bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhancement of kallikrein production and kinin sensitivity in T84 cells by growth in the nude mouse
Authors: Baird A. W. , Miller D. H. , Schwartz D. A. , Margolius H. S.
Published: December 2017

The production of tissue kallikrein and the short-circuit current (SCC) response in the human colonic epithelial cell line T84 to bradykinin have been studied. These cells produce true tissue kallikrein and secrete it into the growth medium, and addition of bradykinin results in a small increase in SCC. After growth of T84 cells as a xenograft in athymic (nude) mice (NuT84), however, the cellular concentration of the enzyme increases 38-fold, and the maximal change in SCC (delta SCC) induced by bradykinin increases 35-fold. The SCC response to prostaglandin E1 is not changed, and the response to forskolin increases eightfold. The bradykinin-induced delta SCC was inhibited by piretanide, suggesting that a secretory movement of chloride was responsible for the change, and the effect was attenuated by an antagonist to kinin receptors. We conclude that both the production of true tissue kallikrein and the chloride secretory response to bradykinin can be regulated in T84 cells by changes in the cell environment.
Dual effects of a phorbol ester on calcium-dependent chloride secretion by T84 epithelial cells
Authors: Kachintorn U. , Vongkovit P. , Vajanaphanich M. , Dinh S. , Barrett K. E. , Dharmsathaphorn K.
Published: December 2017

Ca(2+)-dependent secretagogues (e.g., carbachol, histamine, ionomycin, and 4-bromo-A23187) have relatively transient effects on chloride secretion, even if there is a sustained increase in cytosolic calcium ([Ca2+]i) (as for the ionophores). Because these agents increase both [Ca2+]i and protein kinase C (PKC) activity, chloride secretion might be stimulated by [Ca2+]i and terminated by PKC activity. We tested the effect of a PKC activator, phorbol 12-myristate 13-acetate (PMA), on Cl- secretion by T84 cell monolayers by measuring short-circuit current (Isc). PMA alone had no effect on Isc but potentiated increases in Isc when added 10 min or less before Ca(2+)-dependent secretagogues. Chelation of [Ca2+]i with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid inhibited the increases both in [Ca2+]i and Isc induced by carbachol with or without brief PMA pretreatment. Longer preincubations with PMA inhibited Isc responses to Ca(2+)-dependent secretagogues, even when increased [Ca2+]i was sustained by ionophores. Inhibitors of PKC could reverse the inhibitory effect of PMA but did not reverse the potentiating effect. The effects of PMA on Cl- secretion were reproduced by 1,2-dioctanoyl-sn-glycerol and were mirrored by effects on K+ channel opening. Thus PMA has dual effects on chloride secretion. Initially, it exerts a stimulatory action and subsequently an inhibitory action. The stimulatory effect only occurs if Ca(2+)-dependent secretion is ongoing. The inhibitory effect of PMA is mediated by PKC and cannot be overcome by increasing [Ca2+]i.
Lipid A activation of glomerular mesangial cells: mimicry of the bioactive lipid, phosphatidic acid
Authors: Bursten S. L. , Harris W. E. , Resch K. , Lovett D. H.
Published: December 2017

Lipid A, the active component of bacterial endotoxin, stimulates multiple cell types, including glomerular mesangial cells (MC), and yet the molecular mechanisms of cell activation remain unclear. Lipid A, in its monosaccharyl form, structurally resembles the biologically active lipid phosphatidic acid (PA). Given this, it was postulated that lipid A activates cells by acting as a structural and functional mimetic of PA. Lipid A was found to specifically stimulate an MC lyso-PA acyl transferase activity, leading to enhanced synthesis of sn-2-unsaturated forms of PA. Sn-2-unsaturated PA itself, in contrast to sn-2-saturated PA, also stimulated the lyso-PA acyl transferase activity, a positive feedback feature previously noted with lyso-lecithin acyl transferase. Structure-function correlations demonstrated that the phosphate moieties in both PA and lipid A were necessary to feedback stimulation of lyso-PA acyl transferase (AT), as dephosphorylated lipid A and 2-unsaturated 1,2-sn-diacylglycerol had no stimulatory effect on lyso-PA AT. The biologic relevance of the lipid A and PA-mediated increases in lyso-PA acyl transferase activity was shown, whereby limited exposure to these lipids rapidly induced identical MC morphologic and functional alterations characteristic of cellular activation. By mimicking the stimulatory action of PA, per se, on lyso-PA acyl transferase activity, lipid A may initiate a positive feedback cycle of acylation, yielding increased amounts of PA enriched in unsaturated fatty acids. This newly synthesized PA may subsequently act as the proximal mediator of cellular activation.
Antibodies against the cystic fibrosis transmembrane regulator
Authors: Fuller C. M. , Howard M. B. , Bedwell D. M. , Frizzell R. A. , Benos D. J.
Published: December 2017

Rabbit polyclonal antibodies have been raised against high-performance liquid chromatography purified synthetic peptides corresponding to two discrete regions of the cystic fibrosis transmembrane regulator (CFTR) protein: the R-domain (residues 785-796) and the extreme COOH-terminus (residues 1467-1480). Antibodies (Ab) to each of these peptides were affinity purified either by passage over a peptide-derivatized agarose matrix (Ab 785) to produce monospecific polyclonal antibodies or by protein A affinity chromatography to purify the immunoglobulin G1 fraction free of other serum proteins (Ab 1467). These antibodies recognize a candidate CFTR protein in the colonic cell line T84, as determined by Western blot analysis and by immunoprecipitation and labeling of the precipitate with [gamma-32P]ATP in the presence of protein kinase A. Both antibodies precipitated CFTR-related polypeptides from four mammalian cell types (HeLa, Bsc-40, HEp-2, and Chinese hamster ovary cells) transfected with the full-length CFTR cDNA clone using a vaccinia T7 protein expression system. Similar results were observed using a yeast CFTR expression system. In each case the Mr values of the bands observed were consistent with that expected for the CFTR protein. These antibodies should be useful probes for the immunocytochemical localization, immunoaffinity purification, and ultimately the functional characterization of the CFTR protein.
Whole cell patch clamping of ciliary epithelial cells during anisosmotic swelling
Authors: Yantorno R. E. , Carre D. A. , Coca-Prados M. , Krupin T. , Civan M. M.
Published: December 2017

Anisosmotic cell swelling triggers a regulatory volume decrease (RVD) in cell lines derived from human nonpigmented ciliary epithelium. Measurements of cell volume have indicated that the RVD reflects activation of K+ and/or Cl- channels. We have begun to characterize the putative channels by whole cell patch clamping. The results obtained by altering the external K+ and Cl- concentrations and by adding 20-50 microM quinidine or 1 mM Ba2+ indicate that K+ conductances contribute substantially and Cl- conductances contribute very little to the total membrane conductance (GT) under baseline isotonic conditions. Reducing the external osmolality by 20-50% reversibly and reproducibly increased GT by an order of magnitude. Data obtained from ion substitutions and the channel blockers quinidine and 5-nitro-2-(3-phenylpropylamino)-benzoate indicate that most of the hypotonicity-induced conductance reflects stationary Cl(-)-channel activity. The contribution of new K(+)-channel activity was small at intracellular free Ca2+ concentrations of 10 or 200 nM. We conclude that the RVD triggered by bath hypotonicity primarily reflects increased Cl(-)-channel activity.
Differential expression of SM1 and SM2 myosin isoforms in cultured vascular smooth muscle
Authors: Babij P. , Kawamoto S. , White S. , Adelstein R. S. , Periasamy M.
Published: December 2017

Ribonuclease protection assays were used to measure expression of smooth muscle (SM) specific myosin heavy chain (MHC) isoforms SM1 (204 kDa) and SM2 (200 kDa) and also nonmuscle MHC-A in cultured smooth muscle cells isolated from rat aorta. In cells grown in 10% serum for 3-5 days until subconfluent, SM1 MHC mRNA decreased by 30% and SM2 MHC mRNA decreased by 80%. In cells grown in confluency for 7-11 days, SM1 MHC mRNA decreased by 45% and SM2 MHC mRNA decreased by 80%. Similar reductions were found in passaged cells. Serum withdrawal for 1-2 days from confluent cultures had little or no effect on SM1 or SM2 MHC mRNA levels. In contrast, nonmuscle MHC-A mRNA increased 10-fold in subconfluent cultures but increased only threefold higher than controls in quiescent cells. Myosin protein analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting indicated that SM1 MHC protein was detectable at a reduced level in confluent cultured cells, whereas SM2 MHC protein was absent in confluent cells. The decrease in SM2 was much greater than SM1, indicating differential regulation. An apparently new isoform of SM1 MHC migrating with a mobility similar to SM2 type MHC was detected by immunoblot analysis in cultured cells.
Effects of bradykinin on Na+ and Cl- transport in human nasal epithelium
Authors: Clarke L. L. , Paradiso A. M. , Mason S. J. , Boucher R. C.
Published: December 2017

Human nasal epithelium (HNE) is a Na+ absorptive epithelium but establishes a baseline Cl- secretory current in the presence of amiloride (10(-4) M, luminal). We compared the effects of an inflammatory mediator, bradykinin (BK), on ion transport in primary cultures of HNE using double-barreled Cl(-)-selective microelectrodes. In untreated HNE, BK (10(-5) M) transiently increased the equivalent short-circuit current (Ieq). Maximal Ieq occurred with hyperpolarization of the transepithelial potential difference (Vt), which was associated with hyperpolarization and decreased resistance of the basolateral membrane; a subsequent depolarization of Vt was observed that was associated with depolarization and decreased resistance of the apical membrane. Removal of bath Cl- did not affect the BK-induced Ieq response. In amiloride-treated HNE, the electrical pattern of the BK-induced response was identical, but the magnitude of the Ieq was reduced by 54% and the change in Ieq could be abolished by removal of bath Cl-. Equivalent-circuit analysis of the response in amiloride-treated tissues indicated activation of a hyperpolarizing conductance in the basolateral membrane, followed 20-30 s later by activation of an apical Cl- conductance. We conclude that BK stimulates both Na+ absorption in untreated HNE and Cl- secretion in amiloride-treated HNE by activating a basolateral (K+) conductance. Analysis of the entire Ieq response under both conditions also suggested that BK induces a delayed activation of apical membrane Na+ and Cl- conductances.
Calcium oscillations in parotid acinar cells induced by microsomal Ca(2+)-ATPase inhibition
Authors: Foskett J. K. , Wong D.
Published: December 2017

Previous studies have demonstrated in single rat parotid acinar cells that the microsomal Ca(2+)-ATPase inhibitor thapsigargin mobilizes Ca2+ specifically from the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store, activates plasma membrane Ca2+ permeability, and induces intracellular Ca2+ concentration ([Ca2+]i) oscillations that are quite similar to those activated by carbachol. Nevertheless, the IP3-sensitive Ca2+ store remains continuously depleted during thapsigargin-induced oscillations, indicating that this pool is not involved in the oscillation mechanism. To determine the specificity of thapsigargin's effects, in the present study we have examined the effects on [Ca2+]i in single rat parotid acinar cells of two other microsomal Ca(2+)-ATPase inhibitors, cyclopiazonic acid (CPA) and 2,5-di-tert-butyl-1,4-benzohydroquinone (BHQ), and compared them with the effects of thapsigargin in the same cells. Our results demonstrate that thapsigargin, CPA, and BHQ all similarly deplete the IP3-sensitive Ca2+ store specifically, activate plasma membrane Ca2+ influx, and induce [Ca2+]i oscillations, strongly suggesting that these agents have a specific inhibitory action on microsomal Ca(2+)-ATPase activity. BHQ, in addition, inhibits plasma membrane Ca2+ influx. The data lend strong support to a model in which the state of Ca2+ filling of the IP3-sensitive store regulates plasma membrane Ca2+ influx. These results suggest either that a Ca2+ pump is involved which is insensitive to structurally dissimilar inhibitors or that a Ca2+ pump is not involved in refilling of the Ca2+ pool involved in [Ca2+]i oscillations in these cells.
Association between synthesis and release of cGMP and nitric oxide biosynthesis by hepatocytes
Authors: Billiar T. R. , Curran R. D. , Harbrecht B. G. , Stadler J. , Williams D. L. , Ochoa J. B. , Di Silvio M. , Simmons R. L. , Murray S. A.
Published: December 2017

Hepatocytes are known to synthesize nitric oxide (NO) from L-arginine via an inducible NO synthase. Studies were performed to determine the relationship between hepatocyte NO production and the stimulation of hepatocyte soluble guanylate cyclase. A combination of lipopolysaccharide (LPS), interferon-gamma, tumor necrosis factor, and interleukin-1 stimulates the biosynthesis of large quantities of nitrite and nitrate (NO2- + NO3-). Hepatocyte NO2- + NO3- production was associated with only small increases in intracellular guanosine 3',5'-cyclic monophosphate (cGMP) levels but much greater increases in extracellular cGMP release over an 18-h time period. This cGMP synthesis was dependent on the L-arginine concentration and was inhibited in a reversible manner by NG-monomethyl-L-arginine. The cytokines or LPS added alone induced small increases in nitrogen oxide production and concomitant minor elevations in cGMP release. Atrial natriuretic peptide also stimulated the release of cGMP by hepatocytes which appeared to be independent of the cytokine+LPS-induced cGMP release. The addition of probenecid reduced the cGMP release by 66%, while cell damage was excluded as a cause for the extracellular release. Addition of 3-isobutyl-1-methylxanthine, but not M&B 22948, increased hepatocyte intra- and extracellular cGMP levels after cytokine+LPS stimulation. Induction of nitrogen oxide synthesis by hepatocytes in vivo by injecting rats with killed Corynebacterium parvum resulted in increased cGMP levels in freshly isolated hepatocytes and increased cGMP release by the hepatocytes when placed in culture.(ABSTRACT TRUNCATED AT 250 WORDS)
Basolateral Na-H antiporter: uncoupled Na transport produces an amiloride-sensitive conductance
Authors: Post M. A. , Dawson D. C.
Published: December 2017

An amiloride-inhibitable, Na(+)-H+ antiporter was identified in the basolateral membrane of turtle colon by measuring 22Na+ fluxes across isolated tissues apically permeabilized with the pore-forming antibiotic amphotericin B. In cells shrunken by exposure to Cl(-)-free (gluconate) solutions and treated with ouabain to block the Na-K-ATPase, Na+ movement across the basolateral membrane was due entirely to the antiporter. Elevation of cytosolic Na+ was associated with an amiloride-inhibitable outward current across the basolateral membrane. The sensitivity of the current to various amiloride analogues paralleled that of Na+ exchange rather than that of the apical Na+ channel. Furthermore, cell volume changes altered basolateral Na+ exchange and basolateral Na+ conductance in a parallel fashion. We propose that this amiloride-sensitive basolateral Na+ conductance represents an altered operating mode of a basolateral Na(+)-H+ exchanger.
Interleukin-1 beta alters actin expression and inhibits contraction of rat thoracic aorta
Authors: Trinkle L. A. , Beasley D. , Moreland R. S.
Published: December 2017

Previous studies have indicated that interleukin-1 beta (IL-1) inhibits contraction of rat aortas by activating nitric oxide production in vascular smooth muscle cells, with subsequent increases in guanosine 3',5'-cyclic monophosphate (cGMP). This study determined if the effect of IL-1 involves the primary regulatory event in smooth muscle activation, myosin light chain (MLC) phosphorylation. This study also examined whether IL-1 affects contractile protein content. IL-1 (20 ng/ml) significantly decreased stress in response to 0.1 microM phenylephrine with a concomitant decrease in MLC phosphorylation. Incubation with IL-1 for 3 h or longer decreased alpha-smooth muscle actin and increased gamma-actin isoform, with no change in beta-nonmuscle actin or myosin isozyme content. These results suggest that IL-1 inhibition of a vascular smooth muscle contraction may be due to a decrease in activator calcium, which may account for the resultant decrease in MLC phosphorylation. These results also indicate that IL-1 significantly affects contractile protein content, enhancing gamma-actin isoforms and decreasing the vascular smooth muscle specific alpha-isoactin.
Amino acid transport by the cultured human placental trophoblast: effect of insulin on AIB transport
Authors: Karl P. I. , Alpy K. L. , Fisher S. E.
Published: December 2017

Insulin responsiveness in the human placenta is controversial. This study evaluated insulin stimulation of alpha-aminoisobutyric acid (AIB) uptake in cultured human placental trophoblasts. Both Na(+)-dependent and -independent components of AIB uptake were present in cultured trophoblasts. Na(+)-dependent AIB uptake was significantly stimulated by insulin in a time-dependent manner, as early as 2 h, with a maximum at 12 h of continuous exposure to hormone. Insulin treatment for 4 h increased both the initial uptake rate and the final intracellular concentration. Stimulation was dependent on insulin concentration, with significant stimulation beginning at 10(-9) M. Insulin treatment increased maximum velocity but not the Michaelis constant. Approximately 75% of basal (unstimulated) AIB uptake was inhibited by 10 mM alpha-methylaminoisobutyric acid (MeAIB). The insulin-stimulated increment above basal AIB uptake was completely inhibited by 10 mM MeAIB. Cycloheximide treatment significantly reduced basal and stimulated AIB uptake, although a significant response to insulin persisted. Na(+)-dependent AIB uptake was also stimulated by glucagon, dexamethasone, and 8-bromoadenosine 3',5'-cyclic monophosphate, but not by vasopressin. This study further characterizes amino acid uptake by the human placenta and demonstrates that the Na(+)-dependent component of AIB uptake by the cultured trophoblasts is stimulated by physiological concentrations of insulin.
Regulation of Na(+)-K(+)-ATPase expression in cultured renal cells by incubation in hypertonic medium
Authors: Bowen J. W.
Published: December 2017

To determine whether alterations in cell volume affect Na(+)-K(+)-adenosinetriphosphatase (ATPase) expression, a subclone of the Madin-Darby canine kidney (MDCK) cell line was incubated in anisotonic serum-free medium and alpha- and beta-subunit mRNA, Na(+)-K(+)-ATPase activity, and active K+ transport were measured. In medium adjusted to 500 mosmol/kgH2O by adding NaCl, the alpha-subunit mRNA concentration was 2.93 +/- 0.14 (SE) times control and beta-mRNA was 1.93 +/- 0.27 times control. When sucrose was added to increase osmolality, alpha-subunit mRNA increased to 1.85 +/- 0.18 times control. Na(+)-K(+)-ATPase activity of homogenates from cells incubated in 500 mosmol/kgH2O medium for 24 h increased to 2.62 +/- 0.52 times control when NaCl was added and 2.31 +/- 0.34 times control when sucrose was added. Active K+ transport increased between 60 and 90% after cells were incubated in 450 mosmol/kgH2O medium with either NaCl or sucrose added. Stimulation of Na(+)-K(+)-ATPase expression in renal cells facing hypertonic stress may represent a long-term mechanism that allows cells to maintain cation gradients in a hypertonic environment.
Thapsigargin stimulates Ca2+ entry in vascular smooth muscle cells: nicardipine-sensitive and -insensitive pathways
Authors: Xuan Y. T. , Wang O. L. , Whorton A. R.
Published: December 2017

We have investigated the role of the sarcoplasmic reticulum Ca2+ pool in regulating Ca2+ entry in vascular smooth muscle cells using a receptor-independent means of mobilizing the intracellular Ca2+ pool. Thapsigargin (TG) has been shown to inhibit the endoplasmic reticulum Ca(2+)-ATPase, mobilize intracellular Ca2+, and activate Ca2+ entry in nonmuscle tissues. When smooth muscle cells were treated with 0.2 microM TG, cytosolic Ca2+ concentrations rose gradually over 8 min to a peak value of 365 +/- 18 nM. Cytosolic Ca2+ remained elevated for at least 20 min and was supported by continued entry of extracellular Ca2+. TG also stimulated entry of Mn2+ and 45Ca2+ from outside the cell. Importantly, TG-induced Ca2+ entry and Mn2+ entry were found to occur through mechanisms that were independent of L-type Ca2+ channel activation because influx was not inhibited by concentrations of nicardipine that were found to block either endothelin- or 100 mM extracellular K(+)-induced cation influx. The mechanism through which TG activates cation entry appears to involve mobilization of the inositol 1,4,5-trisphosphate-responsive intracellular Ca2+ pool. In permeabilized cells, TG prevented ATP-stimulated Ca2+ uptake into the sarcoplasmic reticulum and slowly released sequestered Ca2+. The Ca2+ pool involved was responsive to inositol 1,4,5-trisphosphate. However, TG did not initiate the formation of inositol polyphosphates. Thus TG mobilizes the sarcoplasmic reticulum Ca2+ pool and activates Ca2+ entry through a nicardipine-insensitive Ca2+ channel in vascular smooth muscle. The mechanism is independent of inositol polyphosphate formation.
Developmental increases in the inwardly rectifying potassium current of rat ventricular myocytes
Authors: Wahler G. M.
Published: December 2017

The neonatal rat ventricular action potential has a shape similar to that of most adult mammals. However, shortly after birth, the action potential shortens to a spike-like configuration. The contribution of changes in repolarizing currents to the shortening is unclear. Thus the inwardly rectifying potassium current (IK1) was measured in heart cells from rats of varying ages using patch-clamp techniques. In freshly isolated cells, whole cell IK1 currents increased greatly between ages 3 and 9-13 days and remained constant thereafter. In culture, IK1 disappeared preferentially in older cells, obscuring the developmental increase. Age-dependent differences in single-channel activity were also observed. Adult cells had IK1 channels consisting of two populations (30 and 42 pS), whereas neonatal cells exhibited only the lower conductance channel. The appearance of the 42-pS channel may contribute a part of the developmental increase in IK1. It was concluded that IK1 increases during postnatal development of the rat ventricle and that this increase may contribute to the postnatal shortening of the rat ventricular action potential.
Activated neutrophils inhibit Na(+)-K(+)-ATPase in canine renal basolateral membrane
Authors: Julin C. M. , Zimmerman J. J. , Sundaram V. , Chobanian M. C.
Published: December 2017

To examine the effects of activated neutrophils (PMNs) on Na(+)-K(+)-ATPase, phorbol 12-myristate 13-acetate (PMA)-stimulated PMNs were incubated with canine renal cortical basolateral membrane (BLM), and BLM ouabain-sensitive Na(+)-K(+)-ATPase activity was subsequently quantified. Na(+)-K(+)-ATPase activity decreased to 40.0 +/- 8.7% (SE) of control in the presence of activated PMNs, from 0.89 +/- 0.12 to 0.34 +/- 0.05 mumol Pi.mg protein-1.min-1. This inhibition coincided with a decrease in the apparent Michaelis constant (Km) for ATP from 0.18 +/- 0.02 to 0.05 +/- 0.01 mM. Inclusion of catalase (CAT) and superoxide dismutase (SOD) in the BLM/PMN/PMA incubation mixture resulted in partial preservation of enzyme activity, with an increase to 57.0 +/- 4.6% of control with CAT alone and to 70.0 +/- 5.3% with both CAT and SOD. SOD alone had no protective effect. Neither the myeloperoxidase inhibitor azide nor the hypochlorous acid scavenger L-methionine preserved enzyme activity. Hydroxyl radical scavengers and iron chelators were also ineffective in attenuating Na(+)-K(+)-ATPase inhibition by activated PMNs. These results indicate that activated PMNs mediate a decrease in BLM Na(+)-K(+)-ATPase activity characterized by a reduction in maximum velocity and Km for ATP that appears to be mediated in part by reactive oxygen metabolites.
Erythrocytes from magnesium-deficient hamsters display an enhanced susceptibility to oxidative stress
Authors: Freedman A. M. , Mak I. T. , Stafford R. E. , Dickens B. F. , Cassidy M. M. , Muesing R. A. , Weglicki W. B.
Published: December 2017

Previous studies in our laboratory have indicated a role for free radical participation in magnesium deficiency cardiomyopathy. We have demonstrated the ability of various antioxidant drugs and nutrients to protect against magnesium deficiency-induced myocardial injury. In this study, we have examined erythrocytes from normal and magnesium-deficient animals and compared their susceptibility to an in vitro oxidative stress. Syrian male hamsters were placed on either magnesium-deficient or magnesium-supplemented diets. Animals from each group also received vitamin E in doses of 10 and 25 mg as subcutaneous implants. Erythrocytes obtained after 14 days on the diet were exposed to an exogenous hydroxyl (.OH) radical generating system (dihydroxyfumarate not equal to Fe3+ ADP) at 37 degrees C for 20 min. Erythrocyte crenation was observed and quantified by scanning electron microscopy. Lipid peroxidation, hemolysis (%), and intracellular glutathione levels were determined. In addition, serum lipid changes and membrane phospholipids were characterized. Our data demonstrate that erythrocytes from magnesium-deficient animals are more susceptible to free radical injury, supporting our hypothesis that magnesium deficiency reduces the threshold antioxidant capacity.
Alpha-subunits of Gs and Gi in adipocyte plasma membranes of genetically diabetic (db/db) mice
Authors: Begin-Heick N.
Published: December 2017

The adipocyte membrane G protein pattern, beta-adrenergic receptor activity, and adenylyl cyclase were determined in adipocyte membranes of the db/db mouse, a mutant that is a model of diabetes preceded by hyperinsulinemia, hyperglycemia, and extreme obesity. These studies were undertaken to determine whether the alterations already noted in the ob/ob mouse and those in the db/db mouse are similar and related to the hormonal defects, particularly the hyperinsulinemia and hyperglycemia prevalent in these animals (cf. Ref. 11). The ADP ribosylation data show that Gs alpha was more highly labeled in the tissues of the db/db mutant than in the homozygous control, but there was no significant difference in the amount of ADP-ribose incorporated in the Gi alpha-subunits. Quantification of the proteins by immunodetection revealed that the long (46-kDa) form of Gs alpha was significantly less abundant in the db mutant than in its control, whereas there was no difference in the short (42-kDa) form. Gi alpha-peptides corresponding to Gi alpha 2 and Gi alpha 1 were both less abundant in the db mutant than in the homozygous control. These data contrasted with those obtained for ob mutants and their lean homozygous controls reported previously (4) and confirmed here. It is concluded on the basis of these studies that factors other than the hormonal status are responsible for the G protein patterns in the ob and db mutants. Differences in G protein patterns noted in between the control groups (B/Ks or B/6 homozygotes) correlated strongly with the quantitative differences in adenylyl cyclase response in the two strains.(ABSTRACT TRUNCATED AT 250 WORDS)
Regulated expression of monocyte chemoattractant protein-1 in normal human osteoblastic cells
Authors: Williams S. R. , Jiang Y. , Cochran D. , Dorsam G. , Graves D. T.
Published: December 2017

An influx of monocytes is observed in many different physiological and pathophysiological states, including bone remodeling and injury. These cells appear at early stages of bone formation and repair and persist throughout the later stages. In experiments described here, unstimulated normal human osteoblastic cells did not produce detectable levels of monocyte chemotactic activity. However, interleukin-1 (IL-1)-stimulated normal human osteoblastic cells produced a chemoattractant that is similar to monocyte chemoattractant protein-1 (MCP-1) at the levels of mRNA expression, protein production, and chemotactic activity. Northern blot analysis indicates that IL-1 elicits a dose-dependent increase in MCP-1 mRNA in normal human osteoblastic cells. Two proteins of M(r) 9,000 and M(r) 13,000 were specifically immunoprecipitated with MCP-1 antiserum from IL-1-stimulated normal human osteoblastic cells. Monocyte chemotactic activity from IL-1-treated cells was blocked by MCP-1 antiserum. These studies establish that normal human osteoblastic cells can be induced to produce monocyte chemoattractants and that this is accounted for by the induced expression of MCP-1.
The iodide channel of the thyroid: a plasma membrane vesicle study
Authors: Golstein P. , Abramow M. , Dumont J. E. , Beauwens R.
Published: December 2017

The uptake of radioactive iodide or chloride by plasma membrane vesicles of bovine thyroid was studied by a rapid filtration technique. A Na(+)-I- cotransport was demonstrated. When this Na(+)-I- cotransport is inactive (i.e., at 4 degrees C and in the absence of Na+), an uptake of iodide above chemical equilibrium could be induced, driven by the membrane potential. The latter was set up by allowing potassium to diffuse into the membrane vesicles in the presence of valinomycin and of an inward K+ gradient. This potential difference (positive inside) induced the uptake of iodide (or other anion present). The data support the existence of two anionic channels. The first one, observed at low near-physiological iodide concentration (micromolar range), which exhibits a high permeability and specificity for iodide (hence called the iodide channel), has a Km of 70 microM. The other one appears similar to the epithelial anion channel as described by Landry et al. (J. Gen. Physiol. 90: 779-798, 1987); it is still about fourfold more permeable to iodide than to chloride and presents a Km of 33 mM. Under physiological conditions the latter channel would mediate chloride transport, and the iodide channel, which is proposed to be restricted to the apical plasma membrane domain of the thyrocyte, transports iodide from the cytosol to the colloid space.
Beta 2-adrenoceptor density in fibroblast culture correlates with human NaCl sensitivity
Authors: Kotanko P. , Hoglinger O. , Skrabal F.
Published: December 2017

To study salt sensitivity in humans and its relation to expression of adrenoceptors, 20 male normotensive Caucasians were investigated on a diet of 180 mmol NaCl/day followed by 60 mmol NaCl/day over 2 wk and again by 180 mmol NaCl/day over 2 wk, and blood pressure changes were assessed by long-term oscillatory blood pressure monitoring under basal conditions. Individual cell cultures of skin fibroblasts from skin biopsies were also established, and alpha 2- and beta 2-adrenoceptors were measured. Seven subjects were salt sensitive, and the remainder were salt resistant. Cultured skin fibroblasts in salt-sensitive subjects express less than half the number of beta 2-adrenoceptors compared with salt-resistant subjects (65 +/- 12.7 vs. 173 +/- 14.8 fmol/mg, P less than 0.001), and there is a correlation between the absolute rise of blood pressure on a high-salt diet and the density of beta 2-adrenoceptors (r = -0.67, P less than 0.01). It remains to be established whether a reduced in vitro density of beta 2-adrenoceptors in cultured cells is causally related to salt sensitivity in normotensive humans.
Ca2+ influx via Na(+)-Ca2+ exchange in immortalized aortic myocytes. I. Dependence on [Na+]i and inhibition by external Na+
Authors: Lyu R. M. , Smith L. , Smith J. B.
Published: December 2017

We have found that immortalized aortic myocytes have high Na(+)-Ca2+ exchange activity that is similar in amount to that of low-passage myocyte cultures. Replacing all external Na+ with K+ slightly increased 45Ca2+ uptake in cells with basal Na+ and greatly increased 45Ca2+ uptake in cells with increased intracellular Na+. External Na+ competitively inhibited 45Ca2+ uptake (inhibition constant approximately 10 mM). The dependence of exchange activity on intracellular Na+ was sigmoidal. The 50% maximum concentration (K0.5) for Na+ was 33 +/- 1 mmol/l cell water space. The Hill coefficient was 2.9 +/- 0.2, which is consistent with a stoichiometry of 3 Na+/1 Ca2+. Cytosolic free Na+ was estimated from the ratio of sodium-binding benzofuran isophthalate (SBFI) fluorescence and an in vivo calibration curve. Sulfinpyrazone, an inhibitor of anion transport, markedly increased the SBFI content of the cells without affecting cell Na+. Cytosolic free Na+ was 9 mM under basal conditions. Because free Na+ did not differ significantly from total Na+, little or no Na+ is bound or compartmentalized in these cells. The complete replacement of extracellular Na+ with K+ fails to evoke appreciable Ca2+ influx, at least in part, because of the low cell Na+ concentration relative to the K0.5 of the exchanger and its sigmoid dependence on Na+.
Intracardiac detection of angiotensinogen and renin: a localized renin-angiotensin system in neonatal rat heart
Authors: Dostal D. E. , Rothblum K. N. , Chernin M. I. , Cooper G. R. , Baker K. M.
Published: December 2017

There is increasing evidence that the renin-angiotensin system (RAS) modulates cardiovascular function through both blood-borne and tissue-derived components. The existence of a local RAS has been proposed in the heart based on biochemical and molecular biological studies that identify angiotensinogen and renin. We conducted the present study to determine the chamber localization of angiotensinogen and renin mRNA in neonatal rat heart and whether these components could be identified in cultured cardiomyocytes and fibroblasts obtained from neonatal rat heart. Experiments using polymerase chain reaction (PCR) indicated that whole hearts obtained from neonatal rats contained both angiotensinogen and renin mRNA. With the use of radiolabeled cDNA probes and in situ hybridization, angiotensinogen and renin transcripts were localized both in the atria and ventricles of neonatal rat hearts. Relative signal strengths for angiotensinogen were highest in the left and right ventricles. In contrast, renin signal strength was overall much lower and preferentially localized in the left ventricle. To investigate the cellular source of angiotensinogen and renin, cultured neonatal heart cardiomyocytes and ventricular fibroblasts were screened for angiotensinogen and renin messenger RNA and protein using PCR and indirect immunofluorescent staining, respectively. These experiments demonstrated that both cell types produce transcripts and the respective translation products for angiotensinogen and renin. These data suggest that the site of angiotensin II synthesis can occur at the level of the individual cardiomyocyte and fibroblast, where it may serve to directly and/or indirectly regulate cardiac rate, force, growth, and development in the neonate.
Effect of cytochalasin D on the actin cytoskeleton of the toad bladder epithelial cell
Authors: Franki N. , Ding G. , Gao Y. , Hays R. M.
Published: December 2017

Cytochalasins are widely used to determine the role of actin in cellular processes. Their actions include capping of the barbed end of actin filaments as well as dimer formation, nucleation, and polymerization. We determined the effect of cytochalasin D (CD) on F-actin in the toad urinary bladder, an epithelium in which vasopressin depolymerizes F-actin. At a low concentration (0.25 microM), CD depolymerized F-actin in the unstimulated cell; at higher concentrations, there was a progressive reduction of depolymerization until actual polymerization was seen. Vasopressin plus CD produced no greater depolymerization than vasopressin alone, suggesting that CD and vasopressin act to a large extent on the same pool of F-actin. CD plus vasopressin also enhanced the fusion rate of aggrephores compared with vasopressin alone, indicating that intact actin filaments retard aggrephore fusion. Despite the increase in aggrephore fusion, water flow was not enhanced by CD, confirming previous reports that intact actin filaments are required for water channel emergence or stabilization in the apical membrane. Vasopressin plus 1 microM CD produced a striking increase in microvillar length, direct evidence of the polymerizing action of CD in the cell.
Role for diacylglycerol in mediating the actions of ACh on M-current in gastric smooth muscle cells
Authors: Clapp L. H. , Sims S. M. , Singer J. J. , Walsh J. V.
Published: December 2017

The role of the second messenger diacylglycerol (DAG) in mediating muscarinic suppression of M-current, a type of a voltage-gated K+ current that is suppressed by acetylcholine (ACh), was examined in freshly isolated smooth muscle cells from toad stomach. Currents were recorded using a single electrode voltage clamp employing conventional microelectrodes. Extracellular application of 1,2-dioctanoyl-sn-glycerol (DiC8), a synthetic DAG that is a potent activator of protein kinase C (PKC), reversibly suppressed M-current. Current relaxations, representing the voltage-dependent closure of K+ channels underlying M-current, were also decreased by DiC8, although suppression was not always as complete as it was with ACh. In contrast, another DAG analogue, 1,2-dioctanoyl-3-thioglycerol, which has a structure closely related to DiC8 but does not activate PKC, failed to inhibit M-current. Furthermore, M-current induced by the beta-agonist isoproterenol, by a mechanism apparently mediated by adenosine 3',5'-cyclic monophosphate (S. M. Sims, L. H. Clapp, J. V. Walsh, Jr., and J. J. Singer. Pflugers Arch. 417: 291, 1990), was also suppressed by DiC8. Both ACh and DiC8 were found to suppress endogenous and isoproterenol-induced M-current without altering the time course of M-current deactivation, suggesting that these agents act by decreasing the number of channels available to be opened. These results provide evidence that muscarinic regulation of M-current is mediated by DAG.
Muscarinic receptors in MDCK cells are coupled to multiple messenger systems
Authors: Mohuczy-Dominiak D. , Garg L. C.
Published: December 2017

Our studies on Madin-Darby canine kidney (MDCK) cells have demonstrated that high-affinity specific muscarinic receptors coupled to the phosphoinositide system are present in these cells. To determine whether muscarinic receptors in MDCK cells are linked negatively to the adenylate cyclase system, we measured the effect of muscarinic agonists and antagonists on vasopressin-, isoproterenol-, and forskolin-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) formation. Vasopressin produced a maximum stimulation of cAMP formation of 13 pmol.10(6) cells-1.2 min-1 at 10(-7) M. Isoproterenol and forskolin stimulated cAMP formation production to 21 pmol.10(6) cells-1.2 min-1 and 64 pmol.10(6) cells-1.10 min-1, respectively, at 10(-4) M. The effects of vasopressin, isoproterenol, and forskolin were blocked by arecoline, a cholinergic agonist, in a concentration-dependent manner. The arecoline response was blocked by treatment of the cells with pertussis toxin. The inhibition by arecoline of forskolin-stimulated cAMP formation was reversed by various muscarinic antagonists in the following order of potency: 4-diphenyl-acetoxy-N-methylpiperidine > p-fluorohexahydrosiladifenidol > pirenzepine > methoctramine. This order of potency of muscarinic antagonists is similar to that observed in our radioligand binding studies and is consistent with the M3 subtype of muscarinic receptors. Our results indicate that muscarinic receptors in MDCK cells are coupled negatively to the adenylate cyclase system via pertussis toxin-sensitive G protein. It is concluded that this intracellular system may at least be partially responsible for the action of cholinergic agonists in these cells and in the kidney.
Adrenergically activated Ca2+ increases in brown fat cells: effects of Ca2+, K+, and K channel block
Authors: Lee S. C. , Nuccitelli R. , Pappone P. A.
Published: December 2017

We measured intracellular calcium concentration ([Ca2+]i) during adrenergic stimulation using fura-2 ratio imaging of individual cultured neonatal rat brown fat cells. One micromolar norepinephrine (NE) increased [Ca2+]i from an average resting value of 105 nM to 555 nM in approximately 30 s. [Ca2+]i remained elevated as long as NE was present but returned to resting levels within 2-3 min after NE removal. The response was half maximal at approximately 50 nM NE and was primarily alpha-adrenergic. The sustained, but not the initial, increase in [Ca2+]i required extracellular calcium. Cells stimulated in high-K media had [Ca2+]i responses like those in 0 Ca2+, suggesting that depolarization abrogates calcium influx. Parallel perforated-patch recordings showed that the increase in [Ca2+]i activates a calcium-activated K conductance. Blocking K channels with moderate concentrations of tetraethylammonium (TEA) had only small effects on NE-induced changes in [Ca2+]i, but high concentrations of TEA significantly reduced the response. We conclude that cytoplasmic calcium is modulated by fluxes from both intracellular and extracellular sources and that K channels may not be required for normal short-term [Ca2+]i responses to hormone.
1,25-Dihydroxyvitamin D3 inhibits Na(+)-dependent phosphate transport in osteoblastic cells
Authors: Green J. , Luong K. V. , Kleeman C. R. , Ye L. H. , Chaimovitz C.
Published: December 2017

In the present work we investigated the influence of vitamin D3 metabolites on Na(+)-dependent phosphate (Pi) transport in the clonal osteoblastic cell line UMR-106. The vitamin D3 metabolite 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] dose-dependently inhibited Pi transport with a half-maximal concentration of approximately 5 x 10(-11) M. The effect of 1,25(OH)2D3 was first observed after 8 h of preincubation period. Inhibition of phosphate uptake was relatively specific for the 1,25(OH)2D3 analogue of vitamin D3. The potency order was 1,25(OH)2D3 >> 24,25-dihydroxyvitamin D3 > 25-[3H]hydroxyvitamin D3. Kinetically, 1,25(OH)2D3 decreased the maximal velocity of the phosphate uptake system, whereas the affinity for phosphate was unaffected. Activation of protein kinase C (PKC) in UMR-106 cells stimulated Na(+)-dependent Pi transport. Nonetheless, the inhibitory effect of 1,25(OH)2D3 on Pi transport was not related to downregulation of PKC. Chemical determination of intracellular Pi showed a 50% reduction after 24-h preincubation with 10(-8) M 1,25(OH)2D3. We conclude that 1,25(OH)2D3 inhibits Na(+)-dependent phosphate transport in osteoblastic cells. This in turn leads to intracellular Pi depletion. The physiological implication of this phenomenon on the effects of vitamin D on osteoblasts in situ is discussed.
Role of polyamines in the regulation of proliferation and hormone production by insulin-secreting cells
Authors: Sjoholm A.
Published: December 2017

This paper focuses on the mechanisms regulating proliferation and insulin production by normal and tumoral pancreatic beta-cells. In particular, the evidence for involvement of polyamines is reviewed. Pancreatic islet cells contain high levels of polyamines, and based on findings obtained using enzyme-directed inhibitors, it appears that putrescine and spermidine are necessary for proinsulin biosynthesis, whereas spermine may exert a stimulatory or permissive role in RNA transcription-stabilization and long-term insulin release. Islet polyamine content is not altered by short-term secretory stimulation, nor is the acute secretory response impeded by polyamine synthesis inhibitors, making it unlikely that these amines play any major role in short-term insulin release. Various mitogens increase islet polyamine contents and DNA synthesis, but increases in cytosolic polyamines do not seem to mediate their mitogenicity. Nuclear polyamine content is not altered by the inhibitors, suggesting that maintenance of polyamines within this organelle may be sufficient to sustain elevated DNA synthesis. In tumoral RINm5F cells, polyamine depletion results in decreased proliferation and increased cellular content of insulin and insulin secretory granules without affecting insulin mRNA levels or translation. Moreover, polyamine-depleted RINm5F cells display improved substrate metabolism and sensitivity of the stimulus-secretion coupling. Possible levels of polyamine interaction with Ca2+ metabolism are discussed.
Low-affinity mixed acetylcholine-responsive receptors at the apical membrane of frog tadpole skin
Authors: Cox T. C.
Published: December 2017

The larval frog skin has a very high electrical resistance and a corresponding low rate of transepithelial ion transport. Amiloride, a blocker of sodium transport in adult skin, transiently stimulates rather than inhibits short-circuit current (Isc) across larval skin through nonselective cation channels. Acetylcholine (ACh) stimulates Isc like amiloride, although the response is more prolonged. Pretreatment with ACh markedly suppressed amiloride stimulation of Isc; amiloride pretreatment also suppressed ACh stimulation. Half-maximal stimulation of Isc by ACh occurred at 347 microM. Stimulation by ACh was inhibited by both d-tubocurarine [dissociation constant (Kd) = 57 microM] and atropine (Kd = 49 microM). The specific nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium and the specific muscarinic agonist oxotremorine-M both stimulated Isc and were blocked by either atropine or d-tubocurarine. Reciprocal desensitization and blocker cross-reactivity suggest that ACh activates the same population of receptors as amiloride. This ACh-responsive receptor has characteristics of both nicotinic and muscarinic receptors found in other tissues.
Inhibition of Ca2+ transport pathways in thymic lymphocytes by econazole, miconazole, and SKF 96365
Authors: Mason M. J. , Mayer B. , Hymel L. J.
Published: December 2017

Cytochrome P-450 has been proposed to underlie the mechanism of regulation of the plasma membrane Ca2+ permeability by the Ca2+ content of the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool. We have investigated the effects on divalent cation uptake in rat thymic lymphocytes of three structurally related imidazole reagents reported to inhibit redox mechanisms. Changes in intracellular Ca2+ concentration and intracellular Mn2+ concentration were measured fluorimetrically with indo-1 and/or quin-2. Econazole, miconazole, and SKF 96365 were found to be potent blockers of Ca2+ and Mn2+ uptake activated by release of Ca2+ from intracellular stores induced by thapsigargin. Additionally, we found that concentrations of these agents required to abolish divalent cation uptake also released Ca2+ from the thapsigargin-sensitive intracellular stores, consistent with inhibition of the endosomal Ca(2+)-ATPase. In agreement with this suggestion, we have found that all three of these agents are potent inhibitors of isolated sarcoplasmic reticulum Ca(2+)-ATPase. We conclude that econazole, miconazole, and SKF 96365 inhibit cytochrome P-450-independent filling of intracellular Ca2+ pools, as well as store-regulated Ca2+ entry, and caution against the use of these compounds as selective inhibitors of cytochrome P-450.