bluuebunny/crossref_metadata_2025 huggingface dataset:
In vivo analysis of the myosin heavy chain IIB promoter region
Authors: Swoap Steven J.
Published: December 2017

The myosin heavy chain (MHC) IIB gene is preferentially expressed in fast-twitch muscles of the hindlimb, such as the tibialis anterior (TA). The molecular mechanism(s) for this preferential expression are unknown. The goals of the current study were 1) to determine whether the cloned region of the MHC IIB promoter contains the necessary cis-acting element(s) to drive fiber-type-specific expression of this gene in vivo, 2) to determine which region within the promoter is responsible for fiber-type-specific expression, and 3) to determine whether transcription off of the cloned region of the MHC IIB promoter accurately mimics endogenous gene expression in a muscle undergoing a fiber-type transition. To accomplish these goals, a 2.6-kilobase fragment of the promoter-enhancer region of the MHC IIB gene was cloned upstream of the firefly luciferase reporter gene and coinjected with pRL-cytomegalovirus (CMV) (CMV promoter driving the renilla luciferase reporter) into the TA and the slow soleus muscle. Firefly luciferase activity relative to renilla luciferase activity within the TA was 35-fold greater than within the soleus. Deletional analysis demonstrated that only the proximal 295 base pairs (pGL3IIB0.3) were required to maintain this muscle-fiber-type specificity. Reporter gene expression of pGL3IIB0.3 construct was significantly upregulated twofold in unweighted soleus muscles compared with normal soleus muscles. Thus the region within the proximal 295 base pairs of the MHC IIB gene contains at least one element that can drive fiber-type-specific expression of a reporter gene.
Osmotic regulation of intestinal epithelial Na+-K+-Cl− cotransport: role of Cl− and F-actin
Authors: Matthews Jeffrey B. , Smith Jeremy A. , Mun Edward C. , Sicklick Jason K.
Published: December 2017

Previous data indicate that adenosine 3′,5′-cyclic monophosphate activates the epithelial basolateral Na+-K+-Cl−cotransporter in microfilament-dependent fashion in part by direct action but also in response to apical Cl− loss (due to cell shrinkage or decreased intracellular Cl−). To further address the actin dependence of Na+-K+-Cl−cotransport, human epithelial T84 monolayers were exposed to anisotonicity, and isotopic flux analysis was performed. Na+-K+-Cl−cotransport was activated by hypertonicity induced by added mannitol but not added NaCl. Cotransport was also markedly activated by hypotonic stress, a response that appeared to be due in part to reduction of extracellular Cl− concentration and also to activation of K+ and Cl− efflux pathways. Stabilization of actin with phalloidin blunted cotransporter activation by hypotonicity and abolished hypotonic activation of K+ and Cl− efflux. However, phalloidin did not prevent activation of cotransport by hypertonicity or isosmotic reduction of extracellular Cl−. Conversely, hypertonic but not hypotonic activation was attenuated by the microfilament disassembler cytochalasin D. The results emphasize the complex interrelationship among intracellular Cl− activity, cell volume, and the actin cytoskeleton in the regulation of epithelial Cl− transport.
Phosphate transport by the human renal cotransporter NaPi-3 expressed in HEK-293 cells
Authors: Timmer Richard T. , Gunn Robert B.
Published: December 2017

The human renal Na-PO4cotransporter gene NaPi-3 was expressed in human embryonic kidney HEK-293 cells, and the transport characteristics were measured in cells transfected with a vector containing NaPi-3 or with the vector alone (sham transfected). The initial rate of32PO4influx had saturation kinetics for external Na and PO4 with[Formula: see text] of 128 mM (PO4 = 0.1 mM) and[Formula: see text]of 0.084 mM (extracellular Na = 143 mM) in sham- and NaPi-3-transfected cells expressing the transporter. Transfection had no effect on the Na-independent32PO4influx, but transfection increased Na-dependent32PO4influxes 2.5- to 5-fold. Of the alkali cations, only Na significantly supported PO4 influx. Arsenate inhibited flux with an inhibition constant of 0.4 mM. The phosphate transport in sham- and NaPi-3-transfected cells has nearly the same temperature dependence in the absence and presence of extracellular Na. The Na-dependent phosphate flux decreased with pH in sham-transfected cells but was pH independent in transfected cells. The Na-dependent32PO4influx was inhibited by p-chloromercuriphenylsulfonate, phosphonoformate, phloretin, vanadate, and 5-( N-methyl- N-isobutyl)-amiloride but not by amiloride or other amiloride analogs. These functional characteristics are in general agreement with the known behavior of NaPi-3 homologues in the renal tubule of other species and, thus, demonstrate the fidelity of this transfection system for the study of this protein. Commensurate with the increased functional expression, there was an increase in the amount of NaPi-3 protein by Western analysis.
Type II protein kinase A regulates CFTR in airway, pancreatic, and intestinal cells
Authors: Steagall Wendy K. , Kelley Thomas J. , Marsick Rebecca J. , Drumm Mitchell L.
Published: December 2017

The type of protein kinase A (PKA) responsible for cystic fibrosis transmembrane conductance regulator (CFTR) activation was determined with adenosine 3′,5′-cyclic monophosphate analogs capable of selectively activating type I or type II PKA. The type II-selective pair stimulated chloride efflux in airway, pancreatic, and colonic epithelial cells; the type I-selective pair only stimulated a calcium-dependent efflux in airway cells. The type II-selective analogs activated larger increases in CFTR-mediated current than did the type I-selective analogs. Measurement of soluble PKA activity demonstrated similar levels stimulated by type I- and type II-selective analogs, creating an apparent paradox regarding PKA activity and current generated. Also, addition of forskolin after the type I-selective analogs resulted in an increase in current; little increase was seen after the type II-selective analogs. Measurement of insoluble PKA activity stimulated by the analogs resolved this paradox. Type II-selective analogs stimulated three times as much insoluble PKA activity as the type I-selective pair, indicating that differential activation of PKA in cellular compartments is important in CFTR regulation.
Reversible effects of acute hypertension on proximal tubule sodium transporters
Authors: Zhang Yibin , Magyar Clara E. , Norian John M. , Holstein-Rathlou Niels-H. , Mircheff Austin K. , McDonough Alicia A.
Published: December 2017

Acute hypertension provokes a rapid decrease in proximal tubule sodium reabsorption with a decrease in basolateral membrane sodium-potassium-ATPase activity and an increase in the density of membranes containing apical membrane sodium/hydrogen exchangers (NHE3) [Y. Zhang, A. K. Mircheff, C. B. Hensley, C. E. Magyar, D. G. Warnock, R. Chambrey, K.-P. Yip, D. J. Marsh, N.-H. Holstein-Rathlou, and A. A. McDonough. Am. J. Physiol.270 ( Renal Fluid Electrolyte Physiol.39): F1004–F1014, 1996]. To determine the reversibility and specificity of these responses, rats were subjected to 1) elevation of blood pressure (BP) of 50 mmHg for 5 min, 2) restoration of normotension after the first protocol, or 3) sham operation. Systolic hypertension increased urine output and endogenous lithium clearance three- to fivefold within 5 min, but these returned to basal levels only 15 min after BP was restored. Renal cortex lysate was fractionated on sorbitol gradients. Basolateral membrane sodium-potassium-ATPase activity (but not subunit immunoreactivity) decreased one-third to one-half after BP was elevated and recovered after BP was normalized. After BP was elevated, 55% of the apical NHE3 immunoreactivity, smaller fractions of sodium-phosphate cotransporter immunoreactivity, and apical alkaline phosphatase and dipeptidyl-peptidase redistributed to membranes of higher density enriched in markers of the intermicrovillar cleft (megalin) and endosomes (Rab 4 and Rab 5), whereas density distributions of the apical cytoskeleton protein villin were unaltered. After 20 min of normalized BP, all the NHE3 and smaller fractions of the other apical membrane proteins returned to their original distributions. These findings suggest that the dynamic regulation of proximal tubule sodium transport by acute changes in BP may be mediated by rapid reversible regulation of sodium pump activity and relocation of apical sodium transporters.
Retinoids modulate P2Upurinergic receptor-mediated changes in transcervical paracellular permeability
Authors: Gorodeski George I. , Pal Dipika , Rorke Ellen A. , Eckert Richard L. , Burfeind Peter
Published: December 2017

In human cervical cells, extracellular ATP induces an acute decrease in the resistance of the lateral intercellular space, the phase I response, followed by a delayed increase in tight junctional resistance, the phase II response. These responses depend on vitamin A because incubation of cells in retinoid-free medium (RFM) abolished both responses. Treatment with retinoic acid restored the phase I response in full, but the amplitude of the phase II response was restored only partly. Shorter incubations and lower concentrations of retinoic acid [half-maximal effective concentration ( K ½) = 0.1 μM] were required for restoring the phase I response than were required for reversing the phase II response ( K ½ = 1 μM). The phase I response could be restored by ligands that bind to either retinoic acid receptors (RARs) or retinoid X receptors, but only RAR agonists had an effect on phase II response. RFM had no effect on decreases in resistance induced by ionomycin, but it attenuated phase II-like increases in resistance induced by KCl or by 1,2-dioctanoyl- sn-diglycerol (diC8). Actinomycin D blocked phase IIresponse but not phase I response or the responses to ionomycin, KCl, or diC8. These results suggest that retinoids act on cervical cells via distinct retinoid receptor mechanisms and modulate phase I and phase II changes in resistance by regulating distinct signal mechanisms.
Contractile function and low-intensity exercise effects of old dystrophic (mdx) mice
Authors: Hayes Alan , Williams David A.
Published: December 2017

Old mdx mice display a severe myopathy almost identical to Duchenne’s muscular dystrophy. This study examined the contractile properties of old mdxmuscles and investigated any effects of low-intensity exercise. Isometric contractile properties of the extensor digitorum longus (EDL) and soleus muscles were tested in adult (8–10 mo) and old (24 mo, split into sedentary and exercised groups) mdx mice. The EDL and soleus from old mdx mice exhibited decreased absolute twitch and tetanic forces, and the soleus exhibited a >50% decrease in relative forces (13.4 ± 0.4 vs. 6.0 ± 0.9 N/cm2) compared with adult mice. Old mdx muscles also showed longer contraction times and a higher percentage of type I fibers. Normal and mdx mice completed 10 wk of swimming, but mdx mice spent significantly less time swimming than normal animals (7.8 ± 0.4 vs. 15.8 ± 1.1 min, respectively). However, despite their severe dystrophy, mdx muscles responded positively to the low-intensity exercise. Relative tetanic tensions were increased (∼25% and ∼45% for the EDL and soleus, respectively) after the swimming, although absolute forces were unaffected. Thus these results indicate that, even with a dystrophin-deficient myopathy, mdx muscles can still respond to low-intensity exercise. This study shows that the contractile function of muscles of old mdx mice displays many similarities to that of human dystrophic patients and provides further evidence that the use of non-weight-bearing, low-intensity exercises, such as swimming, has no detrimental effect on dystrophic muscle and could be a useful therapeutic aid for sufferers of muscular dystrophy.
Sertoli cell expression of the cystic fibrosis transmembrane conductance regulator
Authors: Boockfor Fredric R. , Morris Rebecca A. , DeSimone Dennis C. , Hunt D. Margaret , Walsh Kenneth B.
Published: December 2017

Mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been associated with a number of male reproductive problems, including testis abnormalities and a reduction in germ cell quality and number. To establish at least one site of functional CFTR expression in the testis, we subjected cultured Sertoli cells to analysis of message, protein, and channel activity for CFTR. With reverse transcription-polymerase chain reaction, we obtained evidence for the presence of CFTR RNA when CFTR primers were used with RNA from cultured Sertoli cells. Western analysis performed with both anti-R and anti-C domain CFTR antibodies revealed immunoreactive material in extracts from primary Sertoli cell cultures that seemed consistent with CFTR previously identified in other cells and tissues. This led us to perform more detailed studies using the whole cell arrangement of the patch-clamp technique. Application of the membrane-soluble cAMP analog, 8-chlorophenylthio-cAMP, resulted in the activation of a Cl− current that displayed a permeability sequence of Br− > I− ≥ Cl− and was blocked by diphenylamine-2-carboxylate and glibenclamide. In addition, a 13-pS conductance Cl− channel was measured in excised membrane patches exposed to the catalytic subunit of protein kinase A. When taken together, our findings of evidence of CFTR message, immunoreactive material that appeared consistent with CFTR, and Cl− channels with properties similar to those reported for CFTR provide strong evidence that Sertoli cells express a functional CFTR-like protein. The presence of CFTR in these cells may be needed to maintain the specific nutritional and fluid balance in the seminiferous tubule that is vital for normal spermatogenesis.
Is cytoskeletal tension a major determinant of cell deformability in adherent endothelial cells?
Authors: Pourati Jacob , Maniotis Andrew , Spiegel David , Schaffer Jonathan L. , Butler James P. , Fredberg Jeffrey J. , Ingber Donald E. , Stamenovic Dimitrijie , Wang Ning
Published: December 2017

We tested the hypothesis that mechanical tension in the cytoskeleton (CSK) is a major determinant of cell deformability. To confirm that tension was present in adherent endothelial cells, we either cut or detached them from their basal surface by a microneedle. After cutting or detachment, the cells rapidly retracted. This retraction was prevented, however, if the CSK actin lattice was disrupted by cytochalasin D (Cyto D). These results confirmed that there was preexisting CSK tension in these cells and that the actin lattice was a primary stress-bearing component of the CSK. Second, to determine the extent to which that preexisting CSK tension could alter cell deformability, we developed a stretchable cell culture membrane system to impose a rapid mechanical distension (and presumably a rapid increase in CSK tension) on adherent endothelial cells. Altered cell deformability was quantitated as the shear stiffness measured by magnetic twisting cytometry. When membrane strain increased 2.5 or 5%, the cell stiffness increased 15 and 30%, respectively. Disruption of actin lattice with Cyto D abolished this stretch-induced increase in stiffness, demonstrating that the increased stiffness depended on the integrity of the actin CSK. Permeabilizing the cells with saponin and washing away ATP and Ca2+ did not inhibit the stretch-induced stiffening of the cell. These results suggest that the stretch-induced stiffening was primarily due to the direct mechanical changes in the forces distending the CSK but not to ATP- or Ca2+-dependent processes. Taken together, these results suggest preexisting CSK tension is a major determinant of cell deformability in adherent endothelial cells.
Modulation of potassium channel gating by coexpression of Kv2.1 with regulatory Kv5.1 or Kv6.1 α-subunits
Authors: Kramer J. W. , Post M. A. , Brown A. M. , Kirsch G. E.
Published: December 2017

We have determined the effects of coexpression of Kv2.1 with electrically silent Kv5.1 or Kv6.1 α-subunits in Xenopus oocytes on channel gating. Kv2.1/5.1 selectively accelerated the rate of inactivation at intermediate potentials (−30 to 0 mV), without affecting the rate at strong depolarization (0 to +40 mV), and markedly accelerated the rate of cumulative inactivation evoked by high-frequency trains of short pulses. Kv5.1 coexpression also slowed deactivation of Kv2.1. In contrast, Kv6.1 was much less effective in speeding inactivation at intermediate potentials, had a slowing effect on inactivation at strong depolarizations, and had no effect on cumulative inactivation. Kv6.1, however, had profound effects on activation, including a negative shift of the steady-state activation curve and marked slowing of deactivation tail currents. Support for the notion that the Kv5.1’s effects stem from coassembly of α-subunits into heteromeric channels was obtained from biochemical evidence of protein-protein interaction and single-channel measurements that showed heterogeneity in unitary conductance. Our results show that Kv5.1 and Kv6.1 function as regulatory α-subunits that when coassembled with Kv2.1 can modulate gating in a physiologically relevant manner.
Testosterone: the culprit for producing splenocyte immune depression after trauma hemorrhage
Authors: Angele Martin K. , Ayala Alfred , Cioffi William G. , Bland Kirby I. , Chaudry Irshad H.
Published: December 2017

Studies indicate that, whereas immune functions in males are depressed, they are enhanced in females after trauma hemorrhage. Moreover, castration of male mice (i.e., androgen depletion) before trauma hemorrhage prevented the depression of cell-mediated immunity. Nonetheless, it remains unknown whether or not testosterone per se is responsible for producing the immune depression. To study this, female C3H/HeN mice ( n = 7 animals/group) were pretreated with 5α-dihydrotestosterone (DHT) or vehicle for 19 days, then subjected to laparotomy (e.g., trauma) and hemorrhagic shock (blood pressure 35 ± 5 mmHg for 90 min) followed by fluid resuscitation or sham operation. Nontreated males underwent either trauma hemorrhage or sham operation. Twenty-four hours thereafter, splenocyte immune functions as well as plasma DHT, estradiol, and corticosterone levels were measured. DHT-pretreated females had significantly ( P < 0.05) increased DHT levels, comparable to those seen in males. Conversely, estradiol levels in such females were similar to control males. Splenocyte proliferation as well as interleukin-2 and interleukin-3 release were not depressed in vehicle-treated females, whereas it was in DHT-treated females after trauma hemorrhage, comparable to hemorrhaged males. Thus high testosterone and/or low estradiol levels appear to be responsible for producing splenocyte immune depression in males after trauma hemorrhage. Agents that block testosterone receptors or increase estradiol levels may therefore be helpful in improving depressed immune functions in male trauma patients.
β-Adrenergic-induced cytosolic redistribution of Rap1 in rat parotid acini: role in secretion
Authors: D’Silva Nisha J. , Jacobson Kerry L. , Ott Sabrina M. , Watson Eileen L.
Published: December 2017

Rap1 has recently been identified on the secretory granule membrane and plasma membrane of rat parotid acinar cells (N. J. D’Silva, D. DiJulio, C. B. Belton, K. L. Jacobson, and E. L. Watson. J. Histochem. Cytochem. 45: 965–973, 1997). In the present study, we examined the cellular redistribution of Rap1 following treatment of acini with isoproterenol (ISO), the β-adrenergic agonist, and determined the relationship between translocation and amylase release. In the presence of ISO, Rap1 translocated to the cytosol in a concentration- and time-dependent manner; this effect was not mimicked by the muscarinic agonist, carbachol. Translocation was maximal at 1 μM ISO and paralleled amylase release immediately after ISO stimulation. Rap1 translocation and amylase release were blocked by the β-adrenergic antagonist, propranolol, whereas okadaic acid, a downstream secretory inhibitor, significantly blocked amylase release but did not inhibit Rap1 redistribution. Results suggest that the translocation of Rap1 is causally related to secretion and that the role of Rap1 in secretion is at a site proximal to the exocytotic event.
Unusual degradation of α-β complexes inXenopus oocytes by β-subunits ofXenopus gastric H-K-ATPase
Authors: Chen Pei-Xian , Mathews Paul M. , Good Peter J. , Rossier Bernard C. , Geering Käthi
Published: December 2017

The catalytic α-subunit of oligomeric P-type ATPases such as Na-K-ATPase and H-K-ATPase requires association with a β-subunit after synthesis in the endoplasmic reticulum (ER) to become stably expressed and functionally active. In this study, we have expressed the β-subunit of Xenopus gastric H-K-ATPase (βHK) in Xenopus oocytes together with α-subunits of H-K-ATPase (αHK) or Na-K-ATPase (αNK) and have followed the biosynthesis, assembly, and cell surface expression of functional pumps. Immunoprecipitations of Xenopus βHK from metabolically labeled oocytes show that it is well expressed and, when synthesized without α-subunits, can leave the ER and become fully glycosylated. Xenopus βHK can associate with both coexpressed αHK and αNK, but the α-β complexes formed are degraded rapidly in or close to the ER and do not produce functional pumps at the cell surface as assessed by86Rb uptake. A possible explanation of these results is that Xenopus βHK may contain a tissue-specific signal that is important in the formation or correct targeting of functional α-β complexes in the stomach but that cannot be recognized in Xenopusoocytes and in consequence leads to cellular degradation of the α-β complexes in this experimental system.
Expression of an AQP2 Cre recombinase transgene in kidney and male reproductive system of transgenic mice
Authors: Nelson Raoul D. , Stricklett Peter , Gustafson Corrine , Stevens Anna , Ausiello Dennis , Brown Dennis , Kohan Donald E.
Published: December 2017

A transgenic mouse approach was used to examine the mechanism of principal cell-specific expression of aquaporin-2 (AQP2) within the renal collecting duct. RT-PCR and immunocytochemistry revealed that murine AQP2 was expressed in principal cells in the renal collecting duct, epithelial cells of the vas deferens, and seminiferous tubules within testis. The vas deferens expression was confirmed in rats. RT-PCR and immunocytochemistry showed that 14 kb of the human 5′-flanking region confers specific expression of a nucleus-targeted and epitope-tagged Cre recombinase in the principal cells within the renal collecting duct, in the epithelial cells of the vas deferens, and within the testis of transgenic mice. These results suggest that cell-specific expression of AQP2 is mediated at the transcriptional level and that 14 kb of the human AQP2 5′-flanking region contain cis elements that are sufficient for cell-specific expression of AQP2. Finally, renal principal cell expression of Cre recombinase is the first step in achieving cell-specific gene knockouts, thereby allowing focused examination of gene function in this cell type.
Mechanical loading regulates NOS expression and activity in developing and adult skeletal muscle
Authors: Tidball James G. , Lavergne Eliane , Lau Kim S. , Spencer Melissa J. , Stull James T. , Wehling Michelle
Published: December 2017

The hypothesis that changes in muscle activation and loading regulate the expression and activity of neuronal nitric oxide (NO) synthase (nNOS) was tested using in vitro and in vivo approaches. Removal of weight bearing from rat hindlimb muscles for 10 days resulted in a significant decrease in nNOS protein and mRNA concentration in soleus muscles, which returned to control concentrations after return to weight bearing. Similarly, the concentration of nNOS in cultured myotubes increased by application of cyclic loading for 2 days. NO release from excised soleus muscles was increased significantly by a single passive stretch of 20% or by submaximal activation at 2 Hz, although the increases were not additive when both stimuli were applied simultaneously. Increased NO release resulting from passive stretch or activation was dependent on the presence of extracellular calcium. Cyclic loading of cultured myotubes also resulted in a significant increase in NO release. Together, these findings show that activity of muscle influences NO production in the short term, by regulating NOS activity, and in the long term, by regulating nNOS expression.
Modulation of K+ currents in monocytes by VCAM-1 and E-selectin on activated human endothelium
Authors: Colden-Stanfield Margaret , Gallin Elaine K.
Published: December 2017

Resting membrane potential (RMP) and whole cell currents were recorded in human THP-1 monocytes adherent to polystyrene, unstimulated human umbilical vein endothelial cells (HUVECs), lipopolysaccharide (LPS)-treated HUVECs, immobilized E-selectin, or vascular cell adhesion molecule 1 (VCAM-1) using the patch-clamp technique. RMP after 5 h on polystyrene was −24.3 ± 1.7 mV ( n = 42) with delayed rectifier K+( I dr) and Cl− currents ( I Cl) present in >75% of the cells. Inwardly rectifying K+ currents ( I ir) were present in only 14% of THP-1 cells. Adherence to unstimulated HUVECs or E-selectin for 5 h had no effect on I ir or I Cl but decreased I dr. Five hours after adherence to LPS-treated HUVECs, outward currents were unchanged, but I ir was present in 81% of THP-1 cells. A twofold increase in I ir and a hyperpolarization (−41.3 ± 3.7 mV, n = 16) were abolished by pretreatment of THP-1 cells with cycloheximide, a protein synthesis inhibitor, or herbimycin A, a tyrosine kinase inhibitor, or by pretreatment of the LPS-treated HUVECs with anti-VCAM-1. Only a brief (15-min) interaction between THP-1 cells and LPS-treated HUVECs was required to induce I irexpression 5 h later. THP-1 cells adherent to VCAM-1 exhibited similar conductances to cells adherent to LPS-treated HUVECs. Thus engagement of specific integrins results in selective modulation of different K+ conductances.
Topological analysis of NHE1, the ubiquitous Na+/H+exchanger using chymotryptic cleavage
Authors: Shrode Lamara D. , Gan Bing Siang , D’Souza Sudhir J. A. , Orlowski John , Grinstein Sergio
Published: December 2017

Proteases, glycosidases, and impermeant biotin derivatives were used in combination with antibodies to analyze the subcellular distribution and transmembrane disposition of the Na+/H+exchanger NHE1. Both native human NHE1 in platelets and epitope-tagged rat NHE1 transfected into antiport-deficient cells were used for these studies. The results indicated that 1) the entire population of exchangers is present on the surface membrane of unstimulated platelets, ruling out regulation by recruitment of internal stores of NHE1; 2) the putative extracellular loops near the NH2 terminus are exposed to the medium and contain all the N- and O-linked carbohydrates; 3) by contrast, the putative extracellular loops between transmembrane domains 9–10 and 11–12 are not readily accessible from the outside and may be folded within the protein, perhaps contributing to an aqueous ion transport pathway; 4) the extreme COOH terminus of the protein was found to be inaccessible to extracellular proteases, antibodies, and other impermeant reagents, consistent with a cytosolic localization; and 5) detachment of ∼150 amino acids from the NH2-terminal end of the protein had little effect on the transport activity of NHE1.
PKC activity modulates availability and long openings of L-type Ca2+ channels in A7r5 cells
Authors: Obejero-Paz C. A. , Auslender M. , Scarpa A.
Published: December 2017

The possibility that protein kinase C (PKC) could control the activity of L-type Ca2+ channels in A7r5 vascular smooth muscle-derived cells in the absence of agonist stimulation was investigated using the patch-clamp technique. Consistent with the possibility that L-type Ca2+ channels are maximally phosphorylated by PKC under these conditions, we show that 1) activation of PKC with the phorbol ester phorbol 12,13-dibutyrate was ineffective in modulating whole cell and single-channel currents, 2) inhibition of PKC activity with staurosporine or chelerythrine inhibited channel activity, 3) inhibition of protein phosphatases by intracellular dialysis of okadaic acid did not affect whole cell currents, and 4) the inhibitory effect of staurosporine was absent in the presence of okadaic acid. The inhibition of Ca2+ currents by PKC inhibitors was due to a decrease in channel availability and long open events, whereas the voltage dependence of the open probability and the single-channel conductance were not affected. The evidence suggests that in resting, nonstimulated A7r5 cells there is a high level of PKC activity that modulates the gating of L-type Ca2+ channels.
Regulation of KCacurrent by store-operated Ca2+influx depends on internal Ca2+release in HSG cells
Authors: Liu Xibao , Rojas Eduardo , Ambudkar Indu S.
Published: December 2017

This study examines the Ca2+ influx-dependent regulation of the Ca2+-activated K+ channel (KCa) in human submandibular gland (HSG) cells. Carbachol (CCh) induced sustained increases in the KCa current and cytosolic Ca2+ concentration ([Ca2+]i), which were prevented by loading cells with 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid (BAPTA). Removal of extracellular Ca2+ and addition of La3+ or Gd3+, but not Zn2+, inhibited the increases in KCa current and [Ca2+]i. Ca2+ influx during refill (i.e., addition of Ca2+ to cells treated with CCh and then atropine in Ca2+-free medium) failed to evoke increases in the KCa current but achieved internal Ca2+ store refill. When refill was prevented by thapsigargin, Ca2+ readdition induced rapid activation of KCa. These data provide further evidence that intracellular Ca2+ accumulation provides tight buffering of [Ca2+]iat the site of Ca2+ influx (H. Mogami, K. Nakano, A. V. Tepikin, and O. H. Petersen. Cell 88: 49–55, 1997). We suggest that the Ca2+ influx-dependent regulation of the sustained KCacurrent in CCh-stimulated HSG cells is mediated by the uptake of Ca2+ into the internal Ca2+ store and release via the inositol 1,4,5-trisphosphate-sensitive channel.
Differences in contractile protein content and isoforms in phasic and tonic smooth muscles
Authors: Szymanski Pawel T. , Chacko Thomas K. , Rovner Arthur S. , Goyal Raj K.
Published: December 2017

The basis of tonic vs. phasic contractile phenotypes of visceral smooth muscles is poorly understood. We used gel electrophoresis and quantitative scanning densitometry to measure the content and isoform composition of contractile proteins in opossum lower esophageal sphincter (LES), to represent tonic muscle, and circular muscle of the esophageal body (EB), to represent phasic smooth muscle. The amount of protein in these two types of muscles is similar: ∼27 mg/g of frozen tissue. There is no difference in the relative proportion of myosin, actin, calponin, and tropomyosin in the two muscle types. However, the EB contains ∼2.4-times more caldesmon than the LES. The relative ratios of α- to γ-contractile isoforms of actin are 0.9 in the LES and 0.3 in EB. The ratio between acidic (LC17a) and basic (LC17b) isoforms of the 17-kDa essential light chain of myosin is 0.7:1 in the LES, compared with 2.7:1 in the EB. There is no significant difference in the ratios of smooth muscle myosin SM1 and SM2 isoforms in the two muscle types. The level of the myosin heavy chain isoform, which contains the seven-amino acid insert in the myosin head, is about threefold higher in the EB compared with LES. In conclusion, the esophageal phasic muscle in contrast to the tonic LES contains proportionally more caldesmon, LC17a, and seven-amino acid-inserted myosin and proportionally less α-actin. These differences may provide a basis for functional differences between tonic and phasic smooth muscles.
Polarity, integrin, and extracellular matrix dynamics in the postischemic rat kidney
Authors: Zuk Anna , Bonventre Joseph V. , Brown Dennis , Matlin Karl S.
Published: December 2017

Acute renal failure (ARF) as a consequence of ischemic injury is a common disease affecting 5% of the hospitalized population. Despite the fact that mortality from ARF is high, there has been little improvement in survival rates over the last 40 years. The pathogenesis of ARF may be related to substantial changes in cell-cell and cell-extracellular matrix interactions mediated by β1-integrins. On the basis of in vitro and in vivo studies, reorganization of β1-integrins from basal to apical surfaces of injured tubular epithelia has been suggested to facilitate epithelial detachment, contributing to tubular obstruction and backleak of glomerular filtrate. In this study, we examine integrin and extracellular matrix dynamics during epithelial injury and repair using an in vivo rat model of unilateral ischemia. We find that, soon after reperfusion, β1-integrins newly appear on lateral borders in epithelial cells of the S3 segment but are not on the apical surface. At later times, as further injury and regeneration coordinately occur, epithelia adherent to the basement membrane localize β1 predominantly to basal surfaces even while the polarity of other marker proteins is lost. At the same time, amorphous material consisting of depolarized exfoliated cells fills the luminal space. Notably, β1-integrins are not detected on exfoliated cells. A novel finding is the presence of fibronectin, a glycoprotein of plasma and the renal interstitium, in tubular spaces of the distal nephron and to a lesser extent S3 segments. These results indicate that β1-integrins dramatically change their distribution during ischemic injury and epithelial repair, possibly contributing to cell exfoliation initially and to epithelial regeneration at later stages. Together with the appearance of large amounts of fibronectin in tubular lumens, these alterations may play a significant role in the pathophysiology of ARF.
Tissue distribution of immunoreactive mouse extracellular superoxide dismutase
Authors: Ookawara Tomomi , Imazeki Nobuo , Matsubara Osamu , Kizaki Takako , Oh-Ishi Shuji , Nakao Chitose , Sato Yuzo , Ohno Hideki
Published: December 2017

Protein content and mRNA expression of extracellular superoxide dismutase (EC-SOD) were investigated in 16 mouse tissues. We developed a double-antibody sandwich ELISA using the affinity-purified IgG against native mouse EC-SOD. EC-SOD could be detected in all of the tissues examined (lung, kidney, testis, brown fat, liver, adrenal gland, pancreas, colon, white fat, thymus, stomach, spleen, heart, skeletal muscle, ileum, and brain, in decreasing order of content measured as μg/g wet tissue). Lung showed a markedly higher value of EC-SOD than other tissues. Interestingly, white fat had a high content of EC-SOD in terms of micrograms per milligram protein, which corresponded to that of lung. Kidney showed the strongest expression of EC-SOD mRNA. Relatively strong expression of the mRNA was observed in lung, white fat, adrenal gland, brown fat, and testis. Heart and brain showed only weak signals, and no such expression could be detected in either digestive organs or skeletal muscle. Immunohistochemically, EC-SOD was localized mainly to connective tissues and vascular walls in the tissues examined. Deep staining in the cytosol was observed in the cortical tubular cells of kidney. These results suggest that EC-SOD is distributed systemically in mice and that the physiological importance of this enzyme may be a compensatory adaptation to oxidative stress, particularly in lung and kidney.
Heme oxygenase-1 induction in skeletal muscle cells: hemin and sodium nitroprusside are regulators in vitro
Authors: Vesely M. J. J. , Exon D. J. , Clark J. E. , Foresti R. , Green C. J. , Motterlini R.
Published: December 2017

The heat shock protein heme oxygenase-1 (HO-1) is regulated by a variety of physiological and pharmacological factors. In skeletal muscle tissue, HO-1 has been shown to be induced only by exercise and electrical stimulation in vivo. Both hemin and sodium nitroprusside (SNP) are potent inducers of HO-1 in other tissues. In this study, we examined the effects of these two agents on HO-1 induction in L6.G8 rat skeletal myoblast cells. Hemin and SNP increased cellular heme oxygenase activity in both a time- and concentration-dependent manner. Increases in the HO-1 mRNA level and protein expression accompanied changes in heme oxygenase activity. The ability of SNP to induce HO-1 in L6.G8 cells was reduced by coincubation with hydroxocobalamin, a known nitric oxide (NO) scavenger, suggesting that NO itself may be involved in HO-1 gene stimulation. These results indicate that HO-1 expression is sensitive to both hemin and SNP in skeletal myoblast cells and may indicate an important regulatory mechanism of heme catabolism in skeletal muscle tissue.
Cytokine-mediated PGE2expression in human colonic fibroblasts
Authors: Kim Edward C. , Zhu Yingting , Andersen Valerie , Sciaky Daniela , Cao H. James , Meekins Heather , Smith Terry J. , Lance Peter
Published: December 2017

We investigated prostanoid biogenesis in human colonic fibroblasts (CCD-18Co and 5 primary fibroblast cultures) and epithelial cell lines (NCM460, T84, HT-29, and LS 174T) and the effect of PGE2 on fibroblast morphology. Cytokine-stimulated PGE2production was measured. PGH synthase-1 and -2 (PGHS-1 and -2) protein and mRNA expression were evaluated. Basal PGE2 levels were low in all cell types (0.15–6.47 ng/mg protein). Treatment for 24 h with interleukin-1β (IL-1β; 10 ng/ml) or tumor necrosis factor-α (50 ng/ml), respectively, elicited maximal 25- and 6-fold inductions of PGE2 synthesis in CCD-18Co cultures and similar results in primary fibroblast cultures; maximal inductions with IL-1β in colonic epithelial cell lines were from zero to fivefold. Treatment of CCD-18Co fibroblasts with IL-1β caused maximal 21- and 53-fold increases, respectively, in PGHS-2 protein and mRNA levels without altering PGHS-1 expression. PGE2 (0.1 μmol/l) elicited a dramatic shape change in selected fibroblasts. Colonic fibroblasts are potentially important as cytokine targets and a source of and target for colonic prostanoids in vivo.
Osmotic pressure regulates αβγ-rENaC expressed inXenopus oocytes
Authors: Ji Hong-Long , Fuller Catherine M. , Benos Dale J.
Published: December 2017

The hypothesis that amiloride-sensitive Na+channels (ENaC) are involved in cell volume regulation was tested. Anisosmotic ND-20 media (ranging from 70 to 450 mosM) were used to superfuse Xenopus oocytes expressing αβγ-rat ENaC (αβγ-rENaC). Whole cell currents were reversibly dependent on external osmolarity. Under conditions of swelling (70 mosM) or shrinkage (450 mosM), current amplitude decreased and increased, respectively. In contrast, there was no change in current amplitude of H2O-injected oocytes to the above osmotic insults. Currents recorded from αβγ-rENaC-injected oocytes were not sensitive to external Cl− concentration or to the K+ channel inhibitor BaCl2. They were sensitive to amiloride. The concentration of amiloride necessary to inhibit one-half of the maximal rENaC current expressed in oocytes ( K i; apparent dissociation constant) decreased in swollen cells and increased in shrunken oocytes. The osmotic pressure-induced Na+ currents showed properties similar to those of stretch-activated channels, including inhibition by Gd3+ and La3+, and decreased selectivity for Na+. αβγ-rENaC-expressing oocytes maintained a nearly constant cell volume in hypertonic ND-20. The present study is the first demonstration that αβγ-rENaC heterologously expressed in Xenopus oocytes may contribute to oocyte volume regulation following shrinkage.
Caco-2 intestinal cell differentiation is associated with G1 arrest and suppression of CDK2 and CDK4
Authors: Ding Qing-Ming , Ko Tien C. , Evers B. Mark
Published: December 2017

The cellular mechanisms regulating intestinal proliferation and differentiation remain largely undefined. Previously, we showed an early induction of the cyclin-dependent kinase (CDK) inhibitor p21Waf1/Cip1 in Caco-2 cells, a human colon cancer line that spontaneously differentiates into a small bowel phenotype. The purpose of our present study was to assess the timing of cell cycle arrest in relation to differentiation in Caco-2 cells and to examine the mechanisms responsible for CDK inactivation. Caco-2 cells undergo a relative G1/S block and cease to proliferate at day 3 postconfluency; an increase in the activity of terminally differentiated brush-border enzymes (sucrase and alkaline phosphatase) was noted at day 6 postconfluency. Cell cycle block was associated with suppression of both CDK2 and CDK4 activities, which are important for G1/S progression. Treatment of the CDK immune complexes with the detergent deoxycholate (DOC) resulted in restoration of CDK2, but not CDK4, activity at day 3postconfluency, suggesting the presence of inhibitory protein(s) binding to the cyclin/CDK2 complex at this time point. An increased binding of p21Waf1/Cip1 to CDK2 complexes at day 3 postconfluency was noted, suggesting a potential role for p21Waf1/Cip1in CDK2 inactivation; however, immunodepletion of p21Waf1/Cip1 from Caco-2 protein extracts demonstrated that p21Waf1/Cip1 is only partially responsible for CDK2 suppression at day 3postconfluency. A decrease in the cyclin E/CDK2 complex appears to contribute to the CDK2 inactivation noted at days 6 and 12 postconfluency. Taken together, our results suggest that multiple mechanisms contribute to CDK suppression during Caco-2 cell differentiation. Inhibition of CDK2 and CDK4 leads to G1 arrest and inhibition of proliferation that precede Caco-2 cell differentiation.
Regulation of cytoskeletal mechanics and cell growth by myosin light chain phosphorylation
Authors: Cai Shuang , Pestic-Dragovich Lidija , O’Donnell Martha E. , Wang Ning , Ingber Donald , Elson Elliot , De Lanerolle Primal
Published: December 2017

The role of myosin light chain phosphorylation in regulating the mechanical properties of the cytoskeleton was studied in NIH/3T3 fibroblasts expressing a truncated, constitutively active form of smooth muscle myosin light chain kinase (tMK). Cytoskeletal stiffness determined by quantifying the force required to indent the apical surface of adherent cells showed that stiffness was increased twofold in tMK cells compared with control cells expressing the empty plasmid (Neo cells). Cytoskeletal stiffness quantified using magnetic twisting cytometry showed an ∼1.5-fold increase in stiffness in tMK cells compared with Neo cells. Electronic volume measurements on cells in suspension revealed that tMK cells had a smaller volume and are more resistant to osmotic swelling than Neo cells. tMK cells also have smaller nuclei, and activation of mitogen-activated protein kinase (MAP kinase) and translocation of MAP kinase to the nucleus are slower in tMK cells than in control cells. In tMK cells, there is also less bromodeoxyuridine incorporation, and the doubling time is increased. These data demonstrate that increased myosin light chain phosphorylation correlates with increased cytoskeletal stiffness and suggest that changing the mechanical characteristics of the cytoskeleton alters the intracellular signaling pathways that regulate cell growth and division.
Molecular dissection of a basic COOH-terminal domain of Cx32 that inhibits gap junction gating sensitivity
Authors: Wang Xiao Guang , Peracchia Camillo
Published: December 2017

Connexin32 (Cx32) mutants were studied by double voltage clamp in Xenopus oocytes to determine the role of basic COOH-terminal residues in gap junction channel gating by CO2 and transjunctional voltage. Replacement of five arginines with N (5R/N) or T residues in the initial COOH-terminal domain (CT1) of Cx32 enhanced CO2 sensitivity. The positive charge, rather than the R residue per se, is responsible for the inhibitory role of CT1, because mutants replacing the five R residues with K (5R/K) or H (5R/H) displayed CO2 sensitivity comparable to that of wild-type Cx32. Mutants replacing R with N residues four at a time (4R/N) showed that CO2 sensitivity is strongly inhibited by R215 and mildly by R219, whereas R220, R223, and R224 may slightly increase sensitivity. Neither the 5R/N nor the 4R/N mutants differed in voltage sensitivity from wild-type Cx32. The possibility that inhibition of gating sensitivity results from electrostatic interactions between CT1 and the cytoplasmic loop is discussed as part of a model that envisions the cytoplasmic loop of Cx32 as a key element of chemical gating.
Effect of serum and mechanical stretch on skeletal α-actin gene regulation in cultured primary muscle cells
Authors: Carson James A. , Booth Frank W.
Published: December 2017

The purpose of this study was to determine whether mechanical stretch or serum availability alters pretranslational regulation of skeletal α-actin (SkA) in cultured striated muscle cells. Chicken primary skeletal myoblasts and cardiac myocytes were plated on collagenized Silastic membranes adherent to nylon supports and stretched 8–20% of initial length 96 h postplating. Serum dependence of SkA gene regulation was determined by maintaining differentiated muscle cells in growth/differentiation (G/D; skeletal myotubes, 10% horse serum-2% chick embryo extract; cardiac myocytes, 10% horse serum) or growth-limiting (G-L; 0.5% horse serum) medium. Skeletal myotubes had higher SkA mRNA and SkA promoter activity in G/D than in G-L medium. Cardiac myocyte SkA mRNA was higher in G-L than in G/D medium. Serum response factor (SRF) protein binding to serum response element 1 (SRE1) of SkA promoter increased in skeletal cultures in G/D compared with G-L medium. Western blot analysis demonstrated that increased SRF-SRE1 binding was due, in part, to increased SRF protein. Stretching skeletal myotubes in G-L medium reduced SkA mRNA and repressed SkA promoter activity. The first 100 bp of SkA promoter were sufficient for stretch-induced repression of SkA promoter activity, and an intact transcriptional enhancer factor 1 (TEF-1) binding site was necessary for this response. Serum and stretch appear to repress SkA promoter activity in skeletal myotubes through different DNA binding elements, the SRE1 and TEF-1 sites, respectively. Stretching increased SkA mRNA in cardiac myocytes in G-L medium but did not alter SkA mRNA level in cardiac cells in G/D medium. These results demonstrate that stretch and serum interact differently to alter SkA expression in cultured cardiac and skeletal muscle cells.