bluuebunny/crossref_metadata_2025 huggingface dataset:
Adult human chondrocytes cultured in alginate form a matrix similar to native human articular cartilage
Authors: Hauselmann H. J. , Masuda K. , Hunziker E. B. , Neidhart M. , Mok S. S. , Michel B. A. , Thonar E. J.
Published: December 2017

The matrix formed by adult human chondrocytes in alginate beads is composed of two compartments: a thin rim of cell-associated matrix that corresponds to the pericellular and territorial matrix of articular cartilage and a more abundant further-removed matrix, the equivalent of the interterritorial matrix in the tissue. On day 30 of culture, the relative and absolute volumes occupied by the cells and each of the two matrix compartments in the beads were nearly identical to those in native articular cartilage. Furthermore, the concentration of aggrecan in the cell-associated matrix was similar to that in adult human articular cartilage and was approximately 40-fold higher than in the further removed matrix compartment. Fluorescence-activated cell sorting revealed that the cell-associated matrix was built on the cell membrane in part via interactions between hyaluronic acid and CD44-like receptors. Approximately 25% of the aggrecan molecules synthesized by the chondrocytes during a 4-h pulse in the presence of [35S]sulfate on day 9 of culture were retained in the cell-associated matrix where they turned over with a half-life (t1/2) = 29 days. Most [35S]aggrecan molecules reached the further removed matrix compartment where they turned over much more slowly (t1/2 > 100 days). These results add support to the contention that aggrecan molecules residing in the pericellular and territorial areas of the adult human articular cartilage matrix are more susceptible to degradation by proteolytic enzymes synthesized by the chondrocytes than those that inhabit the interterritorial areas further removed from the cells.
Adenine nucleotide diphosphates: emerging second messengers acting via intracellular Ca2+ release
Authors: Dousa T. P. , Chini E. N. , Beers K. W.
Published: December 2017

Release of Ca2+ from intracellular stores is a widespread mechanism in regulation of cell function. Two hitherto unknown adenine diphosphonucleotides were recently identified, which trigger Ca2+ release from intracellular stores via channels that are distinct from the well-known receptor/channel controlled by inositol 1,4,5,-trisphosphate (IP3): cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). Here we review synthesis of cADPR from beta-NAD, its hydrolysis to adenosine diphosphoribose (noncyclic) by cADPR glycohydrolase, as well as our knowledge about the metabolism of NAADP. The Ca2+ release triggered by cADPR, NAADP, or IP3 can be distinguished by the action of inhibitors and by desensitization studies. Evidence now emerges that cADPR synthesis from beta-NAD can be stimulated, at least in some cell types by all-trans-retinoic acid as a first messenger. We then review the properties of cADPR and NAADP as potential second messengers in the intracrine regulation of cell functions. Although their exact role in signaling sequences is not yet known, cADPR and NAADP are likely to play important intracellular regulatory functions, as extensively documented for the process of egg fertilization.
Electrolyte transport mechanisms involved in regulatory volume increase in C6 glioma cells
Authors: Mountian I. , Chou K. Y. , Van Driessche W.
Published: December 2017

Volume regulation of C6 glioma cells was studied with an automatic system for monitoring cell thickness, while increasing bath osmolality from 300 to 440 mosmol/kgH2O. At 37 degrees C, tissues incubated in solutions containing active substances (inositol, D-biotin, hydrocortisone, prostaglandin E1, insulin, transferrin, sodium selenite, and 3,5,3'-triiodothyronine) responded to hyperosmotic challenge with a typical regulatory volume increase (RVI). Lowering temperature or removing the active substances inhibited osmoregulation. Bumetanide, amiloride, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, or ouabain significantly reduced RVI. Ion substitutions of Na+, Cl-, NaCl, or HCO3- also importantly affected the process. Extracellular acidification rate (EAR) was studied by microphysiometry. Hyperosmotic shock induced an increase in EAR with a time course that matched volume recovery. This increase in EAR was prevented by amiloride. The data show that under hyperosmotic conditions C6 cells are able to regulate their volume. Ion substitutions and application of blockers demonstrate that Na+/H+ and Cl-/HCO3- exchangers and Na(+)-K(-)-2Cl- cotransporter are involved in RVI. The rise in EAR is due to the enhanced activity of Na+/H+ antiporter, which seems to be volume dependent but not osmotic dependent.
TXA2 agonists inhibit high-voltage-activated calcium channels in rat hippocampal CA1 neurons
Authors: Hsu K. S. , Huang C. C. , Kan W. M. , Gean P. W.
Published: December 2017

Whole cell voltage clamp recordings were used to investigate the effects of thromboxane A2 (TXA2) agonists on the voltage-dependent Ca2+ currents in rat hippocampal CA1 neurons. TXA2 agonists [1S-[1 alpha, 2 beta(5Z), 3 alpha(1E, 3S*)4 alpha ]]-7-[3-[3-hydroxy-4-(4'-iodophenoxy)-1-butenyl]-7-oxabicyclo [2,2,1]heptan-2-yl]-5-heptenoic acid (I-BOP) and U-46619, reversibly suppressed the whole cell Ca2+ currents in a concentration-dependent manner. The effect was blocked by specific TXA2 receptor antagonist, SQ-29548. I-BOP as well as U-46619 inhibited both omega-conotoxin GVIA (CgTx)-sensitive and nimodipine sensitive Ca2+ currents but had no effect on CgTx/nimodipine insensitive Ca2+ currents. The I-BOP and U-46619 inhibition of Ca2+ currents was blocked by internal dialysis of hippocampal neurons with specific protein kinase C (PKC) inhibitors, NPC-15437 and PKC inhibitor-(19-36). Pretreatment of hippocampal neurons with either 5 micrograms/ml pertussis toxin (PTX) or 5 micrograms/ml cholera toxin (CTX) did not significantly affect the suppression of the Ca2+ currents by I-BOP and U-46619. Dialyzing with 1 mM guanosine 5'-O-(3-thiotriphosphate) or 1 mM GDP significantly attenuated the I-BOP or U-46619 action. These results demonstrate that TXA2 agonists inhibit both CgTx- and nimodipine-sensitive Ca2+ currents but not CgTx/nimodipine-insensitive currents in rat hippocampal CA1 neurons via a PTX- and CTX-insensitive G protein-coupled activation of the PKC pathway.
A critical evaluation of resting intracellular free calcium regulation in dystrophic mdx muscle
Authors: Hopf F. W. , Turner P. R. , Denetclaw W. F. , Reddy P. , Steinhardt R. A.
Published: December 2017

There are conflicting reports regarding whether resting free calcium levels ([Ca2+]i) are elevated in dystrophic mouse (mdx) myotubes and adult myofibers. We reinvestigated this question and found several lines of evidence supporting the hypothesis that increased calcium influx via leak channels leads to increases in resting [Ca2+]i. 1) Step calibration of fura 2/free acid in myofibers with use of microinjected Ca(2+)-ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid buffers revealed greater [Ca2+]i in dystrophic cells. Careful calibration of fura PE3-AM, a compartmentalization-resistant derivative of fura 2, also showed elevated [Ca2+]i in mdx myotubes. 2) Chronic, but not acute, application of tetrodotoxin reduced resting [Ca2+]i in dystrophic myotubes, suggesting that elevated resting [Ca2+]i is a consequence of previous long-term contractile activity. 3) Rates of manganese quenching of fura 2 fluorescence, an indirect indicator of calcium influx, were significantly higher in mdx myotubes and were increased by nifedipine, a calcium leak channel agonist. 4) Calcium leak channel activity, measured using patch clamping, was greater in the sarcolemma of adult non-enzyme-treated mdx myofibers.
An equibiaxial strain system for cultured cells
Authors: Lee A. A. , Delhaas T. , Waldman L. K. , MacKenna D. A. , Villarreal F. J. , McCulloch A. D.
Published: December 2017

We developed a device that applies homogeneous equibiaxial strains of 0-10% to a cell culture substrate and quantitatively verified transmission of substrate deformation to cultured cardiac cells. Clamped elastic membranes in both single-well and multiwell versions of the device are uniformly stretched by indentation with a plastic ring, resulting in strain that is directly proportional to the pitch-to-radius ratio. Two-dimensional deformations were measured by tracking fluorescent microspheres attached to the substrate and to cultured adult rat cardiac fibroblasts. For nominal stretches up to 18%, strains along circumferential and radial axes were equal in magnitude and homogeneously distributed with negligible shear. For 5% stretch, circumferential and radial strains in the substrate were 0.046 +/- 0.005 and 0.048 +/- 0.004 [not significant (NS)], respectively, and shear strain was 0.001 +/- 0.003 (NS). Calibration of both single-well and multiwell versions permits strain selection by device rotation. The reproducible application and quantification of homogeneous equibiaxial strain in cultured cells provides a quantitative approach for correlating mechanical stimuli to cellular transduction mechanisms.
Bicarbonate/chloride exchange and intracellular pH throughout preimplantation mouse embryo development
Authors: Zhao Y. , Baltz J. M.
Published: December 2017

HCO3-/Cl- exchanger activity, which regulates intracellular pH (pHi) in the alkaline range, has been shown to be present throughout preimplantation mouse embryo development and to be necessary for embryo viability. We have characterized HCO3-/Cl- exchange activity and its regulation of pHi throughout preimplantation development (1-cell, 2-cell, morula, and blastocyst stages). Embryos at each stage can recover from alkalosis. Recovery was dependent on external [Cl-], activated by increased pHi, and inhibited by the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and external HCO3-. Dependence of exchanger activity on external [Cl-] and pHi remained unchanged during preimplantation development. However, the concentration at which DIDS inhibits the maximal response by 50% increased significantly (by approximately 5-fold) after the one-cell stage. In addition, HCO3-/Cl- exchange activity decreased over the course of development, with significantly lower activity at the morula and blastocyst stages relative to the one- and two-cell stages, coinciding with the movement of embryo from the high pH environment of the oviduct to the lower pH environment of the uterus.
Polarized expression of cAMP-activated chloride channels in isolated epithelial cells
Authors: Torres R. J. , Altenberg G. A. , Cohn J. A. , Reuss L.
Published: December 2017

We have described a preparation of Necturus maculosus gallbladder (NGB) epithelium yielding isolated cells that retain structural and functional polarity ("figure-eight" cells). These cells have a normal membrane voltage and remain polarized for several hours after isolation. Apical and basolateral membrane domains are differentially labeled with hydrophobic fluorescent dyes; freeze-fracture electron microscopy reveals two distinct membrane domains separated by tight junctions; ZO-1, Na+/H+ exchanger (NHE3), and Na(+)-K(+)-ATPase are present in the junctional, apical, and basolateral region, respectively; and cell-attached patch-clamp experiments reveal different K+ currents in the two membrane domains [R. J. Torres, G. A. Altenberg, J. A. Copello, G. Zampighi, and L. Reuss, Am. J. Physiol. 270 (Cell Physiol. 39): C1864-C1874, 1996]. Here, we show that NGB epithelial cells express a protein cross-reactive with an antibody against human cystic fibrosis transmembrane conductance regulator (CFTR). In figure-eight cells, immunoreactivity was restricted to the apical membrane domain. Using intracellular microelectrodes and a novel method of regional superfusion, we found that control cells have high K+ conductances in both membranes and a small basolateral Cl- conductance, similar to findings in the epithelium. Activation of adenylate cyclase with forskolin elicited a large apical membrane Cl- conductance and membrane depolarization. Whole cell patch-clamp studies yielded a forskolin-activated linear Cl- current, with high Cl-/aspartate selectivity. In conclusion, 1) figure-eight cells maintain the conductive membrane properties present in the epithelium, including polarized expression of adenosine 3',5'-cyclic monophosphate (cAMP)-activated Cl- channels, and 2) the cAMP-activated Cl- conductance is underlied by a CFTR homologue.
Characterization of the rabbit renal Na(+)-dicarboxylate cotransporter using antifusion protein antibodies
Authors: Pajor A. M. , Sun N.
Published: December 2017

Polyclonal antibodies were prepared against the rabbit renal Na(+)-dicarboxylate cotransporter, NaDC-1. The antibodies were raised in chickens against a fusion protein consisting of a 60-amino acid peptide from NaDC-1 and glutathione S-transferase. These antibodies specifically recognized the fusion protein in Western blots and could immunoprecipitate the full-length NaDC-1 after in vitro translation. The antifusion protein antibodies specifically recognized a protein of 63 kDa in rabbit renal brush-border membrane vesicles (BBMV), similar to the predicted mass of 66 kDa. Two proteins of 57 and 115 kDa were recognized in rabbit intestinal brush-border membranes. A protein of 66 kDa was recognized in Xenopus oocytes injected with NaDC-1 cRNA. Enzymatic deglycosylation of rabbit renal BBMV resulted in a decrease in mass by 11 kDa, consistent with N-glycosylation at a single site. Site-directed mutagenesis of the two consensus sequences for N-glycosylation in the NaDC-1 cDNA showed that Asn-576, located near the COOH-terminal, is glycosylated. The nonglycosylated mutant of NaDC-1 exhibited 50% of wild-type succinate transport activity when expressed in Xenopus oocytes, suggesting that glycosylation is not essential for function. The revised secondary structure model of NaDC-1 contains 11 putative transmembrane domains and an extracellular glycosylated COOH-terminal.
Carbonic anhydrase and cardiac pH regulation
Authors: Vandenberg J. I. , Carter N. D. , Bethell H. W. , Nogradi A. , Ridderstrale Y. , Metcalfe J. C. , Grace A. A.
Published: December 2017

Membrane-bound carbonic anhydrase (CA) has recently been identified in mammalian cardiac tissue. In this study, we have investigated the histochemical location and functional role of CA in the ferret heart. Heart sections stained by a modified Hansson's technique showed CA to be located on capillary endothelial membranes as well as on sarcolemmal membranes. In the Langendorff-perfused heart, washout of CO2 brought about by switching perfusion between 25 mM HCO3(-)-5% CO2-buffered solution and nominally HCO3(-)-CO2-free solution caused a transient rise in intracellular pH (pHi) measured by the chemical shift of 2-deoxy-D-glucose 6-phosphate with 31P nuclear magnetic resonance spectroscopy. The initial rate of change of pHi, measured over the first 60-75 s of CO2 efflux, was significantly reduced from 0.41 +/- 0.03 pH units/min (n = 9) in control hearts to 0.28 +/- 0.02 pH units/min (n = 5) in the presence of the membrane-permeable CA inhibitor 6-ethoxzolamide (P < 0.05 compared with control) and to 0.22 +/- 0.04 pH units/min (n = 5) in the presence of the membrane-impermeable CA inhibitor CL-11,366 (P < 0.01 compared with control). After reperfusion of the ischemic myocardium, both CA inhibitors caused a significant slowing of initial rate of change in pH (and initial rate of recovery of contractile function) compared with control hearts. These results suggest that CA, by facilitating the hydration-dehydration of CO2-H2CO3, alters the relative concentrations of CO2 inside and outside the cells, thus enhancing the rate of CO2 transfer from the intracellular to extracellular compartments, which contributes significantly to pHi recovery after reperfusion of the ischemic myocardium.
CFTR-dependent membrane insertion is linked to stimulation of the CFTR chloride conductance
Authors: Takahashi A. , Watkins S. C. , Howard M. , Frizzell R. A.
Published: December 2017

We examined the relation between Cl current (Icl) stimulation and cell membrane capacitance (Cm) when cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in Xenopus oocytes. ICl and Cm increased in parallel when oocytes expressing CFTR were stimulated by forskolin (10 microM) and 3-isobutyl-1-methylxanthine (1 mM). The adenosine 3',5'-cyclic monophosphate (cAMP)-induced increase in surface area detected by Cm was confirmed by morphometry in the same oocytes used for the electrical recordings. These increases in ICl and Cm were reversible and were absent from control oocytes not injected with CFTR cRNA. The time to reach peak ICl lagged slightly behind the peak in Cm. ICl was varied by altering CFTR expression level or agonist dose or by expressing different CFTR mutants. In all cases, there was a close correlation between ICl and Cm, and the kinetics of ICl and Cm stimulation were more rapid the larger the magnitude of the stimulated current. The Cm-ICl relation for wild-type CFTR saturated, consistent with a limited capacity of cells to increase their surface area. These results indicate that stimulation of the CFTR ICl is linked closely to increases in membrane area. This suggests that CFTR is present in the membrane vesicles whose insertion is stimulated by cAMP. The contents of these vesicles may provide a link between activation of CFTR and its cAMP-dependent regulation of other channels.
Ischemia-induced alterations in sarcoplasmic reticulum Ca(2+)-ATPase activity in rat soleus and EDL muscles
Authors: Green H. J. , McKee N. H. , Carvalho A. J. , Dossett-Mercer J. C.
Published: December 2017

To investigate the time-dependent effects of ischemia, as modified by muscle fiber type composition, on sarcoplasmic reticulum (SR) function, Ca(2+)-ATPase activity (total minus basal) was measured in homogenates prepared from samples obtained from rat soleus and extensor digitorum longus (EDL) muscle of ischemic and contralateral controls. Ischemia was induced by occlusion of blood flow to one hindlimb for periods of 1, 2, and 3 h (n = 10 per group). In EDL, maximal Ca(2+)-ATPase activity (expressed in mumol.g wet wt-1.min-1) was higher (P < 0.05) in ischemic than in control at 1 h (80 +/- 10 vs. 56.5 +/- 5.3) and increased progressively with ischemia at both 2 h (88 +/- 4.6 vs. 53.1 +/- 2.8) and 3 h (116 +/- 3.8 vs. 67.8 +/- 3.2). In contrast, in soleus, increases (P < 0.05) in Ca(2+)-ATPase activity with ischemia were observed at 2 h (19.2 +/- 0.86 vs. 14.0 +/- 0.56) and 3 h (19.9 +/- 1.4 vs. 12.4 +/- 0.62) but not at 1 h (10.7 +/- 1.5 vs. 10.0 +/- 0.83). In both EDL and soleus, basal Mg(2+)-ATPase was unchanged with ischemia. On the basis of these findings, it can be concluded that ischemia results in an increase in the maximal SR Ca(2+)-ATPase activity but that the time course of the change is dependent on the fiber type composition of the muscle.
Differential regulation of connexin43 and connexin37 in endothelial cells by cell density, growth, and TGF-beta1
Authors: Larson D. M. , Wrobleski M. J. , Sagar G. D. , Westphale E. M. , Beyer E. C.
Published: December 2017

We studied the growth-related expression of the gap junction proteins connexin43 (Cx43) and connexin37 (Cx37) to characterize mechanisms of their differential regulation in cultured bovine aortic endothelial cells. During growth to confluency, Cx43 mRNA levels were high in subconfluent cells and decreased at confluency; Cx37 mRNA was weakly detectable until the cultures became confluent, when Cx37 levels became similar to those of Cx43. Immunoprecipitation, immunoblots, and immunostaining demonstrated that Cx43 synthesis and content paralleled the changes in mRNA levels. These data suggested regulation of connexin expression related to growth status or cell density. We tested this hypothesis by inhibiting growth with transforming growth factor-beta1 (TGF-beta1). TGF-beta1 treatment caused an upregulation of Cx43 synthesis, content, and apparent half-life and an upregulation of mRNA, independent of changes in cell density. Increases in Cx43 synthesis preceded increases in mRNA, suggesting both translational and transcriptional regulation, whereas the increased half-life suggested post-translational regulation, as well. Immunostaining revealed the development of intense vesicular staining in the treated cells, which may explain the increased half-life. TGF-beta1 treatment also suppressed the upregulation of Cx37 expression. These alterations in connexin expression may have implications for endothelial communication under conditions of elevated vascular TGF-beta1 concentrations such as in wound healing.
Interleukin-1beta stimulates phospholipase A2 activity in adult rat ventricular myocytes
Authors: McHowat J. , Liu S.
Published: December 2017

We have examined whether interleukin (IL)-1beta modulates phospholipase A2 (PLA2) activity in ventricular myocytes. PLA2 activity was measured in isolated membrane and cytosol fractions with (16:0,[3H]18:1) plasmenylcholine and (16:0,[3H]18:1) phosphatidylcholine in the absence and presence of Ca2+. When measured in the absence of Ca2+ with plasmenylcholine, exposure to 5 ng/ml IL-1beta caused an increase in membrane-associated PLA2 activity for 10 min that returned to basal levels by 20 min. In the presence of Ca2+ with phosphatidylcholine, IL-1beta had no effect on membrane-associated PLA2 but decreased cytosolic PLA2 activity. Additionally, IL-1beta caused an increase in arachidonic acid release in 20 min. Pretreatment with E-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one, a selective Ca2+-independent PLA2 inhibitor, blocked IL-1beta-induced increases in both PLA2 activity and arachidonic acid release. Exposure to IL-1 receptor antagonist (IL-1RA) alone had no effect on membrane-associated PLA2 activity. When incubated with IL-1beta, IL-1RA inhibited the IL-1beta-enhanced PLA2 activity. These results show that, via activation of its receptors, IL-1beta stimulates specifically membrane-associated Ca2+-independent plasmalogen-selective PLA2 in rat ventricular myocytes.
Different contributions of L- and Q-type Ca2+ channels to Ca2+ signals and secretion in chromaffin cell subtypes
Authors: Lomax R. B. , Michelena P. , Nunez L. , Garcia-Sancho J. , Garcia A. G. , Montiel C.
Published: December 2017

In this study, we investigated the contribution of different subtypes of voltage-dependent Ca2+ channels to changes in cytosolic free Ca2+ ([Ca2+]i) and secretion in noradrenergic and adrenergic bovine chromaffin cells. In single immunocytochemically identified chromaffin cells, [Ca2+]i increased transiently during high K+ depolarization. Furnidipine and BAY K 8644, L-type Ca2+ channel blocker and activator, respectively, affected the [Ca2+]i rise more in noradrenergic than in adrenergic cells. In contrast, the Q-type Ca2+ channel blocker omega-conotoxin MVIIC inhibited the [Ca2+]i rise more in adrenergic cells. omega-Agatoxin IVA (30 nM), which blocks P-type Ca2+ channels, had little effect on the [Ca2+]i signal. The N-type Ca2+ channel blocker omega-conotoxin GVIA similarly inhibited the [Ca2+]i rise in both cell types. The effects of furnidipine, BAY K 8644, and omega-conotoxin MVIIC on K+-evoked norepinephrine and epinephrine release paralleled those effects on [Ca2+]i signals. However, omega-conotoxin GVIA and 30 nM omega-agatoxin IVA did not affect the secretion of either amine. The data suggest that, in the bovine adrenal medulla, the release of epinephrine and norepinephrine are preferentially controlled by Q- and L-type Ca2+ channels, respectively. P- and N-type Ca2+ channels do not seem to control the secretion of either catecholamine.
Effects of aging on shortening velocity and myosin isoform composition in single human skeletal muscle cells
Authors: Larsson L. , Li X. , Frontera W. R.
Published: December 2017

Maximum unloaded shortening velocity (V0) and maximum force normalized to cross-sectional fiber area (specific tension) were determined in 400 single quadriceps muscle cells of young (n = 4, 25-31 yr) and old (n = 4, 73-81 yr) men. Two of the old men were physically very active, and the subjects were divided into young control, old control, and old physically active groups. The expressions of types I, IIa, and IIb myosin heavy chains (MHC) and essential and regulatory myosin light chains were determined by 6 and 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. V0 was highly dependent on the MHC isoform composition, and a gradient from slow to fast was observed in the sequence I, I-IIa, IIa, IIab, and IIb MHC cells. The V0 values and specific tensions of types I and IIa MHC fibers in the young control group were significantly higher than those in the old control and old physically active groups. Thus the present results provide evidence of qualitative changes in contractile properties of human skeletal muscle in old age, which probably play an important role in the age-related impairment of skeletal muscle function.
Activation of the ubiquitin pathway in rat skeletal muscle by catabolic doses of glucocorticoids
Authors: Auclair D. , Garrel D. R. , Chaouki Zerouala A. , Ferland L. H.
Published: December 2017

To evaluate whether catabolic levels of glucocorticoids activate the ubiquitin pathway in conjunction with their known proteolytic effect in skeletal muscle, rats were injected daily with corticosterone (CTC; 10 mg/100 g body wt) for 7 days. Two peaks of urinary excretion of 3-methylhistidine (3-MH), a specific marker of myofibrillar proteolysis, were observed at days 1 and 3 (165 and 295% of controls, respectively). Levels of ubiquitin pathway mRNAs in skeletal muscle were assessed around the 3-MH peaks. In the extensor digitorum longus, a first rise of two polyubiquitin (pUb) mRNAs was seen at day 1 (183 and 162% of control for the UbB and UbC transcripts, respectively, P < 0.01). An accumulation of both E2-14k mRNAs (140%, P < 0.02, and 157% of controls, P < 0.01) and proteasome C8 subunit mRNA (222% of control, P < 0.05) was seen at day 2. A second more important peak of induction of pUb mRNA was seen at day 3 (251 and 217% of controls for the UbB and UbC transcripts, respectively, P< 0.001). All transcripts returned to near control levels by day 4. In the soleus, induction of E2-14k mRNA started at day 3 and reached 216 and 208% of controls at day 4 (P < 0.001), whereas an increase of pUb mRNA was observed at days 3 (213 and 241%, P < 0.05) and 4 (211 and 221%, P < 0.001). A rise of proteasome C8 subunit mRNA accumulation was also seen in the soleus at days 3 (217%, P< 0.05) and 4 (157%, P < 0.05). Reduced ubiquitin conjugate levels, possibly due to their rapid degradation through increased proteasome activity, were observed in both muscle types at day 3. The parallel between the catabolic effects of CTC and activation of the ubiquitin pathway in muscles of CTC-treated rats strongly suggests the involvement of this system in glucocorticoid-induced muscular atrophy.
Four-dimensional imaging of living chondrocytes in cartilage using confocal microscopy: a pragmatic approach
Authors: Errington R. J. , Fricker M. D. , Wood J. L. , Hall A. C. , White N. S.
Published: December 2017

Regulation of cell volume is a fundamental cellular homeostatic mechanism in the face of osmotic stress. In normal articular cartilage, chondrocytes are exposed to a changing osmotic environment. We present a comprehensive protocol for studying the volume regulatory behavior of chondrocytes within intact cartilage tissue using confocal laser-scanning microscopy. Our data acquisition regime optimizes both signal-to-noise and cell viability during time-lapsed three-dimensional (3-D) (x, y, z, t) imaging. The porcine cartilage is treated as an integrated component of the imaging system, and we demonstrate methods for the direct assessment of tissue-induced axial attenuation and image distortion. Parameterized functions describing these two components of image degradation are used to correct experimental data. The current study also highlights the problems associated with the analysis and visualization of four-dimensional (4-D) images. We have devised two new types of data reconstruction. The first compresses each 3-D time point into a single quantitative view, termed a coordinate view. From these reconstructions we are able to simultaneously view and extract cell measurements. A second type, a 4-D reconstruction, uses color to represent relative changes in cell volume, again while maintaining the morphological and spatial information. Both these approaches of image analysis and visualization have been implemented to study the morphology, spatial distribution, and dynamic volume behavior of chondrocytes after osmotic perturbation. We have mapped chondrocyte shape, arrangement, and absolute volume in situ, which vary significantly from the tissue surface through to the underlying bone. Despite the rigid nature of the extracellular matrix, cartilage cells are osmotically sensitive and respond to stimulation of volume regulatory mechanisms. The combined techniques of confocal laser-scanning microscopy and vital cell labeling have enabled us to study, for the first time, the response of chondrocytes in situ to changes in interstitial osmotic pressure.
Effects of arachidonic acid on A-type potassium currents in smooth muscle cells of the guinea pig
Authors: Nagano N. , Imaizumi Y. , Watanabe M.
Published: December 2017

Effects of arachidonic acid (AA) and related fatty acids on Ca2+ -independent transient (A-type) K+ current (I(A)) were examined in single myocytes of guinea pig vas deferens, ureter, and proximal colon as well as in rabbit vas deferens. The peak amplitude of I(A) was reduced by external application of AA (half-maximal inhibitory concentration = approximately 1 microM). The blocking effect was not changed significantly by indomethacin, nordihydroguaiaretic acid, guanosine 5'-O-(2-thiodiphosphate), or guanosine 5'-O-(3-thiotriphosphate). Pharmacological studies suggested that the effect of AA was not mediated by activation of protein kinases A or C or tyrosine kinase. AA (20:4) was the most potent of the four types of cis-eicosanoic acids with two to five double bonds (20:2 to 20:5) that were tested. I(A)-like current in cardiac atrial myocytes of the rabbit was not affected significantly by 30 microM AA. These results indicate that AA itself directly blocks A-type K+ channels. A relationship between stereospecific chemical structure of fatty acids and their blockade of A-type K+ channels is suggested. A-type K+ channels in smooth muscle cells can be clearly resolved from those in atrial myocytes by the responses to AA.
Colonic epithelium-enriched protein A4 is a proteolipid that exhibits ion channel characteristics
Authors: Breitwieser G. E. , McLenithan J. C. , Cortese J. F. , Shields J. M. , Oliva M. M. , Majewski J. L. , Machamer C. E. , Yang V. W.
Published: December 2017

Expression of the human gene A4 is enriched in the colonic epithelium and is transcriptionally activated on differentiation of colonic epithelial cells in vitro (M. M. Oliva, T. C. Wu, and V. W. Yang. Arch. Biochem. Biophys. 302: 183-192, 1993). A4 cDNA contains an open reading frame that predicts a polypeptide of 17 kDa. To determine the function of the A4 protein, we characterized its biochemical and physiological properties. Hydropathy analysis of deduced A4 amino acid sequence revealed four putative membrane-spanning alpha-helices. The hydrophobic nature of A4 was confirmed by its being extractable with organic solvents. Immunocytochemical studies of cells expressing A4 localized it to the endoplasmic reticulum. Moreover, A4 multimerized in vivo as determined by coimmunoprecipitation experiments. The four-transmembrane topology and biophysical characteristics of A4 suggest that it belongs to a family of integral membrane proteins called proteolipids, some of which multimerize to form ion channels. Subsequent electrophysiological studies of nuclei isolated from microinjected Xenopus laevis oocytes transiently expressing A4 showed the appearance of a 28-pS channel. Thus our studies indicate that A4 is a colonic epithelium-enriched protein localized to the endoplasmic reticulum and that, similar to other proteolipids, A4 multimerizes and exhibits characteristics of an ion channel.
Thrombin modulates vectorial secretion of extracellular matrix proteins in cultured endothelial cells
Authors: Papadimitriou E. , Manolopoulos V. G. , Hayman G. T. , Maragoudakis M. E. , Unsworth B. R. , Fenton J. W. , Lelkes P. I.
Published: December 2017

We have identified a novel cellular action of thrombin on cultured rat adrenal medullary endothelial cells (RAMEC). Five-minute incubation of RAMEC with physiological concentrations of thrombin (<1 U/ml) caused within 3 h an increase in the basolateral deposition of the extracellular matrix (ECM) proteins fibronectin, laminin, and collagens IV and I, concomitant with a corresponding decrease in the apical release of these proteins into the medium. This shift in vectorial secretion of ECM proteins, quantitated with enzyme-linked immunoassays, was time dependent. Maximal stimulation of ECM protein deposition was observed after incubation of cells with thrombin for 5-15 min. Prolonged exposure (>1 h) to thrombin resulted in loss of proteins from the ECM. Thrombin-stimulated ECM protein deposition exhibited a bell-shaped dose dependence, peaking for all proteins at 0.25 U/ml of thrombin, and was independent of de novo mRNA or protein synthesis. Maximal amounts of deposited proteins increased between 2.5-fold (fibronectin) and 4-fold (collagen I) over baseline values. Similar results were obtained with thrombin receptor agonist peptide (TRAP), proteolytically active gamma-thrombin, and, to a lesser extent, other serine proteases such as trypsin and plasmin. A scrambled TRAP, proteolytically inactive PPACK-thrombin, DIP-thrombin, and type IV collagenase were ineffective. Together, these results suggest that the thrombin effects are mediated by proteolytic activation of the thrombin receptor. Possible involvement of the phospholipase C-signaling pathway in thrombin-mediated ECM protein deposition was also investigated. Inhibition or downregulation of protein kinase C (PKC) and chelation of intracellular or extracellular Ca2+ did not suppress, but rather enhanced, basal and thrombin-stimulated ECM protein deposition. Quantitative differences in augmentation of basolateral deposition by these treatments suggest differential regulatory pathways for individual ECM proteins. Our data indicate that, in cultured RAMEC, short-term activation of the thrombin receptor causes an increase in amounts of deposited ECM protein by a cellular signaling pathway that is independent of PKC activation and/or elevation of intracellular Ca2+.
Modulation of voltage-dependent Ca2+ conductance by changing Cl- concentration in rat lactotrophs
Authors: Garcia L. , Fahmi M. , Prevarskaya N. , Dufy B. , Sartor P.
Published: December 2017

In pituitary cells, voltage-dependent Ca2+ channels play an important role in such physiological processes as exocytosis, secretion, the cell cycle, and proliferation. Thus mechanisms that modulate voltage-dependent Ca2+ channel activity participate indirectly in regulating intracellular Ca2+ concentration. We have shown a new modulating mechanism for voltage-dependent Ca2+ channels by demonstrating that Ca2+ influx is influenced by Cl-. To evaluate the role of Cl- on Ca2+ conductance coupling, we first measured the intracellular Cl- concentration of rat lactotrophs using the Cl(-)-sensitive fluorescence probe sulfopropylquinolinium by simple microspectrofluorometry or combined with electrophysiology. We found an average intracellular Cl- concentration of rat lactotrophs of approximately 60 mM (n = 39). Using the whole cell tight-seal recording technique, we showed that a reduction in external Cl- concentration ([Cl-]o) and a decrease in Cl- conductances affected Ca2+ conductance as measured by Ba2+ movement through the Ca2+ channels (I(Ba)). Low [Cl-]o (39 mM) induced a decrease in Ca2+ entry via voltage-gated Ca2+ channels (-27.75 +/- 4% of normalized I(Ba)). Similarly, blockade of the Cl- conductance by 1 mM 9-anthracene carboxylic acid induced a decrease in I(Ba) (-26 +/- 6% of normalized I(Ba)). This modulation of I(Ba) was inhibited by 24-h pretreatment of the cells with pertussis toxin (1 microg/ml), suggesting that changes in Cl- concentration induced by low [Cl-]o and 9-anthracene carboxylic acid interfered with the phosphorylation of G proteins involved in Ca2+ channel activation. These results suggest a feedback mechanism based on constant interaction between Ca2+ and Cl-. Finally, they also emphasize the physiological role of Cl- in rat lactotrophs.
A caffeine-sensitive Ca2+ store modulates K+-evoked secretion in chromaffin cells
Authors: Lara B. , Lopez M. G. , Villarroya M. , Gandia L. , Cleeman L. , Morad M. , Garcia A. G.
Published: December 2017

Catecholamine release from bovine adrenal medulla chromaffin cells superfused with a Krebs-N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid solution was monitored on-line with an electrochemical detector. Caffeine (10 mM) progressively depressed the magnitude of secretory responses to depolarizing pulses of 70 mM K+ and 2 mM Ca2+ (70 K+/2 Ca2+) in cells superfused with a Krebs-N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid solution containing 0 mM Ca2+ + 0.5 mM EGTA; blockade reached 80% at the third 70 K+/2 Ca2+ challenge given in the presence of caffeine. A similar effect was obtained when, instead of continuous superfusion, prepulses of caffeine were applied (10 mM for 60 s). The blocking effects of caffeine on K+-induced secretion depended on the time of exposure to the drug: the longer the exposure time the greater the blockade. The recovery of the K+ secretory responses previously impaired by caffeine was always gradual and followed a staircase mode. This contrasts with the effects of caffeine on various parameters measuring Ca2+ entry through Ca2+ channels, which did not parallel its effects on K+-evoked secretion. The secretion data, however, are compatible with the disappearance and recovery of an intracellular Ca2+ concentration signal triggered by K+ in single chromaffin cells loaded with fura 2 and treated with 10 mM caffeine. Thus, contrary to previous views, the depression of secretion by caffeine does not seem to be associated with inhibition of extracellular Ca2+ entry through Ca2+ channels. These functional data are, rather, compatible with the view that the degree of filling of a caffeine-sensitive intracellular Ca2+ store might regulate the extent of exocytosis. When emptied, such a store might act as a sink for the external Ca2+ entering through Ca2+ channels during cell depolarization, thus decreasing the intracellular Ca2+ concentration available for exocytosis.
Expression of CFTR in human and bovine thyroid epithelium
Authors: Devuyst O. , Golstein P. E. , Sanches M. V. , Piontek K. , Wilson P. D. , Guggino W. B. , Dumont J. E. , Beauwens R.
Published: December 2017

The expression of cystic fibrosis transmembrane conductance regulator (CFTR) in the thyroid has not been documented to date, although a role for CFTR in the thyroid follicular epithelium is suggested both clinically, by the occurrence of subclinical hypothyroidism in patients with cystic fibrosis (CF), and physiologically, by the presence of low-conductance, adenosine 3',5'-cyclic monophosphate-activated Cl channels in the follicular cells. Using reverse transcriptase-polymerase chain reaction with nested primers derived from exons 13 and 14 of the human CF gene, we have now documented the presence of CFTR mRNA in the human thyroid. Western blot analyses using six antibodies directed against different domains of human CFTR showed that a 165-kDa band was present in membrane extracts from bovine and human thyroid. This protein has the predicted size of mature CFTR and was not detected with preimmune serum or preadsorbed antiserum. By immunofluorescence and immunoperoxidase, CFTR was located in the follicular cells, with a diffuse, intracellular labeling pattern. Quantitative analysis revealed that 64% of the follicles were CFTR positive, but only 16% of the follicular cells were stained per follicle. The number of CFTR-positive cells was inversely proportional to the size of the follicle. These results 1) demonstrate the expression of CFTR at the mRNA and protein levels in human and bovine thyroid follicular cells and 2) suggest that CFTR expression could be instrumental in follicular enlargement.
Loss of cytoskeletal support is not sufficient for anoxic plasma membrane disruption in renal cells
Authors: Chen J. , Dai J. , Grant R. L. , Doctor R. B. , Sheetz M. P. , Mandel L. J.
Published: December 2017

The goal of this study was to determine whether anoxic membrane disruption is initiated by loss of cytoskeletal support in rabbit renal proximal tubules (PT). We specifically tested 1) whether cytoskeletal perturbation affects membrane integrity under normoxia, 2) whether cytoskeletal perturbation potentiates anoxic membrane damage, and 3) whether the membrane protection by glycine depends on cytoskeletal integrity. Cytoskeletal perturbation was achieved with 10 microM cytochalasin D (CD) because it selectively disturbs F-actin organization and has similar effects as anoxia on the cytoskeleton of PT. During normoxia, CD caused decreased basal F-actin content, microvillar breakdown, and membrane-cytoskeleton dissociation, as revealed by the use of laser tweezers. However, membrane integrity was not altered by CD, as monitored by lactate dehydrogenase release. CD pretreatment of PT did not potentiate anoxic membrane damage. Finally, plasma membrane protection by glycine during anoxia remained in CD-pretreated PT despite loss of cytoskeletal support. These results demonstrate that loss of cytoskeletal support is not sufficient for anoxic plasma membrane disruption.
F-actin disruption attenuates agonist-induced [Ca2+], myosin phosphorylation, and force in smooth muscle
Authors: Tseng S. , Kim R. , Kim T. , Morgan K. G. , Hai C. M.
Published: December 2017

Cytochalasins B and D (at 10 microM) inhibited stress development induced by 1 microM carbachol in bovine tracheal smooth muscle by 55% and 90%, respectively. Glucose depletion was ineffective in inhibiting carbachol-induced contraction, indicating that inhibition of glucose transport was not the cause. Cytochalasin D-treated smooth muscle cells appeared collapsed, with spiky protrusions from the cell membrane. Deconvolution of fluorescent images of fluorescein isothiocyanate-phalloidin-labeled smooth muscle cells revealed concentrations of actin filaments near the cell periphery, including near the spiky protrusions. Cytochalasin B attenuated carbachol-induced intracellular Ca2+ concentration ([Ca2+]), especially the initial peak intracellular [Ca2+]. Cytochalasin B also attenuated carbachol-induced myosin light chain phosphorylation. However, when the myosin phosphorylation data were plotted against time-matched intracellular [Ca2+] data, the two relationships in control and cytochalasin B-treated smooth muscle were similar, suggesting that the changes in myosin phosphorylation could be explained by the changes in intracellular [Ca2+]. These results suggest that actin filaments in smooth muscle cells are dynamic and may be an integral component of Ca2+ regulation and/or signal transduction in receptor-coupled mechanisms.
Survival of human epidermal keratinocytes after short-duration high temperature: synthesis of HSP70 and IL-8
Authors: Bowman P. D. , Schuschereba S. T. , Lawlor D. F. , Gilligan G. R. , Mata J. R. , DeBaere D. R.
Published: December 2017

Thermal injury by short pulses (1-30 s) of relatively high temperature (50-68 degrees C) was investigated in normal human epidermal keratinocytes (NHEK). NHEK were cultured on plastic cover-slips and dipped in medium held at various temperatures. Survival assessed by methylthiazol tetrazolium reduction assay at 6 days postheating demonstrated an inverse time-temperature relationship that indicated that most cells could survive after a 1-s, 60 degrees C exposure or a 30-s, 55 degrees C exposure. Arrhenius plots of the data indicated major transition points for cell injury at 50 and 60 degrees C. Heat shock protein 70 (HSP70) and interleukin-8 (IL-8) were both induced by elevation of temperature between 50 and 60 degrees C for as short a time as 1 s. HSP70 synthesis stimulated by short, high pulses of heat appeared to induce thermotolerance. These results demonstrate that brief exposure to relatively high temperature can induce HSP70 and IL-8 synthesis in keratinocytes.
Length-dependent potentiation and myosin light chain phosphorylation in rat gastrocnemius muscle
Authors: Rassier D. E. , Tubman L. A. , MacIntosh B. R.
Published: December 2017

Changes in muscle length affect the degree of staircase potentiation in skeletal muscle, but the mechanism by which this occurs is unknown. In this study, we tested the hypothesis that length-dependent change in staircase is modulated by phosphorylation of the myosin regulatory light chains (RLC), since this is believed to be the main mechanism of potentiation. In situ isometric contractile responses of rat gastrocnemius muscle during 10 s of repetitive stimulation at 10 Hz were analyzed at optimal length (Lo), Lo - 10%, and Lo + 10%. The degree of enhancement of developed tension during 10 s of repetitive stimulation was observed to be length dependent, with increases of 118.5 +/- 7.8, 63.1 +/- 3.9, and 45.6 +/- 4.1% (means +/- SE) at Lo - 10%, Lo, and Lo + 10%, respectively. Staircase was accompanied by increases in the average rate of force development of 105.6 +/- 7.7, 55.6 +/- 4.1, and 37.2 +/- 4.4% for Lo - 10%, Lo, and Lo + 10%, respectively. RLC phosphorylation after 10 s of 10-Hz stimulation was higher than under resting conditions but not different among Lo - 10% (40 +/- 3.5%), Lo (35 +/- 3.5%), and Lo + 10% (41 +/- 3.5%). This study shows that there is a length dependence of staircase potentiation in mammalian skeletal muscle that may not be directly modulated by RLC phosphorylation. Interaction of RLC phosphorylation with length-dependent changes in Ca2+ release and intermyofilament spacing may explain these observations.
Na+/Ca2+ exchanger in Drosophila: cloning, expression, and transport differences
Authors: Ruknudin A. , Valdivia C. , Kofuji P. , Lederer W. J. , Schulze D. H.
Published: December 2017

cDNAs for the Na+/Ca2+ exchanger from Drosophila melanogaster (Dmel/Nck) have been cloned by homology screening using the human heart Na+/Ca2+ exchanger cDNA. The overall deduced protein structure for Dmel/Nck is similar to that of mammalian Na+/Ca2+ exchanger genes NCX1 and NCX2, having six hydrophobic regions in the amino terminus separated from six at the carboxy-terminal end by a large intracellular loop. Sequence comparison of the Drosophila exchanger cDNAs with NCX1 and NCX2 Na+/Ca2+ exchangers are approximately 46% identical at the deduced amino acid level. Consensus phosphorylation sites for both protein kinase C and protein kinase A are present on the intracellular loop region of the Dmel/Nck. Alternative splicing for the Dmel/Nck gene is suggested in the same intracellular loop region as demonstrated for NCX1. Functionally, the Drosophila Na+/ Ca2+ exchanger expressed in oocytes differs from expressed mammalian NCX1 with regard to Ca2+ transport in Ca2+/ Ca2+ exchange and the effect of monovalent-dependent Ca2+/ Ca2+ exchange. The Dmel/Nck gene maps to chromosome 3 (93A-B) using in situ hybridization to polytene chromosomes, the same position as the Na(+)-K(+)-ATPase, a related transporter. We conclude that, although extracellular Na+ concentration-dependent Ca2+ transport is subserved by both human and Drosophila Na+/Ca2+ exchangers, there are clear and important differences in the transporters, which should be useful in deducing how the Na+/Ca2+ exchanger protein function depends on its structure.