Madin-Darby canine kidney (MDCK) cells. Virally expressed M2–901/CFTR was functional and was readily detected on the apical surface of FSK-stimulated polarized MDCK cells. Interestingly, at low multiplicity of infection, we observed FSK-stimulated insertion of M2901/CFTR into the apical PM, whereas at higher M2–901/CFTR expression levels, no increase in surface expression was detected using indirect immunofluorescence. Immunoelectron microscopy of unstimulated and FSK-stimulated cells confirmed the M2–901/CFTR redistribution to the PM upon FSK stimulation and demonstrates that the apically inserted M2–901/CFTR originates from a population of subapical vesicles. Our observations may reconcile previous conflicting reports regarding the effect of cAMP stimulation on CFTR trafficking.
Actin filament disruption inhibits L-type Ca2+ channel current in cultured vascular smooth muscle cells
Authors: Nakamura Mariko , Sunagawa Masanori , Kosugi Tadayoshi , Sperelakis Nicholas
Published: December 2017

To clarify interactions between the cytoskeleton and activity of L-type Ca2+ (CaL) channels in vascular smooth muscle (VSM) cells, we investigated the effect of disruption of actin filaments and microtubules on the L-type Ca2+ current [ I Ba(L)] of cultured VSM cells (A7r5 cell line) using whole cell voltage clamp. The cells were exposed to each disrupter for 1 h and then examined electrophysiologically and morphologically. Results of immunostaining using anti-α-actin and anti-α-tubulin antibodies showed that colchicine disrupted both actin filaments and microtubules, cytochalasin D disrupted only actin filaments, and nocodazole disrupted only microtubules. I Ba(L) was greatly reduced in cells that were exposed to colchicine or cytochalasin D but not to nocodazole. Colchicine even inhibited I Ba(L) by about 40% when the actin filaments were stabilized by phalloidin or when the cells were treated with phalloidin plus taxol to stabilize both cytoskeletal components. These results suggest that colchicine must also cause some inhibition of I Ba(L) due to another unknown mechanism, e.g., a direct block of CaLchannels. In summary, actin filament disruption of VSM cells inhibits CaL channel activity, whereas disrupting the microtubules does not.
Connexin46 mutations linked to congenital cataract show loss of gap junction channel function
Authors: Pal Jay D. , Liu Xiaoqin , Mackay Donna , Shiels Alan , Berthoud Viviana M. , Beyer Eric C. , Ebihara Lisa
Published: December 2017

Human connexin46 (hCx46) forms gap junctional channels interconnecting lens fiber cells and appears to be critical for normal lens function, because hCx46 mutations have been linked to congenital cataracts. We studied two hCx46 mutants, N63S, a missense mutation in the first extracellular domain, and fs380, a frame-shift mutation that shifts the translational reading frame at amino acid residue 380. We expressed wild-type Cx46 and the two mutants in Xenopus oocytes. Production of the expressed proteins was verified by SDS-PAGE after metabolic labeling with [35S]methionine or by immunoblotting. Dual two-microelectrode voltage-clamp studies showed that hCx46 formed both gap junctional channels in paired Xenopus oocytes and hemi-gap junctional channels in single oocytes. In contrast, neither of the two cataract-associated hCx46 mutants could form intercellular channels in paired Xenopus oocytes. The hCx46 mutants were also impaired in their ability to form hemi-gap-junctional channels. When N63S or fs380 was coexpressed with wild-type connexins, both mutations acted like “loss of function” rather than “dominant negative” mutations, because they did not affect the gap junctional conductance induced by either wild-type hCx46 or wild-type hCx50.
Regulation of secretion of PTHrP by Ca2+-sensing receptor in human astrocytes, astrocytomas, and meningiomas
Authors: Chattopadhyay Naibedya , Evliyaoglu Cetin , Heese Oliver , Carroll Rona , Sanders Jennifer , Black Peter , Brown Edward M.
Published: December 2017

Parathyroid hormone-related protein (PTHrP) is the major mediator of the humoral hypercalcemia of malignancy and of malignant osteolysis associated with skeletal metastases of common epithelial cancers. PTHrP secretion is regulated by the extracellular calcium concentration ([Ca2+]o) in several types of normal and malignant cells. Because the [Ca2+]o-sensing receptor (CaR) is a key mediator of [Ca2+]o-regulated hormone secretion [e.g., of parathyroid hormone (PTH) by parathyroid chief cells], we investigated the expression of the CaR and PTHrP in normal and neoplastic glial cells and studied the effects of [Ca2+]o on PTHrP secretion. Our results show that primary embryonic human astrocytes (HPA) express CaR mRNA and protein as detected by RT-PCR and Western analysis, respectively. Furthermore, astrocytomas and meningiomas also express the CaR at similar levels as assessed by RT-PCR and Northern and Western blot analyses. HPA and astrocytomas express transcripts encoding all three known isoforms of PTHrP [PTHrP(139), PTHrP(141), and PTHrP(173), comprising 139, 141, and 173 predicted amino acid residues, respectively] as assessed by RT-PCR, whereas meningiomas express only the first two of these. Finally, elevated levels of [Ca2+]o and other polycationic CaR agonists dose dependently stimulate PTHrP secretion from HPA, astrocytomas, and meningiomas, although both basal and high [Ca2+]o-stimulated rates of PTHrP secretion are ∼2.5-fold higher in HPA than in the glial tumors studied here. Therefore, our results show that HPA, astrocytomas, and meningiomas express both the CaR and PTHrP and that CaR agonists stimulate PTHrP secretion.
Decreased adenylyl cyclase protein and function in airway smooth muscle by chronic carbachol pretreatment
Authors: Emala Charles W. , Clancy-Keen Judith , Hirshman Carol A.
Published: December 2017

Cellular levels of cAMP are an important determinant of airway smooth muscle tone. We have previously shown that chronic (18 h) but not acute (30 min or 2 h) pretreatment with the muscarinic receptor agonist carbachol resulted in decreased adenylyl cyclase activity in response to GTP, isoproterenol, or forskolin via a pathway blocked by the protein kinase C inhibitor staurosporine. The present study was designed to determine if carbachol-induced decreases in adenylyl cyclase activity were due to regulatory events at the level of either Gsα or adenylyl cyclase. Detergent-solubilized Gsα from control or carbachol-pretreated bovine airway smooth muscle had similar adenylyl cyclase activity in response to either NaF or guanosine 5′- O-(3-thiotriphosphate) (GTPγS) when reconstituted into S49 cyc− membranes that lack endogenous Gsα (carbachol pretreated: GTPγS, 93 ± 13% of control; NaF/AlCl3, 99 ± 8.6% of control; n = 4). Exogenous Gsα solubilized from red blood cells failed to restore normal adenylyl cyclase activity when reconstituted into carbachol-pretreated bovine airway smooth muscle (carbachol pretreated: GTP, 36 ± 10% of control; NaF/AlCl3, 54 ± 11% of control; n = 4). [3H]forskolin radioligand saturation binding assays revealed a decreased quantity of total adenylyl cyclase protein after carbachol pretreatment (maximal binding: 152 ± 40 and 107 ± 31 fmol/mg protein in control and carbachol-pretreated airway smooth muscle, respectively). These results suggest that chronic activation of muscarinic receptors downregulates the expression of adenylyl cyclase protein in bovine airway smooth muscle.
Splice variants of a ClC-2 chloride channel with differing functional characteristics
Authors: Cid L. Pablo , Niemeyer María-Isabel , Ramírez Alfredo , Sepúlveda Francisco V.
Published: December 2017

We identified two ClC-2 clones in a guinea pig intestinal epithelial cDNA library, one of which carries a 30-bp deletion in the NH2 terminus. PCR using primers encompassing the deletion gave two products that furthermore were amplified with specific primers confirming their authenticity. The corresponding genomic DNA sequence gave a structure of three exons and two introns. An internal donor site occurring within one of the exons accounts for the deletion, consistent with alternative splicing. Expression of the variants gpClC-2 and gpClC-2Δ77–86 in HEK-293 cells generated inwardly rectifying chloride currents with similar activation characteristics. Deactivation, however, occurred with faster kinetics in gpClC-2Δ77–86. Site-directed mutagenesis suggests that a protein kinase C-mediated phosphorylation consensus site lost in gpClC-2Δ77–86 is not responsible for the observed change. The deletion-carrying variant is found in most tissues examined, and it appears more abundant in proximal colon, kidney, and testis. The presence of a splice variant of ClC-2 modified in its NH2-terminal domain could have functional consequences in tissues where their relative expression levels are different.
Effects of fatty acids on BK channels in GH3cells
Authors: Denson Donald D. , Wang Xiaoping , Worrell Roger T. , Eaton Douglas C.
Published: December 2017

Ca2+-activated K+ (BK) channels in GH3 cells are activated by arachidonic acid (AA). Because cytosolic phospholipase A2 can produce other unsaturated free fatty acids (FFA), we examined the effects of FFA on BK channels in excised patches. Control recordings were made at several holding potentials. The desired FFA was added to the bath solution, and the voltage paradigm was repeated. AA increased the activity of BK channels by 3.6 ± 1.6-fold. The cis FFA, palmitoleic, oleic, linoleic, linolenic, eicosapentaenoic, and the triple bond analog of AA, eicosatetraynoic acid, all increased BK channel activity, whereas stearic (saturated) or the trans isomers elaidic, linolelaidic, and linolenelaidic had no effect. The cisunsaturated FFA shifted the open probability vs. voltage relationships to the left without a change in slope, suggesting no change in the sensitivity of the voltage sensor. Measurements of membrane fluidity showed no correlation between the change of membrane fluidity and the change in BK channel activation. In addition, AA effects on BK channels were unaffected in the presence of N-acetylcysteine. Arachidonyl-CoA, a membrane impermeable analog of AA, activates channels when applied to the cytosolic surface of excised patches, suggesting an effect of FFAs from the cytosolic surface of BK channels. Our data imply a direct interaction between cis FFA and the BK channel protein.
Protein kinase C-ζ mediates TNF-α-induced ICAM-1 gene transcription in endothelial cells
Authors: Rahman Arshad , Anwar Khandaker N. , Malik Asrar B.
Published: December 2017

We addressed the role of protein kinase C (PKC) isozymes in mediating tumor necrosis factor-α (TNF-α)-induced oxidant generation in endothelial cells, a requirement for nuclear factor-κB (NF-κB) activation and intercellular adhesion molecule-1 (ICAM-1) gene transcription. Depletion of the conventional (c) and novel (n) PKC isozymes following 24 h exposure of human pulmonary artery endothelial (HPAE) cells with the phorbol ester, phorbol 12-myristate 13-acetate (500 nM), failed to prevent TNF-α-induced oxidant generation. In contrast, inhibition of PKC-ζ synthesis by the antisense oligonucleotide prevented the oxidant generation following the TNF-α stimulation. We observed that PKC-ζ also induced the TNF-α-induced NF-κB binding to the ICAM-1 promoter and the resultant ICAM-1 gene transcription. We showed that expression of the dominant negative mutant of PKC-ζ prevented the TNF-α-induced ICAM-1 promoter activity, whereas overexpression of the wild-type PKC-ζ augmented the response. These data imply a critical role for the PKC-ζ isozyme in regulating TNF-α-induced oxidant generation and in signaling the activation of NF-κB and ICAM-1 transcription in endothelial cells.
The calcineurin-NFAT pathway and muscle fiber-type gene expression
Authors: Swoap Steven J. , Hunter R. Bridge , Stevenson Eric J. , Felton Heather Mitchell , Kansagra Nilesh V. , Lang Joshua M. , Esser Karyn A. , Kandarian Susan C.
Published: December 2017

To test for a role of the calcineurin-NFAT (nuclear factor of activated T cells) pathway in the regulation of fiber type-specific gene expression, slow and fast muscle-specific promoters were examined in C2C12 myotubes and in slow and fast muscle in the presence of calcineurin or NFAT2 expression plasmids. Overexpression of active calcineurin in myotubes induced both fast and slow muscle-specific promoters but not non-muscle-specific reporters. Overexpression of NFAT2 in myotubes did not activate muscle-specific promoters, although it strongly activated an NFAT reporter. Thus overexpression of active calcineurin activates transcription of muscle-specific promoters in vitro but likely not via the NFAT2 transcription factor. Slow myosin light chain 2 (MLC2) and fast sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1) reporter genes injected into rat soleus (slow) and extensor digitorum longus (EDL) (fast) muscles were not activated by coinjection of activated calcineurin or NFAT2 expression plasmids. However, an NFAT reporter was strongly activated by overexpression of NFAT2 in both muscle types. Calcineurin and NFAT protein expression and binding activity to NFAT oligonucleotides were different in slow vs. fast muscle. Taken together, these results indicate that neither calcineurin nor NFAT appear to have dominant roles in the induction and/or maintenance of slow or fast fiber type in adult skeletal muscle. Furthermore, different pathways may be involved in muscle-specific gene expression in vitro vs. in vivo.
Carbachol inhibits Na+-K+-ATPase activity in choroid plexus via stimulation of the NO/cGMP pathway
Authors: Ellis Dorette Z. , Nathanson James A. , Sweadner Kathleen J.
Published: December 2017

Secretion of cerebrospinal fluid by the choroid plexus can be inhibited by its cholinergic innervation. We demonstrated that carbachol inhibits the Na+-K+-ATPase in bovine choroid tissue slices and investigated the mechanism. Many of the actions of cholinergic agents are mediated by nitric oxide (NO), which plays important roles in fluid homeostasis. The inhibition of Na+-K+-ATPase was blocked by the NO synthase inhibitor [ N ω-nitro-L-arginine methyl ester] and was quantitatively mimicked by the NO agonists sodium nitroprusside (SNP) and diethylenetriamine NO. Inhibition by SNP correlated with an increase in tissue cGMP and was abolished by 1 H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylate cyclase. Inhibition was mimicked by the protein kinase G activator 8-bromo-cGMP and by okadaic acid, an inhibitor of protein phosphatases 1 and 2A. cGMP-dependent protein kinase inhibitors Rp-8-pCPT-cGMP (0.5–5 μM) and KT-5823 (2.0 μM) did not block the effects of SNP, but higher concentrations of the more selective inhibitor (Rp-8-pCPT-cGMP) had a pharmacological inhibitory effect on Na+-K+-ATPase. The data suggest that cholinergic regulation of the Na+-K+-ATPase is mediated by NO and involves activation of guanylate cyclase and elevation of cGMP.
MAP kinase upregulation after hematopoietic differentiation: role of chemotaxis
Authors: Lehman Jason A. , Paul Cassandra C. , Baumann Michael A. , Gómez-Cambronero Julian
Published: December 2017

Mitogen-activated protein kinase (MAPK) isoform p42 is known to be active in exponentially growing cells at several points of the cell cycle. A high basal activity was present in three cell lines representative of immature myeloid cells tested: uHL-60, AML-14, and MPD. However, DMSO-induced differentiation of HL-60 cells (dHL-60) and subsequent expression of the neutrophilic phenotype occurred with a concomitant reduction on the basal level of MAPK activity. Simultaneously, extracellular stimuli like the cytokine granulocyte/macrophage colony-stimulating factor (GM-CSF) induced a fast (<10 min) and robust response. In terms of MAPK activity, the more mature the cell was, the higher the corresponding activity, in the three differentiation series considered: AML-14 < 3D10; MPD < G-MPD; uHL-60 < dHL-60 < neutrophils. Interestingly, peripheral blood neutrophils expressed the highest (16-fold) MAPK activation level in response to GM-CSF. Finally, using the specific MAPK inhibitor PD-98059, we demonstrated that MAPK activation is needed for neutrophil chemotaxis toward interleukin-8 and its priming by GM-CSF. Since neutrophils are terminally differentiated cells, GM-CSF does not serve a purpose in proliferation, and it must trigger the recruitment of selective signal transduction pathways particular to that final stage that includes enhanced physiological functions such as chemotaxis.
L-type calcium channels regulate epithelial fluid transport in Drosophila melanogaster
Authors: MacPherson Matthew R. , Pollock Valerie P. , Broderick Kate E. , Kean Laura , O'Connell Fiona C. , Dow Julian A. T. , Davies Shireen A.
Published: December 2017

The neuropeptide CAP2b stimulates fluid transport obligatorily via calcium entry, nitric oxide, and cGMP in Drosophila melanogaster Malpighian (renal) tubules. We have shown by RT-PCR that the Drosophila L-type calcium channel α1-subunit genes Dmca1D and Dmca1A ( nbA) are both expressed in tubules. CAP2b-stimulated fluid transport and cytosolic calcium concentration ([Ca2+]i) increases are inhibited by the L-type calcium channel blockers verapamil and nifedipine. cGMP-stimulated fluid transport is verapamil and nifedipine sensitive. Furthermore, cGMP induces a slow [Ca2+]i increase in tubule principal cells via verapamil- and nifedipine-sensitive calcium entry; RT-PCR shows that tubules express Drosophila cyclic nucleotide-gated channel ( cng). Additionally, thapsigargin-induced [Ca2+]i increase is verapamil sensitive. Phenylalkylamines bind with differing affinities to the basolateral and apical surfaces of principal cells in the main segment; however, dihydropyridine binds apically in the tubule initial segment. Immunocytochemical evidence suggests localization of α1-subunits to both basolateral and apical surfaces of principal cells in the tubule main segment. We suggest roles for L-type calcium channels and cGMP-mediated calcium influx in both calcium signaling and fluid transport mechanisms in Drosophila.
Creatine transporter protein content, localization, and gene expression in rat skeletal muscle
Authors: Murphy R. , McConell G. , Cameron-Smith D. , Watt K. , Ackland L. , Walzel B. , Wallimann T. , Snow R.
Published: December 2017

The present study examined the gene expression and cellular localization of the creatine transporter (CreaT) protein in rat skeletal muscle. Soleus (SOL) and red (RG) and white gastrocnemius (WG) muscles were analyzed for CreaT mRNA, CreaT protein, and total creatine (TCr) content. Cellular location of the CreaT protein was visualized with immunohistochemical analysis of muscle cross sections. TCr was higher ( P ≤ 0.05) in WG than in both RG and SOL, and was higher in RG than in SOL. Total CreaT protein content was greater ( P ≤ 0.05) in SOL and RG than in WG. Two bands (55 and 70 kDa) of the CreaT protein were found in all muscle types. Both the 55-kDa (CreaT-55) and the 70-kDa (CreaT-70) bands were present in greater ( P ≤ 0.05) amounts in SOL and RG than in WG. SOL and RG had a greater amount ( P ≤ 0.05) of CreaT-55 than CreaT-70. Immunohistochemical analysis revealed that the CreaT was mainly associated with the sarcolemmal membrane in all muscle types. CreaT mRNA expression per microgram of total RNA was similar across the three muscle types. These data indicate that rat SOL and RG have an enhanced potential to transport Cr compared with WG, despite a higher TCr in the latter.
Effects of KCNQ channel blockers on K+ currents in vestibular hair cells
Authors: Rennie Katherine J. , Weng Tianxiang , Correia Manning J.
Published: December 2017

Linopirdine and XE991, selective blockers of K+ channels belonging to the KCNQ family, were applied to hair cells isolated from gerbil vestibular system and to hair cells in slices of pigeon crista. In type II hair cells, both compounds inhibited a slowly activating, slowly inactivating component of the macroscopic current recruited at potentials above −60 mV. The dissociation constants for linopirdine and XE991 block were <5 μM. A similar component of the current was also blocked by 50 μM capsaicin in gerbil type II hair cells. All three drugs blocked a current component that showed steady-state inactivation and a biexponential inactivation with time constants of ∼300 ms and 4 s. Linopirdine (10 μM) reduced inward currents through the low-voltage-activated K+ current in type I hair cells, but concentrations up to 200 μM had little effect on steady-state outward K+ current in these cells. These results suggest that KCNQ channels may be present in amniote vestibular hair cells.
Contractile activity-induced oxidative stress: cellular origin and adaptive responses
Authors: McArdle A. , Pattwell D. , Vasilaki A. , Griffiths R. D. , Jackson M. J.
Published: December 2017

Previous studies have reported that oxidizing free radical species are generated during exercise, and there has been considerable interest in the potential effects of these on exercising tissues. We hypothesized that contracting skeletal muscle was a major source of oxidizing free radical species and that untrained skeletal muscle would adapt to the oxidative stress of a single short period of contractile activity by upregulation of the activity of cytoprotective proteins in the absence of overt cellular damage. Fifteen minutes of aerobic contractile activity was found to induce a rapid release of superoxide anions from mouse skeletal muscle in vivo, and studies with contracting cultured skeletal muscle myotubes confirmed that this was due to release from myocytes rather than other cell types present within muscle tissue in vivo. This increased oxidant production caused a rapid, transient reduction in muscle protein thiol content, followed by increases in the activities of superoxide dismutase and catalase and in content of heat shock proteins. These changes occurred in the absence of overt damage to the muscle cells.
Mutation of the IIB myosin heavy chain gene results in muscle fiber loss and compensatory hypertrophy
Authors: Allen David L. , Harrison Brooke C. , Sartorius Carol , Byrnes William C. , Leinwand Leslie A.
Published: December 2017

The fast skeletal IIb gene is the source of most myosin heavy chain (MyHC) in adult mouse skeletal muscle. We have examined the effects of a null mutation in the IIb MyHC gene on the growth and morphology of mouse skeletal muscle. Loss in muscle mass of several head and hindlimb muscles correlated with amounts of IIb MyHC expressed in that muscle in wild types. Decreased mass was accompanied by decreases in mean fiber number, and immunological and ultrastructural studies revealed fiber pathology. However, mean cross-sectional area was increased in all fiber types, suggesting compensatory hypertrophy. Loss of muscle and body mass was not attributable to impaired chewing, and decreased food intake as a softer diet did not prevent the decrease in body mass. Thus loss of the major MyHC isoform produces fiber loss and fiber pathology reminiscent of muscle disease.
K+ channels and the microglial respiratory burst
Authors: Khanna Rajesh , Roy Lipi , Zhu Xiaoping , Schlichter Lyanne C.
Published: December 2017

Microglial activation following central nervous system damage or disease often culminates in a respiratory burst that is necessary for antimicrobial function, but, paradoxically, can damage bystander cells. We show that several K+ channels are expressed and play a role in the respiratory burst of cultured rat microglia. Three pharmacologically separable K+ currents had properties of Kv1.3 and the Ca2+/calmodulin-gated channels, SK2, SK3, and SK4. mRNA was detected for Kv1.3, Kv1.5, SK2, and/or SK3, and SK4. Protein was detected for Kv1.3, Kv1.5, and SK3 (selective SK2 and SK4 antibodies not available). No Kv1.5-like current was detected, and confocal immunofluorescence showed the protein to be subcellular, in contrast to the robust membrane localization of Kv1.3. To determine whether any of these channels play a role in microglial activation, a respiratory burst was stimulated with phorbol 12-myristate 13-acetate and measured using a single cell, fluorescence-based dihydrorhodamine 123 assay. The respiratory burst was markedly inhibited by blockers of SK2 (apamin) and SK4 channels (clotrimazole and charybdotoxin), and to a lesser extent, by the potent Kv1.3 blocker agitoxin-2.
Na+/Ca2+ exchange and its role in intracellular Ca2+ regulation in guinea pig detrusor smooth muscle
Authors: Wu C. , Fry C. H.
Published: December 2017

The role of Na+/Ca2+ exchange in regulating intracellular Ca2+ concentration ([Ca2+]i) in isolated smooth muscle cells from the guinea pig urinary bladder was investigated. Incremental reduction of extracellular Na+ concentration resulted in a graded rise of [Ca2+]i; 50–100 μM strophanthidin also increased [Ca2+]i. A small outward current accompanied the rise of [Ca2+]i in low-Na+ solutions (17.1 ± 1.8 pA in 29.4 mM Na+). The quantity of Ca2+ influx through the exchanger was estimated from the charge carried by the outward current and was ∼30 times that which is necessary to account for the rise of [Ca2+]i, after correction was made for intracellular Ca2+ buffering. Ca2+ influx through the exchanger was able to load intracellular Ca2+ stores. It is concluded that the level of resting [Ca2+]i is not determined by the exchanger, and under resting conditions (membrane potential −50 to −60 mV), there is little net flux through the exchanger. However, a small rise of intracellular Na+ concentration would be sufficient to generate significant net Ca2+ influx.
Differential effects of growth hormone and insulin-like growth factor I on human endothelial cell migration
Authors: Ikeo Sachiko , Yamauchi Keishi , Shigematsu Satoshi , Nakajima Koji , Aizawa Toru , Hashizume Kiyoshi
Published: December 2017

Effects of growth hormone (GH), insulin-like growth factor I (IGF-I), and endothelin-1 (ET-1) on endothelial cell migration and the underlying molecular mechanisms were explored using a human umbilical cord endothelial cell line, ECV304 cells, in vitro. Treatment of the cells with IGF-I or ET-1, but not GH, stimulated the cell migration. Interestingly, however, ET-1-induced, but not IGF-I-induced, migration of the cells was inhibited by GH. Both ET-1 and IGF-I caused activation of mitogen-activated protein kinase (MAPK) in the cells, and GH eliminated the MAPK activation produced by ET-1 but not that produced by IGF-I. On the other hand, migration of the cells was stimulated by protein kinase C (PKC) agonist, phorbol 12-myristate 13-acetate. ET-1 promoted PKC activity, and a PKC inhibitor, GF-109203X, blocked ET-1-induced cell migration. Although GH inhibited ET-1-induced cell migration and MAPK activity, it did not block ET-1-induced PKC activation. Thus ET-1 stimulation of endothelial cell migration appears to be mediated by PKC/MAPK pathway, and GH may inhibit the MAPK activation by ET-1 at the downstream of PKC.
Role of IP3 in modulation of spontaneous activity in pacemaker cells of rabbit urethra
Authors: Sergeant G. P. , Hollywood M. A. , McCloskey K. D. , McHale N. G. , Thornbury K. D.
Published: December 2017

Isolated interstitial (“pacemaker”) cells from rabbit urethra were examined using the perforated-patch technique. Under voltage clamp at −60 mV, these cells fired large spontaneous transient inward currents (STICs), averaging −860 pA and >1 s in duration, which could account for urethral pacemaker activity. Spontaneous transient outward currents (STOCs) were also observed and fell into two categories, “fast” (<100 ms in duration) and “slow” (>1 s in duration). The latter were coupled to STICs, suggesting that they shared the same mechanism, while the former occurred independently at faster rates. All of these currents were abolished by cyclopiazonic acid, caffeine, or ryanodine, suggesting that they were activated by Ca2+ release. Whend- myo-inositol 1,4,5-trisphosphate (IP3)-sensitive stores were blocked with 2-aminoethoxydiphenyl borate, the STICs and slow STOCs were abolished, but the fast STOCs remained. In contrast, the fast STOCs were more nifedipine sensitive than the STICs or the slow STOCs. These results suggest that while fast STOCs are mediated by a mechanism similar to STOCs in smooth muscle, STICs and slow STOCs are driven by IP3. These results support the hypothesis that pacemaker activity in the urethra is driven by the IP3-sensitive store.
Deficiency in parvalbumin increases fatigue resistance in fast-twitch muscle and upregulates mitochondria
Authors: Chen Gaoping , Carroll Stefanie , Racay Peter , Dick Jim , Pette Dirk , Traub Irmtrud , Vrbova Gerta , Eggli Peter , Celio Marco , Schwaller Beat
Published: December 2017

The soluble Ca2+-binding protein parvalbumin (PV) is expressed at high levels in fast-twitch muscles of mice. Deficiency of PV in knockout mice (PV −/−) slows down the speed of twitch relaxation, while maximum force generated during tetanic contraction is unaltered. We observed that PV-deficient fast-twitch muscles were significantly more resistant to fatigue than were the wild type. Thus components involved in Ca2+ homeostasis during the contraction-relaxation cycle were analyzed. No upregulation of another cytosolic Ca2+-binding protein was found. Mitochondria are thought to play a physiological role during muscle relaxation and were thus analyzed. The fractional volume of mitochondria in the fast-twitch muscle extensor digitorum longus (EDL) was almost doubled in PV −/− mice, and this was reflected in an increase of cytochrome coxidase. A faster removal of intracellular Ca2+concentration ([Ca2+]i) 200–700 ms after fast-twitch muscle stimulation observed in PV −/− muscles supports the role for mitochondria in late [Ca2+]iremoval. The present results also show a significant increase of the density of capillaries in EDL muscles of PV −/− mice. Thus alterations in the dynamics of Ca2+ transients detected in fast-twitch muscles of PV −/− mice might be linked to the increase in mitochondria volume and capillary density, which contribute to the greater fatigue resistance of these muscles.
Functional aspects of creatine kinase isoenzymes in endothelial cells
Authors: Decking Ulrich K. M. , Alves Christiane , Wallimann Theo , Wyss Markus , Schrader Jürgen
Published: December 2017

To characterize the isoenzyme distribution of creatine kinase (CK) in endothelial cells (ECs) and its functional role during substrate depletion, ECs from aorta (AECs) and microvasculature (MVECs) of pig and rat were studied. In addition, high- energy phosphates were continuously monitored by 31P NMR spectroscopy in pig AECs attached to microcarrier beads. CK activity per milligram of protein in rat AECs and MVECs (0.08 ± 0.01 and 0.15 ± 0.08 U/mg, respectively) was <3% of that of cardiomyocytes (6.46 ± 1.02 U/mg). Rat and pig AECs and MVECs displayed cytosolic BB-CK, but no MM-CK. Gel electrophoresis of mitochondrial fractions of rat and pig ECs indicated the presence of mitochondrial Mi-CK, mostly in dimeric form. The presence of Mia-CK was demonstrated by indirect immunofluorescence staining using Mia-CK antibodies. When perifused with creatine-supplemented medium, phosphocreatine (PCr) continuously increased with time (1.2 ± 0.6 nmol · h−1 · mg protein−1), indicating creatine uptake and CK activity. Glucose withdrawal from the medium induced a rapid decrease in PCr, which was fully reversible on glucose addition, demonstrating temporal buffering of an energy deficit. Because both cytosolic and mitochondrial CK isoforms are present in ECs, the CK system may also contribute to energy transduction (“shuttle hypothesis”).
Hypertonicity-induced expression of aquaporin 3 in MDCK cells
Authors: Matsuzaki Toshiyuki , Suzuki Takeshi , Takata Kuniaki
Published: December 2017

Aquaporins (AQPs) are water channel proteins that participate in water transport. In the principal cells of the kidney collecting duct, water reabsorption is mediated by the combined action of AQP2 in the apical membrane and both AQP3 and AQP4 in the basolateral membrane, and the expression of AQP2 and AQP3 is regulated by antidiuretic hormone and water restriction. The effect of hypertonicity on AQP3 expression in Madin-Darby canine kidney (MDCK) epithelial cells was investigated by exposing the cells to hypertonic medium containing raffinose or NaCl. Northern blot and immunoblot analyses revealed that the amounts of AQP3 mRNA and AQP3 protein, respectively, were markedly increased by exposure of cells to hypertonicity. These effects were maximal at 12 and 24 h, respectively. Immunofluorescence and immunoelectron microscopy also demonstrated that the abundance of AQP3 protein was increased in cells incubated in hypertonic medium and that the protein was localized at the basolateral plasma membrane. These results indicate that the expression of AQP3 is upregulated by hypertonicity.
Energy dependence of restitution in the gastric mucosa
Authors: Cheng Aaron M. , Morrison Sarah W. , Yang David X. , Hagen Susan J.
Published: December 2017

Rapid epithelial repair (restitution) after injury is required to maintain barrier function of the gastrointestinal mucosa and skin and is thought to be a highly ATP-dependent process that would be inhibited under hypoxic conditions. However, little is known about the metabolic pathways required for restitution. Thus, this study was undertaken to evaluate, in vitro, the role of oxidative respiration and glycolysis in restitution after injury. To this end, restitution of the bullfrog gastric mucosa was evaluated under the following conditions: 1) blockade of mitochondrial respiration; 2) blockade of glycolysis; or 3) absence of glucose. The extent of mucosal repair after injury was evaluated by electrophysiology and morphology. Cell migration, repolarization, and the formation of tight junctions after injury occurred during blockade of mitochondrial respiration, whereas the recovery of mucosal barrier function did not. In contrast, glycolytic inhibition completely blocked all aspects of restitution by inhibiting the migration of surface epithelial cells. Restitution occurred in tissues incubated with glucose-free solutions, suggesting that cells contain sufficient glucose (glycogen) to drive glycolysis for many hours. Our results demonstrate that the glycolytic pathway is essential for restitution after injury in the bullfrog gastric mucosa and that all but complete repair of barrier function occurs in the absence of mitochondrial respiration.
Rho activation in excitatory agonist-stimulated vascular smooth muscle
Authors: Sakurada Sotaro , Okamoto Hiroyuki , Takuwa Noriko , Sugimoto Naotoshi , Takuwa Yoh
Published: December 2017

Small GTPase Rho and its downstream effector, Rho kinase, have been implicated in agonist-stimulated Ca2+ sensitization of 20-kDa myosin light chain (MLC20) phosphorylation and contraction in smooth muscle. In the present study we demonstrated for the first time that excitatory receptor agonists induce increases in amounts of an active GTP-bound form of RhoA, GTP-RhoA, in rabbit aortic smooth muscle. Using a pull-down assay with a recombinant RhoA-binding protein, Rhotekin, we found that a thromboxane A2 mimetic, U-46619, which induced a sustained contractile response, induced a sustained rise in the amount of GTP-RhoA in a dose-dependent manner with an EC50 value similar to that for the contractile response. U-46619-induced RhoA activation was thromboxane A2 receptor-mediated and reversible. Other agonists including norepinephrine, serotonin, histamine, and endothelin-1 (ET-1) also stimulated RhoA, albeit to lesser extents than U-46619. In contrast, ANG II and phorbol 12,13-dibutyrate failed to increase GTP-RhoA. The tyrosine kinase inhibitor genistein substantially inhibited RhoA activation by these agonists, except for ET-1. Thus excitatory agonists induce Rho activation in an agonist-specific manner, which is thought to contribute to stimulation of MLC20 phosphorylation Ca2+ sensitivity.