bluuebunny/crossref_metadata_2025 huggingface dataset:
Chloride is required for fluid transport by the rabbit corneal endothelium
Authors: Winkler B. S. , Riley M. V. , Peters M. I. , Williams F. J.
Published: December 2017

The role of chloride in fluid transport of the rabbit corneal endothelium was examined by measuring changes in corneal thickness following ion substitutions or addition of ion transport inhibitors in media superfusing the isolated tissue. Normal fluid transport is indicated by maintenance of constant thickness in a fresh cornea or thinning (deturgescence) of a preswollen deepithelialized cornea to its initial thickness at approximately 40 microns/h. These patterns are seen when tissues are superfused with HCO(3-)-Ringer containing 114 mM Cl-. When Cl- was substituted with gluconate, glucuronate, or SO4(2-) fresh and preswollen corneas immediately thinned at greater than 150 microns/h to a value less than 300 microns and then began to swell at 30 microns/h to above their original thickness. Substitution of Cl- with NO3- or Br- had a negligible immediate thinning effect, but fresh corneas subsequently swelled and preswollen corneas failed to deturgesce fully. The rapid thinning (called a "downtransient") observed with gluconate, glucuronate, and SO4(2-) also occurred in these media when ion and fluid transport were completely inhibited with ouabain or stilbenes or by absence of HCO3-, indicating that the thinning results from osmotic gradients induced by ionic reflection coefficients different from that of Cl-. When the downstransient was avoided in deepithelialized corneas by preswelling with the same Cl(-)-free media on both sides of the cornea, corneas maintained a constant but swollen thickness in gluconate and in NO3- or Br- deturgesced slowly and incompletely; ouabain or stilbenes caused further swelling in all media. We conclude that absence of Cl- partially impairs fluid transport, most probably via its role in a Cl(-)-HCO3- exchanger which has been proposed in a recent model of endothelial fluid transport.
Altered coupling of alpha 1-adrenergic receptor-G protein in rat parotid during aging
Authors: Miyamoto A. , Villalobos-Molina R. , Kowatch M. A. , Roth G. S.
Published: December 2017

A possible role for altered signal transduction mechanisms in impaired alpha 1-adrenergic-stimulated secretory function during aging was investigated in parotid cells prepared from adult (6 mo) and old (24 mo) rats. Compared with adults, epinephrine-stimulated 45Ca2+ efflux and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] production were reduced 31 and 36% in cells of old rats, respectively. There was a highly significant correlation between 45Ca2+ efflux and Ins(1,4,5)P3 production. In saponin-permeabilized cells, no significant differences in Ins(1,4,5)P3-stimulated 45Ca2+ efflux in adult and old preparations were observed. When G proteins were stimulated by guanosine 5'-O-(3-thiotriphosphate) or NaF, no age differences in Ins(1,4,5)P3 production were detected. Stimulation of phosphoinositide-specific phospholipase C (PLC) by CaCl2 in adult and old cells was also comparable. Moreover, no differences in immunolabeled common alpha (GTP binding site), Gi alpha, PLC-gamma, or PLC-delta could be detected in either cytosol or membranes of adult and old preparations. In the absence of 5'-guanylylimidodiphosphate [Gpp(NH)p], no age-related changes in epinephrine competition for [3H]prazosin binding sites were observed. Approximately 30% of the agonist binding sites existed in a high-affinity form at both ages. Gpp(NH)p caused large rightward shifts of epinephrine displacement curves in adult membranes (converting all binding sites to the low-affinity form), but not old. Moreover, epinephrine was much more effective in stimulating G protein low-Km GTPase in parotid membranes from adult than old rats. These data suggest that age-related impairments in alpha 1-adrenergic responsiveness are mediated, at least in part, by the functional alterations in the coupling of G proteins with alpha 1-adrenergic receptors.
Phorbol ester inhibits erythropoietin production in human hepatoma cells (Hep G2)
Authors: Kurtz A. , Eckardt K. U. , Pugh C. , Corvol P. , Fabbro D. , Ratcliffe P.
Published: December 2017

Using the human hepatoma cell line Hep G2, we have studied a possible role of protein kinase C (PKC) activity for regulation of erythropoietin (EPO) production. During a 72-h incubation, EPO production by the cells was stimulated sevenfold by exposure to low oxygen tension (1%) and threefold by exposure to cobaltous chloride (100 microM). The phorbol ester phorbol 12-myristate-13 acetate (PMA) led to a concentration-dependent inhibition of basal and stimulated EPO formation (ED50 10 nM). This decrease of EPO production, which was apparent already after 1 h of incubation with PMA, reached its maximal effect after 24 h and held on for 72 h. It was paralleled by an inhibition of the increase of EPO mRNA levels in response to stimulation. A 24-h preincubation of the cells with PMA (100 nM) virtually blunted the effect of hypoxia on EPO formation. Recovery of EPO synthesis after removal of PMA took 48-72 h. The effect of PMA on EPO production was mimicked by phorbol 12,13-dibutyrate (ED50 1 microM) but not by 4 alpha-phorbol 12,13-didecanoate. The synthetic diacylglycerol analogues oleolyl-acetylglycerol and dioctanoylglycerol (2-200 microM) also had no effect on either basal or stimulated EPO production. Treatment with PMA caused a translocation of the alpha-isoenzyme of PKC from the cytosol to the membrane after 1 h and a disappearance of the membrane-bound form after 24 h of incubation. Staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, two structurally different inhibitors of PKC activity, inhibited basal and stimulated EPO production with ED50 values of 9 nM and 50 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Goblet cell degranulation in isolated canine tracheal epithelium: response to exogenous ATP, ADP, and adenosine
Authors: Davis C. W. , Dowell M. L. , Lethem M. , Van Scott M.
Published: December 2017

Mucin secretion by goblet cells was determined by quantifying degranulation events (DE) in isolated, superficial epithelium from canine trachea. The epithelium was isolated and explanted to a novel transparent, permeable support, and the goblet cells were visualized by video microscopy. Baseline degranulation events were quantified at 0.05 DE/min. Luminal ATP (10(-4) M, n = 10) stimulated a biphasic secretory response; a burst, maximum rate = 87.9 +/- 25.3, was followed by a plateau, rate = 1.9 +/- 0.3 DE/min. Serosal ATP elicited a complex set of responses: 9 cells failed to respond, 13 exhibited a trivial response, and 31 responded vigorously but with highly variable patterns of degranulation. Nonhydrolyzable 5'-adenylylimidodiphosphate caused degranulation from both sides of the epithelium. Luminal ADP and adenosine were ineffective. Serosal ADP and adenosine elicited a range of responses that was similar in diversity and magnitude to the ATP response. Our conclusions were as follows: 1) goblet cells in the superficial epithelium of the airway can be studied at the single-cell level in explants; 2) nucleotides stimulate goblet cells to secrete mucin; and 3) the goblet cell expresses different nucleotide receptors on its apical and basolateral membranes.
Swelling-induced and depolarization-induced ClŐ channels in normal and cystic fibrosis epithelial cells
Authors: Solc C. K. , Wine J. J.
Published: December 2017

Pages C658–C674: C. K. Sole and J. J. Wine. “Swelling-induced and depolarization-induced Cl- channels in normal and cystic fibrosis epithelial cells.” Page C672, four lines were dropped in printing from the top of the left-hand column; they should read as follows: with time as a cell is perfused with standard saline containing 10 mM EGTA. Conversely, the current is probably also not due to a simple disinhibition by a diffusible factor because activated channels can be read-. Note: at the bottom of the right-hand column, four lines have been repeated.
Steady-state dependence of stress on cross-bridge phosphorylation in the swine carotid media
Authors: Di Blasi P. , Van Riper D. , Kaiser R. , Rembold C. M. , Murphy R. A.
Published: December 2017

Tonic contractions of the swine carotid media are typically characterized by initial transients in myoplasmic [Ca2+] and cross-bridge phosphorylation followed by force maintenance with reduced intracellular [Ca2+] and cross-bridge phosphorylation (“latch”). The presence of effective mechanisms in the carotid media to limit steady-state myoplasmic [Ca2+] and cross-bridge phosphorylation to modest increases over resting values has limited experimental attempts to determine the dependence of active stress (force/tissue cross-sectional area) on cross-bridge phosphorylation. In this study, we employed stimulation protocols that combined effective contractile agonists with inhibitors of Ca2+ extrusion or sequestration to achieve high steady-state levels of cross-bridge phosphorylation (up to 60%). Increases in cross-bridge phosphorylation from 30 to 60% were not associated with significant increases in stress in agreement with the predictions of Hai and Murphy [Am. J. Physiol. 254 (Cell Physiol. 23): C99-C106, 1988] four-state cross-bridge model for the carotid media. Thus cross-bridge phosphorylation may suffice to determine force generation in vascular smooth muscle if both phosphorylated and dephosphorylated attached cross bridges (or latch bridges) contribute to active stress.
Glucocorticoid-induced skeletal muscle atrophy in vitro is attenuated by mechanical stimulation
Authors: Chromiak J. A. , Vandenburgh H. H.
Published: December 2017

Glucocorticoids induce rapid atrophy of fast skeletal myofibers in vivo, and either weight lifting or endurance exercise reduces this atrophy by unknown mechanisms. We examined the effects of the synthetic glucocorticoid dexamethasone (Dex) on protein turnover in tissue-cultured avian fast skeletal myofibers and determined whether repetitive mechanical stretch altered the myofiber response to Dex. In static cultures after 3-5 days, 10(-8) M Dex decreased total protein content 42-74%, total protein synthesis rates 38-56%, mean myofiber diameter 35%, myosin heavy chain (MHC) content 86%, MHC synthesis rate 44%, and fibronectin synthesis rate 29%. Repetitive 10% stretch-relaxations of the cultured myofibers for 60 s every 5 min for 3-4 days prevented 52% of the Dex-induced decrease in protein content, 42% of the decrease in total protein synthesis rate, 77% of the decrease in MHC content, 42% of the decrease in MHC synthesis rate, and 67% of the decrease in fibronectin synthesis rate. This in vitro model system will complement in vivo studies in understanding the mechanism by which mechanical activity and glucocorticoids interact to regulate skeletal muscle growth.
Sepsis-induced changes in protein synthesis: differential effects on fast- and slow-twitch muscles
Authors: Vary T. C. , Kimball S. R.
Published: December 2017

Sepsis is associated with severe muscle wasting. Mechanisms responsible for sepsis-induced alterations in muscle protein metabolism were investigated in vivo and compared with changes induced by nonseptic inflammation. The rate of protein synthesis in mixed hindlimb muscles was not altered in inflammation but was inhibited 50% in sepsis. This inhibition did not result from a decreased RNA content. Instead, the translational efficiency was significantly reduced by 50% in skeletal muscle of septic animals compared with control. The effect of sepsis to lower the rate of protein synthesis was further examined using individual muscles containing different fiber types. Both the protein concentration and protein synthetic rate in fast-twitch muscles were reduced by sepsis, whereas neither of these parameters was affected in slow-twitch muscles or heart. The decreased translational efficiency did not result from a change in the rate of peptide-chain elongation. Instead, the sepsis-induced inhibition of protein synthesis resulted from a restraint in peptide-chain initiation because sepsis caused a 1.6-fold increase in free ribosomal subunits. Overall, sepsis, but not inflammation, caused an inhibition of protein synthesis primarily in muscles composed of fast-twitch fibers. The mechanism involved in the reduced rates of protein synthesis in muscles resulted from an inhibition of peptide-chain initiation, with no change in peptide-chain elongation.
Mechanism of heptanol-induced uncoupling of cardiac gap junctions: a perforated patch-clamp study
Authors: Takens-Kwak B. R. , Jongsma H. J. , Rook M. B. , Van Ginneken A. C.
Published: December 2017

The influence of heptanol on gap junctional and non-junctional membrane currents was studied in cultured neonatal rat heart cells using both the whole cell and perforated patch voltage-clamp method. With both methods, exposure to heptanol produced a dose-dependent decrease in the junctional current (dissociation constant = 0.54 and 1.20 mM for whole cell and perforated patch experiments, respectively). Heptanol-induced uncoupling was reversible. In the same concentration range, heptanol reduced all nonjunctional membrane ionic currents examined. This suggests that heptanol does not act specifically on gap junction channels but rather on the structure of the lipid membrane. This hypothesis is strengthened by the observation that in monolayer cultures of neonatal rat heart cells fluorescence steady-state anisotropy decreased proportional with increasing the heptanol cxoncentration in the bath. Single-channel conductances (gamma j) were identical with both recording methods (21 and 40-45 pS); heptanol did not alter gamma j. Under conditions of reduced junctional coupling induced by heptanol, junctional conductance (gj) displayed voltage sensitivity at values of gj at which no voltage sensitivity could be observed under control conditions. These results suggest that heptanol-dependent uncoupling was caused by a decrease in open probability of the gap junction channels.
CFTR!
Authors: Fuller C. M. , Benos D. J.
Published: December 2017

Cystic fibrosis (CF) is a fatal genetic disease primarily affecting Caucasians, although cases have been reported from other ethnic groups. CF has a complex etiology, but it is chiefly a disease of electrolyte transport and is characterized by defects in fluid secretion by several epithelia, including the sweat duct, exocrine pancreas, and the pulmonary airways. The link between CF and a defect in cAMP-mediated Cl- transport in secretory epithelia was established in the early 1980s. Since then, numerous electrophysiological studies have focused on the characterization and regulation of individual Cl- channels underlying the macroscopic Cl- currents of secretory epithelia in the airways, sweat ducts, and gut. In this review the results of these studies in the light of current knowledge of the function of the CF gene product, the CF transmembrane conductance regulator (CFTR) protein, will be analyzed. The CFTR protein is a member of a family of ATP-binding proteins that act as unidirectional solute pumps. These proteins are membrane spanning, are found in both prokaryotic and eukaryotic cells, and have two ATP-binding domains. The family includes the p-glycoproteins that are involved with the expression of multidrug resistance in certain tumor cells. The majority of CF chromosomes (70%) have a single codon deletion that translates to a missing phenylalanine residue at position 508 (delta F508) of the protein. Unique for this family of proteins, the CFTR protein possesses an additional highly charged domain (the R domain) containing several consensus polypeptide sequences for kinase phosphorylation. Although CFTR bears structural resemblance to this family of ATP-dependent pumps, overexpression of the protein in a variety of different cell types is associated with the appearence of a cAMP-sensitive Cl- channel. We critically examine current information concerning the structure-function relationships of the CFTR protein obtained from both electrophysiological and biochemical approaches. We also summarize recent evidence suggesting that the CFTR protein may act as a pump and a channel, a hypothesis in keeping with the multifaceted nature of the disease.
Gap junction-mediated intercellular diffusion of Ca2+ in cultured human corporal smooth muscle cells
Authors: Christ G. J. , Moreno A. P. , Melman A. , Spray D. C.
Published: December 2017

Ratio imaging using the calcium-sensitive probe fura-2 was employed to study intracellular calcium concentrations and intercellular calcium flux through gap junctions in homogeneous vascular smooth muscle cell cultures derived from the human corpora cavernosa. Microinjection techniques demonstrated that fura-2 free acid was freely diffusible through gap junctions between cultured cells. The resting intracellular calcium level in fura-2-loaded cells was 176.9 +/- 10.5. A robust increase in intracellular calcium was seen in response to both phenylephrine and the calcium ionophore A23187. Microinjection of Ca2+ into individual smooth muscle cells always resulted in significant, although temporally delayed, increases in intracellular calcium levels in adjacent cells; this intercellular calcium flux was reversibly blocked by inhibition of gap junctional communication with 2 mM heptanol. However, although microinjection of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] into individual smooth muscle cells always produced significant increases in intracellular calcium levels in the injected cell, the intercellular spread of Ca2+ in response to Ins(1,4,5)P3 was more variable than for Ca2+ injections. These studies demonstrate that Ca2+, and perhaps Ins(1,4,5)P3 as well, can diffuse between smooth muscle cells through gap junction channels.
Volume regulation during recovery from chronic hypertonicity in brain glial cells
Authors: Strange K. , Morrison R.
Published: December 2017

Rat C6 glial cells undergo rapid regulatory volume increase (5-10 min) via electrolyte uptake when exposed to a hypertonic medium. With chronic exposure to hypertonicity (greater than 8 h), accumulated electrolyte is replaced partly by inositol. Inositol accumulation is brought about by upregulation of Na(+)-dependent inositol transport. When C6 cells acclimated chronically to hypertonic NaCl medium were returned to isotonic conditions, inositol levels dropped slowly from 478 nmol/mg protein towards control values (117 nmol/mg protein) in 18-24 h. Inositol loss occurred in part by efflux to the external medium via a pathway distinct from the uptake mechanism. Laser light-scattering measurements demonstrated that regulatory volume decrease (RVD) is slow under these experimental conditions. In contrast, cells exposed acutely to hypertonicity swell and then undergo a rapid and nearly complete RVD when returned to isotonic medium. These results suggest that slow inositol loss is rate limiting for RVD during recovery from chronic hypertonic stress. The slow inositol loss and RVD may be due to slow turnover of the efflux mechanism and/or slow downregulation of the hypertonically stimulated inositol uptake pathway.
Abundance, localization, and insulin-induced translocation of glucose transporters in red and white muscle
Authors: Marette A. , Richardson J. M. , Ramlal T. , Balon T. W. , Vranic M. , Pessin J. E. , Klip A.
Published: December 2017

D-Glucose protectable cytochalasin B (CB) binding to subcellular membrane fractions was used to determine glucose transporter number in red (quadriceps-gastrocnemius-soleus) and white (quadriceps-gastrocnemius) rat muscle. CB binding was twofold higher in isolated plasma membranes of red than of white muscle. In contrast, the number of transporters in an isolated insulin-sensitive intracellular membrane organelle was similar in the two muscle groups. Immunoblotting and immunofluorescence microscopy with anti-GLUT4 and anti-GLUT1 antibodies indicated that both GLUT1 and GLUT4 transporter isoforms account for the higher abundance of CB binding sites in plasma membranes of red than of white muscle. Immunofluorescence localized GLUT4 to both the surface and the interior of the muscle cell and demonstrated that type I (slow twitch oxidative) and type IIa (fast twitch oxidative-glycolytic) fibers are enriched in GLUT4 protein compared with type IIb (fast twitch glycolytic) fibers. In contrast, GLUT1 reactivity was restricted to the surface of the muscle cell and was also highly enriched in the perineurial sheaths of peripheral nerves and the capsules of muscle spindles present in both red and white muscles. Insulin caused a twofold increase in CB binding in isolated plasma membranes of red or white muscles with a corresponding 40-50% decrease in CB binding in isolated intracellular membranes. These changes in CB binding were paralleled by similar changes in the membrane distribution of the GLUT4 glucose transporter isoform and in glucose transport activity measured after insulin perfusion of hindquarter muscles. In contrast, insulin did not change the distribution of either GLUT1 glucose transporters or Na(+)-K(+)-ATPase alpha 1-subunits. The molar ratio of GLUT4 to GLUT1 in red and white muscle plasma membranes was found to be 4:1 in the basal state and 7:1 in the insulin-stimulated state. These results indicate that red muscle contains a higher amount of GLUT1 and GLUT4 transporters at the plasma membrane than white muscle in the basal and insulin-stimulated states but that GLUT4 translocation does not differ between muscle types. In addition, GLUT4 expression correlates with the metabolic nature (oxidative vs. glycolytic) of skeletal muscle fibers, rather than with their contractile properties (slow twitch vs. fast twitch).
Insulin-like growth factors decrease oxygen-regulated erythropoietin production by human hepatoma cells (Hep G2)
Authors: Scholz H. , Baier W. , Ratcliffe P. , Eckardt K. , Zapf J. , Kurtz A. , Bauer C.
Published: December 2017

We examined the effects of insulin-like growth factors (IGFs) and insulin on erythropoietin (EPO) production by human hepatoma cells (Hep G2). Compared with normoxia (20% O2), EPO production by Hep G2 cells during a 72-h incubation was stimulated fivefold by exposure to low oxygen tension (1% O2) and nearly threefold by exposure to cobalt chloride (100 microM). IGF-I caused a concentration-dependent attenuation of EPO formation under normoxic conditions and inhibited (maximally 50%) EPO production stimulated by either low oxygen tension or cobalt [half-maximal effect (ED50) approximately 5 nM]. The increase of EPO mRNA levels in response to hypoxia was significantly reduced by IGF-I. Similarly to IGF-I, IGF-II (ED50 approximately 8 nM) and insulin (ED50 approximately 80 nM) also inhibited EPO formation in Hep G2 cells. IGF-I (100 pM-100 nM) stimulated the incorporation of radiolabeled alanine as a measure for total protein synthesis, 3H-labeled thymidine incorporation into DNA, and glycogen synthesis at 20 and 1% O2 in a concentration-dependent fashion. IGF-I exhibited a high affinity for the IGF-I receptor (apparent Kd approximately 3 nM). Unlabeled insulin was greater than 100-fold less potent than IGF-I in competing for 125I-IGF-I binding (apparent Kd approximately 360 nM). Conversely, insulin bound to the insulin receptor with high affinity (apparent Kd approximately 0.3 nM), whereas IGF-I was less than 1% as potent in competing for 125I-insulin binding. In summary, IGFs and insulin exert a negative control function on oxygen-regulated EPO production in Hep G2 cells. The inhibitory effect of IGFs and insulin on EPO formation appears to be mediated via the IGF-I receptor.
Immunofluorescence localization of the Na-Ca exchanger in heart cells
Authors: Kieval R. S. , Bloch R. J. , Lindenmayer G. E. , Ambesi A. , Lederer W. J.
Published: December 2017

We investigated the localization of the Na-Ca exchanger in fixed, isolated heart cells from rat and guinea pig using immunocytochemical methods with epifluorescence and confocal microscopy. We found that the Na-Ca exchanger is distributed throughout all membranes in contact with the extracellular space, including the sarcolemma, the transverse tubules (T-tubules), and the intercalated disks. Microscopic nonuniformities in the fluorescent labeling appear to reflect varying views of the membranes containing Na-Ca exchanger protein. Confocal thin-section imaging reveals a regular grid of discrete foci of fluorescence, which represent Na-Ca exchanger in T-tubules viewed en face. These foci are 1.80 +/- 0.01 microns apart from sarcomere to sarcomere and are aligned with the Z-line. Along each Z-line, these foci are spaced at 1.22 +/- 0.11-microns intervals. Longitudinal sections of the sarcolemma-T-tubule junction show a comblike appearance, with T-tubules extending inward from the heavily labeled sarcolemma. Our finding that the Na-Ca exchanger is widely distributed over the cell surface may provide further insight into the role of Na-Ca exchange in the heart.
Stimulation of intestinal Cl- transport by heat-stable enterotoxin: activation of cAMP-dependent protein kinase by cGMP
Authors: Forte L. R. , Thorne P. K. , Eber S. L. , Krause W. J. , Freeman R. H. , Francis S. H. , Corbin J. D.
Published: December 2017

Heat-stable enterotoxins activate guanylate cyclase, whereas heat-labile enterotoxins stimulate adenylate cyclase. Both classes of toxins cause secretory diarrhea at least in part by stimulating Cl- secretion in the intestine. The mechanism for regulation of Cl- secretion by guanosine 3',5'-cyclic monophosphate (cGMP) was investigated using cultured T84 intestinal cells as a model for intestinal crypt cells. Escherichia coli heat-stable enterotoxin (ST) markedly stimulated cGMP production in T84 cells. Cl- secretion across T84 cell monolayers cultured on permeable filters was stimulated by E. coli ST, cholera toxin, or 8-BrcAMP, but 8-BrcGMP was ineffective. cGMP analogues that are known to be potent and specific activators of cGMP-dependent protein kinase (cG-kinase) also had little effect on 36Cl- uptake by T84 cells cultured in plastic dishes. E. coli ST, forskolin, cholera toxin, or membrane-permeant cAMP analogues markedly increased 36Cl- uptake into T84 cells. The general protein kinase inhibitor, staurosporine, inhibited the stimulation of Cl- permeability elicited by E. coli ST, vasoactive intestinal peptide (VIP), or 8-BrcAMP. DEAE-Sephacel chromatography revealed a predominant type II isoform of cAMP-dependent protein kinase (cA-kinase) in T84 cells, whereas little or no cytosolic cG-kinase activity was found. Treatment of T84 cells with E. coli ST or VIP resulted in an increase in the cA-kinase activity ratio (-cAMP/+cAMP) if the cytosolic enzyme was assayed at reduced temperature (on ice).(ABSTRACT TRUNCATED AT 250 WORDS)
Protein kinase A phosphorylation enhances sodium channel currents in Xenopus oocytes
Authors: Smith R. D. , Goldin A. L.
Published: December 2017

The voltage-sensitive rat brain sodium channel is known to be phosphorylated by adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA), but the functional significance of that phosphorylation is unknown. We have shown that rat brain sodium channel currents expressed in Xenopus oocytes were enhanced by induction of PKA activity. Stimulation of the beta 2-adrenergic receptor or treatment with dibutyryl cAMP resulted in increased sodium current amplitudes without affecting the voltage dependence of channel activation or inactivation. These increases were completely blocked by preinjection of protein kinase inhibitor, a specific inhibitor of PKA. Injection of phosphatase into the oocytes resulted in a significant decrease in sodium current amplitude, indicating that phosphorylation is important for basal levels of sodium channel activity in oocytes. The enhancement was specific for the rat brain IIA sodium channel, because currents expressed from the rat muscle microI sodium channel were not enhanced by the same procedures. These data demonstrate a modulatory role of PKA phosphorylation on brain sodium channel function and suggest a means by which the electrical excitability of cells may be regulated.
Intracellular signal transduction in four dimensions: from molecular design to physiology
Authors: Tsien R. Y.
Published: December 2017

Designed fluorescent indicators are the basis for a major new technique in cell physiology, the quantitative measurement and dynamic imaging of intracellular concentrations of important ions and messengers such as Ca2+, Na+, H+, and adenosine 3',5'-cyclic monophosphate. Molecular engineering has now produced indicators with quite good selectivity and sensitivity for these analytes. In many cases, these probes can be introduced into large populations of cells by means of membrane-permeant chemical derivatives, so that the plasma membrane need never be disrupted or physically breached at any point. Like many other optical microscopic techniques, fluorescent indicators are readily applied to study living cells and tissues, with an unparalleled combination of spatial and temporal resolution. They offer one of the few methods for continuous nondestructive monitoring of dynamic intracellular biochemistry and signal transduction in single cells or subregions of cells.
Induction of ICAM-1 by TNF-alpha, IL-1 beta, and LPS in human endothelial cells after downregulation of PKC
Authors: Myers C. L. , Wertheimer S. J. , Schembri-King J. , Parks T. , Wallace R. W.
Published: December 2017

The intercellular adhesion molecule 1 (ICAM-1) is induced on endothelial cells by tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and lipopolysaccharide (LPS). We have reported the sensitivity of cytokine-induced ICAM-1 expression to protein kinase inhibitors, including inhibitors of protein kinase C (PKC) [C. L. Myers, S. N. Desai, J. Schembri-King, G. L. Letts, and R. W. Wallace. Am. J. Physiol. 262 (Cell Physiol. 31): C365-C373, 1992]. To directly investigate the role of PKC in ICAM-1 induction, we downregulated PKC by pretreatment of human umbilical vein endothelial cells with phorbol 12-myristate 13-acetate (PMA) and assessed ICAM-1 protein and mRNA induction elicited by subsequent exposure to inflammatory stimuli. PMA treatment results in ICAM-1 protein induction that declines to basal levels by 3 days. Western blots of endothelial cell lysates reveal a nearly complete loss of immunologically reactive PKC. Subsequent activation with cytokine or LPS leads to reinduction of ICAM-1 protein and mRNA; however, the cells no longer produced substantial amounts of ICAM-1 protein or mRNA in response to PMA stimulation. Cross desensitization is observed with phorbol dibutyrate, while 4 alpha-phorbol has no desensitizing effect. The data indicate that PKC activation, while capable of inducing ICAM-1 expression, is not essential for ICAM-1 induction by the inflammatory mediators TNF-alpha, IL-1 beta, or LPS.
Adrenalectomy reduces alpha 1 and not beta 1 Na(+)-K(+)-ATPase mRNA expression in rat distal nephron
Authors: Farman N. , Coutry N. , Logvinenko N. , Blot-Chabaud M. , Bourbouze R. , Bonvalet J. P.
Published: December 2017

The abundance of mRNA of alpha 1-, alpha 2-, alpha 3-, beta 1-, and beta 2-isoforms of Na(+)-K(+)-ATPase was examined in several renal structures of normal and adrenalectomized (ADX) rats. In situ hybridization with 35S-labeled cRNA probes was performed on kidney sections from adult rats. The number of silver grains per unit surface area was quantified over cells of the glomerulus, proximal convoluted tubules (PCT), early distal tubules (EDT), and cortical collecting ducts (CCD). In normal rat kidney, alpha 1- and beta 1-mRNA was detected in PCT, EDT, and CCD, with the following range of magnitude: EDT > CCD > PCT > glomerulus. The amount of alpha 1- and beta 1-mRNA was equivalent. A large abundance of these two mRNA species was also found in the medullary thick ascending limb of the loop of Henle. Expression of alpha 2, alpha 3, and beta 2 was very low and evenly distributed over any cell type. In ADX, a significant decrease in alpha 1-mRNA (30%) was observed in EDT and CCD, with no change in PCT. beta 1-mRNA abundance was unaffected by adrenalectomy. These results indicate that 1) in the rat kidney alpha 1- and beta 1-mRNA are coexpressed at a similar level that varies along the renal tubule according to the cell type, 2) minute expression of alpha 2-, alpha 3-, and beta 2-mRNA is present in the kidney, and 3) corticosteroid depletion reduces the expression of alpha 1- and not beta 1-mRNA in the corticosteroid-sensitive tubular cells.
Detection of angiotensin I and II in cultured rat cardiac myocytes and fibroblasts
Authors: Dostal D. E. , Rothblum K. N. , Conrad K. M. , Cooper G. R. , Baker K. M.
Published: December 2017

Angiotensin II (ANG II) is a stimulus for positive chronotropic and inotropic effects, protein synthesis, and hypertrophic growth in cardiac tissue. These short- and long-term effects of ANG II are mediated through specific plasma membrane receptors. Indirect evidence suggests that ANG II synthesized in the myocardium may be important in regulating cardiac function. The cell types in the myocardium that produce components of the renin-angiotensin system have not been determined. In this study, we evaluated whether cultured cardiomyocytes and fibroblasts obtained from ventricles of neonatal rat hearts were capable of synthesizing ANG I and II. Both cardiomyocytes and fibroblasts were found to have immunofluorescent staining for ANG I, ANG II, and angiotensin-converting enzyme (ACE). The amounts of ANG I and II in cell extracts and conditioned media obtained from cardiomyocytes and fibroblasts were quantified by radioimmunoassay. The amounts of ANG I and II detected in cardiomyocyte cultures (1.48 x 10(6) cells/dish) were 32.2 +/- 16.2 (n = 4) and 6.2 +/- 2.9 (n = 4) ng/10(6) cells, respectively. The amounts of ANG I and II detected in the media conditioned by a 48-h exposure to cardiomyocytes were 5.2 +/- 1.2 (n = 3) and 2.1 +/- 1.2 (n = 3) ng/10(6) cells, respectively. The amounts of ANG I and II detected in fibroblast cultures (5.38 x 10(6) cells/dish) were 34.8 +/- 4.9 (n = 4) and 8.0 +/- 3.5 (n = 4) ng/10(6) cells, respectively. The amounts of ANG I and II obtained from media conditioned by a 48-h exposure to fibroblasts were 4.7 +/- 0.6 (n = 4) and 3.3 +/- 2.1 (n = 4) ng/10(6) cells, respectively. The identity of the radioimmunoassayable materials as ANG I and II peptides was confirmed in cardiomyocytes using an in vitro bioassay based on displacement of 125I-ANG II from receptor binding sites in cardiac membranes prepared from neonatal pig heart. Identification of ANG I and II and ACE in vitro in cultures of cardiac myocytes and fibroblasts supports the hypothesis that there is an intracardiac renin-angiotensin system that produces these peptides.
C-type natriuretic peptide inhibits growth factor-dependent DNA synthesis in smooth muscle cells
Authors: Porter J. G. , Catalano R. , McEnroe G. , Lewicki J. A. , Protter A. A.
Published: December 2017

We have examined the ability of C-type natriuretic peptide (CNP) to interact with guanylate cyclase-coupled natriuretic peptide receptors by measuring its ability to stimulate intracellular guanosine 3',5'-cyclic monophosphate (cGMP) accumulation in cultured bovine aortic endothelial (BAE) and bovine aortic smooth muscle (BASM) cells. Our experiments indicate that CNP is unable to stimulate the production of cGMP in BAE cells, whereas both atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) markedly elevate cGMP levels in these cells (ANP = BNP >> CNP). In contrast, CNP is the most effective of the three peptides with respect to the stimulation of cGMP levels in BASM cells, fetal human vascular smooth muscle cells, and rat A10 cells (CNP >> ANP > BNP), with the maximal level of stimulation being approximately 5- to 10-fold over that observed for ANP. We have also shown that CNP is able to inhibit serum- and growth factor-induced DNA synthesis in BASM cells. Low concentrations of CNP (20 x 10(-9) M) inhibit up to 80% of the [3H]-thymidine incorporation induced by basic fibroblast growth factor, platelet derived growth factor, epidermal growth factor (EGF), and heparin binding EGF-like growth factor. These data indicate that, although CNP has been detected only in the central nervous system and not in the circulation, it may possess multiple effects on vascular tissue.
Calcium signaling in endothelia: cellular heterogeneity and receptor internalization
Authors: Weintraub W. H. , Negulescu P. A. , Machen T. E.
Published: December 2017

The vasoactive factors thrombin, bradykinin (BK), and ATP are released in response to tissue damage and inflammation and act on endothelium to modulate vascular perfusion. We have investigated the second messenger response of endothelium activated by these agonists and, in particular, the mechanism of desensitization to BK. Fura-2 fluorescence ratio imaging of calf pulmonary artery endothelial cells (CPAE) revealed 5- to 10-fold increases on intracellular Ca (Cai) in response to these agents. Maximal doses caused Cai to increase from 52 to 248 nM (thrombin), 556 nM (BK), and 643 nM (ATP). Agonists elicited a rapid (within 30 s) increase of Cai due to release of Ca from intracellular stores followed by a secondary elevation of Cai dependent on entry of external Ca. The temporal characteristics of the Cai responses to all agonists were heterogeneous from cell to cell, and, interestingly, repeated stimulation gave identical signature responses from individual cells, although the amplitude of the Cai response decreased to thrombin and especially bradykinin but not for ATP. This decrease was agonist specific because ATP elicited large increases of Cai after thrombin or BK desensitization. Maximal desensitization was obtained with BK applied for 5-10 min followed by a rest of < 10 min before restimulation. Although desensitization primarily reduced the elevation of Cai due to the release of the internal store, entry of extracellular Ca was also reduced. Cells responded heterogeneously to desensitization in that those with prominent extracellular Ca entry responded most strongly upon a second stimulation with BK. Because desensitized cells still responded to ATP with an increase of Cai, the desensitization was controlled at a step prior to the activation of phospholipase C. Desensitization occurred by a reduction of BK receptor number; a 10-min BK pretreatment reduced [3H]BK binding to receptors by 70% (from 14,600 receptors/cell, Km = 5 nM, to 5,300). As surface receptor numbers decreased, internalized receptors increased as assayed by an acetic acid wash. The time course of the receptor internalization was similar to the decrease in Cai response to BK. We conclude that the vasoactive agonists thrombin, BK, and ATP increase the second messenger Cai in endothelial cells and that a desensitized Cai response occurs with BK, but not with ATP, due to downregulation and endocytosis of the BK receptor.
Amino acid loss during volume regulatory decrease in cultured chick heart cells
Authors: Rasmusson R. L. , Davis D. G. , Lieberman M.
Published: December 2017

Mechanisms of volume regulation in hyposomotically treated cultured chick heart cell preparations were studied using optical, biochemical, and nuclear magnetic resonance methods. This approach afforded the resolution of time-dependent responses that might ordinarily be obscured by the complex morphology of intact cardiac muscle preparations. In hyposmotic solutions, cells swelled to a peak volume within 3 min and slowly regulated toward original volume (regulatory volume decrease, RVD). Upon return of the cells to isosmotic solution following hyposmotic treatment, the cells shrank to a steady-state volume that was substantially less than the initial volume in control solution. A vigorous RVD could also be elicited by hyposmotic swelling under Cl(-)-free conditions. Measurement of both inorganic cation loss via atomic absorption spectroscopy and organic solute loss via 1H-nuclear magnetic resonance and high-pressure liquid chromatographic techniques revealed that the RVD observed following exposure to hyposomotic solutions was mediated in part by a substantial loss of taurine, glutamate, aspartate, and glycine as well as loss of inorganic ions (Na+,K+). The hyposmotically activated transport of amino acids was also associated with the production of glutamate and aspartate. The volume regulatory release and production of amino acids have significant implications for the metabolic and functional integrity of cardiac cells.
Inhibition of cAMP- and Ca-dependent Cl- secretion by phorbol esters: inhibition of basolateral K+ channels
Authors: Reenstra W. W.
Published: December 2017

Pretreating confluent T84 cells with the phorbol ester phorbol 12-myristate 13-acetate (PMA) inhibits adenosine 3',5'-cyclic monophosphate (cAMP)- and carbachol-induced Cl secretion. Both a sustained short-circuit current (Isc), seen after the addition of 50 microM 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) and 100 microM 3-isobutyl-1-methylxanthine (IBMX), and a transient current, seen after the subsequent addition of 100 microM carbachol, are inhibited by 80% following pretreatment with 100 nM PMA for 2 h. Pretreatment with PMA has no effect on the level of cystic fibrosis transmembrane conductance regulator protein or apical cAMP-dependent Cl conductance. Carbachol does not induce an increase in apical Cl conductance. Basolateral K conductance was measured in monolayers treated with apical nystatin and exposed to a K gradient. Agonist-independent K conductance is 10-fold greater in Cl media than in gluconate media. Pretreatment with PMA inhibits agonist-independent K conductance by 57% in Cl media but stimulates K conductance by 1.9-fold in gluconate media. The addition of carbachol induces a transient increase in basolateral K conductance, and pretreatment with PMA inhibits the agonist-dependent K conductance by 66% in Cl media and by 92% in gluconate media. In Cl media, serosal barium, at 3 mM, inhibits agonist-independent K conductance but has no significant effect on the carbachol-induced conductance. In nonpermeabilized monolayers, serosal barium inhibits the cAMP-dependent Isc by 56% but has no effect on the carbachol-induced Isc. These results demonstrate that the primary effect of PMA on Cl secretion is not inhibition of apical Cl channels but inhibition of basolateral K channels.(ABSTRACT TRUNCATED AT 250 WORDS)
Cation transport and volume regulation in sickle red blood cells
Authors: Joiner C. H.
Published: December 2017

Cellular dehydration is one of several pathological features of the sickle cell. Cation depletion is quite severe in certain populations of sickle cells and contributes to the rheological dysfunction that is the root cause of vascular occlusion in this disease. The mechanism of dehydration of sickle cells in vivo has not been ascertained, but three transport pathways may play important roles in this process. These include the deoxygenation-induced pathway that permits passive K+ loss and entry of Na+ and Ca2+; the K(+)-Cl- cotransport pathway, activated by acidification or cell swelling; and the Ca(2+)-activated K+ channel, or Gardos pathway, presumably activated by deoxygenation-induced Ca2+ influx. Recent evidence suggests that these pathways may interact in vivo. Heterogeneity exists among sickle cells as to the rate at which they become dense, suggesting that other factors may affect the activity or interactions of these pathways. Understanding the mechanism of dehydration of sickle cells may provide opportunities for pharmacological manipulation of cell volume to mitigate some of the symptoms of sickle cell disease.
Properties of skeletal muscle mitochondria isolated from subsarcolemmal and intermyofibrillar regions
Authors: Cogswell A. M. , Stevens R. J. , Hood D. A.
Published: December 2017

Two mitochondrial fractions, termed intermyofibrillar (IMF) and subsarcolemmal (SS), were isolated from skeletal muscle, and their biochemical properties were related to differences in respiration and mitochondrial protein synthesis. State III respiration was 2.3- to 2.8-fold greater in IMF than in SS mitochondria. Site 1 inhibition of respiration with rotenone reduced this difference to 1.4-fold. When sites 1 and 2 were inhibited with antimycin, the 1.4-fold differences remained. The activities of cytochrome-c oxidase (CYTOX) and succinate dehydrogenase (SDH) could account for some of these differences, since CYTOX was 20% greater (P < 0.05) in IMF mitochondria, and SDH was 40% greater (P < 0.05) in SS mitochondria. Cytochromes a, b, c, and c1 contents were similar in the two fractions. Cardiolipin (CL) content was higher (P < 0.05) in SS mitochondria, indicating a less dense mitochondrial fraction with respect to CL. In vitro [3H]leucine incorporation was 1.8-fold higher (P < 0.05) in IMF than in SS mitochondria. Thus compositional differences between IMF and SS fractions exist, perhaps representing mitochondria at different stages of biogenesis. The biochemical and functional differences could not solely be due to differences in mitochondrial protein synthesis but could also be due to nuclear-directed protein synthesis specific to each mitochondrial fraction.
Perchlorate potentiation of excitation-contraction coupling in mammalian skeletal muscles
Authors: Gallant E. M. , Taus N. S. , Fletcher T. F. , Lentz L. R. , Louis C. F. , Mickelson J. R.
Published: December 2017

The action of perchlorate (ClO4-), an agonist of the voltage sensor in excitation-contraction (EC) coupling, has been examined using bundles of intact muscle cells, isolated membrane vesicles [sarcoplasmic reticulum (SR) and transverse tubule (TT)], and cultured myotubes. The effect of ClO4- on mechanical parameters was investigated in isolated murine limb muscles. The presence of ClO4- (5 or 10 mM) greatly increased twitch tension ( > 250%), slightly enhanced tetanic tension, and increased K contracture tension. K contracture thresholds of extensor digitorum longus (EDL, 40 mM K+) and soleus (30 mM K+) muscles were not altered by ClO4-. However, in whole cell patch clamp studies of mouse myotubes, contractile activation was shifted by approximately -10 mV by 10 mM ClO4-. To further define the site of alteration of EC coupling by ClO4-, studies were conducted with isolated porcine SR and TT vesicles and with cultured mouse myotubes. The rate constant of Ca-induced 45Ca release from SR vesicles was significantly increased by ClO4-. However, neither the affinity nor level of [3H]PN200-110 binding to TT vesicles was significantly affected by ClO4- concentrations that increased twitch tension. Furthermore, slow plasmalemmal Ca currents of myotubes recorded in the whole cell patch-clamp mode were enhanced by 10 mM ClO4-, and the current-voltage relationship was shifted approximately -7mV. Thus, in enhancing EC coupling in mammalian muscle, ClO4- may act at multiple sites including the SR Ca release channel and the TT Ca channel-voltage sensor.
Intracellular tetanic calcium signals are reduced in fatigue of whole skeletal muscle
Authors: Baker A. J. , Longuemare M. C. , Brandes R. , Weiner M. W.
Published: December 2017

Force and intracellular calcium signals were monitored in whole bullfrog semitendinosus muscles during fatigue produced by intermittent tetanic stimulation. Intracellular calcium signals were monitored using the fluorescent calcium-sensitive indicator indo-1 from the ratio of fluorescence intensities (R) at 400 and 470 nm. Fatiguing stimulation caused 1) proportional decreases of tetanic force and R, suggesting a component of the decreased force during fatigue of whole muscle may be due to insufficient calcium to activate contraction; 2) a progressive slowing of the relaxation of both force and R, suggesting slowed force relaxation may be mediated by slowed calcium removal from the myoplasm; 3) an increase of resting level R, suggesting impaired calcium removal from, or increased leakage to the cytosol; 4) prolongation of the twitch contraction, which was paralleled by changes in R. These findings are consistent with previous single fiber studies and suggest that changes in whole muscle contractility with fatigue may be partially mediated by changes in calcium handling by the cell.