Showing papers in "American Journal of Physiology-cell Physiology in 1996"

Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly.

Abstract: Nitric oxide contrasts with most intercellular messengers because it diffuses rapidly and isotropically through most tissues with little reaction but cannot be transported through the vasculature due to rapid destruction by oxyhemoglobin. The rapid diffusion of nitric oxide between cells allows it to locally integrate the responses of blood vessels to turbulence, modulate synaptic plasticity in neurons, and control the oscillatory behavior of neuronal networks. Nitric oxide is not necessarily short lived and is intrinsically no more reactive than oxygen. The reactivity of nitric oxide per se has been greatly overestimated in vitro because no drain is provided to remove nitric oxide. Nitric oxide persists in solution for several minutes in micromolar concentrations before it reacts with oxygen to form much stronger oxidants like nitrogen dioxide. Nitric oxide is removed within seconds in vivo by diffusion over 100 microns through tissues to enter red blood cells and react with oxyhemoglobin. The direct toxicity of nitric oxide is modest but is greatly enhanced by reacting with superoxide to form peroxynitrite (ONOO-). Nitric oxide is the only biological molecule produced in high enough concentrations to out-compete superoxide dismutase for superoxide. Peroxynitrite reacts relatively slowly with most biological molecules, making peroxynitrite a selective oxidant. Peroxynitrite modifies tyrosine in proteins to create nitrotyrosines, leaving a footprint detectable in vivo. Nitration of structural proteins, including neurofilaments and actin, can disrupt filament assembly with major pathological consequences. Antibodies to nitrotyrosine have revealed nitration in human atherosclerosis, myocardial ischemia, septic and distressed lung, inflammatory bowel disease, and amyotrophic lateral sclerosis.

Hypoxia-inducible factor 1 levels vary exponentially over a physiologically relevant range of O2 tension.

Abstract: Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix protein implicated in the transcriptional activation of genes encoding erythropoietin, glycolytic enzymes, and vascular endothelial growth factor in hypoxic mammalian cells. In this study, we have quantitated HIF-1 DNA-binding activity and protein levels of the HIF-1 alpha and HIF-1 beta subunits in human HeLa cells exposed to O2 concentrations ranging from 0 to 20% in the absence or presence of 1 mM KCN to inhibit oxidative phosphorylation and cellular O2 consumption. HIF-1 DNA-binding activity, HIF-1 alpha protein and HIF-1 beta protein each increased exponentially as cells were subjected to decreasing O2 concentrations, with a half maximal response between 1.5 and 2% O2 and a maximal response at 0.5% O2, both in the presence and absence of KCN. The HIF-1 response was greatest over O2 concentrations associated with ischemic/hypoxic events in vivo. These results provide evidence for the involvement of HIF-1 in O2 homeostasis and represent a functional characterization of the putative O2 sensor that initiates hypoxia signal transduction leading to HIF-1 expression.

Cellular and molecular physiology of volume-sensitive anion channels

Abstract: Maintenance of a constant cell volume in the face of osmotic stress is an evolutionarily ancient homeostatic process. Over the last two decades physiologists have gained an impressive understanding of the "volume-sensitive" channels, cotransporters, exchangers, metabolic pathways, and genes that are responsible for modulating intracellular solute content and cell volume. This review focuses on one part of this story, the characteristics and osmoregulatory functions of volume-sensitive anion channels. Three distinct types of swelling-activated anion channels have been observed and studied extensively in animal cells. These channels include 1) ClC-2, which is a member of the ClC family of voltage-gated anion channels, 2) an outwardly rectifying intermediate conductance channel, and 3) a large-conductance or "maxi" channel. In addition to these three channels, several other less well-characterized anion channels have been observed. This review discusses the electrophysiological and molecular biological characteristics and regulation of these channels. The possible roles different types of anion channels might play in cell volume homeostasis are also discussed.

Calcium sparks and [Ca2+]i waves in cardiac myocytes

Abstract: Local elevations in intracellular calcium ("Ca2+ sparks") in heart muscle are elementary sarcoplasmic reticulum (SR) Ca(2+)-release events. Ca2+ sparks occur at a low rate in quiescent cells but can also be evoked by electrical stimulation of the cell to produce the cell-wide Ca2+ transient. In this study we investigate how Ca2+ sparks are related to propagating waves of elevated cytosolic Ca2+ induced by "Ca2+ overload." Single ventricular myocytes from rat were loaded with the Ca(2+)-sensitive indicator fluo 3 and imaged with a confocal microscope. After extracellular Ca2+ concentration was increased from 1 to 10 mM to produce Ca2+ overload, the frequency of spontaneous Ca2+ sparks, which occur at the t tubule/SR junction, increased approximately 4-fold, whereas the spark amplitude and spatial size increased 4.1-and 1.7-fold, respectively. In addition, a spectrum of larger subcellular events, including propagating Ca2+ waves, was observed. Ca2+ sparks were seen to occur at the majority (65%) of the sites of wave initiation. For slowly propagating Ca2+ waves, discrete Ca(2+)-release events, similar to Ca2+ sparks, were detected in the wave front. These Ca2+ sparks appeared to recruit other sparks along the wave front so that the wave progressed in a saltatory manner. We conclude that Ca2+ sparks are elementary events that can explain both the initiation and propagation of Ca2+ waves. In addition, we show that Ca2+ waves and electrically evoked Ca2+ transients have the same time course and interact with each other in a manner that is consistent with both phenomena having the same underlying mechanism(s). These results suggest that SR Ca2+ release during Ca2+ waves, like that during normal excitation-contraction coupling, results from the spatial and temporal summation of Ca2+ sparks.

Characteristics and physiological role of the Ca(2+)-activated Cl- conductance in smooth muscle

Abstract: In this review we discuss the properties of the Ca(2+)-activated Cl- current [ICl(Ca)] recorded in isolated smooth muscle cells with electrophysiological techniques and speculate on the possible physiological role(s) of ICl(Ca) in smooth muscle function. In particular, we concentrate on 1) the Ca2+ dependence of ICl(Ca), 2) the mechanisms that link pharmacological receptor stimulation on the cell surface membrane to activation of ICl(Ca), 3) the biophysical properties of ICl(Ca), and 4) the pharmacology of ICl(Ca). It is evident that a diverse array of pharmacological agonists can evoke ICl(Ca) in many types of smooth muscle, and it seems that the well-established G protein-phosphoinositide metabolism (inositol 1,4,5-trisphosphate)-intracellular Ca2+ store pathway couples the receptor to the membrane channels. Also, the results indicate that the biophysical and pharmacological properties of ICl(Ca) are not only similar in the various smooth muscle types studied so far but, possibly, are also similar to ICl(Ca) in non-smooth muscle tissue. Evidence is presented that the Ca(2+)-activated Cl- channel exists in two states, open and closed, with a relatively long mean open time and that some of the agents that inhibit ICl(Ca) interact directly with the open channel. It is suggested that the most likely role of ICl(Ca) in smooth muscle is to produce membrane depolarization and contraction to neurotransmitters and local mediators.

Water transport across mammalian cell membranes.

Abstract: This review summarizes recent progress in water-transporting mechanisms across cell membranes. Modern biophysical concepts of water transport and new measurement strategies are evaluated. A family of water-transporting proteins (water channels, aquaporins) has been identified, consisting of small hydrophobic proteins expressed widely in epithelial and nonepithelial tissues. The functional properties, genetics, and cellular distributions of these proteins are summarized. The majority of molecular-level information about water-transporting mechanisms comes from studies on CHIP28, a 28-kDa glycoprotein that forms tetramers in membranes; each monomer contains six putative helical domains surrounding a central aqueous pathway and functions independently as a water-selective channel. Only mutations in the vasopressin-sensitive water channel have been shown to cause human disease (non-X-linked congenital nephrogenic diabetes insipidus); the physiological significance of other water channels remains unproven. One mercurial-insensitive water channel has been identified, which has the unique feature of multiple overlapping transcriptional units. Systems for expression of water channel proteins are described, including Xenopus oocytes, mammalian and insect cells, and bacteria. Further work should be directed at elucidation of the role of water channels in normal physiology and disease, molecular analysis of regulatory mechanisms, and water channel structure determination at atomic resolution.

Contribution of mitochondrial proton leak to skeletal muscle respiration and to standard metabolic rate

Abstract: We have tested the hypothesis that the leak of protons across the mitochondrial inner membrane (proton leak) is a significant contributor to standard metabolic rate (SMR). We found that proton leak accounts for around one-half of the resting respiration rate of perfused rat skeletal muscle. Proton leak is known to make a significant (26%) contribution to the resting respiration rate of isolated rat hepatocytes (M. D. Brand, L.-F. Chien, E. K. Ainscow, D. F. S. Rolfe, and R. K. Porter. Biochim. Biophys. Acta 1187: 132-139, 1994). If the importance of proton leak in these isolated and perfused systems is similar to its importance in vivo, then using literature values for the contribution of liver and skeletal muscle to SMR, we can calculate that proton leak in liver and skeletal muscle alone accounts for 11-26% (mean 20%) of the SMR of the rat. If proton leak activity in the other tissues of the rat is similar to that in liver cells, then the contribution of proton leak to rat SMR would be 16-31% (mean 25%).

Aldosterone-induced increase in the abundance of Na+ channel subunits.

Abstract: The highly selective, amilorideblockable Na+ channel is a major target to the natriferic action of the mineralocorticoid aldosterone. This rat epithelial Na+ channel (rENaC) has been recently cloned from colon and is composed of three homologous subunits denoted alpha-, beta-, and gamma-rENaC (C. M. Canessa, L. Schild, G. Buell, B. Thorens, L. Gautschi, J.-D. Horisberger, and B. C. Rossier. Nature Lond. 367: 463-467, 1994). We have tested the effects of corticosteroids on the abundance of mRNA coding for each subunit in kidney cortex and distal colon. Chronic treatment of rats with aldosterone or dexamethasone evoked in kidney cortex a small induction of alpha-rENaC and no change in beta- and gamma-rENaC. In distal colon, however, beta- and gamma-rENaC were strongly induced by either aldosterone or dexamethasone, whereas alpha-rENaC was constitutively expressed. Most of the aldosterone-induced increase in beta- and gamma-rENaC mRNA took place during 3-24 h after plasma aldosterone was elevated. A similar differential induction of rENaC subunits in kidney and colon was also evoked by a Na(+)-free diet. The effects of salt deprivation were reversed by resalinating rats with a half time of < 2 h, suggesting a high turnover rate of at least beta- and gamma-rENaC. The data are consistent with the possibility that induction of channel subunits contributes to the chronic but not the acute response to aldosterone in the colon. Such a mechanism is not likely to play a major role in cortical collecting ducts.

Adult human chondrocytes cultured in alginate form a matrix similar to native human articular cartilage.

Abstract: The matrix formed by adult human chondrocytes in alginate beads is composed of two compartments: a thin rim of cell-associated matrix that corresponds to the pericellular and territorial matrix of articular cartilage and a more abundant further-removed matrix, the equivalent of the interterritorial matrix in the tissue. On day 30 of culture, the relative and absolute volumes occupied by the cells and each of the two matrix compartments in the beads were nearly identical to those in native articular cartilage. Furthermore, the concentration of aggrecan in the cell-associated matrix was similar to that in adult human articular cartilage and was approximately 40-fold higher than in the further removed matrix compartment. Fluorescence-activated cell sorting revealed that the cell-associated matrix was built on the cell membrane in part via interactions between hyaluronic acid and CD44-like receptors. Approximately 25% of the aggrecan molecules synthesized by the chondrocytes during a 4-h pulse in the presence of [35S]sulfate on day 9 of culture were retained in the cell-associated matrix where they turned over with a half-life (t1/2) = 29 days. Most [35S]aggrecan molecules reached the further removed matrix compartment where they turned over much more slowly (t1/2 > 100 days). These results add support to the contention that aggrecan molecules residing in the pericellular and territorial areas of the adult human articular cartilage matrix are more susceptible to degradation by proteolytic enzymes synthesized by the chondrocytes than those that inhabit the interterritorial areas further removed from the cells.

Absence of volume regulatory mechanisms contributes to the rapid activation of apoptosis in thymocytes

Abstract: A common event that occurs during apoptosis is a loss of cell volume, but little information is available on its role in the cell death process. Lymphocytes undergo apoptosis in response to glucocorticoids and exhibit cell shrinkage, nuclear condensation, internucleosomal DNA fragmentation, and apoptotic body formation. Interestingly, only cells that exhibit a loss in cell volume degrade their DNA. To determine if physical shrinkage was sufficient to initiate apoptosis, S49 Neo lymphocytes were cultured in hypertonic medium. The normal osmolarity (approximately 300 mosM) of tissue culture medium was increased to either 550 or 800 mosM, using impermeant sugars such as mannitol and sucrose or NaCl. These hypertonic conditions led to a rapid killing of S49 Neo cells. Evaluation of the mode of cell death revealed that these hypertonic conditions resulted in apoptosis. Unlike glucocorticoid-induced cell death, hypertonically induced apoptosis did not require protein synthesis. When S49 Neo cells were cultured under hypotonic conditions, the cells swelled but apoptosis did not occur. Analysis of several cell types revealed that all lymphoid cells examined (S49 Neo, CEM-C7, primary thymocytes) undergo apoptosis in response to hypertonic conditions, whereas several other cell types (L cells, COS, HeLa, GH3) did not. Although these nonlymphoid cells showed a similar initial reduction in cell volume in response to hypertonic conditions, they subsequently maintained volume or regulated back to a near normal cell volume. These data indicate that thymic lymphoid cells have the machinery in place for rapid induction of apoptosis in response to physical shrinkage, whereas other cell types resist shrinkage-induced apoptosis by the activation of cell volume regulatory mechanisms.

Cytochrome P-450 metabolites mediate extracellular Ca(2+)-induced inhibition of apical K+ channels in the TAL

Abstract: We used the patch-clamp technique to study the effect of extracellular Ca2+ (Ca2+o) on the activity of the apical 70-pS K+ channel in the isolated split-open thick ascending limb (TAL) of the rat kidney. Raising Ca2+o from 1.1 to 5 mM reversibly reduced the activity of the 70-pS K+ channel in cell-attached patches to 16 +/- 2% of the control value within 300 s. In addition, 50 microM neomycin mimicked the effect of an increase in Ca2+o on channel activity in cell-attached patches and completely inhibited channel activity. The effect of neomycin on the channel activity in cell-attached patches is an indirect effect, since addition of 50 microM neomycin on the 70-pS K+ channel in inside-out patches reduced only the apparent amplitude of the channel current without changing channel open probability. We examined further the role of protein kinase C (PKC) and the cytochrome P-450-dependent metabolites of arachidonic acid in mediating the Ca2+o -induced inhibition of channel activity. Addition of phorbol 12-myristate 13-acetate (2 microM) reversibly blocked channel activity in cell-attached patches to 4 +/- 1% of the control value, whereas 75 nM calphostin C increased the channel activity by 115 +/- 10%. Moreover, addition of 1 nM exogenous PKC reversibly and completely inhibited the 70-pS K+ channel. However, inhibition of PKC with calphostin C (75 nM) only slightly prolonged the time course of the effect of Ca2+o on channel activity (370 +/- 40 s) and failed to abolish the inhibitory effect of 5 mM Ca2+o on channel activity in cell-attached patches, indicating that PKC was not mainly responsible for the effect of Ca2+o on channel activity. In contrast, the effect of 5 mM Ca2+o on the apical 70-pS K+ channel was completely abolished when TAL tubules were first incubated in the 17-octadecynoic acid (5 microM)-containing solution, an agent that specifically blocks cytochrome P-450 monooxygenase. In conclusion, these data indicate that Ca2+o is an important regulator of the apical 70-pS K+ channel and that a cytochrome P-450-dependent metabolite of arachidonic acid is involved in mediating this inhibitory effect.

Mechanisms of inhibition of insulin release

Abstract: Several agonists including norepinephrine, somatostatin, galanin, and prostaglandins inhibit insulin release. The inhibition is sensitive to pertussis toxin, indicating the involvement of heterotrimeric Gi and/or Go proteins. Receptors for the different agonists have different selectivity for these G proteins. After G protein activation, the alpha- and beta gamma-subunits dissociate and interact with multiple targets to inhibit release. These include 1) the ATP-sensitive K+ channel and perhaps other K+ channels, 2) L-type voltage-dependent Ca2+ channels, 3) adenylyl cyclase, and 4) a "distal" site late in stimulus-secretion coupling. The latter effect, which may be exerted close to the final stage of exocytosis, is the most powerful of the individual inhibitory mechanisms. G protein action on the target molecules is determined by the individual G proteins activated and their specificity for the targets. The L-type Ca2+ channel is inhibited by G(o)-1. Adenylyl cyclase is inhibited by Gi-2 and Gi-3. The distal inhibition can be exerted by Gi-1, Gi-2, Gi-3, and G(o)-2. Thus there is both selectivity and promiscuity in G protein action in the beta-cell. These characteristics allow an inhibitory ligand to be effective at multiple targets and to act differentially from other inhibitory ligands.

Relationship between replication and differentiation in cultured human adipocyte precursor cells

Abstract: The aim of this study was to investigate the role of cell replication for the differentiation of human adipocyte precursor cells in primary culture. When cells were seeded in a medium supplemented with 10% fetal bovine serum, they started to proliferate within 48 h after exposure, as assessed by cell counting and [3H]thymidine autoradiography. When cells were inoculated in the absence of serum, a significant degree of cell proliferation was not detectable. Histochemical investigations using bromodeoxyuridine incorporation demonstrated that cells replicating their DNA did not accumulate lipid droplets. Inoculating adipocyte precursor cells under completely serum-free conditions resulted in a 30-50% higher expression of lipogenic enzymes such as glycerol-3-phosphate dehydrogenase and lipoprotein lipase than keeping cells in serum-supplemented medium for the initial 16 h. Addition of cytosine arabinoside at concentrations that effectively block mitosis did not interfere with adipocyte development. In conclusion, adipocyte precursor cells from human adipose tissue do not require cell division to enter the differentiation process in vitro. These cells may have already undergone possibly critical cell divisions in vivo and may be in a late stage of adipocyte development.

Regulatory phosphorylation of the secretory Na-K-Cl cotransporter: modulation by cytoplasmic Cl

Abstract: The effect of cytoplasmic Cl concentration ([Cl]i) on the activation state ([3H]benzmetanide binding rate) and phosphorylation state (32P incorporation) of the Na-K-Cl cotransporter was evaluated i...

An equibiaxial strain system for cultured cells.

Abstract: We developed a device that applies homogeneous equibiaxial strains of 0-10% to a cell culture substrate and quantitatively verified transmission of substrate deformation to cultured cardiac cells. Clamped elastic membranes in both single-well and multiwell versions of the device are uniformly stretched by indentation with a plastic ring, resulting in strain that is directly proportional to the pitch-to-radius ratio. Two-dimensional deformations were measured by tracking fluorescent microspheres attached to the substrate and to cultured adult rat cardiac fibroblasts. For nominal stretches up to 18%, strains along circumferential and radial axes were equal in magnitude and homogeneously distributed with negligible shear. For 5% stretch, circumferential and radial strains in the substrate were 0.046 +/- 0.005 and 0.048 +/- 0.004 [not significant (NS)], respectively, and shear strain was 0.001 +/- 0.003 (NS). Calibration of both single-well and multiwell versions permits strain selection by device rotation. The reproducible application and quantification of homogeneous equibiaxial strain in cultured cells provides a quantitative approach for correlating mechanical stimuli to cellular transduction mechanisms.

Regulation of ion channels in smooth muscles by calcium.

Abstract: Intracellular Ca2+ concentration ([Ca2+]i) plays a central role in regulating tone and contractility of smooth muscle cells. In contrast to the “classic” model of electromechanical coupling where m...

Parvalbumin relaxes frog skeletal muscle when sarcoplasmic reticulum Ca(2+)-ATPase is inhibited.

Abstract: Inhibition of sarcoplasmic reticulum (SR) Ca(2+)-adenosinetriphosphatase (ATPase) with 2,5-di-(tert-butyl)-1,4-benzohydroquinone (TBQ) in frog skeletal muscle fibers at 10 degrees C prolonged the half time of the fall of the Ca2+ transient by 62% and twitch force by 100% and increased peak force by 120% without increasing the amplitude of the Ca2+ signal. In the presence of TBQ the rate of relaxation and the rate of fall of Ca2+ became progressively slower in a series of twitches until relaxation failed. Relaxation rate decreased with a time course (approximately 2 s-1) similar to the Mg2+ off rate from purified parvalbumin (PA; 3.6 s-1). TBQ slowed the rate of fall of Ca2+ (5-fold) and force (8-fold) in a 0.3-s tetanus so that the rate of fall of Ca2+ (approximately 2.5 s-1) was similar to the Mg2+ off rate from PA. TBQ caused a near total failure of both Ca2+ sequestration and relaxation in a 1.1-s tetanus, during which PA would be saturated with Ca2+ and could not contribute to relaxation. Thus, when the SR Ca(2+)-ATPase is inhibited, Mg(2+)-PA can sequester Ca2+ and produce relaxation at a rate that is defined by the Mg2+ off rate from PA.

Effects of acidosis on phosphorylation of phospholamban and troponin I in rat cardiac muscle

Abstract: Acidosis inhibits Ca2+ transport by the sarcoplasmic reticulum of cardiac muscle and decreases the Ca2+ sensitivity of the contractile proteins, although the mechanisms underlying these changes are...

Characteristics of mitochondria isolated from type I and type IIb skeletal muscle

Abstract: Mitochondria isolated from rabbit soleus (98% type I) and gracilis (99% type IIb) skeletal muscle were compared for compositional differences. Whole muscle mitochondrial contents were 14.5 +/- 1.2 ...

Contraction-induced injury to single fiber segments from fast and slow muscles of rats by single stretches

Abstract: Susceptibility to contraction-induced injury was investigated in single permeabilized muscle fiber segments from fast extensor digitorum longus and slow soleus muscles of rats. We tested the hypotheses that, after single stretches of varying strains and under three conditions of Ca2+ activation (none, submaximum, and maximum), 1) the magnitude of the deficit in maximum isometric force is dependent on the work done to stretch the fiber, and 2) for each condition of activation and strain, fast fibers incur greater force deficits than slow fibers. When all data on force deficits were analyzed together, the best predictors of the overall force deficits for both fast and slow muscle fibers were linear regression models that introduced the simultaneous but independent effects of strain and average force (r2 = 0.52 and 0.63, respectively). Under comparable conditions, greater force deficits were produced in fast than slows fibers. Despite differences in the strain required to produce injury in fast and slow muscle fibers, for a given force deficit, the ultrastructural damage was strikingly similar.

cAMP motifs regulating transcription in the aquaporin 2 gene.

Abstract: Genomic clones including the 5' flanking regions of the AQP2 (aquaporin 2) gene were isolated, and the promoter region was examined by transiently transfecting a promoter-luciferase reporter fusion...

A role for CFTR in human autosomal dominant polycystic kidney disease.

Abstract: Human autosomal dominant polycystic kidney disease (ADPKD) is the most common lethal dominant hereditary disorder characterized by enormous renal enlargement and the development of multiple cysts originating from nephrons. We investigated the pathogenesis of cyst formation in ADPKD by using patch-clamp and immunocytochemical techniques. Adenosine 3',5'-cyclic monophosphate-activated Cl- currents are present in primary cultures of ADPKD cells and have characteristics such as a linear current-voltage relation, insensitivity to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, sensitivity to glibenclamide and diphenylamine carboxylic acid, and an anion selectivity sequence of Br- > Cl- > I- > glutamate, all of which are identical to cystic fibrosis transmembrane conductance regulator (CFTR). With the use of CFTR antibodies raised against the regulatory and first nucleotide-binding domains, CFTR was detected in primary cultures of ADPKD cells. Similar results were obtained in vivo in cyst-lining epithelial cells in ADPKD kidneys, where staining was seen associated with the apical membrane regions. These data indicate that the CFTR Cl- channel exists in apical membranes of ADPKD cells and may play an important role in cyst formation or enlargement.

Effect of ablation of phospholamban on dynamics of cardiac myocyte contraction and intracellular Ca2

Abstract: We compared mechanical activity and Ca2+ transients of ventricular myocytes isolated from wild-type and phospholamban (PLB)-deficient mouse hearts in control conditions and during beta-adrenergic stimulation. Compared with wild-type controls, cells isolated from PLB-deficient mouse hearts showed 1) a 2-fold increase in extent of cell shortening, 2) a 3-fold increase in maximal shortening velocity, and 3) a 3.4-fold increase in maximal relengthening velocity. PLB-deficient myocytes also demonstrated significant increases in the peak amplitude of the fura 2 fluorescence ratio and the rates of rising and falling phases of the Ca2+ transient. The fura 2 diastolic ratios were similar in both groups, suggesting no change in intracellular Ca2+ during diastole. In PLB-deficient myocytes, 0.05 microM isoproterenol induced an increase in the twitch amplitude by 152 +/- 11% (n = 6) compared with 290 +/- 31% (n = 6) in wild-type cells. Maximal shortening velocity was increased by 183 +/- 10% (n = 6) in PLB-deficient myocytes, compared with 398 +/- 62% (n = 6) in wild-type cells. The isoproterenol-induced increase in maximum relengthening velocity was increased by 168 +/- 8% (n = 6) in PLB-deficient cells compared with 445 +/- 71% (n = 6) in wild-type myocytes. In both groups, these changes in contractile parameters were accompanied by changes in the Ca2+ transient. Our results indicate that phosphorylation of sites other than PLB may play an important role in regulation of contraction-relaxation dynamics of heart cells responding to beta-adrenergic stimulation.

Transimmortalized mouse intestinal cells (m-ICc12) that maintain a crypt phenotype

Abstract: This study describes the properties of a clone of immortalized cells (m-ICc12 cells) derived from the bases of small intestinal villi from 20-day-old fetuses of L-type pyruvate kinase (L-PK)/ TAg1 transgenic mice. The mice harbor the simian virus 40 large T antigen under the control of the 5' regulatory sequence from the L-PK gene. m-ICc12 cells expressed nuclear large T antigen, had a prolonged life span, and were nontumorigenic when injected into nude mice. They formed confluent monolayers of cuboid cells separated by tight junctions, developed dense, short apical microvilli, and formed domes. They also possessed cytokeratins, villin, aminopeptidase N, dipeptidyl-peptidase IV, and glucoamylase and retained crypt cell features, including intracellular sucrase isomaltase and alpha-L-fucose glycoconjugates accumulation and expression of the polymeric immunoglobulin receptor and the cystic fibrosis transmembrane conductance regulator gene. Thus the m-ICc12 cell line obtained by targeted oncogenesis in transgenic mice maintained in culture several important properties and differentiated functions of intestinal crypt cells.

Tyrosine phosphorylation of pp125FAK and paxillin in aortic endothelial cells induced by mechanical strain

Abstract: The objective of this study was to determine whether focal adhesion proteins pp125FAK (focal adhesion kinase) and paxillin are phosphorylated on tyrosine and might play a role in the morphological change and cell migration induced by strain. Bovine aortic endothelial cells (EC) were subjected to 10% average strain at 60 cycles/min. Cyclic strain increased the tyrosine phosphorylation of pp125FAK at 30 min (3.4-fold) and 4 h (5.9-fold) and the tyrosine phosphorylation of paxillin at 4 h (2.0-fold). Confocal microscopy showed that, after 4-h exposure to strain, EC began to elongate and F-actin, pp125FAK, and paxillin aligned, although they randomly distributed in static condition. Tyrosine kinase inhibitor tyrphostin A25 (100 microM) inhibited not only the tyrosine phosphorylation of pp125FAK and paxillin but also the redistribution of pp125FAK and paxillin, morphological change, and migration of EC induced by strain. These data demonstrate that cyclic strain induced tyrosine phosphorylation and reorganization of pp125FAK and paxillin and suggest that these focal adhesion proteins play a specific role in cyclic strain-induced morphological change and migration.

Alternate stimulation of apical CFTR by genistein in epithelia.

Abstract: The cystic fibrosis transmembrane regulator (CFTR) is a Cl- channel regulated by adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A. A cAMP-independent activation has been recently shown for the protein tyrosine kinase inhibitor genistein in CFTR-transfected NIH/3T3 fibroblasts. We further studied the role of genistein on Cl- secretion in HT-29/B6 and T84 colonic epithelial cells, which express native CFTR in their apical membranes. Transepithelial Cl- secretion was more effectively stimulated in T84 cells when compared with HT-29/B6 cells by mucosal perfusion with 50 microM genistein. Genistein, like the cAMP agonist forskolin, stimulated CFTR activity in cell-attached patches of single cells with similar slope conductances of 8.5 +/- 0.5 and 9.2 +/- 0.3 pS, respectively. Monolayers in Ussing chambers were basolaterally permeabilized with the pore former alpha-toxin, and gradient-driven Cl- current across the apical membrane (ICl) was measured. ICl was stimulated by serosal (i.e., cytosolic) cAMP (half-maximal stimulatory concentration = 9.8 +/- 1.9 microM). In the presence of cAMP (> 5 microM), subsequent mucosal, but not serosal, addition of genistein further increased Icl by approximately 16%; in the absence of cytosolic cAMP, genistein had no effect on ICl. The inactive analogue daidzein had no effect. When cAMP agonists were removed in the continued presence of genistein, ICl remained elevated in both permeabilized and intact monolayers as well as in cell-attached patches of single cells. In addition, genistein blocked K- currents across the basolateral membrane in apically amphotericin B-permeabilized monolayers (half maximal inhibitory concentration = 44.2 +/- 8.1 microM). Therefore, in intact epithelia, the overall secretory response to genistein is composed of stimulatory effects on the apical CFTR and inhibitory effects on the basolateral K+ conductance. We propose that genistein blocks a phosphatase, which regulates CFTR during cAMP-dependent stimulation.

Pharmacological activation changes stiffness of cultured human airway smooth muscle cells

Abstract: Using magnetic twisting cytometry (MTC), we measured the cytoskeletal stiffness of adherent human airway smooth muscle (HASM) cells. We hypothesized that modulation of actin-myosin interactions by ...

Delta F508-CFTR channels: kinetics, activation by forskolin, and potentiation by xanthines

Abstract: Trafficking, activation, and kinetics of delta F508-cystic fibrosis transmembrane conductance regulator (CFTR) and CFTR were compared in stably transduced C127I mouse mammary epithelial cells. Western blots detected a small amount of fully glycosylated delta F508-CFTR Efflux of 125I was stimulated by forskolin with the same mean effective concentration (EC50; approximately 0.5 microM) for CFTR and delta F508-CFTR cells, but the maximum response was reduced more than fivefold and its latency increased approximately threefold in delta F508-CFTR cells. In delta F508-CFTR cells, 3-isobutyl-1-methylxanthine (IBMX; EC50 = 1.45 microM) and 8-cyclopentyl-1,3-dipropylxanthine (CPX; EC50 = 58 microM) increased the peak forskolin-stimulated efflux rate approximately 2.5-fold and decreased the time to peak. A sevenfold increase in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels accompanied potentiation of forskolin-induced 125I efflux by IBMX but not by CPX. Elevation of intracellular cAMP increased linear voltage-independent whole cell currents 30-fold in CFTR and 4-fold in delta F508-CFTR cells; the response rate in delta F508-CFTR cells was much slower. Single-channel currents were detected in 57 of 68 cell-attached patches from forskolin-prestimulated CFTR cells vs. 6 of 35 patches in delta F508-CFTR cells. Mean number of active channels per patch was 4.1 for CFTR [open probability (Po) = 0.34] and 0.2 for delta F508-CFTR (Po = 0.11). The lower Po of delta F508-CFTR resulted from an approximately threefold longer mean interburst interval. We estimate that forskolin-stimulated chloride conductance of delta F508-CFTR C127I cells is < 5% of CFTR cells. CPX is approximately 25-fold more potent than IBMX in potentiating delta F508-CFTR and may operate by a mechanism other than elevation of cAMP.

IL-1 stimulates intestinal myofibroblast COX gene expression and augments activation of Cl- secretion in T84 cells.

Abstract: Because interleukin-1 (IL-1) is an important mediator in the inflamed intestine, its effects on enterocyte-subepithelial myofibroblast (SEMF) interaction were investigated in vitro. Acutely juxtaposing T84 cells with 18Co or P2JF SEMF preincubated with IL-1 alpha significantly enhanced T84 short-circuit current (Isc) responsiveness to secretagogues in comparison to SEMF not activated by IL-1 alpha. The sensitivity of T84 cell Isc to Ca(2+)-dependent, but not adenosine 3',5'-cyclic monophosphate-dependent, secretagogues was augmented by IL-1 alpha-treated SEMF. These effects of IL-1 alpha are directly correlated with SEMF prostaglandin E2 (PGE2) production. Both IL-1 alpha augmentation of Cl secretagogue responsiveness and PGE2 formation were inhibited by IL-1 receptor antagonist. Within 5 h, IL-1 alpha stimulated a 10-fold increase in cyclooxygenase (COX)-2 steady-state mRNA levels in 18Co cells. In contrast, COX-1 message levels increased more slowly to two- to threefold above control levels after 24 h incubation. These results demonstrate that the proinflammatory cytokine IL-1 alpha accentuates intestinal SEMF augmentation of enterocyte responsiveness to Ca(2+)-dependent CI-secretagogues. PGE2 is an important mediator of SEMF-enterocyte interaction. The effects of IL-1 alpha on SEMF PGE2 productions are, at least in part, due to stimulation of COX gene expression.

Effect of low-protein diet-induced intrauterine growth retardation on rat placental amino acid transport

Abstract: Given the central role of the placenta in nutrient transport to the fetus, one might propose that maternal nutrition would have a regulatory effect on this nutrient delivery. We have examined the e...