bluuebunny/crossref_metadata_2025 huggingface dataset:
Cyclopiazonic acid-induced changes in the contraction and Ca2+ transient of frog fast-twitch skeletal muscle
Authors: Même William , Huchet-Cadiou Corinne , Léoty Claude
Published: December 2017

The effects of cyclopiazonic acid (CPA) were investigated on isolated skeletal muscle fibers of frog semitendinosus muscle. CPA (0.5–10 μM) enhanced isometric twitch but produced little change in resting tension. At higher concentrations (10–50 μM), CPA depressed twitch and induced sustained contracture without affecting resting and action potentials. In Triton-skinned fibers, CPA had no significant effect on myofibrillar Ca2+ sensitivity but decreased maximal activated force at concentrations >5 μM. In intact cells loaded with the Ca2+fluorescence indicator indo 1, CPA (2 μM) induced an increase in Ca2+-transient amplitude (10 ± 2.5%), which was associated with an increase in time to peak and in the time constant of decay. Consequently, peak force was increased by 35 ± 4%, and both time to peak and the time constant of relaxation were prolonged. It is concluded that CPA effects, at a concentration of up to 2 μM, were associated with specific inhibition of sarcoplasmic reticulum Ca2+-adenosinetriphosphatase in intact skeletal muscle and that inhibition of the pump directly affected the handling of intracellular Ca2+ and force production.
PKA inhibitor, H-89, affects the intracellular transit of regulated secretory proteins in rat lacrimal glands
Authors: Robin P. , Rossignol B. , Raymond M. N.
Published: December 2017

We tested the effect of H-89, a protein kinase A (PKA) inhibitor, on the intracellular transit of the regulated secretory proteins in rat lacrimal glands. We show that H-89, by itself, induces the secretion of newly synthesized proteins trafficking in its presence but not of proteins already stored in the mature secretory granules. This secretion does not depend on the presence of extracellular Ca2+. The proteins released are identical to those secreted after cholinergic stimulation or under the action of the ionophore A-23187, but the secretion level is ∼40% lower. The effect of H-89 seems to be due to PKA inhibition because other protein kinase inhibitors (calphostin C, chelerythrine, H-85) do not induce secretion. We further show that H-89 does not modify the rate of glycoprotein galactosylation but induces the secretion of newly galactosylated glycoproteins. Finally, we used a “20°C block” procedure to show that H-89 affects a trans-Golgi network (TGN) or post-TGN step of the secretory pathway. Our results demonstrate that, in lacrimal cells, H-89 affects the intracellular trafficking of secretory proteins, suggesting a role for PKA in this process.
ANG II-induced translocation of cytosolic PLA2 to the nucleus in vascular smooth muscle cells
Authors: Freeman Ernest J. , Ruehr Mary L. , Dorman Robert V.
Published: December 2017

The accumulation of radiolabeled arachidonic acid (AA), immunoblot analysis of subcellular fractions, and immunofluorescence tagging of proteins in intact cells were used to examine the coupling of ANG II receptors with the activity and location of a cytosolic phospholipase A2(cPLA2) in vascular smooth muscle cells (VSMC). ANG II induced the accumulation of AA, which peaked by 10 min and was downregulated by 20 min. A large proportion of the AA released in response to ANG II was due to the activation of a Ca2+-dependent lipase coupled to an AT1 receptor. However, regulation of Ca2+ availability failed to completely block AA release, and a small but significant reduction in ANG II-mediated AA release was observed in the presence of an AT2 antagonist. These findings, coupled with a 25% reduction in the ANG II-induced AA release by an inhibitor specific for a Ca2+-independent PLA2, are consistent with the presence and activation of a Ca2+-independent PLA2. In contrast, immunoblot analysis and immunofluorescence detection showed that the ANG II-mediated translocation of cPLA2to a membrane fraction was exclusively AT1 dependent and regulated by Ca2+ availability. Furthermore, the nucleus was the membrane target. We conclude that ANG II regulates the Ca2+-dependent activation and translocation of cPLA2 through an AT1 receptor and that this event is targeted at the nucleus in VSMC.
Aldosterone responsiveness of A6 cells is restored by cloned rat mineralocorticoid receptor
Authors: Chen Sei-Yu , Wang Jian , Liu Weihong , Pearce David
Published: December 2017

A6 cells, derived from Xenopus laevis renal tubule, form a high-resistance ion-transporting monolayer when grown on permeable supports and can generate a short-circuit current (SCC) that is stimulated by high levels of the mineralocorticoid aldosterone. Surprisingly, A6 SCC is more responsive to glucocorticoids than to mineralocorticoids, suggesting the possibility that these cells do not contain transcriptionally active mineralocorticoid receptor (MR) and that glucocorticoid receptor (GR) mediates MR-like responses in these collecting duct-like cells. We have examined the response of both SCC and a transfected reporter gene to mineralocorticoids and glucocorticoids in the presence and absence of transfected rat MR (rMR). We found that, in the absence of transfected MR, a reporter gene that can be activated by MR or GR was more responsive to glucocorticoids such as dexamethasone and RU-28362 than to mineralocorticoids such as aldosterone. Transfected rMR underwent mineralocorticoid-dependent nuclear localization and restored both transcriptional sensitivity of a reporter gene and SCC response to mineralocorticoids. These data demonstrate that A6 cells contain transcriptionally active GR but not MR and thus suggest a molecular basis for the defect in A6 cell SCC response to aldosterone. Our results also demonstrate that GR is capable of mediating hormone stimulation of SCC, a classic mineralocorticoid response. Finally, the observation that heterologous expression of rMR can localize normally to the A6 nucleus in a hormone-dependent fashion and restore both the transcriptional and SCC response to mineralocorticoids suggests that MR function is conserved in species as distantly related as toads and mammals.
Hyperexpression of recombinant CFTR in heterologous cells alters its physiological properties
Authors: Mohammad-Panah Raha , Demolombe Sophie , Riochet David , Leblais Veronique , Loussouarn Gildas , Pollard Helene , Baró Isabelle , Escande Denis
Published: December 2017

We investigated whether high levels of expression of the cystic fibrosis transmembrane conductance regulator (CFTR) would alter the functional properties of newly synthesized recombinant proteins. COS-7, CFPAC-1, and A549 cells were intranuclearly injected with a Simian virus 40-driven pECE-CFTR plasmid and assayed for halide permeability using the 6-methoxy- N-(3-sulfopropyl)quinolinium fluorescent probe. With increasing numbers of microinjected pECE-CFTR copies, the baseline permeability to halide dose dependently increased, and the response to adenosine 3′,5′-cyclic monophosphate (cAMP) stimulation decreased. In cells hyperexpressing CFTR, the high level of halide permeability was reduced when a cell metabolism poisoning cocktail was applied to decrease intracellular ATP and, inversely, was increased by orthovanadate. In CFPAC-1 cells investigated with the patch-clamp technique, CFTR hyperexpression led to a time-independent nonrectifying chloride current that was not sensitive to cAMP stimulation. CFPAC-1 cells hyperexpressing CFTR exhibited no outward rectifying chloride current nor inward rectifying potassium current either spontaneously or under cAMP stimulation. We conclude that hyperexpression of recombinant CFTR proteins modifies their properties inasmuch as 1) CFTR channels are permanently activated and not susceptible to cAMP regulation and 2) they lose their capacity to regulate heterologous ionic channels.
Molecular and kinetic alterations of muscle AMP deaminase during chronic creatine depletion
Authors: Rush James W. E. , Tullson Peter C. , Terjung Ronald L.
Published: December 2017

We examined a possible mechanism to account for the maintenance of peak AMP deamination rate in fast-twitch muscle of rats fed the creatine analog β-guanidinopropionic acid (β-GPA), in spite of reduced abundance of the enzyme AMP deaminase (AMPD). AMPD enzymatic capacity (determined at saturating AMP concentration) and AMPD protein abundance (Western blot) were coordinately reduced ∼80% in fast-twitch white gastrocnemius muscle by β-GPA feeding over 7 wk. Kinetic analysis of AMPD in the soluble cell fraction demonstrated a single Michaelis-Menten constant ( K m; ∼1.5 mM) in control muscle extracts. An additional high-affinity K m (∼0.03 mM) was revealed at low AMP concentrations in extracts of β-GPA-treated muscle. The kinetic alteration in AMPD reflects increased molecular activity at low AMP concentrations; this could account for high rates of deamination in β-GPA-treated muscle in situ, despite the loss of AMPD enzyme protein. The elimination of this kinetic effect by treatment of β-GPA-treated muscle extracts with acid phosphatase in vitro suggests that phosphorylation is involved in the kinetic control of skeletal muscle AMPD in vivo.
Direct inhibitory effect of CCCP on the Cl−-H+ symporter of the guinea pig ileal brush-border membrane
Authors: Alvarado Francisco , Vasseur Monique
Published: December 2017

The effect of carbonyl cyanide- m-chlorophenylhydrazone (CCCP) on Cl− uptake across the brush-border membrane (BBM) was quantified using36Cl and BBM vesicles from guinea pig ileum. CCCP inhibited only partially both the pH gradient-activated Cl− uptake and Cl−/Cl−exchange activities present in these vesicles. In contrast, CCCP had no effect on the initial (2–30 s) decay rate of an imposed proton gradient, as determined using the pH-sensitive fluorophore pyranine. Taken together, these results strongly indicate that the main action of CCCP does not consist of dissipating any imposed pH gradient but rather in inhibiting directly the pH gradient-activated Cl− uptake and Cl−/Cl−exchange activities characterizing the intestinal BBM. Because these two activities can be explained in terms of a single (homogeneous) random, nonobligatory two-site Cl−-H+symporter, in which Cl−/Cl−exchange occurs by counterflow [F. Alvarado and M. Vasseur. Am. J. Physiol. 271 ( Cell Physiol. 40): C1612–C1628, 1996], we developed a new, more general three-site symport model that fully explains the Cl−uptake inhibitions caused by CCCP. This new model postulates the existence of a third, allosteric, inhibitory CCCP-binding site separate from either of the two substrate-binding sites of the Cl−-H+symporter, the Cl−-binding and the H+-binding sites. Finally, we show that, to explain the partial inhibitions observed, it is necessary to postulate that all the substrate-bound carrier complexes, =C-S, I=C-S, A=C-S, and IA=C-S, where C is carrier, I is inhibitor, S is substrate, and A is activator, can form and be translocated.
Capacitative Ca2+ entry is involved in cAMP synthesis in mouse parotid acini
Authors: Watson Eileen L. , Wu Zhiliang , Jacobson Kerry L. , Storm Daniel R. , Singh Jean C. , Ott Sabrina M.
Published: December 2017

Muscarinic receptor interaction leading to augmentation of isoproterenol-stimulated cAMP accumulation in mouse parotid acini involves Ca2+ (28). The effectiveness of capacitative Ca2+entry and intracellular Ca2+release on this response was determined in time course studies by using three independent tools to manipulate the free intracellular Ca2+ concentration: the muscarinic agonist carbachol, thapsigargin, and ionomycin. Time course studies revealed that Ca2+ release from intracellular stores by carbachol produced an early rapid increase (0.25–0.5 min) in stimulated cAMP levels, whereas capacitative Ca2+ entry resulted in a sustained increase in stimulated cAMP levels that was blocked by La3+. Capacitative Ca2+ entry, alone, was involved in thapsigargin and ionomycin augmentation of stimulated cAMP accumulation. The inability of phosphodiesterase inhibitors, 3-isobutyl-1-methylxanthine and milrinone, to prevent agonist augmentation of cAMP levels, as well as the finding that the type VIII adenylyl cyclase (ACVIII) is expressed in parotid acini, suggests that capacitative Ca2+ entry augments stimulated cAMP accumulation, at least in part, via activation of this adenylyl cyclase isoenzyme.
A cationic nonselective stretch-activated channel in the Reissner’s membrane of the guinea pig cochlea
Authors: Yeh Te-Huei , Herman Philippe , Tsai Ming-Cheng , Huy Patrice Tran Ba , Van Den Abbeele Thierry
Published: December 2017

The Reissner’s membrane (RM) separates in the mammalian cochlea the K+-rich endolymph from the Na+-rich perilymph. The patch-clamp technique was used to investigate the transport mechanisms in epithelial cells of RM freshly dissected from the guinea pig cochlea. This study shows a stretch-activated nonselective cationic channel (SA channel) with a linear current-voltage relationship (23 pS) highly selective for cations over anions [K+ ≈ Na+ (1) > Ba2+ (0.65) > Ca2+ (0.32) ≫ Cl− (0.14)] and activated by the intrapipette gradient pressure. The open probability-pressure relationship is best fitted by a Boltzmann distribution (half-maximal pressure = 37.8 mmHg, slope constant = 8.2 mmHg). SA channels exhibit a strong voltage dependency and are insensitive to internal Ca2+, ATP, and fenamates but are blocked by 1 μM GdCl3 in the pipette. They are reversibly activated by in situ superfusion of the cell with hyposmotic solutions. Kinetic studies show that depolarization and mechanical or osmotic stretch modify the closed and open time constants probably by a different mechanism. These channels could participate in pressure-induced modifications of ionic permeability of the RM.
Aldosterone stimulates intestinal Na+ absorption in rats by increasing NHE3 expression of the proximal colon
Authors: Cho Judy H. , Musch Mark W. , Bookstein Crescence M. , McSwine Rebecca L. , Rabenau Karen , Chang Eugene B.
Published: December 2017

Na+ retention by the colon in response to salt deprivation is mediated in part by the resulting secondary hyperaldosteronism. We show that experimental hyperaldosteronism, to levels seen with salt deprivation, causes an increase in the selective expression and activity of NHE3, an apically located isoform of the Na+/H+exchange family that functions in transepithelial Na+ absorption. The effect of aldosterone on NHE3 expression is tissue specific, occurring in intestine and not in kidney. Within the intestine, these effects are regional, being observed only in proximal colon, and different in distribution from that observed with glucocorticoids, where the predominant effect occurs in ileum. Although glucocorticoids are well known to exert many effects via regulation of transcript levels, the present study demonstrates that aldosterone stimulates intestinal Na+ absorption by increasing cellular NHE3 expression, a response that is tissue and region specific.
α-Cardiac-like myosin heavy chain as an intermediate between MHCIIa and MHCIβ in transforming rabbit muscle
Authors: Peuker Heidemarie , Conjard Agnès , Pette Dirk
Published: December 2017

To elucidate the sequence of myosin heavy chain (MHC) transitions in fast-to-slow transforming rabbit muscle, direct reverse transcriptase-polymerase chain reaction was applied for detecting mRNAs specific to five MHC isoforms in single fibers from control and low-frequency-stimulated tibialis anterior muscles. The detection of MHCIIb, MHCIId(x), MHCIα, and MHCIβ mRNAs was based on previously published methods. The RT-PCR assay for MHCIIa mRNA was based on the identification of a cDNA sequence in the 3′-region from which specific primers were derived. Comparisons between rat, rabbit, and human MHCIIa sequences revealed high degrees of sequence identities. MHC mRNA isoform patterns in single fibers from stimulated muscles showed hybrid fibers expressing the following combinations: MHCIId(x) + MHCIIa, MHCIId(x) + MHCIIa + MHCIα, MHCIId(x) + MHCIIa + MHCIα + MHCIβ, MHCIIa + MHCIα, MHCIIa + MHCIα + MHCIβ, and MHCIα + MHCIβ. The combination MHCIIa + MHCIβ without MHCIα was never seen. These coexpression patterns suggest that the fast-to-slow fiber transition results from sequential isoform expressions in the order MHCIId(x) → MHCIIa → MHCIα → MHCIβ. The allocation of MHCIα between MHCIIa and MHCIβ seems to be in line with graded differences in sequence identity of the 3′-regions of these mRNA isoforms.
Imaging caffeine-induced Ca2+transients in individual fast-twitch and slow-twitch rat skeletal muscle fibers
Authors: Pagala Murali K. D. , Taylor Stuart R.
Published: December 2017

Fast-twitch and slow-twitch rat skeletal muscles produce dissimilar contractures with caffeine. We used digital imaging microscopy to monitor Ca2+ (with fluo 3-acetoxymethyl ester) and sarcomere motion in intact, unrestrained rat muscle fibers to study this difference. Changes in Ca2+ in individual fibers were markedly different from average responses of a population. All fibers showed discrete, nonpropagated, local Ca2+ transients occurring randomly in spots about one sarcomere apart. Caffeine increased local Ca2+ transients and sarcomere motion initially at 4 mM in soleus and 8 mM in extensor digitorum longus (EDL; ∼23°C). Ca2+release subsequently adapted or inactivated; this was surmounted by higher doses. Motion also adapted but was not surmounted. Prolonged exposure to caffeine evidently suppressed myofilament interaction in both types of fiber. In EDL fibers, 16 mM caffeine moderately increased local Ca2+ transients. In soleus fibers, 16 mM caffeine greatly increased Ca2+ release and produced propagated waves of Ca2+(∼1.5–2.5 μm/s). Ca2+waves in slow-twitch fibers reflect the caffeine-sensitive mechanism of Ca2+-induced Ca2+ release. Fast-twitch fibers possibly lack this mechanism, which could account for their lower sensitivity to caffeine.
A divergent CFTR homologue: highly regulated salt transport in the euryhaline teleost F. heteroclitus
Authors: Singer Thomas D. , Tucker Stephen J. , Marshall William S. , Higgins Christopher F.
Published: December 2017

The killifish, Fundulus heteroclitus, is a euryhaline teleost fish capable of adapting rapidly to transfer from freshwater (FW) to four times seawater (SW). To investigate osmoregulation at a molecular level, a 5.7-kilobase cDNA homologous to human cystic fibrosis transmembrane conductance regulator (hCFTR) was isolated from a gill cDNA library from SW-adapted killifish. This cDNA encodes a protein product (kfCFTR) that is 59% identical to hCFTR, the most divergent form of CFTR characterized to date. Expression of kfCFTR in Xenopus oocytes generated adenosine 3′,5′-cyclic monophosphate-activated, Cl−-selective currents similar to those generated by hCFTR. In SW-adapted killifish, kfCFTR was expressed at high levels in the gill, opercular epithelium, and intestine. After abrupt exposure of FW-adapted killifish to SW, kfCFTR expression in the gill increased severalfold, suggesting a role for kfCFTR in salinity adaptation. Under similar conditions, plasma Na+levels rose significantly after 8 h and then fell, although it is not known whether these changes are directly responsible for the changes in kfCFTR expression. The killifish provides a unique opportunity to understand teleost osmoregulation and the role of CFTR.
Activation of p42mapk in human umbilical vein endothelial cells by interleukin-1α and tumor necrosis factor-α
Authors: May Michael J. , Wheeler-Jones Caroline P. D. , Houliston Rebecca A. , Pearson Jeremy D.
Published: December 2017

Work from this and other laboratories has identified a role for protein tyrosine kinases in interleukin-1α (IL-1α)- and tumor necrosis factor-α (TNF-α)-induced responses in endothelial cells. In this study, we show that activation of human umbilical vein endothelial cells (HUVEC) by IL-1α leads to increased tyrosine phosphorylation of several proteins including one with a molecular mass of ∼42 kDa. This protein was identified as p42mapk by Western blot analysis. Tyrosine phosphorylation and catalytic activation of p42mapk by IL-1α was transient, reaching maximal levels after 30 min and returning to basal levels by 120–300 min. Activation of p42mapk in HUVEC was also observed in response to TNF-α or to the protein kinase C (PKC)-activating phorbol ester phorbol 12-myristate 13-acetate (PMA). Pretreatment of HUVEC with IL-1α or TNF-α prevented reactivation of p42mapk by either cytokine but did not affect subsequent activation in response to PMA. Activation of p42mapk by PMA was significantly reduced by the PKC inhibitor Ro-31-8220 and completely inhibited by the protein tyrosine kinase inhibitor genistein. Genistein, but not Ro-31-8220, attenuated IL-1α- and TNF-α-induced p42mapk activation. Taken together, the results of this study demonstrate 1) that p42mapk is transiently activated in HUVEC by IL-1α and TNF-α, 2) that this activation is PKC independent, and 3) that a genistein-inhibitable tyrosine kinase may be an upstream regulator of cytokine-induced p42mapk activation in human endothelium.
Electrodiffusional ATP movement through the cystic fibrosis transmembrane conductance regulator
Authors: Cantiello Horacio F. , Jackson George R. , Grosman Claudio F. , Prat Adriana G. , Borkan Steven C. , Wang Yihan , Reisin Ignacio L. , O’Riordan Catherine R. , Ausiello Dennis A.
Published: December 2017

Expression of the cystic fibrosis transmembrane conductance regulator (CFTR), and of at least one other member of the ATP-binding cassette family of transport proteins, P-glycoprotein, is associated with the electrodiffusional movement of the nucleotide ATP. Evidence directly implicating CFTR expression with ATP channel activity, however, is still missing. Here it is reported that reconstitution into a lipid bilayer of highly purified CFTR of human epithelial origin enables the permeation of both Cl− and ATP. Similar to previously reported data for in vivo ATP currents of CFTR-expressing cells, the reconstituted channels displayed competition between Cl− and ATP and had multiple conductance states in the presence of Cl− and ATP. Purified CFTR-mediated ATP currents were activated by protein kinase A and ATP (1 mM) from the “intracellular” side of the molecule and were inhibited by diphenylamine-2-carboxylate, glibenclamide, and anti-CFTR antibodies. The absence of CFTR-mediated electrodiffusional ATP movement may thus be a relevant component of the pleiotropic cystic fibrosis phenotype.
Identification of clathrin and clathrin adaptors on tubulovesicles of gastric acid secretory (oxyntic) cells
Authors: Okamoto Curtis T. , Karam Sherif M. , Jeng Young Y. , Forte John G. , Goldenring James R.
Published: December 2017

γ-Adaptin and clathrin heavy chain were identified on tubulovesicles of gastric oxyntic cells with the anti-γ-adaptin monoclonal antibody (MAb) 100/3 and an anti-clathrin heavy chain MAb (MAb 23), respectively. In Western blots, crude gastric microsomes from rabbit and rat and density gradient-purified, H-K-ATPase-rich microsomes from these same species were immunoreactive for γ-adaptin and clathrin. In immunofluorescent labeling of isolated rabbit gastric glands, anti-γ-adaptin and anti-clathrin heavy chain immunoreactivity appeared to be concentrated in oxyntic cells. In primary cultures of rabbit oxyntic cells, the immunocytochemical distribution of γ-adaptin immunoreactivity was similar to that of the tubulovesicular membrane marker in oxyntic cells, the H-K-ATPase. Further biochemical characterization of the tubulovesicular γ-adaptin-containing complex suggested that it has a subunit composition that is typical of that for a clathrin adaptor: in addition to the γ-adaptin subunit, it contains a β-adaptin subunit and other subunits of apparent molecular masses of 50 kDa and 19 kDa. From solubilized gastric microsomes from rabbit, γ-adaptin could be copurified with the major cargo protein of tubulovesicles, the H-K-ATPase. Thus this tubulovesicular coat may bind directly to the H-K-ATPase and may thereby mediate the regulated trafficking of the H-K-ATPase at the apical membrane of the oxyntic cell during the gastric acid secretory cycle. Given the similarities of the regulated trafficking of the H-K-ATPase with recycling of cargo through the apical recycling endosome of many epithelial cells, we propose that tubulovesicular clathrin and adaptors may regulate some part of an apical recycling pathway in other epithelial cells.
Myogenic NOS in canine lower esophageal sphincter: enzyme activation, substrate recycling, and product actions
Authors: Salapatek Anne Marie F. , Wang Yu-Fang , Mao Yu-Kang , Mori Masataka , Daniel Edwin E.
Published: December 2017

Depolarization elicited outward K+ currents from canine lower esophageal sphincter (LES) muscle cells, primarily through iberiotoxin (IbTX)- and tetraethylammonium-sensitive Ca2+-dependent K+ channels. Current magnitudes varied with pipette Ca2+concentration (EC50 = 108.5 nM). N G-nitro-l-arginine (l-NNA, 10−4 M), IbTX (10−8 M), or buffering intracellular Ca2+ to 8 nM decreased outward currents >80%. Sodium nitroprusside (NaNP, 10−4 M) restoredl-NNA-inhibited or low intracellular Ca2+ concentration (not IbTX)-inhibited currents.l-NNA or IbTX application depolarized LES cells from −43 to −35 mV. NaNP restored the membrane potential to −46 mV afterl-NNA but not after IbTX application. Nifedipine (30 μM) reduced outward currents and abolished or reduced l-NNA or NaNP effects, respectively. Immunocytochemistry revealed the presence of both argininosuccinate synthetase and argininosuccinate lyase in LES muscle cells. l-Citrulline, likel-arginine, reversedl-NNA inhibition of outward currents; only l-arginine reversed inhibition of outward currents by an antibody to argininosuccinate synthetase. Therefore, endogenous nitric oxide production, activated by Ca2+entrance involving l-type Ca2+ channels, may continuously enhance outward currents to modulate LES muscle cell membrane potential and excitability.
Serum deprivation induces a unique hypercontractile phenotype of cultured smooth muscle cells
Authors: Ma Xuefei , Wang Ying , Stephens Newman L.
Published: December 2017

Chronic asthma is characterized by hypertrophy and hyperplasia of airway smooth muscle cells (SMC) that limit airflow by a geometric effect. Whether contractility of airway SMC is altered is not clear. Cultured cells were used as a model of hyperplasia. Phenotypic changes seen indicated conversion to a synthetic, weakly contractile type. At confluence, although limited reversal of protein changes was seen, no restoration in contractility occurred. Phenotypic modulation of postconfluent cultured airway SMC under prolonged serum deprivation (arrested cells) is reported here. Two phenotypically distinct groups of cells were identified in primary airway SMC cultures: 1) elongated spindle-shaped cells, which expressed large amounts of smooth muscle contractile and regulatory proteins, and 2) flat and stellate cells, which expressed very little. The first group showed a surprising shortening capacity and a velocity that was even greater than that of the freshly isolated cells, whereas the second group became spherical and noncontractile. Even more surprising was that the myosin heavy chain (MHC) isoform (SM-B) generally said to be associated with the higher shortening velocity disappeared from the cell, while the content of the key rate-limiting regulating enzyme, myosin light chain kinase (MLCK), increased 30-fold. We conclude that a functional, contractile phenotype of airway SMC can be obtained by prolonged serum deprivation. We speculate that the increased contractility could be the result of increased phosphorylation of the 20-kDa myosin light chain resulting from increased content of smooth muscle MLCK rather than any increase in endogenous MHC ATPase activity. This model may be useful for study of SMC differentiation and contraction.
Contractile activity is required for sarcomeric assembly in phenylephrine-induced cardiac myocyte hypertrophy
Authors: Eble Diane M. , Qi Ming , Waldschmidt Stephanie , Lucchesi Pamela A. , Byron Kenneth L. , Samarel Allen M.
Published: December 2017

Agonist-induced hypertrophy of cultured neonatal rat ventricular myocytes (NRVM) has been attributed to biochemical signals generated during receptor activation. However, NRVM hypertrophy can also be induced by spontaneous or electrically stimulated contractile activity in the absence of exogenous neurohormonal stimuli. Using single-cell imaging of fura 2-loaded myocytes, we found that low-density, noncontracting NRVM begin to generate intracellular Ca2+ concentration ([Ca2+]i) transients and contractile activity within minutes of exposure to the α1-adrenergic agonist phenylephrine (PE; 50 μM). However, NRVM pretreated with verapamil and then stimulated with PE failed to elicit [Ca2+]itransients and beating. We therefore examined whether PE-induced [Ca2+]itransients and contractile activity were required to elicit specific aspects of the hypertrophic phenotype. PE treatment (48–72 h) increased cell size, total protein content, total protein-to-DNA ratio, and myosin heavy chain (MHC) isoenzyme content. PE also stimulated sarcomeric protein assembly and prolonged MHC half-life. However, blockade of voltage-gated L-type Ca2+ channels with verapamil, diltiazem, or nifedipine (10 μM) blocked PE-induced total protein and MHC accumulation and prevented the time-dependent assembly of myofibrillar proteins into sarcomeres. Inhibition of actin-myosin cross-bridge cycling with 2,3-butanedione monoxime (7.5 mM) also prevented PE-induced total protein and MHC accumulation, indicating that mechanical activity, rather than [Ca2+]itransients per se, was required. In contrast, blockade of [Ca2+]itransients and contractile activity did not prevent the PE-induced increase in cell surface area, activation of the mitogen-activated protein kinases ERK1 and ERK2, or upregulation of atrial natriuretic factor gene expression. Thus contractile activity is required to elicit some but not all aspects of the the hypertrophic phenotype induced by α1-adrenergic receptor activation.
A second enzyme protecting mineralocorticoid receptors from glucocorticoid occupancy
Authors: Morris David J. , Latif Syed A. , Rokaw Michael D. , Watlington Charles O. , Johnson John P.
Published: December 2017

We have confirmed that A6 cells (derived from kidney of Xenopus laevis), which contain both mineralocorticoid and glucocorticoid receptors, do not normally possess 11β-hydroxysteroid dehydroxgenase (11β-HSD1 or 11β-HSD2) enzymatic activity and so are without apparent “protective” enzymes. A6 cells do not convert the glucocorticoid corticosterone to 11-dehydrocorticosterone but do, however, possess steroid 6β-hydroxylase that transforms corticosterone to 6β-hydroxycorticosterone. This hydroxylase is cytochrome P-450 3A (CYP3A). We have now determined the effects of 3α,5β-tetrahydroprogesterone and chenodeoxycholic acid (both inhibitors of 11β-HSD1) and 11-dehydrocorticosterone and 11β-hydroxy-3α,5β-tetrahydroprogesterone (inhibitors of 11β-HSD2) and carbenoxalone, which inhibits both 11β-HSD1 and 11β-HSD2, on the actions and metabolism of corticosterone and active Na+ transport [short-circuit current ( I sc)] in A6 cells. All of these 11β-HSD inhibitory substances induced a significant increment in corticosterone-induced I sc, which was detectable within 2 h. However, none of these agents caused an increase in I sc when incubated by themselves with A6 cells. In all cases, the additional I sc was inhibited by the mineralocorticoid receptor (MR) antagonist, RU-28318, whereas the original I scelicited by corticosterone alone was inhibited by the glucocorticoid receptor antagonist, RU-38486. In separate experiments, each agent was shown to significantly inhibit metabolism of corticosterone to 6β-hydroxycorticosterone in A6 cells, and a linear relationship existed between 6β-hydroxylase inhibition and the MR-mediated increase in I scin the one inhibitor tested. Troleandomycin, a selective inhibitor of CYP3A, inhibited 6β-hydroxylase and also significantly enhanced corticosterone-induced I sc at 2 h. These experiments indicate that the enhanced MR-mediated I sc in A6 cells may be related to inhibition of 6β-hydroxylase activity in these cells and that this 6β-hydroxylase (CYP3A) may be protecting the expression of corticosterone-induced active Na+ transport in A6 cells by MR-mediated mechanism(s).
Structure and in vitro function of human subcutaneous small arteries in mild heart failure
Authors: Stephens Nicola , Drinkhill Mark J. , Hall Alistair S. , Ball Stephen G. , Heagerty Anthony M.
Published: December 2017

The structure and function of subcutaneous small arteries from patients with mild heart failure ( n = 27) 6–43 mo after myocardial infarction were compared with vessels from healthy control subjects ( n = 10). Patients were randomized to treatment with placebo or the angiotensin-converting enzyme inhibitor ramipril starting 3–10 days after myocardial infarction. Dissected arterial vessels were mounted on a wire myograph for measurement of morphology and isometric tension. Morphology was not different in arteries from the three groups. Responses to norepinephrine, angiotensin II, and electrical field stimulation were similar in arteries from placebo-treated patients with mild heart failure and control subjects. Similarly, endothelium-dependent and -independent relaxation was normal in arteries from patients with mild heart failure. Ramipril therapy was associated with functional alterations: vasoconstrictor responses to norepinephrine and angiotensin II were significantly enhanced compared with placebo ( P < 0.001). These data suggest that vascular structure and function are not different in vitro in subcutaneous arteries from placebo-treated patients with mild heart failure. Angiotensin-converting enzyme inhibitor therapy is associated with enhanced vasoconstriction to norepinephrine and angiotensin II, which may reflect upregulation of receptor-mediated events.
Milrinone inhibits contractility in skinned skeletal muscle fibers
Authors: Seow C. Y. , Morishita L. , Bressler B. H.
Published: December 2017

Direct action of the cardiotonic bipyridine milrinone on the cross bridges of single fibers of skinned rabbit skeletal muscle was investigated. At 10°C and pH 7.0, milrinone reduced isometric tension in a logarithmically concentration-dependent manner, with a 55% reduction in force at 0.6 mM. Milrinone also reduced Ca2+ sensitivity of skinned fibers in terms of force production; the shift in the force-pCa curve indicated a change in the pCa value at 50% maximal force from 6.10 to 5.94. The unloaded velocity of shortening was reduced by 18% in the presence of 0.6 mM milrinone. Parts of the transient tension response to step change in length were altered by milrinone, so that the test and control transients could not be superimposed. The results indicate that milrinone interferes with the cross-bridge cycle and possibly detains cross bridges in low-force states. The results also suggest that the positive inotropic effect of milrinone on cardiac muscle is probably not due to the drug’s direct action on the muscle cross bridges. The specific and reversible action of the bipyridine on muscle cross bridges makes it a potentially useful tool for probing the chemomechanical cross-bridge cycle.
Modulation of human neuronal α1E-type calcium channel by α2δ-subunit
Authors: Qin Ning , Olcese Riccardo , Stefani Enrico , Birnbaumer Lutz
Published: December 2017

Calcium channels are composed of a pore-forming subunit, α1, and at least two auxiliary subunits, β- and α2δ-subunits. It is well known that β-subunits regulate most of the properties of the channel. The function of α2δ-subunit is less understood. In this study, the effects of the calcium channel α2δ-subunit on the neuronal α1E voltage-gated calcium channel expressed in Xenopus oocytes was investigated without and with simultaneous coexpression of either the β1b- or the β2a-subunit. Most aspects of α1E function were affected by α2δ. Thus α2δ caused a shift in the current-voltage and conductance-voltage curves toward more positive potentials and accelerated activation, deactivation, and the installation of the inactivation process. In addition, the efficiency with which charge movement is coupled to pore opening assessed by determining ratios of limiting conductance to limiting charge movement was decreased by α2δ by factors that ranged from 1.6 ( P < 0.01) for α1E-channels to 3.0 ( P < 0.005) for α1Eβ1b-channels. These results indicate that α2δ facilitates the expression and the maturation of α1E-channels and converts these channels into molecules responding more rapidly to voltage.
Chronic exposure to TPA depletes PKCα and augments Ca-dependent insulin secretion from cultured rat islets
Authors: Zawalich Walter S. , Bonnet-Eymard Marc , Zawalich Kathleen C. , Yaney Gordon C.
Published: December 2017

The insulin secretory responses of rat islets to glucose (15 mM), 12- O-tetradecanoylphorbol 13-acetate (TPA; 500 nM), and potassium (30 mM) were determined from perifused islets cultured for 22–24 h in CMRL-1066 medium (control cultured) or islets cultured in the additional presence of 500 nM TPA. Islet content of protein kinase C α (PKCα) and serine and threonine phosphoprotein patterns were also monitored after the culture period. Compared with freshly isolated islets, culturing alone had no adverse effect on the capacity of TPA or 30 mM potassium to stimulate secretion or on the islet content of PKCα. In agreement with previous studies, culturing in TPA reduced the islet content of immunoreactive PKCα by >95% and abolished the capacity of the phorbol ester to stimulate secretion during a subsequent dynamic perifusion. Culturing in TPA slightly improved the insulin secretory response to 15 mM glucose compared with control-cultured islets; however, sustained rates of 15 mM glucose-induced secretion from these islets were significantly less than the responses of freshly isolated islets. Islets cultured in TPA responded to 30 mM potassium with a markedly amplified insulin secretory response that was abolished by nitrendipine. Enhanced phosphorylation of several islet proteins was also observed in TPA-cultured islets compared with control-cultured islets. These findings demonstrate that culturing alone impairs glucose-induced secretion, a response that is improved but still subnormal compared with freshly isolated islet responses, if TPA is included in the culture medium. Sustained phosphorylation of several islet proteins in TPA-cultured islets may account, at least in part, for augmented calcium-dependent secretion.
Equibiaxial strain and strain rate stimulate early activation of G proteins in cardiac fibroblasts
Authors: Gudi Siva R. P. , Lee Ann A. , Clark Craig B. , Frangos John A.
Published: December 2017

Cardiac fibroblasts are responsible for the production of the extracellular matrix of the heart, with alterations of fibroblast function implicated in myocardial infarction and cardiac hypertrophy. Here the role of heterotrimeric GTP-binding proteins (G proteins) in the mechanotransduction of strain in rat cardiac fibroblasts was investigated. Cells in an equibiaxial stretch device were incubated with the photoreactive GTP analog azidoanalido [α-32P]GTP (AAGTP) and were subjected to various regimens of strain. Autoradiographic analysis showed a 42-kDa protein labeled for cells exposed to 12 cycles of 3% strain or 6 cycles of 6% strain over 60 s (strain rate of 1.2%/s), whereas 6 cycles of 3% strain (0.6%/s) elicited no measurable response. To further investigate the role of strain rate, a single 6% cycle over 10 or 60 s (1.2% and 0.2%/s, respectively) was applied, with the more rapid cycle stimulating AAGTP binding, whereas the lower strain rate showed no response. In cells subjected to a single 6% cycle/10 s, immunoprecipitation identified the AAGTP-labeled 42-kDa band as the G protein subunits Gαq and Gαi1. These results demonstrate that G protein activation represents one of the early mechanotransduction events in cardiac fibroblasts subjected to mechanical strain, with the rate at which the strain is applied modulating this response.
Synergistic induction of ornithine decarboxylase by asparagine and gut peptides in intestinal crypt cells
Authors: Wang Jian-Ying , Li Ji , Patel Anami R. , Summers Stephen , Li Li , Bass Barbara L.
Published: December 2017

The objective of this study was to determine whether the amino acid asparagine stimulated the activity of ornithine decarboxylase (ODC) synergistically with epidermal growth factor (EGF) or gastrin in IEC-6 cells, a line of normal rat small intestinal crypt cells. Cells were grown in DMEM containing 5% dialyzed fetal bovine serum, and serum was deprived for 24 h before experiments. Exposure to EGF or gastrin alone increased ODC activity 4.5- to 6-fold. Asparagine alone increased the enzyme activity 10- to 13-fold in IEC-6 cells. Simultaneous addition of asparagine and EGF or gastrin, however, increased ODC activity more than 40-fold. In contrast, there was no synergistic induction of ODC activity when gastrin and EGF were added together. Increased ODC activity in cells treated with asparagine and EGF or gastrin was associated with an increase in ODC mRNA and protein levels. The rate of transcription of the ODC gene was significantly increased by exposure to EGF or gastrin. Asparagine alone had little or no effect on the rate of transcription of the ODC gene. When given together with EGF or gastrin, asparagine also had no additional effect on the transcription rate of the ODC gene. The half-life of mRNA for ODC in unstimulated IEC-6 cells was ∼30 min and increased to more than 2 h in cells exposed to asparagine, although neither gastrin nor EGF prolonged the stability of ODC mRNA. The half-life of mRNA for ODC after combined addition of asparagine and EGF or gastrin was extended to ∼2 h, similar to asparagine alone. Combined addition of asparagine and EGF or gastrin also significantly increased DNA synthesis compared with cells exposed to each of the three agents alone. In conclusion, 1) simultaneous addition of asparagine and EGF or gastrin increases ODC activity in a synergistic manner and 2) asparagine increases ODC mRNA levels through completely distinct mechanisms from EGF or gastrin. EGF or gastrin specifically stimulates transcription of the ODC gene, whereas asparagine affects a posttranscriptional process.
Pharmacological characterization of swelling-induced d-[3H]aspartate release from primary astrocyte cultures
Authors: Rutledge Eric M. , Aschner Michael , Kimelberg Harold K.
Published: December 2017

During stroke or head trauma, extracellular K+concentration increases, which can cause astrocytes to swell. In vitro, such swelling causes astrocytes to release excitatory amino acids, which may contribute to excitotoxicity in vivo. Several putative swelling-activated channels have been identified through which such anionic organic cellular osmolytes can be released. In the present study, we sought to identify the swelling-activated channel(s) responsible ford-[3H]aspartate release from primary cultured astrocytes exposed to either KCl or hypotonic medium. KCl-inducedd-[3H]aspartate release was inhibited by the anion channel inhibitors 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), dideoxyforskolin, L-644711, ATP, ITP, 3′-azido-3′-deoxythymidine, DIDS, and tamoxifen but not by cAMP. The cell swelling caused by raised KCl was not inhibited by extracellular ATP or tamoxifen as measured by an electrical impedance method, which suggests that these anion channel inhibitors directly blocked the channel responsible for efflux. Extracellular nucleotides and DIDS, however, had no or only partial effects ond-[3H]aspartate release from cells swollen by hypotonic medium, but such release was inhibited by NPPB, dideoxyforskolin, and tamoxifen. Of the swelling-activated channels so far identified, our data suggest that a volume-sensitive outwardly rectifying channel is responsible ford-[3H]aspartate release from primary cultured astrocytes during raised extracellular K+ and possibly during hypotonic medium-induced release.
Growth factor-like action of lysophosphatidic acid on human B lymphoblasts
Authors: Rosskopf Dieter , Daelman Wim , Busch Stefan , Schürks Markus , Hartung Kathrin , Kribben Andreas , Michel Martin C. , Siffert Winfried
Published: December 2017

Proliferation and immunoglobulin secretion of B lymphocytes are regulated by specific antigens and numerous accessory immunomodulatory factors. Lysophosphatidic acid (LPA) is a glycerophospholipid mediator that is released from activated blood platelets, attains high levels in serum, and exerts potent stimulatory effects on, e.g., neutrophils, monocytes, and T lymphocytes. LPA is also generated by a secretory, cytokine-inducible phospholipase A2 present in high concentrations in inflammatory exudates and septic states. We investigated effects of LPA on human Epstein-Barr virus-immortalized B lymphoblasts, a model for immunoglobulin-secreting B cells. Intracellular Ca2+ was determined with fura 2 and the formation of inositol 1,4,5-trisphosphate by anion-exchange chromatography. LPA stimulated an increase in inositol 1,4,5-trisphosphate levels and induced a transient rise in intracellular free Ca2+concentration from 105 ± 17 to 226 ± 21 nM. This Ca2+ signal resulted from Ca2+ mobilization and Ca2+ influx and was subject to homologous desensitization. Pertussis toxin inhibited these responses by ∼70%. Furthermore, LPA stimulated a 27.5% increase in guanosine 5′- O-(3-thiotriphosphate) binding to permeabilized B lymphoblasts, which suggests the direct activation of pertussis toxin-sensitive G proteins by LPA. LPA stimulated a strong increase in the specific phosphorylation of the mitogen-activated protein kinase (immunoblot analysis) that was prevented by the MEK inhibitor PD-98059. Finally, LPA triggered a 2-fold increase in DNA synthesis ([3H]thymidine incorporation) and a 2-fold increase in B lymphoblast number and evoked a 20- to 50-fold increase in immunoglobulin formation. By RT-PCR we detected specific mRNA transcripts for the recently cloned human LPA receptor. Thus our data suggest that LPA behaves as a B cell growth factor.
Length-dependent modulation of smooth muscle activation: effects of agonist, cytochalasin, and temperature
Authors: Youn Thomas , Kim Son A. , Hai Chi-Ming
Published: December 2017

We tested the hypothesis that mechanical strain modulates agonist sensitivity of smooth muscle by measuring myosin phosphorylation and contractile force in bovine tracheal smooth muscle activated by various concentrations of the muscarinic receptor agonist carbachol and at various muscle lengths. Increasing carbachol concentration by 10,000-fold did not restore myosin phosphorylation levels at shorter muscle lengths to the level at optimal length ( L o). Maximum levels of myosin phosphorylation induced by carbachol at 0.6, 0.8, and 1.0 L o were similar but became lower at <0.6 L o. Cytochalasin D significantly attenuated carbachol-induced contraction by 54%. In addition, cytochalasin D treatment induced a parallel downward shift in the length-myosin phosphorylation relation. Lowering temperature from 37 to 23°C did not significantly change the length dependencies of carbachol-induced active force and myosin phosphorylation. These results have led us to conclude that 1) agonist sensitivity and maximum level of activation (as measured by myosin phosphorylation) are targets of length-dependent modulation, 2) actin filaments involved in contraction and length-dependent modulation are distinct in sensitivity to cytochalasin D, and 3) length-dependent modulation is relatively temperature insensitive.