bluuebunny/crossref_metadata_2025 huggingface dataset:
Generation of a cell line with smooth muscle phenotype from hypertrophied urinary bladder
Authors: Zheng Yongmu , Weber Wilfried T. , Wang Shuqin , Wein Alan J. , Zderic Stephen A. , Chacko Samuel , DiSanto Michael E.
Published: May 2013

We have established a cell line from hypertrophied rabbit urinary bladder smooth muscle (SM) that stably expresses SM myosin (SMM). These cells, termed BSM, are spindle shaped and form swirls, similar to the “hills and valleys” described for cultured aortic SM cells. Western blotting revealed that BSM expresses the amino-terminal SMM heavy chain isoform SM-B, the carboxy-terminal SM1 and SM2 isoforms, and SM α-actin. In addition, they express cGMP-dependent protein kinase G, made by contractile SM cells in vitro but not by noncontractile cells synthesizing extracellular matrix. Immunofluorescence studies indicate a homogeneous population of cells expressing α-actin and SMM, including the SM-B isoform, and karyotyping demonstrates a stable 4N chromosomal pattern. These cells also express calcium-dependent myosin light chain kinase and phosphatase activity and contract in response to the muscarinic agonist bethanechol. To our knowledge, BSM is the first visceral SM cell line that expresses the SM-B isoform and might serve as a useful model to study the transcriptional regulation of tissue-specific SMM isoforms in differentiation and pathological SM.
Role of gap junctions in fluid secretion of lacrimal glands
Authors: Walcott Benjamin , Moore Leon C. , Birzgalis Aija , Claros Nidia , Valiunas Virginijus , Ott Thomas , Willecke Klaus , Brink Peter R.
Published: May 2013

In glands such as the liver and pancreas, gap junctions containing connexin 26 and 32 (Cx26 and Cx32, respectively) couple the secretory cells. Uncoupling these junctions compromises the secretory function of these glands. Lacrimal glands also contain extensive arrays of gap junctions consisting of Cx26 and Cx32. We wanted to determine the role of these junctions in fluid secretion. In Cx32-deficient mice, immunocytochemistry showed that, in the male lacrimal gland, the remaining Cx26 was found evenly distributed in the membrane whereas there was little in the membranes of female glands. Western blot analysis of Cx26 showed that female Cx32-deficient mice expressed Cx26. Patch-clamp analyses of acinar cell coupling showed that the cell pairs from male glands were coupled whereas those from female glands were not. Stimulated fluid production by the glands from Cx32-deficient mice was abnormally low in female glands compared with controls at low topical doses of carbachol. The protein secretory response to different doses of carbachol was the same in all animals. These data suggest that gap junctions are essential for optimal fluid secretion in lacrimal glands.
Force generation, but not myosin ATPase activity, declines with age in rat muscle fibers
Authors: Lowe Dawn A. , Thomas David D. , Thompson LaDora V.
Published: May 2013

We tested the hypothesis that age-associated decline in muscle function is related to a change in myosin ATPase activity. Single, glycerinated semimembranosus fibers from young (8–12 mo) and aged (32–37 mo) Fischer 344 × Brown Norway male rats were analyzed simultaneously for force and myosin ATPase activity over a range of Ca2+ concentrations. Maximal force generation was ∼20% lower in fibers from aged animals ( P = 0.02), but myosin ATPase activity was not different between fibers from young and aged rats: 686 ± 46 ( n = 30) and 697 ± 46 μM/s ( n = 33) ( P = 0.89). The apparent rate constant for the dissociation of strong-binding myosin from actin was calculated to be ∼30% greater in fibers from aged animals ( P = 0.03), indicating that the lower force produced by fibers from aged animals is due to a greater flux of myosin heads from the strong-binding state to the weak-binding state during contraction. This is in agreement with our previous electron paramagnetic resonance experiments that showed a reduced fraction of myosin heads in the strong-binding state during a maximal isometric contraction in fibers from older rats.
Coordinate upregulation of guanylin and uroguanylin expression by hypertonicity in HT29-18-N2 cells
Authors: Steinbrecher Kris A. , Rudolph Jeffrey A. , Luo Guangju , Cohen Mitchell B.
Published: May 2013

Guanylin and uroguanylin are particulate guanylate cyclase-activating peptides that are secreted from the epithelia of the intestine, kidney, pancreas, and salivary gland. These peptides elicit chloride and bicarbonate secretion via the cystic fibrosis transmembrane conductance regulator. To test the hypothesis that hypertonicity mediates an increase in guanylin and uroguanylin mRNA, we subjected HT29-18-N2 to osmotic stress. Guanylin and uroguanylin RNA were increased substantially in the presence of hypertonicity but only with solutes that were relatively impermeable to the cell membrane. This hypertonicity-mediated increase was transcriptional and did not require protein synthesis. Herbimycin A and mitogen-activated protein kinase inhibitors SB-203580 and PD-98059 had no effect on basal or induced levels of guanylin or uroguanylin. Both staurosporine and prolonged exposure to phorbol ester reduced basal levels and completely blocked hypertonicity-related increases in guanylin or uroguanylin RNA. These data suggest that serine/theonine protein kinases, possibly protein kinase C (PKC), mediate the hypertonicity-associated increase in guanylin and uroguanylin RNA. We conclude that guanylin and uroguanylin are released in response to hypertonic stress and that regulation of these genes may be mediated by PKC isoforms.
Inhibition of fibroblast proliferation in cardiac myocyte cultures by surface microtopography
Authors: Boateng Samuel Y. , Hartman Thomas J. , Ahluwalia Neil , Vidula Himabindu , Desai Tejal A. , Russell Brenda
Published: May 2013

Cardiac myocyte cultures usually require pharmacological intervention to prevent overproliferation of contaminating nonmyocytes. Our aim is to prevent excessive fibroblast cell proliferation without the use of cytostatins. We have produced a silicone surface with 10-μm vertical projections that we term “pegs,” to which over 80% of rat neonatal cardiac fibroblasts attach within 48 h after plating. There was a 50% decrease in cell proliferation by 5 days of culture compared with flat membranes ( P < 0.001) and a concomitant 60% decrease ( P < 0.01) in cyclin D1 protein levels, suggesting a G1/S1 cell cycle arrest due to microtopography. Inhibition of Rho kinase with 5 or 20 μM Y-27632 reduced attachment of fibroblasts to the pegs by over 50% ( P < 0.001), suggesting that this signaling pathway plays an important role in the process. Using mobile and immobile 10-μm polystyrene spheres, we show that reactive forces are important for inhibiting fibroblast cell proliferation, because mobile spheres failed to reduce cell proliferation. In primary myocyte cultures, pegs also inhibit fibroblast proliferation in the absence of cytostatins. The ratio of aminopropeptide of collagen protein from fibroblasts to myosin from myocytes was significantly reduced in cultures from pegged surfaces ( P < 0.01), suggesting an increase in the proportion of myocytes on the pegged surfaces. Connexin43 protein expression was also increased, suggesting improved myocyte-myocyte interaction in the presence of pegs. We conclude that this microtextured culture system is useful for preventing proliferation of fibroblasts in myocyte cultures and may ultimately be useful for tissue engineering applications in vivo.
Mechanism and role of high-potassium-induced reduction of intracellular Ca2+ concentration in rat osteoclasts
Authors: Kajiya Hiroshi , Okamoto Fujio , Fukushima Hidefumi , Takada Keisuke , Okabe Koji
Published: May 2013

Osteoclasts are multinucleated, bone-resorbing cells that show structural and functional differences between the resorbing and nonresorbing (motile) states during the bone resorption cycle. In the present study, we measured intracellular Ca2+ concentration ([Ca2+]i) in nonresorbing vs. resorbing rat osteoclasts. Basal [Ca2+]i in osteoclasts possessing pseudopodia (nonresorbing/motile state) was around 110 nM and significantly higher than that in actin ring-forming osteoclasts (resorbing state, around 50 nM). In nonresorbing/motile osteoclasts, exposure to high K+ reduced [Ca2+]i, whereas high K+ increased [Ca2+]i in resorbing state osteoclasts. In nonresorbing/motile cells, membrane depolarization and hyperpolarization applied by the patch-clamp technique decreased and increased [Ca2+]i, respectively. Removal of extracellular Ca2+ or application of 300 μM La3+ reduced [Ca2+]i to ∼50 nM in nonresorbing/motile osteoclasts, and high-K+-induced reduction of [Ca2+]i could not be observed under these conditions. Neither inhibition of intracellular Ca2+ stores or plasma membrane Ca2+ pumps nor blocking of L- and N-type Ca2+ channels significantly reduced [Ca2+]i. Exposure to high K+ inhibited the motility of nonresorbing osteoclasts and reduced the number of actin rings and pit formation in resorbing osteoclasts. These results indicate that in nonresorbing/motile osteoclasts, a La3+-sensitive Ca2+ entry pathway is continuously active under resting conditions, keeping [Ca2+]i high. Changes in membrane potential regulate osteoclastic motility by controlling the net amount of Ca2+ entry in a “reversed” voltage-dependent manner, i.e., depolarization decreases and hyperpolarization increases [Ca2+]i.
Modulation of BKCa channel activity by fatty acids: structural requirements and mechanism of action
Authors: Clarke Alison L. , Petrou Steven , Walsh John V. , Singer Joshua J.
Published: May 2013

To determine the mechanism of fatty acid modulation of rabbit pulmonary artery large-conductance Ca2+-activated K+(BKCa) channel activity, we studied effects of fatty acids and other lipids on channel activity in excised patches with patch-clamp techniques. The structural features of the fatty acid required to increase BKCa channel activity (or average number of open channels, NP o) were identified to be the negatively charged head group and a sufficiently long (C > 8) carbon chain. Positively charged lipids like sphingosine, which have a sufficiently long alkyl chain (C ≥ 8), produced a decrease in NP o. Neutral and short-chain lipids did not alter NP o. Screening of membrane surface charge with high-ionic-strength bathing solutions (330 mM K+ or 130 mM K+, 300 mM Na+) did not alter the modulation of the BKCa channel NP oby fatty acids and other charged lipids, indicating that channel modulation is unlikely to be due to an alteration of the membrane electric field or the attraction of local counterions to the channel. Fatty acids and other negatively charged lipids were able to modulate BKCa channel activity in bathing solutions containing 0 mM Ca2+, 20 mM EGTA, suggesting that calcium is not required for this modulation. Together, these results indicate that modulation of BKCa channels by fatty acids and other charged lipids most likely occurs by their direct interaction with the channel protein itself or with some other channel-associated component.
A novel method to quantify the turnover and release of monocytes from the bone marrow using the thymidine analog 5′-bromo-2′-deoxyuridine
Authors: Goto Yukinobu , Hogg James C. , Suwa Tatsushi , Quinlan Kevin B. , van Eeden Stephan F.
Published: May 2013

The present study was designed to develop methods to study the production and release of monocytes from the bone marrow using the thymidine analog 5′-bromo-2′-deoxyuridine (BrdU). Dividing monocytes in bone marrow were labeled with BrdU (MOBrdU), and their release into the blood and disappearance from the circulation were monitored using a double immunostaining method. The first MOBrdU appeared in the circulation 4 h after labeling with BrdU and peaked at 18 h when 34.3 ± 5.8% of monocytes were labeled. The calculated transit time of monocytes through bone marrow was 38.1 ± 3.1 h in control rabbits with a half-life ( T 1/2) of 12.7 h. Instillation of Streptococcus pneumoniae into the lung accelerated the release of monocytes from bone marrow (peak at 10 h) and shortened their bone marrow transit time (27.1 ± 1.8 vs. 22.6 ± 0.6, vehicle vs. pneumonia; P < 0.05). We conclude that this nonradioisotope method provides a novel way to monitor monocyte kinetics and confirmed previous reports that a focal pneumonia shortens monocyte marrow transit and increases their release into the circulation.
Intracellular calcium measurements as a method in studies on activity of purinergic P2X receptor channels
Authors: He Mu-Lan , Zemkova Hana , Koshimizu Taka-aki , Tomić Melanija , Stojilkovic Stanko S.
Published: May 2013

Extracellular nucleotide-activated purinergic receptors (P2XRs) are a family of cation-permeable channels that conduct small cations, including Ca2+, leading to the depolarization of cells and subsequent stimulation of voltage-gated Ca2+ influx in excitable cells. Here, we studied the spatiotemporal characteristics of intracellular Ca2+ signaling and its dependence on current signaling in excitable mouse immortalized gonadotropin-releasing hormone-secreting cells (GT1) and nonexcitable human embryonic kidney cells (HEK-293) cells expressing wild-type and chimeric P2XRs. In both cell types, P2XR generated depolarizing currents during the sustained ATP stimulation, which desensitized in order (from rapidly desensitizing to nondesensitizing): P2X3R > P2X2b + X4R > P2X2bR > P2X2a + X4R > P2X4R > P2X2aR > P2X7R. HEK-293 cells were not suitable for studies on P2XR-mediated Ca2+ influx because of the coactivation of endogenously expressed Ca2+-mobilizing purinergic P2Y receptors. However, when expressed in GT1 cells, all wild-type and chimeric P2XRs responded to agonist binding with global Ca2+ signals, which desensitized in the same order as current signals but in a significantly slower manner. The global distribution of Ca2+ signals was present independently of the rate of current desensitization. The temporal characteristics of Ca2+ signals were not affected by voltage-gated Ca2+ influx and removal of extracellular sodium. Ca2+ signals reflected well the receptor-specific EC50 values for ATP and the extracellular Zn2+ and pH sensitivities of P2XRs. These results indicate that intracellular Ca2+ measurements are useful for characterizing the pharmacological properties and messenger functions of P2XRs, as well as the kinetics of channel activity, when the host cells do not express other members of purinergic receptors.
Molecular identification of Ca2+-activated K+ channels in parotid acinar cells
Authors: Nehrke Keith , Quinn Claire C. , Begenisich Ted
Published: May 2013

We used molecular biological and patch-clamp techniques to identify the Ca2+-activated K+ channel genes in mouse parotid acinar cells. Two types of K+ channels were activated by intracellular Ca2+ with single-channel conductance values of 22 and 140 pS (in 135 mM external K+), consistent with the intermediate and maxi-K classes of Ca2+-activated K+ channels, typified by the mIK1 ( Kcnn4) and mSlo ( Kcnma1) genes, respectively. The presence of mIK1 mRNA was established in acinar cells by in situ hybridization. The electrophysiological and pharmacological properties of heterologously expressed mIK1 channels matched those of the native current; thus the native, smaller conductance channel is likely derived from the mIK1 gene. We found that parotid acinar cells express a single, uncommon splice variant of the mSlo gene and that heterologously expressed channels of this Slo variant had a single-channel conductance indistinguishable from that of the native, large-conductance channel. However, the sensitivity of this expressed Slo variant to the scorpion toxin iberiotoxin was considerably different from that of the native current. RT-PCR analysis revealed the presence of two mSlo β-subunits ( Kcnmb1 and Kcnmb4) in parotid tissue. Comparison of the iberiotoxin sensitivity of the native current with that of parotid mSlo expressed with each β-subunit in isolation and measurements of the iberiotoxin sensitivity of currents in cells from β1 knockout mice suggest that parotid acinar cells contain approximately equal numbers of homotetrameric channel proteins from the parotid variant of the Slo gene and heteromeric proteins composed of the parotid Slo variant in combination with the β4-subunit.
Regulation of Fas (CD95)-induced apoptosis by nuclear factor-κB and tumor necrosis factor-α in macrophages
Authors: Lu Bin , Wang Liying , Medan Djordje , Toledo David , Huang Chuanshu , Chen Fei , Shi Xianglin , Rojanasakul Yon
Published: May 2013

The APO-1/Fas ligand (FasL) and tumor necrosis factor-α (TNF-α) are two functionally related molecules that induce apoptosis of susceptible cells. Although the two molecules have been reported to induce apoptosis via distinct signaling pathways, we have shown that FasL can also upregulate the expression of TNF-α, raising the possibility that TNF-α may be involved in FasL-induced apoptosis. Because TNF-α gene expression is under the control of nuclear factor-κB (NF-κB), we investigated whether FasL can induce NF-κB activation and whether such activation plays a role in FasL-mediated cell death in macrophages. Gene transfection studies using NF-κB-dependent reporter plasmid showed that FasL did activate NF-κB promoter activity. Gel shift studies also revealed that FasL mobilized the p50/p65 heterodimeric form of NF-κB. Inhibition of NF-κB by a specific NF-κB inhibitor, caffeic acid phenylethyl ester, or by dominant expression of the NF-κB inhibitory subunit IκB caused an increase in FasL-induced apoptosis and a reduction in TNF-α expression. However, neutralization of TNF-α by specific anti-TNF-α antibody had no effect on FasL-induced apoptosis. These results indicate that FasL-mediated cell death in macrophages is regulated through NF-κB and is independent of TNF-α activation, suggesting the antiapoptotic role of NF-κB and a separate death signaling pathway mediated by FasL.
Differential expression of TGF-β isoforms by normal and inflammatory bowel disease intestinal myofibroblasts
Authors: McKaig B. C. , Hughes K. , Tighe P. J. , Mahida Y. R.
Published: May 2013

First published September 5, 2001; 10.1152/ajpcell. 00048.2001.—Intestinal strictures are frequent in Crohn's disease but not ulcerative colitis. We investigated the expression of transforming growth factor (TGF)-β isoforms by isolated and cultured primary human intestinal myofibroblasts and the responsiveness of these cells and intestinal epithelial cells to TGF-β isoforms. Normal intestinal myofibroblasts released predominantly TGF-β3 and ulcerative colitis myofibroblasts expressed both TGF-β1 and TGF-β3, whereas in myofibroblast cultures from fibrotic Crohn's disease tissue, there was significantly lower expression of TGF-β3 but enhanced release of TGF-β2. These distinctive patterns of TGF-β isoform release were sustained through several myofibroblast passages. Proliferation of Crohn's disease myofibroblasts was significantly greater than that of myofibroblasts derived from normal and ulcerative colitis tissue. In contrast to cells from normal and ulcerative colitis tissue, neutralization of the three TGF-β isoforms did not affect the proliferation of Crohn's disease intestinal myofibroblasts. Studies on the effect of recombinant TGF-β isoforms on epithelial restitution and proliferation suggest that TGF-β2 may be the least effective of the three isoforms in intestinal wound repair. In conclusion, the enhanced release of TGF-β2 but reduced expression of TGF-β3 by Crohn's disease intestinal myofibroblasts, together with their enhanced proliferative capacity, may lead to the development of intestinal strictures.
HSP72 inhibits apoptosis-inducing factor release in ATP-depleted renal epithelial cells
Authors: Ruchalski Kathleen , Mao Haiping , Singh Satish K. , Wang Yihan , Mosser Dick D. , Li Fanghong , Schwartz John H. , Borkan Steven C.
Published: May 2013

Inhibition of the mitochondrial release and nuclear translocation of apoptosis-inducing factor (AIF) by heat stress protein (HSP)72 may ameliorate apoptosis in renal epithelial cells exposed to a metabolic inhibitor. To evaluate this hypothesis, cells were transiently exposed to 5 mM sodium cyanide in the absence of medium glucose, a maneuver known to induce apoptosis. ATP depletion for 1-2 h resulted in the progressive accumulation of mitochondrial AIF in the cytosol of samples obtained by selectively permeabilizing the plasma membrane with digitonin. During recovery from ATP depletion, time-dependent nuclear AIF accumulation (but not cytochrome c, an F0F1 ATP synthase subunit, or talin) was observed in isolated nuclei. Nuclear AIF accumulation was associated with peripheral chromatin condensation and DNA degradation. Prior heat stress (HS) significantly reduced AIF leakage into the cytosol, decreased nuclear accumulation of AIF, and inhibited DNA degradation. HS also increased the interaction between AIF and HSP72 detected by immunoprecipitation. In ATP depleted cells, selective overexpression of human HSP72 reduced the leakage of mitochondrial AIF in a dose-dependent manner ( r = 0.997). This study suggests that mitochondrial membrane injury and subsequent AIF release contribute to nuclear injury and apoptosis in ATP-depleted renal cells. HSP72, an antiapoptotic protein, inhibits cell injury in part by preventing mitochondrial AIF release and perhaps by decreasing its nuclear accumulation.
Fluid pressure in human dermal fibroblast aggregates measured with micropipettes
Authors: Stuhr L. E. B. , Reith A. , Lepsøe S. , Myklebust R. , Wiig H. , Reed R. K.
Published: May 2013

Previous studies indicated that connective tissue cells in dermis are involved in control of interstitial fluid pressure (Pif). We wanted to develop and characterize an in vitro model representative of loose connective tissue to study dynamic changes in fluid pressure (Pf) over a time course of a few minutes. Pf was measured with micropipettes in human dermal fibroblast cell aggregates of varying size (<100- and >100-μm diameter) and age ( days 1-4) kept at different temperatures (∼15, 25, and 35°C). Pressures were measured at different depths of micropipette penetration and after treatment with prostaglandin E1 isopropyl ester (PGE1), latanoprost (PGF2α), and ouabain. Pf was positive (more than +2 mmHg) during control conditions and increased with increasing aggregate size ( day 2), age ( day 4 vs. day 1), temperature, and depth of micropipette penetration. Pf decreased from 2.9 to 2.0 mmHg during the first 10 min after application of 10 μl of 1 mM PGE1 ( P < 0.001). Pf increased from 3.0 to 4.8 mmHg ( P < 0.01) after administration of 10 μl of 1.4 μM ouabain and from 3.1 to 4.4 mmHg after addition of 5 μl of 1.42 mM PGF2α ( P > 0.05). In conclusion, we have developed and validated a new in vitro method for studying fluid pressure in loose connective tissue elements with the advantage of allowing reliable and rapid screening of substances that have a potential to modify Pf and studying in more detail specific cell types involved in control of Pf. This study also provides evidence that fibroblasts in the connective tissue can actively modulate Pf.
Stretch-induced Ca2+ release via an IP3-insensitive Ca2+ channel
Authors: Mohanty Madhumita Jena , Li Xingli
Published: May 2013

Various mechanical stimuli increase the intracellular Ca2+ concentration ([Ca2+]i) in vascular smooth muscle cells (VSMC). A part of the increase in [Ca2+]i is due to the release of Ca2+ from intracellular stores. We have investigated the effect of mechanical stimulation produced by cyclical stretch on the release of Ca2+ from the intracellular stores. Permeabilized VSMC loaded with45Ca2+ were subjected to 7.5% average (15% maximal) cyclical stretch. This resulted in an increase in45Ca2+ rate constant by 0.126 ± 0.0035. Inhibition of inositol 1,4,5-trisphosphate (IP3), ryanodine, and nicotinic acid adenine dinucleotide phosphate channels (NAADP) with 50 μg/ml heparin, 50 μM ruthenium red, and 25 μM thio-NADP, respectively, did not block the increase in45Ca2+ efflux in response to cyclical stretch. However, 10 μM lanthanum, 10 μM gadolinium, and 10 μM cytochalasin D but not 10 μM nocodazole inhibited the increase in45Ca2+ efflux. This supports the existence of a novel stretch-sensitive intracellular Ca2+ store in VSMC that is distinct from the IP3-, ryanodine-, and NAADP-sensitive stores.
Distribution of Glut1 in detergent-resistant membranes (DRMs) and non-DRM domains: effect of treatment with azide
Authors: Rubin Darrell , Ismail-Beigi Faramarz
Published: May 2013

We have previously shown that the acute stimulation of glucose transport in Clone 9 cells in response to azide is mediated by activation of Glut1 and that stomatin, a Glut1-binding protein, appears to inhibit Glut1 function. In Clone 9 cells under basal conditions, ∼38% of Glut1, ∼70% of stomatin, and the bulk of caveolin-1 was localized in the detergent-resistant membrane (DRM) fraction; a significant fraction of Glut1 is also present in DRMs of 3T3-L1 fibroblasts and human red blood cells (RBCs). Acute exposure to azide resulted in 40 and 50% decreases in the content of Glut1 in DRMs of Clone 9 cells and 3T3-L1 fibroblasts, respectively, whereas the distribution of stomatin and caveolin-1 in Clone 9 cells remained unchanged. In addition, treatment of Clone 9 cells with azide resulted in a ∼50% decrease in the content of Glut1 in the DRM fraction of plasma membranes. We conclude that 1) a significant fraction of Glut1 is localized in DRMs, and 2) treatment of cells with azide results in a partial redistribution of Glut1 out of the DRM fraction.
Differential COX localization and PG release in Thy-1+ and Thy-1− human female reproductive tract fibroblasts
Authors: Koumas Laura , Phipps Richard P.
Published: May 2013

A key role exists for prostaglandins (PGs) in reproductive health, including fertility and parturition. However, the cellular sources and regulation of PG production by cyclooxygenase (COX) in the human female reproductive tract remain poorly understood. We recently reported that human female reproductive tract fibroblasts are divisible into distinct subsets based on their Thy-1 surface expression. Herein, we demonstrate that the expression, induction, and subcellular localization of COX-1 and COX-2 and the downstream PG biosynthesis are markedly different between these subsets. Specifically, Thy-1+ fibroblasts highly express COX-1, which is responsible for high-level PGE2 production, a feature usually attributed to the COX-2 isoenzyme. In contrast, COX-2, generally considered an inducible isoform, is constitutively expressed in the Thy-1− subset, which only minimally produces PGE2. The intracellular signaling pathways for COX regulation also differ between the subsets. Determination of differences in signal transduction, COX expression and localization, and PG production by human reproductive fibroblast subtypes supports the concept of fibroblast heterogeneity and the possibility that these subsets may play unique roles in tissue homeostasis and in inflammation.
Fibroblast fiber contraction: role of C and Rho kinase in activation by thromboxane A2
Authors: Nobe Hiromi , Nobe Koji , Paul Richard J.
Published: May 2013

We investigated the mechanisms underlying regulation of contraction with measurements of isometric force and intracellular Ca2+ concentration ([Ca2+]i) in NIH 3T3 fibroblast reconstituted into fibers with the use of a collagen matrix. Treatment with the major phospholipids, neurotransmitters, and growth factors had little effect on baseline isometric force. However, U-46619, a thromboxane A2 (TxA2) analog, increased force and [Ca2+]i; EC50 values were 11.0 and 10.0 nM, respectively. The time courses were similar to those induced by calf serum (CS), and the maximal force was 65% of a CS-mediated contraction. The selective TxA2 receptor antagonist SQ-29548 abolished the U-46619-induced responses. CS-induced contractions are dependent on an intracellular Ca2+ store function; however, the U-46619 response depended not only on intracellular Ca2+ stores, but also on Ca2+ influx from the extracellular medium. Inhibition of Rho kinase suppressed U-46619- and CS-induced responses; in contrast, inhibition of C kinase (PKC) reduced only the U-46619 response. Moreover, addition of U-46619 to a CS contracture enhanced force and [Ca2+]i responses. These results indicate that U-46619-induced responses involve PKC and Rho kinase pathways, in contrast to activation by CS. Thus TxA2 may have a role in not only the initial step of wound repair as an activator of blood coagulation, but also in fibroblast contractility in later stages.
Plasma membrane delivery of the gastric H,K-ATPase: the role of β-subunit glycosylation
Authors: Vagin O. , Denevich S. , Sachs G.
Published: May 2013

The factors determining trafficking of the gastric H,K-ATPase to the apical membrane remain elusive. To identify such determinants in the gastric H,K-ATPase, fusion proteins of yellow fluorescent protein (YFP) and the gastric H,K-ATPase β-subunit (YFP-β) and cyan fluorescent protein (CFP) and the gastric H,K-ATPase α-subunit (CFP-α) were expressed in HEK-293 cells. Then plasma membrane delivery of wild-type CFP-α, wild-type YFP-β, and YFP-β mutants lacking one or two of the seven β-subunit glycosylation sites was determined using confocal microscopy and surface biotinylation. Expression of the wild-type YFP-β resulted in the plasma membrane localization of the protein, whereas the expressed CFP-α was retained intracellularly. When coexpressed, both CFP-α and YFP-β were delivered to the plasma membrane. Removing each of the seven glycosylation sites, except the second one, from the extracellular loop of YFP-β prevented plasma membrane delivery of the protein. Only the mutant lacking the second glycosylation site (Asn103Gln) was localized both intracellularly and on the plasma membrane. A double mutant lacking the first (Asn99Gln) and the second (Asn103Gln) glycosylation sites displayed intracellular accumulation of the protein. Therefore, six of the seven glycosylation sites in the β-subunit are essential for the plasma membrane delivery of the β-subunit of the gastric H,K-ATPase, whereas the second glycosylation site (Asn103), which is not conserved among the β-subunits from different species, is not critical for plasma delivery of the protein.
Translated Alu sequence determines nuclear localization of a novel catalytic subunit of casein kinase 2
Authors: Hilgard Philip , Huang Tianmin , Wolkoff Allan W. , Stockert Richard J.
Published: May 2013

Casein kinase 2 (CK2) is a tetrameric enzyme constitutively expressed in all eukaryotic tissues. The two known isoforms of the catalytic subunit, CK2α and CK2α′, have been reported to have distinct tissue-dependent subcellular distributions. We recently described a third isoform of the catalytic subunit, designated CK2α", which is highly expressed in liver. Immunoblot analysis of HuH-7 human hepatoma cell fractions as well as immunofluorescent microscopy revealed that CK2α" was exclusively localized to the nucleus and preferentially associated with the nuclear matrix. CK2α and CK2α′ were found in nuclear, membrane, and cytosolic compartments. Deletion of the carboxy-terminal 32 amino acids from the CK2α" sequence resulted in release of the truncated green fluorescent protein fusion protein from the nuclear matrix and redistribution to both the nucleus and the cytoplasm. Demonstration that the carboxy terminus is necessary but not sufficient for nuclear retention indicates that the underlying mechanism of CK2α" nuclear localization is dependent on the secondary structure of the holoenzyme directed by the carboxy-terminal sequence.
Essential role for Ca2+ in regulation of IL-1β secretion by P2X7 nucleotide receptor in monocytes, macrophages, and HEK-293 cells
Authors: Gudipaty Lalitha , Munetz Jonathan , Verhoef Philip A. , Dubyak George R.
Published: May 2013

Interleukin (IL)-1β is a proinflammatory cytokine that elicits the majority of its biological activity extracellularly, but the lack of a secretory signal sequence prevents its export via classic secretory pathways. Efficient externalization of IL-1β in macrophages and monocytes can occur via stimulation of P2X7 nucleotide receptors with extracellular ATP. However, the exact mechanisms by which the activation of these nonselective cation channels facilitates secretion of IL-1β remain unclear. Here we demonstrate a pivotal role for a sustained increase in cytosolic Ca2+ to potentiate secretion of IL-1β via the P2X7 receptors. Using HEK-293 cells engineered to coexpress P2X7 receptors with mature IL-1β (mIL-1β), we show that activation of P2X7 receptors results in a rapid secretion of mIL-1β by a process(es) that is dependent on influx of extracellular Ca2+ and a sustained rise in cytosolic Ca2+. Moreover, reduction in extracellular Ca2+ attenuates ∼90% of P2X7 receptor-mediated IL-1β secretion but has no effect on enzymatic processing of precursor IL-1β (proIL-1β) to mIL-1β by caspase-1. Similar experiments with THP-1 human monocytes and Bac1.2F5 murine macrophages confirm the unique role of Ca2+ in P2X7 receptor-mediated secretion of IL-1β. In addition, we report that cell surface expression of P2X7 receptors in the absence of external stimulation also results in enhanced release of IL-1β and that this can be repressed by inhibitors of P2X7 receptors. We clarify an essential role for Ca2+ in ATP-induced IL-1β secretion and indicate an additional role of P2X7 receptors as enhancers of the secretory apparatus by which IL-1β is released.
A role for PYK2 in ANG II-dependent regulation of the PHAS-1-eIF4E complex by multiple signaling cascades in vascular smooth muscle
Authors: Rocic Petra , Jo Hanjoong , Lucchesi Pamela A.
Published: May 2013

Regulation of the PHAS-1-eukaryotic initiation factor-4E (eIF4E) complex is the rate-limiting step in the initiation of protein synthesis. This study characterized the upstream signaling pathways that mediate ANG II-dependent phosphorylation of PHAS-1 and eIF4E in vascular smooth muscle. ANG II-dependent PHAS-1 phosphorylation was maximal at 10 min (2.47 ± 0.3 fold vs. control). This effect was completely blocked by the specific inhibitors of phosphatidylinositol 3-kinase (PI3-kinase, LY-294002), mammalian target of rapamycin, and extracellular signal-regulated kinase 1/2 (ERK1/2, U-0126) or by a recombinant adenovirus encoding dominant-negative Akt. PHAS-1 phosphorylation was followed by dissociation of eIF4E. Increased ANG II-induced eIF4E phosphorylation was observed at 45 min (2.63 ± 0.5 fold vs. control), was maximal at 90 min (3.38 ± 0.3 fold vs. control), and was sustained at 2 h. This effect was blocked by inhibitors of the ERK1/2 and p38 mitogen-activated protein (MAP) kinase pathways, but not by PI3-kinase inhibition, and was dependent on PKC, intracellular Ca2+, and tyrosine kinases. Downregulation of proline-rich tyrosine kinase 2 (PYK2) by antisense oligonucleotides led to a near-complete inhibition of PHAS-1 and eIF4E phosphorylation in response to ANG II. Therefore, PYK2 represents a proximal signaling intermediate that regulates ANG II-induced vascular smooth muscle cell protein synthesis via regulation of the PHAS-1-eIF4E complex.
Channels formed with a mutant prion protein PrP(82-146) homologous to a 7-kDa fragment in diseased brain of GSS patients
Authors: Bahadi Randa , Farrelly Peter V. , Kenna Bronwyn L. , Kourie Joseph I. , Tagliavini Fabrizio , Forloni Gianluigi , Salmona Mario
Published: May 2013

A major prion protein (PrP) mutant that forms amyloid fibrils in the diseased brain of patients with Gerstmann-Sträussler-Scheinker syndrome (GSS) is a fragment of 7 kDa spanning from residues 81-82 to 144-153 of PrP. Analysis of ionic membrane currents, recorded with a libid bilayer technique, revealed that the wild-type fragment PrP(82-146) WT and the partially scrambled PrP(82-146) (127-146) SC are capable of forming heterogenous ion channels that are similar to those channels formed with PrP(106-126). In contrast, PrP(82-146) peptides in which the region from residue 106 to 126 had been scrambled (SC) showed a reduction in interaction with lipid membranes and did not form channels. The PrP(82-146) WT- and PrP(82-146) (127-146) SC-formed cation channels with fast kinetics are Cu2+ sensitive and rifampicin (RIF) insensitive, whereas the time-dependent inactivating channels formed by these same peptides are both Cu2+ and RIF insensitive. The presence of RIF in the solution before the addition of PrP(82-146) WT or PrP(82-146) (127-146) SC affected their incorporation into the lipid bilayers. PrP(82-146) WT and PrP(82-146) (127-146) SC fast cation channels formed in the presence of RIF appeared in an electrically semisilent state or an inactivated state. Increasing [Cd2+] cis enhanced the incorporation of PrP(82-146) WT and PrP(82-146) (127-146) SC channels formed in the presence of RIF. We conclude that the major PrP mutant fragment in the diseased brain of GSS patients is prone to form channels in neuronal membranes, causing their dysfunction. We propose that Cd2+ may accentuate the neurotoxicity of this channel-forming PrP fragment by enhancing its incorporation into the membrane.
Acid-base effects on intestinal Na+ absorption and vesicular trafficking
Authors: Charney Alan N. , Egnor Richard W. , Alexander-Chacko Jesline , Cassai Nicholas , Sidhu Gurdip S.
Published: May 2013

We examined for vesicular trafficking of the Na+/H+ exchanger (NHE) in pH-stimulated ileal and CO2-stimulated colonic Na+absorption. Subapical vesicles in rat distal ileum were quantified by transmission electron microscopy at ×27,500 magnification. Internalization of ileal apical membranes labeled with FITC-phytohemagglutinin was assessed using confocal microscopy, and pH-stimulated ileal Na+ absorption was measured after exposure to wortmannin. Apical membrane protein biotinylation of ileal and colonic segments and Western blots of recovered proteins were performed. In ileal epithelial cells incubated in HCO[Formula: see text]/Ringer or HEPES/Ringer solution, the number of subapical vesicles, the relative quantity of apical membrane NHE isoforms 2 and 3 (NHE2 and NHE3, respectively), and apical membrane fluorescence under the confocal microscope were not affected by pH values between 7.1 and 7.6. Wortmannin did not inhibit pH-stimulated ileal Na+ absorption. In colonic epithelial apical membranes, NHE3 protein content was greater at a Pco 2 value of 70 than 21 mmHg, was internalized when Pco 2 was reduced, and was exocytosed when Pco 2 was increased. We conclude that vesicle trafficking plays no part in pH-stimulated ileal Na+absorption but is important in CO2-stimulated colonic Na+ absorption.
CCK-A receptor activates RhoA through Gα12/13 in NIH3T3 cells
Authors: Le Page Sophie L. , Bi Yan , Williams John A.
Published: May 2013

Cholecystokinin (CCK) is a major regulator of pancreatic acinar cells and was shown previously to be capable of inducing cytoskeletal changes in these cells. In the present study, using NIH3T3 cells stably transfected with CCK-A receptors as a model cell, we demonstrate that CCK can induce actin stress fibers through a G13- and RhoA-dependent mechanism. CCK induced stress fibers within minutes similar to those induced by lysophosphatidic acid (LPA), the active component of serum. The effects of CCK were mimicked by active RhoV14 and blocked by dominant-negative RhoN19, Clostridium botulinum C3 transferase, and the Rho-kinase inhibitor Y-27632. CCK rapidly induced active Rho in cells as shown with a pull-down assay using the Rho binding domain of rhotekin and by a serum response element (SRE)-luciferase reporter assay. To evaluate the G protein mediating the action of CCK, cells were transfected with active α-subunits; Gα13 and Gα12 but not Gαq induced stress fibers and in some cases cell rounding. A p115 Rho guanine nucleotide exchange factor (GEF) regulator of G protein signaling (RGS) domain known to interact with G12/13 inhibited active α12/13-and CCK-induced stress fibers, whereas RGS2 and RGS4, which are known to inhibit Gq, had no effect. Cotransfection with plasmids coding for the G protein α-subunit carboxy-terminal peptide from α13 and, to a lesser extent α12, also inhibited the effect of CCK, whereas the peptide from αq did not. These results show that in NIH3T3 cells bearing CCK-A receptors, CCK activates Rho primarily through G13, leading to rearrangement of the actin cytoskeleton.
Hyperosmotic stress activates Rho: differential involvement in Rho kinase-dependent MLC phosphorylation and NKCC activation
Authors: Ciano-Oliveira Caterina Di , Sirokmány Gábor , Szászi Katalin , Arthur William T. , Masszi András , Peterson Mark , Rotstein Ori D. , Kapus András
Published: May 2013

Hyperosmotic stress initiates adaptive responses, including phosphorylation of myosin light chain (MLC) and concomitant activation of Na+-K+-Cl– cotransporter (NKCC). Because the small GTPase Rho is a key regulator of MLC phosphorylation, we investigated 1) whether Rho is activated by hyperosmotic stress, and if so, what the triggering factors are, and 2) whether the Rho/Rho kinase (ROK) pathway is involved in MLC phosphorylation and NKCC activation. Rho activity was measured in tubular epithelial cells by affinity pulldown assay. Hyperosmolarity induced rapid (<1 min) and sustained (>20 min) Rho activation that was proportional to the osmotic concentration and reversed within minutes upon restoration of isotonicity. Both decreased cell volume at constant ionic strength and elevated total ionic strength at constant cell volume were capable of activating Rho. Changes in [Na+] and [K+] at normal total salinity failed to activate Rho, and Cl– depletion did not affect the hyperosmotic response. Thus alterations in cellular volume and ionic strength but not individual ion concentrations seem to be the critical triggering factors. Hyperosmolarity induced mono- and diphosphorylation of MLC, which was abrogated by the Rho-family blocker Clostridium toxin B. ROK inhibitor Y-27632 suppressed MLC phosphorylation under isotonic conditions and prevented its rise over isotonic levels in hypertonically stimulated cells. ML-7 had a smaller inhibitory effect. In contrast, it abolished the hypertonic activation of NKCC, whereas Y-27632 failed to inhibit this response. Thus hyperosmolarity activates Rho, and Rho/ROK pathway contributes to basal and hyperosmotic MLC phosphorylation. However, the hypertonic activation of NKCC is ROK independent, implying that the ROK-dependent component of MLC phosphorylation can be uncoupled from NKCC activation.
Monochloramine induces reorganization of nuclear speckles and phosphorylation of SRp30 in human colonic epithelial cells: role of protein kinase C
Authors: Zhu Ya-Qin , Lu Yu , Tan Xiao-Di
Published: May 2013

Intestinal epithelial cells are constantly stimulated by reactive oxidant metabolites (ROMs) in inflamed mucosa. Monochloramine (NH2Cl), a cell-permeant ROM, is particularly relevant to the pathogenesis of inflammation in the gastrointestinal tract. Nuclear speckles, a unique nuclear subcompartment, accumulate a family of proteins, namely, serine- and arginine-rich (SR) proteins. They play important roles in regulation of pre-mRNA splicing. Currently, little is known about the link between inflammatory stimulation and the pre-mRNA splicing process, although gene expression is changed in inflamed tissues. The present study was designed to investigate whether stimulation of human colonic epithelial cells (HT-29 and Caco-2 cell lines) with NH2Cl affects nuclear speckles and their components. By indirect immunofluorescence, nuclear speckles have been shown to undergo rapid aggregation after NH2Cl stimulation. By utilizing Western blotting, SRp30 (a subset of SR proteins) in intestinal epithelial cells was found to be phosphorylated after NH2Cl treatment, whereas other SR proteins were not responsive to NH2Cl stimulation. The cytotoxic effect of NH2Cl was excluded by both negative lactate dehydrogenase assay and propidium iodide staining. Therefore, NH2Cl-induced morphological changes on nuclear speckles and phosphorylated SRp30 do not result from intestinal epithelial injury. Furthermore, the effect of NH2Cl on nuclear speckles and SRp30 was blocked by bisindolylmaleimide I, a selective PKC inhibitor. Together, the available data suggest that stimulation of intestinal epithelial cells with NH2Cl results in a consequent change on pre-mRNA splicing machinery via a distinctive signal pathway involving activation of PKC. This effect may contribute to oxidant-induced pathophysiological changes in the gastrointestinal tract.
Duality of G protein-coupled mechanisms for β-adrenergic activation of NKCC activity in skeletal muscle
Authors: Gosmanov Aidar R. , Wong Jennifer A. , Thomason Donald B.
Published: May 2013

Skeletal muscle Na+-K+-2Cl− cotransporter (NKCC) activity provides a potential mechanism for regulated K+uptake. β-Adrenergic receptor (β-AR) activation stimulates skeletal muscle NKCC activity in a MAPK pathway-dependent manner. We examined potential G protein-coupled pathways for β-AR-stimulated NKCC activity. Inhibition of Gs-coupled PKA blocked isoproterenol-stimulated NKCC activity in both the slow-twitch soleus muscle and the fast-twitch plantaris muscle. However, the PKA-activating agents cholera toxin, forskolin, and 8-bromo-cAMP (8-BrcAMP) were not sufficient to activate NKCC in the plantaris and partially stimulated NKCC activity in the soleus. Isoproterenol-stimulated NKCC activity in the soleus was abolished by pretreatment with pertussis toxin (PTX), indicating a Gi-coupled mechanism. PTX did not affect the 8-BrcAMP-stimulated NKCC activity. PTX treatment also precluded the isoproterenol-mediated ERK1/2 MAPK phosphorylation in the soleus, consistent with NKCC's MAPK dependency. Inhibition of isoproterenol-stimulated ERK activity by PTX treatment was associated with an increase in Akt activation and phosphorylation of Raf-1 on the inhibitory residue Ser259. These results demonstrate a novel, muscle phenotype-dependent mechanism for β-AR-mediated NKCC activation that involves both Gs and Giprotein-coupled mechanisms.
Early activation of p160ROCK by pressure overload in rat heart
Authors: Torsoni Adriana S. , Fonseca Priscila M. , Crosara-Alberto Daniela P , Franchini Kleber G.
Published: May 2013

We investigated the effects of acute pressure overload on activation of p160ROCK in rat myocardium. Constriction of transverse aorta, controlled to increase peak systolic pressure of ascending aorta by ∼40 mmHg, induced a rapid association of RhoA with Dbl-3 and p160ROCK. The binding of p160ROCK to RhoA was rapidly increased, peaking at 30 min (∼3.5-fold), but reduced to lower levels (∼1.9-fold) by 60 min of pressure overload. The activity of immunoprecipitated p160ROCK toward myosin light chain increased ∼2.5-fold within 10 min but decreased to lower levels (∼1.6-fold) after 60 min of pressure overload. Confocal microscopic analysis indicated that pressure overload induced the formation of aggregates of p160ROCK and RhoA along the longitudinal axis of cardiac myocytes. Immunoelectron microscopic analysis showed that pressure overload induced the association of p160ROCK and RhoA to Z-line, T-tubule, and subsarcolemmal areas. The rapid activation of p160ROCK by pressure overload and its aggregation in subcellular structures involved in transmission of mechanical force suggest a role for this enzyme in the mechanobiochemical transduction in the myocardium.