bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of Na(+)-K(+)-ATPase and its subunit mRNAs by serum in a rat liver cell line
Authors: Bhutada A. , Perez C. , Chon D. Y. , Ismail-Beigi F.
Published: December 2017

Addition of serum to confluent Clone 9 cells increased the protein, RNA, and DNA content per plate of cells; the increments became manifest within 3-6 h and were sustained for the 48-h duration of study. After the addition of serum, Na(+)-K(+)-ATPase activity was stimulated 1.25- and 1.45-fold at 6 and 12 h, respectively. In cells preincubated in the absence of serum for 24 h, addition of serum increased the abundances of Na(+)-K(+)-ATPase subunit mRNAs, mRNA alpha 1 and mRNA beta, coordinately by approximately 2- and 2.7-fold at 3 h, an effect that preceded the stimulation of Na(+)-K(+)-ATPase activity. The serum-induced increments in subunit mRNA abundances were further enhanced by the combined presence of serum and cycloheximide; mRNA alpha 1 and mRNA beta abundances were also augmented by cycloheximide alone (approximately 2.5- and 9.2-fold at 6 h, respectively). In cells incubated in the absence of serum, the half-lives of mRNA alpha 1 and mRNA beta, estimated from decrements in their abundances after the addition of actinomycin D, were 12 and 10 h, respectively. These data demonstrate that serum enhances Na(+)-K(+)-ATPase subunit mRNA abundance and enzyme activity in Clone 9 cells. Comparison of the estimated half-lives of Na(+)-K(+)-ATPase subunit mRNAs with the observed increments in their abundances at 3 h suggests that the serum-induced increases in mRNA alpha 1 and mRNA beta abundances are in large part due to enhanced synthesis of these mRNAs.
Myosin-product complex in the resting state and during relaxation of smooth muscle
Authors: Butler T. M. , Siegman M. J. , Mooers S. U. , Narayan S. R.
Published: December 2017

Previous findings suggested that in resting smooth muscle ADP is bound to myosin and that phosphorylation of the myosin, and its subsequent interaction with actin, increases the rate of ADP release. We have now extended these studies to include measurements of bound Pi as well as bound ADP in permeabilized rabbit portal vein. We report that in resting smooth muscle that has been exposed to [3H]ATP and [gamma-32P]ATP, followed by a chase in an unlabeled relaxing solution, the ratio of bound [3H]ADP to bound [32P]Pi is close to unity, and both are released at approximately the same rate. This suggests that myosin exists predominantly with both ADP and Pi bound under resting conditions and that the release of one is quickly followed by the release of the other. In contrast, there is a significant 30% excess of bound Pi over ADP in a muscle during relaxation from an isometric contraction. Under these conditions, while force output is slowly decreasing, both light chain phosphorylation and adenosinetriphosphatase (ATPase) activity have decreased to near-resting values. The time course of relaxation is similar to the time course of Pi release from both the resting and relaxing muscle. We propose that during relaxation the dephosphorylated cross bridges which are bearing force have Pi but not ADP bound and that detachment of the cross bridge (and thus force decay) is limited by Pi release from myosin which occurs at the same rate as in the resting muscle.
Polarity and kinetics of Na(+)-H+ exchange in cultured opossum kidney cells
Authors: Montrose M. H. , Murer H.
Published: December 2017

Opossum kidney (OK) cells (an established cell line) were loaded with 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF; a fluorescent dye with a pH-sensitive spectrum), and intracellular pH (pHi) was examined by microfluorometry. Single cells, within a confluent monolayer and grown on a permeant support, were examined for the mechanism of recovery from an acid load as imposed by exposure to ammonium chloride (NH4 prepulse). The Na(+)-dependent recovery of pHi from an acid load (Na(+)-H+ exchange) is examined in terms of the Na+ activation kinetics of the recovery and the polarity of the response. In 80% of the cells examined (33/41), both apical and basolateral Na+ cause recovery from an acid load. The response of cells to apical Na+ is well fit by Michaelis-Menten kinetics [Kt(Na) = 35 mM], but the response to basolateral Na+ is not. The response to basolateral Na+ addition is modeled in terms of variable transepithelial leak of Na+ and variable amounts of basolateral Na(+)-H+ exchange. Despite an average response to basolateral (145 mM) Na+ that is 34% of the response to apical Na+, modeling suggests that basolateral Na(+)-H+ exchange must be less than 10% of the cellular total to fit the basolateral Na+ activation kinetics. The model, and experiments using ordered addition of Na+ from the apical vs. basolateral medium, also suggest that transepithelial leak (of basolateral Na+ to the apical compartment) is required to explain the pHi recovery observed due to addition of basolateral Na+. Direct estimation of (basolateral to apical) transepithelial leak demonstrates that the response due to basolateral Na+ addition is explained by transepithelial leak and a Na(+)-H+ exchange that is expressed solely in the apical membrane.
Ba2+, TEA+, and quinine effects on apical membrane K+ conductance and maxi K+ channels in gallbladder epithelium
Authors: Segal Y. , Reuss L.
Published: December 2017

The apical membrane of Necturus gallbladder epithelium contains a voltage-activated K+ conductance [Ga(V)]. Large-conductance (maxi) K+ channels underlie Ga(V) and account for 17% of the membrane conductance (Ga) under control conditions. We examined the Ba2+, tetraethylammonium (TEA+), and quinine sensitivities of Ga and single maxi K+ channels. Mucosal Ba2+ addition decreased resting Ga in a concentration-dependent manner (65% block at 5 mM) and decreased Ga(V) in a concentration- and voltage-dependent manner. Mucosal TEA+ addition also decreased control Ga (60% reduction at 5 mM). TEA+ block of Ga(V) was more potent and less voltage dependent that Ba2+ block. Maxi K+ channels were blocked by external Ba2+ at millimolar levels and by external TEA+ at submillimolar levels. At 0.3 mM, quinine (mucosal addition) hyperpolarized the cell membranes by 6 mV and reduced the fractional apical membrane resistance by 50%, suggesting activation of an apical membrane K+ conductance. At 1 mM, quinine both activated and blocked K(+)-conductive pathways. Quinine blocked maxi K+ channel currents at submillimolar concentrations. We conclude that 1) Ba2+ and TEA+ block maxi K+ channels and other K+ channels underlying resting Ga; 2) parallels between the Ba2+ and TEA+ sensitivities of Ga(V) and maxi K+ channels support a role for these channels in Ga(V); and 3) quinine has multiple effects on K(+)-conductive pathways in gallbladder epithelium, which are only partially explained by block of apical membrane maxi K+ channels.
High-affinity paroxetine binding to the human placental serotonin transporter
Authors: Cool D. R. , Leibach F. H. , Ganapathy V.
Published: December 2017

We investigated the interaction of paroxetine, a nontricyclic antidepressant, with the serotonin transporter of the human placental brush-border membrane. Paroxetine bound to the purified placental brush-border membranes with a high affinity [dissociation constant (Kd) = 72 pM]. The maximal binding capacity (Bmax) was 3.9 pmol/mg protein. Imipramine, desipramine, and serotonin inhibited the binding in a dose-dependent manner with inhibition constant (Ki) values of 4.4 nM, 48.7 nM, and 1.77 microM, respectively, whereas reserpine, ketanserin, and 5-hydroxytryptophan did not have any effect. Imipramine and serotonin inhibited paroxetine binding by increasing the Kd with essentially no effect on Bmax. Binding of paroxetine to the membranes increased hyperbolically with increasing concentrations of Na+ in the assay medium. Cl- had little effect on the binding. The effect of Na+ was primarily to increase the affinity of the transporter for paroxetine with no effect on Bmax. The association constant (Ka) increased hyperbolically as the concentration of Na+ increased, indicating a 1Na+:1paroxetine stoichiometry. The maximal value for Ka was 12.1 +/- 2.5 x 10(12) M-1, and Kd for Na+ was 10.0 +/- 3.5 mM. Treatment of the membranes with tyrosyl group-specific reagents reduced the Na(+)-dependent binding, suggesting the involvement of tyrosyl residues in the binding process. This inhibition was, however, significantly reduced when treatment with the reagent was performed in the presence of Na+, suggesting that the reactive tyrosyl residues were located at or near the Na(+)-binding site. Paroxetine inhibited NaCl gradient-dependent serotonin uptake in placental brush-border membrane vesicles both at pH 6.5 and 7.5.(ABSTRACT TRUNCATED AT 250 WORDS)
Elevated extracellular K+ enhances arachidonic acid release in MDCK-D1 cells
Authors: Howard M. J. , Insel P. A.
Published: December 2017

Depolarization can alter the expression of membrane receptors and change certain receptor-mediated events, but previous studies have not assessed the impact of depolarization on generation of arachidonic acid and its metabolites (AA) in nonexcitable tissues. We assessed AA generation in Madin-Darby canine kidney (MDCK) cells grown for 3 days in increased extracellular [K+], which is known to acutely depolarize these cells. Growth under these conditions resulted in decreases in the number of alpha 1-adrenergic receptors (alpha 1 AR), a small decrease in receptor-mediated phosphoinositide hydrolysis, but increases in alpha 1 AR-mediated prostaglandin E2 formation and AA release. Calcium ionophore (A23187)-, melittin-, and bradykinin-stimulated AA release were also enhanced. The reduction in alpha 1 AR number and increased AA release were substantially reduced or eliminated when K(+)-treated cells were grown in the absence of extracellular calcium. The results provide evidence that hormone-stimulated AA release and prostaglandin production can be enhanced by chronic exposure to elevated extracellular K+ concentration, perhaps as a consequence of a depolarization-induced enhancement in phospholipase A2 activity. The results provide evidence for the parallel and independent regulation of the pathways for receptor-mediated phosphoinositide hydrolysis (phospholipase C activation) and AA release (phospholipase A2 activation) in MDCK cells.
Role of protein kinase C in renal vasoconstriction caused by angiotensin II
Authors: Scholz H. , Kurtz A.
Published: December 2017

In this study we have examined the subcellar pathways along which angiotensin II (ANG II) causes renal vasoconstriction. Using the isolated perfused rat kidney model, we found that renal vasoconstriction produced by ANG II (100 pM) was not altered by the calmodulin antagonists calmidazolium (1 microM) and N-(6-aminohexyl)-5-chloro-1-naphthalensulfonamide (W-7, 10 microM) but was blunted by staurosporine (100 nM) and 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7, 50 microM), two structurally distinct putative protein kinase C inhibitors. The phorbol ester 4 alpha-phorbol 12,13-didecanoate (1-100 nM) did not alter renal vascular resistance, whereas phorbol 12-myristate 13-acetate (PMA, 1-100 nM) caused potent and dose-dependent vasoconstriction that was prevented by staurosporine (100 nM) and H-7 (50 microM). The vasoconstrictory effects of ANG II and PMA were attenuated by the calcium channel blockers verapamil (5 microM) and nifedipine (5 microM) and were reversibly inhibited when cobaltous chloride (2 mM) was added to the perfusate. Taken together, our findings support the concept that the renal vasoconstrictory effect of ANG II is essentially mediated by protein kinase C activation, which either requires or enhances the entrance of extracellular calcium.
Quasi-simultaneous measurement of ionized calcium and alpha-granule release in individual platelets
Authors: Oda A. , Daley J. F. , Kang J. , Smith M. , Ware J. A. , Salzman E. W.
Published: December 2017

The effect of alpha-thrombin and ADP on calcium mobilization and alpha-granule release in individual platelets was investigated by flow cytometry. alpha-Thrombin (4.5 nM) caused a uniform rise of free cytosolic calcium ([Ca2+]i) among indo-1-loaded human platelets. Despite the uniformity of this effect, approximately 20% of the cells failed to secrete alpha-granule content, as shown by binding of fluorescein isothiocyanate (FITC)-conjugated S12 monoclonal antibody. ADP (10 microM) caused a similar brisk and uniform rise of calcium but did not increase S12 binding to any platelets. On the other hand, with alpha-thrombin (0.5 nM), calcium mobilization was heterogeneous and paralleled granule release. [Ca2+]i increased rapidly in some platelets, while only slowly in others. When an electronic gate was set according to FITC-S12 fluorescence, cells with a greater secretory response proved to be those with a higher calcium level. With both alpha-thrombin and ADP, chelation of external calcium by EGTA (2 mM) reduced calcium response of individual cells. NiCl2 (1 mM) also inhibited calcium rise of individual platelets to the same extent as EGTA (2 mM) in spite of the presence of 1 mM CaCl2 in the extracellular media. The effects of EGTA and NiCl2 were not limited to a particular subpopulation of cells. These data suggest that the putative Ni2(+)-inhibitable divalent cation channel(s) may be responsible for the increased influx of calcium that occurs during platelet activation by alpha-thrombin and ADP. It appears that these calcium channels contribute to the elevation of [Ca2+]i among virtually all the platelets.
Distinct epitopes on amiloride. II. Variably restricted epitopes defined by monoclonal anti-amiloride antibodies
Authors: Kleyman T. R. , Zebrowitz J. R.
Published: December 2017

Specific regions of amiloride appear to participate in binding to receptors on amiloride-sensitive transport proteins. Previous studies characterizing epitopes on amiloride recognized by anti-amiloride antibodies have demonstrated that antibodies recognize specific domains on amiloride and that these epitopes are determined, in part, by the site on amiloride used to couple to carrier protein. The 3,5-diaminopyrazinyl and guanidinocarbonyl moieties were identified as distinct epitopes. Since Na(+)-selective transport proteins are sensitive to changes of the halide on the amiloride molecule, additional monoclonal anti-amiloride antibodies were raised to determine whether the C-6 halo group of amiloride could be identified as an important site for drug-antibody binding. The epitopes recognized by a series of three monoclonal antibodies raised against amiloride coupled to rabbit serum albumin through its C-5 NH2-group were defined. Two antibodies recognize extensive regions on the amiloride molecule, including both the acylguanidino and pyrazinyl groups. In addition, both antibodies are sensitive to changes in the C-6 halo group on amiloride. A third antibody was relatively insensitive to changes in the halide in the C-6 position of the pyrazine ring of amiloride and recognized a more restricted epitope on amiloride.
Effect of diabetes and insulin treatment of diabetic rats on total RNA, poly(A)+ RNA, and mRNA in skeletal muscle
Authors: Kent J. D. , Kimball S. R. , Jefferson L. S.
Published: December 2017

We have assessed the time course of alterations in several biochemical parameters and expression of specific mRNAs in gastrocnemius muscle following both the induction of diabetes and the administration of insulin to diabetic rats. Muscle mass, total RNA, and total protein were reduced, whereas poly(A)+ RNA relative to total RNA was increased following the induction of diabetes. All the above parameters, with the exception of poly(A)+ RNA, were reciprocally and rapidly altered following administration of insulin to 3-day diabetic animals. These changes suggest that during the induction of diabetes 1) total cellular protein is reduced at a rate that is less than the reduction in gastrocnemius mass, whereas RNA is reduced at a rate 1.5 times the reduction in tissue mass, and 2) poly(A)+ RNA is elevated relative to total RNA. After insulin administration, there appears to be coordinate synthesis of both poly(A)+ RNA and ribosomal RNA, assuming 85% of total RNA is ribosomal. Therefore, we conclude that poly(A)+ RNA is more stable than ribosomal RNA during diabetes, whereas the amounts of poly(A)+ RNA and ribosomal RNA are increased at the same rates following insulin administration to diabetic animals. Analysis of expression of specific gene products over the same time course, as assessed by in vitro translation of total RNA followed by two-dimensional gel analysis, suggests that there are a few mRNAs that are very rapidly altered in response to insulin administration. The mRNAs that are altered demonstrate variable temporal patterns of either repression or full or transient expression. These rapid, but limited, alterations in gene expression may prove important in the development of the defects that occur in skeletal muscle in response to diabetes.
Lysophosphatidylcholine and sodium-calcium exchange in cardiac sarcolemma: comparison with ischemia
Authors: Bersohn M. M. , Philipson K. D. , Weiss R. S.
Published: December 2017

Lysophosphoglyceride accumulation in ischemic myocardium has been hypothesized to be a mechanism for altered sarcolemmal properties that underlie electrophysiological changes and Ca2+ accumulation in ischemia. We find that in vitro application of lysophosphatidylcholine to normal canine sarcolemmal vesicles at a concentration of 0.3 mumol/mg sarcolemmal protein inhibits Na(+)-Ca2+ exchange. Both maximum velocity (Vmax) for Ca2+ transport and Ca2+ affinity are reduced by lysophosphatidylcholine, whereas in ischemia only Vmax is reduced [M. M. Bersohn, K. D. Philipson, and J. Y. Fukushima. Am. J. Physiol. 242 (Cell Physiol. 11): C288-C295, 1982]. This amount of lysophosphatidylcholine does not affect sarcolemmal passive permeability to either Ca2+ or Na+. Treatment of sarcolemma with phospholipase A2 sufficient to inhibit Na(+)-Ca2+ exchange velocity by 50% causes large increases in sarcolemmal lysophosphatidylcholine and lysophosphatidylethanolamine. On the other hand, 1 h of ischemia in rabbit hearts does not affect sarcolemmal phospholipid composition. Thus, although in vitro treatment with lysophosphatidylcholine or phospholipase A2 has profound effects on sarcolemmal properties, sarcolemmal accumulation of lysophosphatidylcholine cannot account for the effects of ischemia as measured in highly purified sarcolemmal vesicles from ischemic hearts.
Mitogenic signals for platelet-derived growth factor isoforms in liver fat-storing cells
Authors: Pinzani M. , Knauss T. C. , Pierce G. F. , Hsieh P. , Kenney W. , Dubyak G. R. , Abboud H. E.
Published: December 2017

Platelet-derived growth factor (PDGF), a key mitogen for liver fat-storing cells (FSC), is a dimeric molecule that occurs as homodimers or heterodimers of related polypeptide chains (PDGF-BB, -AB, and -AA). In chronic inflammation of the liver lobule, any of the three dimeric forms of PDGF derived from multiple sources could potentially interact with FSC. We explored the effects of the three different PDGF isoforms on DNA synthesis and early signal transduction pathways potentially related to PDGF mitogenicity in rat liver FSC. PDGF-BB homodimer and -AB heterodimer induced a marked increase in DNA synthesis, whereas the effect of PDGF-AA homodimer was considerably lower. Moreover, the mitogenicity of each isoform proportionally correlated with their effects on phosphoinositide turnover and intracellular Ca2+. Both the PDGF-BB and -AB dimers likely interact with the PDGF-beta-receptor, although PDGF-AB requires at least one alpha-receptor. The low responsiveness to PDGF-AA could not be accounted for by downregulation of the PDGF-alpha-receptor because FSC expressed very low levels of PDGF-A- and B-chain mRNAs and did not secrete detectable amounts of PDGF activity in the conditioned media. In addition, preincubation of FSC with suramin, a potent inhibitor of PDGF binding to its receptor, failed to increase PDGF-AA-induced DNA synthesis. These results are consistent with a predominant expression of PDGF-beta-receptor in liver FSC, that is linked to phospholipase C activation.
Foreign anions modulate volume set point of sheep erythrocyte K-Cl cotransport
Authors: Lauf P. K.
Published: December 2017

Preequilibration at 37 degrees C in isosmotic media with Cl replaced by lyotropic (foreign) anions reversibly increased Cl-dependent K efflux and Rb influx, the inhibition by furosemide, and thus K-Cl cotransport in low-K but not in high-K sheep erythrocytes with the following order of effectiveness: SCN greater than I greater than NO3 greater than Cl = Br. This effect depended on time, temperature, and anion concentration and was reversible. Preincubation in isosmotic SCN at 37 degrees C stimulated K-Cl flux in anisosmotic Cl media (370-240 mosM) by increasing the volume sensitivity through shifting the point of zero K-Cl flux by approximately 100 mosmol. Thus even shrunken cells exhibited K-Cl cotransport. Preincubation in hyperosmotic SCN or Cl (440 mosM) followed by K flux in hyposmotic Cl (240 mosM) caused a 30-min lag phase that was absent when cells were swollen only. Hence, foreign anions increased the K flux rate in Cl, suggesting upregulation of K-Cl cotransport through new sites or higher turnover per transporter. The anions must act directly on proteins and/or lipids as the accompanying intracellular pH (pHi) changes were too small to attribute the K-Cl flux activation to cellular acidification. After thiol alkylation, which also activates K-Cl cotransport, SCN preexposure at 37 degrees C became ineffective. Carbethoxylation significantly reduced the foreign anion enhancement of K-Cl cotransport and abolished K efflux in Br. It is concluded that interaction of anions through carbethoxylation-sensitive sites with thiols may determine the level of K-Cl cotransport activity.
Imaging of filter-grown epithelial cells: MDCK Na(+)-H+ exchanger is basolateral
Authors: Rosenberg S. O. , Berkowitz P. A. , Li L. , Schuster V. L.
Published: December 2017

We report a simple method for growing epithelial cells on permeable supports and for imaging the cells from the apical side using an inverted microscope. Madin-Darby canine kidney (MDCK) cells were either seeded onto the conventional side of Millipore-CM filters or onto “inverted” filters. The peak transepithelial resistance of confluent monolayers was the same with either system. Cells on inverted filters that were stained with various dyes and imaged by epifluorescence exhibited more distinct intercellular spaces, cell margins, nuclei, and subapical vesicles. We also perfused both sides of inverted filters with HCO3/CO2-free saline and measured intracellular pH (pHi) using 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) and digital imaging. The intrinsic buffer capacity of MDCK cells increased exponentially as pHi decreased. After an NH4Cl load, the H+ extrusion rate (JH+) in control saline was 2.42 +/- 0.62 mM/min. JH+ was completely blocked by 1 mM basolateral amiloride. In contrast, 1 mM apical amiloride had no effect. We conclude that 1) growth of epithelial cells on an inverted filter system is useful for the microspectrofluorimetric determination of pHi in single cells and for the imaging of apical/subapical structures, and 2) the Na(+)-H+ exchanger of MDCK cells is functionally polarized to the basolateral membrane.
TGF-beta stimulates insulin secretion and blocks mitogenic response of pancreatic beta-cells to glucose
Authors: Sjoholm A. , Hellerstrom C.
Published: December 2017

The long-term influence of transforming growth factor-beta (TGF-beta) on replication and insulin secretion by insulin-producing pancreatic beta-cells was investigated. For this purpose, fetal rat pancreatic islets containing a high proportion of beta-cells were isolated and maintained in tissue culture for 3 days at different concentrations of TGF-beta. TGF-beta (5-500 pM) at a glucose concentration of 11.1 mM did not affect the replication of the beta-cells or their insulin content but enhanced secretion of insulin from these cells. TGF-beta (500 pM) counter-acted the mitogenic action of 16.7 mM glucose but failed to affect the glucose-induced increase in islet insulin content or secretion. Growth hormone (GH) also stimulated beta-cell DNA synthesis and insulin secretion, but TGF-beta was unable to prevent these effects. It was found, moreover, that TGF-beta did not prevent the increase in islet polyamine content which occurred in response to glucose or GH, indicating that the effects of TGF-beta are not mediated through this pathway. Addition of neutralizing antibodies to TGF-beta did not affect the mitogenic or secretory responses to glucose or GH, suggesting that TGF-beta does not exert any autocrine or paracrine function in islets.
Norepinephrine and iloprost improve barrier function of human endothelial cell monolayers: role of cAMP
Authors: Langeler E. G. , van Hinsbergh V. W.
Published: December 2017

The barrier function of human artery endothelial cells was improved by addition of agents that increase the cellular adenosine 3',5'-cyclic monophosphate (cAMP) concentration. Together with a decrease in the passage rate of peroxidase, an increase in the transendothelial electrical resistance was observed. A direct correlation was found between the relative increases in cellular cAMP concentration and the relative decrease in peroxidase passage after incubation of the cells with forskolin (0.25 and 2.5 microM), the beta-adrenergic agonist isoproterenol (10 microM), and the stable prostacyclin analogue iloprost (10 microM). Norepinephrine (10 microM) reduced the peroxidase passage to a much larger extent (40% reduction) than might be expected on the basis of a small increase of cAMP concentration. This small increase in cAMP (44%) was the result of interactions of norepinephrine with beta-adrenergic receptors, which increase cAMP, and alpha-adrenergic receptors, which decrease cAMP. The relatively strong reduction in permeability (also found in the presence of the alpha-adrenergic antagonist phentolamine) suggests that an additional cAMP-independent mechanism underlaid the barrier-improving effect of norepinephrine. A marked elevation of cAMP by forskolin was accompanied by a disappearance of F-actin and myosin from stress fibers. They were found diffusely spread over the cell, and F-actin in the cell periphery became prominently visible.
Macromolecular conjugates of transport inhibitors: new tools for probing topography of anion transport proteins
Authors: Eidelman O. , Yani P. , Englert H. C. , Lang H. G. , Greger R. , Cabantchik Z. I.
Published: December 2017

Macromolecular-conjugated, water-soluble, membrane-impermeant compounds were designed and assessed as topological probes for chloride-transporting agencies. The novel compounds were derivatives of either disulfonic stilbene (DS) and benzylaminoethylsulfonate (BS), "classical" inhibitors of erythrocyte chloride-bicarbonate exchange, or of phenylanthranilates (PA), high-affinity blockers of epithelial chloride channels. Covalent reactive derivatives of various DS, BS, and PA were synthesized and coupled either directly to polyethylene glycol or via spacer arms of different lengths to dextrans. The macromolecular conjugates were demonstrably inhibitory to red blood cell anion exchange when the ligands were appropriately coupled: inhibitory efficacy strongly depended on the chemical structure of the coupled ligand and the spacer length between the inhibitory moiety and the macromolecule. Mechanistic studies indicated that impermeant DS and PA derivatives acted exofacially on sites, which although different in their affinity for chloride, shared geographical proximity. BS derivatives were unique in that they affected transport from either surface. The results suggest asymmetric aqueous access routes leading to the functional domain of the anion transporter from either membrane surface.
Cellular mechanisms in proximal tubular reabsorption of inorganic phosphate
Authors: Murer H. , Werner A. , Reshkin S. , Wuarin F. , Biber J.
Published: December 2017

Filtered inorganic phosphate (Pi) is largely reabsorbed in the proximal tubule. Na-Pi cotransport, with a stoichiometry of at least 2:1, mediates uphill transport at the apical membrane; at the basolateral membrane different types of transport systems can be involved in efflux and uptake of Pi from the interstitium. Regulation of transcellular Pi flux involves alteration of the apical Na-Pi cotransport; at least three different cellular control/sensing systems seem to participate in this regulation and are exemplified by parathyroid hormone (PTH)-dependent inhibition, Pi deprivation-dependent increase, and insulin-like growth factor I (IGF-I)-dependent increase in Na-Pi cotransport. For PTH inhibition, recent evidence suggests a role of the phospholipase C/protein kinase C-dependent regulatory cascade in inhibition of Na-Pi cotransport, at least at low PTH concentrations. In addition, an endocytic mechanism seems to be involved in this PTH action. Little is known of the cellular mechanisms in Pi deprivation-dependent and/or IGF-I-dependent increases in Na-Pi cotransport; they are dependent on de novo protein synthesis. Recent experiments involving an expression in Xenopus laevis oocytes led to the identification of an approximately 50 kDa membrane protein that is a good candidate for being involved in brush-border membrane Na-Pi cotransport activity.
Intracellular ramification of endothelin signal
Authors: Iijima K. , Lin L. , Nasjletti A. , Goligorsky M. S.
Published: December 2017

Effects of porcine 1-21 endothelin (ET-1) on [Ca2+]i, [Na+]i, and [Cl-]i and on membrane potential were studied in individual mesangial (MC) and vascular smooth muscle (VSMC) cells using microspectrofluorimetry of fura-2, SBFI, SPQ, and bis-oxonol, respectively. ET-1 increased [Ca2+]i by fivefold, showing an immediate and a sustained phase of response. Ca(2+)-free medium and nifedipine pretreatment significantly curtailed the sustained phase of response to ET-1. These findings were confirmed in studies of vascular ring preparations, demonstrating that Ca2+ influx may account for at least 50% of contraction. ET-1 caused immediate and sustained depolarization of MC and VSMC. This could not be attributed to Na+ influx, since fluorescence of SBFI was not affected by ET-1 and Na(+)-free medium did not abolish the ET-1-induced membrane depolarization. Studies of SPQ fluorescence changes induced by ET-1 revealed an increase in fluorescence intensity consistent with the decrease in [Cl-]i. A Cl- channel blocker, IAA-94, abolished changes in SPQ fluorescence and curtailed sustained phases of membrane depolarization and [Ca2+]i elevation in response to ET-1, but did not affect KCl-induced [Ca2+]i transients. IAA-94 also attenuated the ET-1-induced contraction of aortic rings. Microinjection of either calcium gluconate or inositol 1,4,5-trisphosphate (IP3) in SPQ-loaded cells resulted in an increase in fluorescence mimicking the effect of ET-1. These changes were blunted by pretreatment of cells with BAPTA and incubation in Ca(2+)-free medium. When IP3 was microinjected into fura-2-loaded MC, this resulted in immediate and sustained elevation of [Ca2+]i. In conclusion, generation of IP3 results in mobilization of intracellular Ca2+ stores and activation of Cl- channels. Ensuing Cl- efflux causes membrane depolarization and, in turn, activation of voltage-dependent Ca2+ channels, resulting in sustained elevation of [Ca2+]i which is indispensable for the full-scale contraction produced by ET-1.
Non-insulin-dependent diabetes-induced defects in cardiac cellular calcium regulation
Authors: Allo S. N. , Lincoln T. M. , Wilson G. L. , Green F. J. , Watanabe A. M. , Schaffer S. W.
Published: December 2017

Non-insulin-dependent diabetic (NIDD) male Wistar rats develop a cardiomyopathy approximately 9 mo after the onset of the diabetic condition. This cardiomyopathy is characterized by reduced contractility, relaxation, cardiac work, and diastolic compliance. Although the basis for these defects is not completely understood, altered cellular Ca2+ regulation appears to play a major role in their development. In both isolated sarcolemmal membrane and cardiomyocytes, significant diabetes-linked defects in Ca2+ metabolism were observed. A small, but significant, decrease in the rate of sarcolemmal ATP-dependent Ca2+ transport of the diabetic heart was observed. Also evident was a major defect in sarcolemmal Na(+)-Ca2+ exchange as determined by reduced Na(+)-dependent Ca2+ transport into vesicles and Na(+)-dependent Ca2+ efflux from 45Ca(2+)-loaded cardiomyocytes from diabetic rats. In isolated cardiomyocytes, it was observed that the relative fluorescence of fura-2 at 502 nm was higher in cells from NIDD hearts, suggestive of a higher cytosolic free Ca2+. Consistent with diabetes-linked defects in Ca(2+)-transporter activities, the accumulation of Ca2+ after depolarization with KCl was greater in the diabetic. This study demonstrates that diabetes-induced defects in Ca2+ movement by the various Ca2+ transporters lead to abnormal cytosolic Ca2+ regulation by the diabetic cardiomyocytes. This observation supports the notion that abnormal Ca2+ regulation contributes to the development of the NIDD cardiomyopathy.
Stimulation of Ca2+ influx by endothelin-1 is subject to negative feedback by elevated intracellular Ca2+
Authors: Muldoon L. L. , Enslen H. , Rodland K. D. , Magun B. E.
Published: December 2017

Endothelin-1 (ET-1) has been shown to require Ca2+ influx for activation of vascular smooth muscle in vivo, but in vitro models show that ET-1 mobilizes intracellular Ca2+ and is independent of extracellular Ca2+. We present data that suggest ET-1 modulates cellular responses through a dual mechanism involving both phosphatidylinositol turnover and Ca2+ channel activation. Addition of low concentrations of ET-1 (less than 10(-9) M) to serum-deprived quiescent Rat-1 cells stimulated Ca2+ influx while having little effect on diacylglycerol (DG) release or intracellular Ca2+ levels. In contrast, higher concentrations of ET-1 (greater than 10(-9) M) stimulated intracellular Ca2+ transients and release of inositol trisphosphate (IP3) and DG but did not activate Ca2+ uptake. Stimulation of Ca2+ influx at low [ET-1] could not be accounted for by depletion of intracellular IP3-sensitive pools. Neither the stimulation of Ca2+ influx at low [ET-1] nor the inhibitory actions of high [ET-1] could be mimicked by the activation of protein kinase C. We tested the hypothesis that elevated intracellular Ca2+ was inhibitory for Ca2+ influx. When intracellular Ca2+ transients were maintained below approximately 165 nM by chelation with BAPTA or BAPTA derivatives with altered affinity for Ca2+, Ca2+ influx was stimulated over the entire range of ET-1 concentrations. In addition, experimentally elevating intracellular Ca2+ levels with the tumor promoter thapsigargin abolished ET-1-stimulated Ca2+ influx. These data suggest that the biological consequences of ET-1 release may be determined by local concentration differences. Thus in vascular smooth muscle cells ET-1 may act either to mobilize intracellular Ca2+ or to promote Ca2+ influx, depending on the distance from the endothelial cell source in the vascular wall. The activation of different processes by low and high ET-1 concentrations may determine the physiological response to ET-1 stimulation in vivo.
Toad urinary bladder epithelial cells contain an analogue of cytoskeletal protein 4.1
Authors: Guay-Woodford L. M. , Platt O. , Harris H. W.
Published: December 2017

Epithelial cell polarity and vectorial transport require cytoskeletal proteins that maintain local cell membrane structure and mediate cytoplasmic vesicle movement. The cytoskeleton of leaky epithelia, such as the intestinal mucosa and renal proximal tubule cells, has been extensively studied. However, cytoskeletal studies in tight epithelia such as the mammalian collecting duct and toad urinary bladder generally have been confined to ultrastructural investigation. Recent research in nonepithelial cell types has identified an interesting family of cytoskeletal proteins. Present in multiple cell types, these protein 4.1 analogues share a number of similar functional characteristics, yet are structurally diverse. They are multiply phosphorylated by several different kinases, and phosphorylation regulates their associations with other cytoskeletal constituents, integral membrane components, and cytoplasmic vesicles. Using a combination of immunochemical and immunofluorescent techniques, we have demonstrated that toad bladder epithelial cells contain a 65-kDa analogue of human erythrocyte protein 4.1. Toad bladder epithelial cell protein 4.1 is structurally similar to its erythrocyte counterpart and is phosphorylated. This protein 4.1 species is present throughout the toad bladder granular cell cytoplasm, suggesting that it participates in multiple granular cell functions.
A simple method for evaluation of Rb+ transport and Na(+)-K+ pump stoichiometry in adherent cells
Authors: Longo N. , Griffin L. D. , Elsas L. J.
Published: December 2017

This report describes a method based on flame photometry for the evaluation of transmembrane Rb+ transport and Na(+)-K+ pump stoichiometry in adherent cells. In monolayers of cultured fibroblasts, the rates of 86Rb+, an isotope widely used as a K+ congener in transport studies, and nonradioactive Rb+ influx were equivalent when measured in the absence and presence of the transport inhibitors ouabain and bumetanide. Ouabain- and bumetanide-sensitive Rb+ fluxes were also equal with the two methods. Flame photometry allowed the simultaneous determination of intracellular [Na+] in the same sample in which Rb was measured. The incubation of human fibroblasts with ouabain for 5 min promoted a significant increase in intracellular [Na+]. Under appropriate experimental conditions, the ratio between the rate of ouabain-promoted increase in intracellular [Na+] and ouabain-sensitive Rb+ influx was 1.4, close to the theoretical value of 1.5 corresponding to a Na(+)-K+ pump stoichiometry of 3 Na+ extruded from the cell in exchange for 2 K+.
Carbohydrate supplementation attenuates IMP accumulation in human muscle during prolonged exercise
Authors: Spencer M. K. , Yan Z. , Katz A.
Published: December 2017

The effect of carbohydrate (CHO) ingestion on metabolic responses to exercise has been investigated. Subjects cycled at approximately 70% of maximal oxygen uptake to fatigue [135 +/- 17 (+/- SE) min] on the first occasion (control, CON) and at the same work load and duration on the second occasion but with addition of ingestion of CHO during the exercise. Biopsies were taken from the quadriceps femoris muscle before and after exercise. The sum of the hexose monophosphates (HMP), as well as lactate and alanine, in muscle was higher after CHO exercise (P less than or equal to 0.05, P less than or equal to 0.05, and P less than or equal to 0.01, respectively). Acetylcarnitine increased during exercise but was not significantly different between treatments after exercise (CON, 6.6 +/- 1.7; CHO, 10.0 +/- 1.2 mmol/kg dry wt; P = NS). The sum of the tricarboxylic acid cycle intermediates (TCAI; citrate + malate + fumarate) was increased during exercise and was higher after CHO exercise (2.34 +/- 0.32 vs. 1.68 +/- 0.17 mmol/kg dry wt; P less than or equal to 0.05). IMP was less than 0.1 mmol/kg dry wt at rest and increased to 0.77 +/- 0.26 (CON) and 0.29 +/- 0.11 mmol/kg dry wt (CHO) (P less than or equal to 0.05) during exercise. It was recently found that during prolonged exercise there is initially a rapid and large expansion of TCAI and glycogenolytic intermediates in human muscle followed by a continuous decline in TCAI and glycogenolytic intermediates [K. Sahlin, A. Katz, and S. Broberg. Am. J. Physiol. 259 (Cell Physiol. 28): C834-C841, 1990].(ABSTRACT TRUNCATED AT 250 WORDS)
Divalent cations modulate the transient outward current in rat ventricular myocytes
Authors: Agus Z. S. , Dukes I. D. , Morad M.
Published: December 2017

The modulation of the transient outward K+ current (Ito) by divalent cations was studied in enzymatically isolated rat ventricular myocytes with the whole cell patch-clamp technique. At holding potentials negative to -70 mV, 1 mM Cd2+ suppressed Ito, whereas, at potentials positive to -50 mV, the current was augmented. These effects were caused by shifts in the voltage dependence of both activation and inactivation of Ito toward more positive potentials. Cd2+ also slowed the activation kinetics of Ito by shifting the voltage dependence of its rate of activation, but the rate of inactivation was unaffected. Other divalent cations produced similar shifts but at markedly different concentrations. Thus, in the millimolar range, a rightward shift of approximately 20 mV was produced by 3 Co2+, 5 Ni2+, and 10 Ca2+, whereas 10 microM concentrations of Cu2+ and Zn2+ produced equivalent shifts. Similar effects were seen in hippocampal neurons with micromolar concentrations of Zn2+. Thus divalent cations have marked and specific effects on the kinetics and voltage dependence of Ito and may serve as a regulatory mechanism in its activation, particularly in cells with resting potentials positive to -60 mV.
Viscoelastic properties of the contracting detrusor. I. Theoretical basis
Authors: Venegas J. G.
Published: December 2017

This paper presents the theoretical basis for estimating the detrusor's viscoelastic properties using the small-amplitude oscillatory perturbations technique. Three possible configurations of the simplest second-order lumped-parameter model of the bladder were analyzed to derive equations of the parameters incremental resistance (R) and incremental elastance (K) in terms of the experimentally measurable magnitude and phase of hydrodynamic stiffness. In model I, single viscous, elastic, and inertial elements were assumed to to be connected in series. In model III the elastic and viscous elements were connected in series, but the inertial element was connected in parallel. With the assumption of a spherical geometry of the bladder, equations were also derived to obtain the bladder wall mechanical properties, spring incremental constant (S), and muscle incremental viscosity (b) as functions of bladder volume and the hydrodynamic properties R and K. Integration of the incremental equation describing the viscous component yields an expression that fits well the force-velocity experimental data from bladder strips reported by others. This finding suggests that muscle viscosity measured with the small-amplitude oscillations and analyzed with the proper theoretical model may be related to the force-velocity characteristics of the muscle. The equations delivered here form the basis for analyzing the experimental data described in the companion paper.
Viscoelastic properties of the contracting detrusor. II. Experimental approach
Authors: Venegas J. G. , Woll J. P. , Woolfson S. B. , Cravalho E. G. , Resnick N. , Yalla S. V.
Published: December 2017

Mechanical properties of detrusor muscle were studied with small-amplitude oscillatory volume perturbations in isometrically contracting bladders of anesthetized dogs. Contractions were studied at oscillatory frequencies (f) of 2 and 4 Hz and at bladder volumes (Vbl) ranging from 30 to 110 ml. The magnitude of bladder hydrodynamic stiffness (magnitude of G) increased linearly with mean detrusor pressure (Pdet) while the phase angle remained relatively constant during contraction. The slope (mG) of magnitude of G-Pdet relations had a positive dependence on f and a negative dependence on Vbl. Analysis of oscillatory data, described in the companion paper, was performed using incremental lumped-parameter models consisting of a spring with incremental constant (S = dF/dL), a viscous element with incremental viscosity (b = dF/du), and a mass (m). Only the model where elastic and viscous elements were placed in series with each other and in parallel with mass was compatible with the experimental data. Both S and b increased linearly with effective force (F), defined as Pdet times the cross-sectional area of the intravesical cavity. Slopes of the S-F and b-F relationships (ms and mb) were independent of Vbl and varied only slightly with f. The importance of this finding stems from recognizing that ms and mb correspond to the exponential coefficients of nonlinear series elastic and internal viscosity elements. These parameters, when normalized by resting muscle length, represent fundamental muscle properties independent of muscle cross-sectional area, stretch, or level of activation and compare well with parameters derived from other muscle systems using techniques such as quick releases and isotonic contractions.
Inhibition of ion conductances by osmotic shrinkage of Madin-Darby canine kidney cells
Authors: Ritter M. , Steidl M. , Lang F.
Published: December 2017

Osmotic swelling of Madin-Darby canine kidney (MDCK) cells enhances the ion conductances of the cell membrane, which allows release of cellular ions and subsequent regulatory cell volume decrease. The present study has been performed to test whether cell shrinkage similarly affects the ion conductances of MDCK cell membranes. Increase of extracellular osmolarity by addition of 50 mM NaCl or 100 mM mannitol leads within 3 min to a hyperpolarization of the cell membrane, a marked increase of cell membrane resistance [by 223 +/- 38% (n = 8) and 228 +/- 21% (n = 5), respectively], as well as a moderate increase of the K+ selectivity of the cell membrane (by 37 +/- 13%, n = 9). Thus exposure to hypertonic extracellular fluid decreases the cell membrane conductances including the K+ conductance. Cell volume measurements reveal a regulatory cell volume increase, which is sensitive to both furosemide and dimethylamiloride. Extracellular ATP (10 microM), which activates calcium-sensitive K+ channels, hyperpolarizes the cell membrane close to the K+ equilibrium potential. The respective values are -69.9 +/- 3.1 mV (n = 9) in isotonic fluid, -79.4 +/- 1.8 mV (n = 9) within 3 min, and -76.4 +/- 1.8 mV (n = 7) within 16-h exposure to hypertonic extracellular fluid. This observation points to a sustained increase of intracellular K+ activity after exposure to hypertonic extracellular fluid.
Serum and growth factor induction of Na(+)-K(+)-ATPase subunit mRNAs in Clone 9 cells: role of protein kinase C
Authors: Bhutada A. , Ismail-Beigi F.
Published: December 2017

In a previous study, we found that addition of serum to confluent Clone 9 cells, a nontransformed rat liver cell line, increased the abundance of mRNA alpha 1 and mRNA beta 1 at 3 h by 2- and 2.7-fold, respectively [Bhutada et al. Am. J. Physiol. 258 (Cell Physiol. 27): C1044-C1050, 1990]. We now report that exposure of these cells to 160 nM 12-O-tetradecanoylphorbol 13-acetate (TPA) for 6 h increases mRNA alpha 1 and mRNA beta 1 by 1.7 +/- 0.2- and 2.1 +/- 0.3-fold, respectively. Incubation in the presence of 160 nM TPA for 24 h reduced high-affinity phorbol dibutyrate-binding sites [dissociation constant (Kd) = 5 nM; maximum binding (Bmax) = 1.2 pmol/mg protein] to undetectable levels. In such cells, exposure to 10% serum for 6 h still resulted in two- and fourfold increment in mRNA alpha 1 and mRNA beta 1 abundances, respectively, while further addition of TPA to these protein kinase C (PKC)-depleted cells resulted in no change in the subunit mRNA abundances. The increments in mRNA alpha 1 content in response to 10% serum and 160 nM TPA at 6 h were additive, whereas the increments in mRNA beta 1 were not. The following agents increased mRNA alpha 1 and mRNA beta 1 abundance in both control and PKC-depleted cells: epidermal growth factor, platelet-derived growth factor, basic fibroblast growth factor, insulin, dexamethasone, and hypothyroid calf serum. In contrast, N6,2'-O-dibutyryl-adenosine 3',5'-cyclic monophosphate and aldosterone had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)