bluuebunny/crossref_metadata_2025 huggingface dataset:
Electrogenic Na+ absorption of rat distal colon is confined to surface epithelium: a voltage-scanning study
Authors: Kockerling A. , Sorgenfrei D. , Fromm M.
Published: December 2017

There is no quantitative assignment of large intestinal electrogenic Na+ absorption to surface epithelium and crypts so far. We determined the spatial distribution of electrogenic Na+ absorption to crypts and surface epithelium of rat late distal colon using a modified voltage-scanning technique. Voltage deflections resulting from external 30-Hz current were sensed by an extracellular microelectrode stepping at 0.7 Hz above crypt openings or surface epithelium. Local conductances were calculated applying a planar model of electrical field distribution to surface epithelium and a electrostatic disk source model to the crypts. These models were confirmed by methodological experiments where the electrode position was varied in vertical and horizontal direction. Electrogenic Na+ absorption was detected by blocking apical Na+ channels by mucosal 0.1 mM amiloride. Under control conditions surface epithelium contributed 44% (2.0 +/- 0.2 mS/cm2) and crypts 56% (2.6 +/- 0.2 mS/cm2) to the total conductance of 4.6 +/- 0.4 mS/cm2. Electrogenic Na+ absorption was induced by 6 h in vitro incubation in a medium containing 3 nM aldosterone. This caused a short-circuit current (ISC) of 12.1 +/- 0.8 mumol.h-1.cm-2, which was paralleled by a 2.5-fold increase in surface epithelial conductance to 5.1 +/- 0.4 mS/cm2, whereas crypt conductance was not significantly altered (3.0 +/- 0.2 mS/cm2). Amiloride reversed ISC to -0.8 +/- 0.1 mumol.-1.cm-2 and decreased surface epithelium conductance to 2.3 +/- 0.3 mS/cm2 but again had no significant effect on crypt conductance (2.5 +/- 0.3 mS/cm2). Sham incubation (no hormones added) for 6 h neither induced electrogenic transport nor altered local epithelial conductances.(ABSTRACT TRUNCATED AT 250 WORDS)
Origin of cAMP-dependent Cl- secretion from both crypts and surface epithelia of rat intestine
Authors: Kockerling A. , Fromm M.
Published: December 2017

Adenosine 3',5'-cyclic monophosphate (cAMP)-dependent Cl- secretion provides the ionic basis for secretory diarrhea. We quantified the spatial distribution of this process by measuring local ion conductance in crypts and surface epithelium or villi of rat late distal colon and ileum. By use of an improved voltage-scanning technique, the tissue was clamped to a 30-Hz sine-wave current and the electrical field above the respective structures was sensed by a stepping glass microelectrode. Under control conditions, crypts and surface epithelium contributed 61 and 39%, respectively, to the total ion conductance of distal colon. Theophylline (10 mM) increased crypt conductance (Gc) by 64% from 2.5 +/- 0.2 to 4.1 +/- 0.3 mS/cm2 and surface epithelium conductance (Gs) by 69% from 1.6 +/- 0.1 to 2.7 +/- 0.1 mS/cm2. These changes in local conductances were completely Cl- dependent, since theophylline had no effect when Cl- was replaced by gluconate. Similar results were obtained when Cl- secretion was elicited by prostaglandin E1 (1 microM) or by dibutyryl-cAMP (DBcAMP, 1 mM). After stimulation, the Cl- channel blocker 5-nitro-2-(3-phenyl-propylamino)benzoic acid (1 mM) decreased both Gc and Gs. In rat ileum, theophylline plus DBcAMP caused an increase in total conductance of 19% only because of its large paracellular conductance. The ratio of scanning signals above villi and intervillous spaces was unaffected, indicating that Cl- conductance is induced in both crypts and villi. We conclude that in distal large intestine cAMP-dependent Cl- secretion is not confined to crypts but is evenly performed also by surface cells. A similar distribution exists in small intestine.
Regulation of sarcoplasmic reticulum calcium pump gene expression by hindlimb unweighting
Authors: Schulte L. M. , Navarro J. , Kandarian S. C.
Published: December 2017

Hindlimb unweighting (HU) causes upregulation of several muscle-specific genes responsible for the slow-to-fast transition in soleus skeletal muscle properties despite the profound muscle atrophy. The purpose of this study was to examine the expression of the fast and slow isoforms of the sarcoplasmic reticulum Ca(2+)-ATPase at the mRNA and protein level in the soleus muscle over a time course of HU and relate them to Ca(2+)-dependent ATPase activity and selected contractile properties. mRNA levels of the acetylcholine receptor (AChR) were measured to compare the signal of unweighting with denervation. Atrophy of the soleus muscles from tail-suspended rats was observed at all time points with muscle mass decreased by 52% at 28 days of HU (P < 0.05). Northern blot analysis showed the relative expression of the fast Ca2+ pump mRNA increased by 0, 250, 910, 1,340, and 4,050% over control levels at 1, 4, 7, 14, and 28 days of HU, respectively, whereas changes in slow mRNA were variable and modest in comparison. For the same time points, Western blot analysis showed relative expression of the fast Ca2+ pump protein increased by 30, 110, 320, 280, and 300% over control levels, whereas the slow-pump protein expression was unchanged except for a 75% decrease at 28 days of HU. Specific Ca(2+)-dependent ATPase activity was increased (P < 0.05) by 170% at 28 days of HU. Contractile properties measured in vitro at 14 and 28 days revealed time to peak tension and one-half relaxation time were shortened (P < 0.05) and a rightward shift in the tension-frequency curves in unloaded soleus muscles.(ABSTRACT TRUNCATED AT 250 WORDS)
Physiological effects of endogenous ouabain: control of intracellular Ca2+ stores and cell responsiveness
Authors: Blaustein M. P.
Published: December 2017

Ouabain is a well-known compound but a newly discovered adrenal cortical hormone that plays a role in cell Na+ regulation and in whole body salt and water balance. Ouabain may also be a paracrine hormone and may be secreted by some central nervous system neurons as well as by other types of cells. This article focuses on the cellular mechanisms that underlie the physiological (and pathophysiological) effects of ouabain. Ouabain directly inhibits the plasmalemmal Na+ pump in a variety of cell types. Low ouabain concentrations cause, in the steady state, a modest rise in the cytosolic Na+ concentration but only a minimal decline in membrane potential. All Na+ gradient-dependent processes may thereby be affected, albeit to only a small extent. Most important, however, is the secondary redistribution of Ca2+, mediated by Na(+)-Ca2+ exchange, that should slightly increase the cytosolic free Ca2+ concentration ([Ca2+]cyt). As a result of Ca2+ sequestration in intracellular stores [the endoplasmic and/or sarcoplasmic reticulum (ER/SR)], however, a new steady state is achieved with a slightly increased [Ca2+]cyt but a substantially augmented Ca2+ store; thus the ER/SR effectively acts as a Ca2+ amplifier. This extra stored Ca2+ is then available for mobilization whenever the cells are activated. Cytosolic Ca2+ is a key signaling mechanism in virtually all cells: it controls numerous physiological processes such as contraction, secretion, and excitability. Thus ouabain may modulate cell responsiveness via its influence on ER/SR Ca2+ stores. With these principles in mind, we examine evidence that endogenous ouabain may play a role in numerous physiological and pathophysiological processes associated with altered fluid and electrolyte metabolism and deviations from the normal blood pressure-blood volume relationship. We discuss the possible participation of ouabain in the regulation of vascular tone and then consider the putative role of ouabain in several forms of hypertension, congestive heart failure, thyroid and adrenocortical dysfunction, and diabetes mellitus, as well as in the adaptation to high altitude.
Active oxygen species in the liver of rats submitted to chronic hypobaric hypoxia
Authors: Costa L. E. , Llesuy S. , Boveris A.
Published: December 2017

The spontaneous in situ liver chemiluminescence of female rats submitted to 4,400 m (simulated altitude) for 2 mo and of their corresponding controls at sea level was determined as an approach to the measurement of the intracellular steady-state concentrations of singlet oxygen and oxygen free radicals. Spontaneous liver chemiluminescence was decreased by approximately 40% in hypoxic rats, whereas CCl4-induced chemiluminescence was unchanged. Liver mitochondria isolated from hypoxic rats showed a 53% decreased rate of H2O2 production and an increased content of cytochrome b (36%), with normal content of cytochromes c1, c, and a-a3. Superoxide dismutase showed a 26% decrease in activity, whereas catalase and glutathione peroxidase activities were not significantly decreased by this extent of hypoxia. Cytochrome P-450 and glutathione contents were unchanged. There were no significant differences in the hydroperoxide-initiated chemiluminescence (an estimation of tissue chain-breaker antioxidants) of homogenates, mitochondria, and microsomes. Results suggest that in chronic hypoxia there is a lower rate of generation of active oxygen species in liver, leading to a decreased steady-state concentration of singlet oxygen.
Hydroperoxide-induced oxidative stress alters pyridine nucleotide metabolism in neonatal heart muscle cells
Authors: Janero D. R. , Hreniuk D. , Sharif H. M. , Prout K. C.
Published: December 2017

An oxidant burden established by hydrogen peroxide (H2O2) overload may elicit postischemic myocardial damage. We assess herein the influence of H2O2-induced oxidative stress on heart muscle pyridine nucleotide metabolism. Exposure of neonatal rat cardiomyocytes to 50 microM-1.0 mM H2O2 bolus rapidly shifted their pyridine-nucleotide redox balance toward oxidation. At least 30% of the observed NADPH oxidation was independent of glutathione cycle activity and appeared chemical in nature with H2O2 itself, and not a radical metabolite, acting as oxidant. Cell exposure to H2O2 also depleted cardiomyocyte pyridine nucleotides as a consequence of enhanced utilization. The oxidative stress activated one major route of pyridine nucleotide catabolism (i.e., protein ADP-ribosylation) without acute inhibitory effect upon the other (cleavage by NAD glycohydrolase). The limited NAD sparing by metal chelators and inhibitors of ADP-ribosylation reflected pyridine nucleotide utilization for repair of single-strand DNA breaks caused by hydroxyl-like radicals formed intracellularly through iron-dependent H2O2 reduction. Cardiomyocyte NAD depletion during H2O2-induced oxidative stress was independent of cell integrity and lipid peroxidation. The NAD lost after a discrete H2O2 "pulse" was only partly replenished over a 24-h postinjury period. These data demonstrate that cardiomyocyte pyridine nucleotide metabolism is a nonperoxidative injury target that is chronically affected by H2O2 overload. Derangement of myocardial pyridine nucleotide pools due to oxidative stress may contribute to ischemic heart injury in vivo by interfering with cardiac hydrogen metabolism and redox balance.
Inhibitory effect of EGF on secretory response of rat parietal cells is associated with an induction of ODC
Authors: Ostrowski J. , Wojciechowski K. , Konturek S. J. , Butruk E.
Published: December 2017

The present studies were designed to evaluate the parietal cell acid production in response to short-time stimulation by epidermal growth factor (EGF). Studies were performed in vitro using isolated cells from rat stomachs, and acid production was indirectly determined by [14C]aminopyrine accumulation. EGF inhibited histamine-stimulated aminopyrine accumulation from standard incubation medium (K+ = 5 mM) but not from that with increased K+ concentration (K+ = 70 mM). EGF significantly stimulated ornithine decarboxylase (ODC) activity, an effect that was blocked by the specific ODC inhibitor, difluoromethylornithine (DFMO). In the presence of DFMO, EGF failed to inhibit histamine-stimulated aminopyrine uptake. Like EGF, the polyamine spermine, which is a direct product of enhanced ODC activity, also inhibited histamine-stimulated aminopyrine uptake. Unlike EGF, the spermine-induced inhibition of aminopyrine accumulation was not altered by DFMO. Thus the DFMO effect was specific to EGF. Taken together, these results are consistent with the postulate that EGF inhibits parietal cell secretory response through the induction of ODC activity and increased synthesis of polyamines.
Effect of glutathione on endothelial prostacyclin synthesis after anoxia
Authors: Hempel S. L. , Wessels D. A. , Spector A. A.
Published: December 2017

We previously observed decreased prostacyclin (PGI2) formation after reoxygenation of anoxic endothelium. In the present study, the effects of glutathione on endothelial prostaglandin (PG) H synthase activity after reoxygenation were explored. Intracellular glutathione content decreased 70% after 24 h of anoxia; reoxygenation did not produce any additional decrease in glutathione content. Intracellular glutathione was maintained in the reduced state by the endothelium even during the oxidant stress caused by reoxygenation or the addition of peroxide. Glutathione depletion produced by DL-buthionine-(S,R)-sulfoximine (BSO), 1,3-bis(chloroethyl)1-nitrosourea (BCNU), or incubation in a sulfhydryl-free medium resulted in increased sensitivity of PGH synthase to the effects of added H2O2. However, glutathione depletion resulting from BSO or culture in sulfhydryl-free medium during anoxia did not increase the sensitivity of PGH synthase to reoxygenation. In addition, anoxia did not make the endothelium more sensitive to H2O2. Glutathione peroxidase and glutathione reductase activities were preserved after anoxia-reoxygenation. When glutathione reductase was inhibited with BCNU during reoxygenation, PGI2 release was decreased further. These findings demonstrate that, although anoxia decreases endothelial glutathione content, the endothelium is able to utilize its remaining glutathione to protect against additional oxidant stress because glutathione peroxidase and glutathione reductase retain their activity.
Single-channel characteristics of a purified bovine renal amiloride-sensitive Na+ channel in planar lipid bilayers
Authors: Oh Y. , Benos D. J.
Published: December 2017

We have purified an amiloride-inhibitable Na+ channel protein from bovine renal papillae using ion-exchange and immunoaffinity chromatography. In the present study, these purified Na+ channels were reconstituted into planar lipid bilayers, and their single-channel characteristics were studied. We observed both large- and small-conductance Na(+)-selective ion channels in planar lipid bilayers. Single-channel conductance for the large- and small-conductance channels saturated as a function of Na+ concentration. These relations could be fitted by a simple Langmuir isotherm with a Michaelis constant of 55 and 45 mM and a maximum open-state conductance of 56 or 8.4 pS, respectively. Both channels were perfectly cation selective, with a Na(+)-to-K+ permeability ratio of 6.7:1 for the large channel and 7.8:1 for the small channel, and their open single-channel current-voltage relations were linear when bathed with symmetrical Na+ solutions. The percent open time of the reconstituted large or small channels varied between 10 and 50% or 1 and 20%, respectively. After application of amiloride, both the large- and small-conductance Na+ channels were inhibited in a dose-dependent manner.
cAMP levels in fast- and slow-twitch skeletal muscle after an acute bout of aerobic exercise
Authors: Sheldon A. , Booth F. W. , Kirby C. R.
Published: December 2017

The present study examined whether exercise duration was associated with elevated and/or sustained elevations of postexercise adenosine 3',5'-cyclic monophosphate (cAMP) by measuring cAMP levels in skeletal muscle for up to 4 h after acute exercise bouts of durations that are known to either produce (60 min) or not produce (10 min) mitochondrial proliferation after chronic training. Treadmill-acclimatized, but untrained, rats were run at 22 m/min for 0 (control), 10, or 60 min and were killed at various postexercise (0, 0.5, 1, 2, and 4 h) time points. Fast-twitch white and red (quadriceps) and slow-twitch (soleus) muscles were quickly excised, frozen in liquid nitrogen, and assayed for cAMP with a commercial kit. Unexpectedly, cAMP contents in all three muscles were similar to control (nonexercise) at most (21 of 30) time points after a single 10- or 60-min run. Values at 9 of 30 time points were significantly different from control (P < 0.05); i.e., 3 time points were significantly higher than control and 6 were significantly less than control. These data suggest that the cAMP concentration of untrained skeletal muscle after a single bout of endurance-type exercise is not, by itself, associated with exercise duration.
Role of local Ca2+ domains in activation of Ca(2+)-induced Ca2+ release in crayfish muscle fibers
Authors: Gyorke S. , Palade P.
Published: December 2017

Simultaneous measurements were made of crayfish muscle Ca2+ currents (ICa) and the intracellular Ca2+ transients they elicit due to Ca(2+)-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR). Ca2+ concentration ([Ca2+]) elevations produced by Ca2+ entry via ICa were much more effective in triggering CICR than were ongoing release or homogeneous elevations of Ca2+ produced by photolysis of caged Ca2+. This suggests that [Ca2+] gradients exist when Ca2+ is elevated by ICa and that, during Ca2+ entry, [Ca2+] at the activation site of the release channels must be much greater than spatially averaged [Ca2+] reported by the indicator. Analysis of voltage dependencies of ICa inactivation and SR Ca2+ release suggest that both Ca(2+)-dependent processes are controlled by ICa via the nearest T tubule Ca2+ channel rather than by total ICa entry. The contribution of SR Ca2+ release to ICa inactivation studied with a two-pulse protocol was less than predicted if Ca2+ derived from SR Ca2+ release and from T tubule Ca2+ channels have equal access to the Ca2+ binding site controlling ICa inactivation. These results can be explained in terms of a scheme where sites for release activation and ICa inactivation are located in the same junctional gap subdomain, closer to the cytoplasmic mouth of the T tubule Ca2+ channel than to the cytoplasmic mouth of the SR Ca2+ release channels. Such a scheme could provide an explanation for the graded nature and selective control of CICR in this preparation as well as in vertebrate cardiac muscle.
Intracellular free Na+ in resting and activated A7r5 vascular smooth muscle cells
Authors: Borin M. L. , Goldman W. F. , Blaustein M. P.
Published: December 2017

Regulation of intracellular Na+ ([Na+]i) in cultured vascular smooth muscle cells (A7r5 line) was studied with Na(+)-sensitive fluorescent dye sodium-binding benzofuran isophthalate. Digital imaging microscopy was used to study single-cell fluorescence. Na+ was distributed uniformly in cytoplasm and nucleus; mean Na+ concentration in resting cells was 4.4 +/- 0.3 mM in cytoplasmic areas ([Na+]cyt) and 4.5 +/- 0.4 mM in nuclear areas ([Na+]n). Na+ pump inhibition and cell activation evoked uniform changes in [Na+]cyt and [Na+]n. Inhibition of Na+ pump with 1 mM ouabain or K(+)-free medium caused a rise in [Na+]cyt; in the latter case, [Na+]cyt fell rapidly when external K+ was later restored. Exposure to Ca(2+)-free medium also caused [Na+]cyt to rise; this effect was augmented by Na+ pump inhibition and was reversed by 10(-5) M verapamil or nitrendipine or by restoration of external Ca2+. The implication is that this Na+ entry in absence of external Ca2+ is mediated by Ca2+ channels. Activation by 10(-9) M arginine vasopressin (AVP) and 10(-6) M serotonin (5-HT) caused [Na+]cyt to increase, but response to 5-HT was small (0.6 mM on average) and transient, whereas response to AVP was larger (2.4 mM on average) and was maintained as long as AVP was present (to 20 min). AVP and, to a much smaller extent, 5-HT stimulated Na+ influx; this could be detected when Na+ pump was inhibited by ouabain. Both AVP and 5-HT activated the Na+ pump, as detected by ouabain-sensitive decrease in [Na+]cyt when Na+ influx was inhibited. Agonist-evoked increases in [Na+]cyt were dependent on a rise in cytosolic Ca2+ concentration ([Ca2+]cyt); these [Na+]cyt responses were abolished by prolonged exposure to Ca(2+)-free media, when cytoplasmic Ca2+ was chelated with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, or when Ca2+ mobilization was blocked with thapsigargin. Raising [Ca2+]cyt with 40 mM K+ or with thapsigargin did not increases in [Na+]cyt. We conclude that 1) AVP- and 5-HT-evoked increases in [Na+]cyt are agonist specific and depend on the balance between stimulated Na+ influx and efflux; 2) AVP and 5-HT activate the Na+ pump; this is, at least in part, independent of agonist-induced rise in [Na+]cyt; and 3) a rise in [Ca2+]cyt is necessary but not sufficient to trigger agonist-evoked rise in [Na+]i.
ANP receptors in neurons and astrocytes from spontaneously hypertensive rat brain
Authors: Tang W. , Paulding W. R. , Sumners C.
Published: December 2017

In this study we compared the levels and responsiveness of atrial natriuretic peptide (ANP) receptors in neuronal and astrocyte glial cultures from spontaneously hypertensive (SH) and normotensive (Wistar-Kyoto: WKY) rat brain. Both neuronal and astrocyte glial cultures from the hypothalamus and brain stem of 1-day-old SH and WKY rats display specific high-affinity binding sites for 125I-labeled ANP. The presence of a large population of ANP-C receptors in each type of culture is indicated by the strong competition of 125I-ANP binding by the ring-deleted analogue of ANP [C-ANF-(4-23)]. In neuronal cultures from both strains, C-type natriuretic peptide (CNP-22) was the most effective natriuretic peptide in stimulating guanosine 3',5'-cyclic monophosphate (cGMP) levels, suggesting the presence of ANP-B receptors in these cells. By contrast, ANP was the most effective stimulator of cGMP levels in SH and WKY rat astrocyte glial cultures, suggesting the presence of ANP-A receptors. Here, we have determined that there is a decrease in the maximum binding capacity for 125I-ANP-specific binding in both SH rat neuronal and astrocyte glial cultures compared with their respective control cells. The stimulatory effects of CNP-22 on cGMP levels in SH rat neurons and of ANP on cGMP levels in SH rat astrocytes were significantly reduced compared with their respective WKY rat cultures. Our data suggest that the lower number of ANP receptors in SH rat neuronal and astrocyte glial cultures includes a reduction in the guanylate cyclase-coupled ANP receptors.
Cell volume regulation in human neutrophils: 2-(aminomethyl)phenols as Cl- channel inhibitors
Authors: Simchowitz L. , Textor J. A. , Cragoe E. J.
Published: December 2017

When subjected to hypotonic stress, human peripheral neutrophils initially swell due to rapid water entry and thereafter recover toward the normal cell size (approximately 330 microns 3). Neutrophils do not behave as perfect osmometers: when resuspended in half-isotonic medium (150 mosM), they swell by only approximately 40% rather than doubling in size as predicted for ideal behavior. As with lymphocytes, restoration to the normal cell size involves the net loss of K+ and Cl- from the cytosol through independent conductance pathways. Volume regulation is sensitive to 0.4-1 mM of quinine, UK-5099, 3,5-diiodosalicylate (DISA), MK-473 (an indanyloxyacetate derivative), and to MK-447 [a 2-(aminomethyl)phenol]. From correlation of drug effects on the time course of cell volume recovery and the associated volume-activated 86Rb+ and 36Cl- fluxes, it was evident that quinine blocked only K+ channels, whereas MK-447 acted as a selective inhibitor of Cl- channels. In contrast, UK-5099, DISA, and MK-473 were nonspecific in that the compounds displayed comparable suppressive effects on all three parameters. Structure-activity relationships in the MK-447 series revealed the critical elements of the molecule responsible for drug potency. In particular, the importance of the neighboring ionizable 1-hydroxyl and 2-aminomethyl groups and the formation of secondary ring structures for biological activity is emphasized. The most potent derivative thus far identified, termed analogue A [inhibitor constant (Ki) approximately 16 microM], had a potency approximately sixfold greater than that of the parent compound (Ki approximately 90 microM). These findings define the mechanism of action of a relatively new class of agents that behave as inhibitors of swelling-activated Cl- channels in these cells.
Effect of the thiol-oxidizing agent diamide on NH2Cl-induced rat colonic electrolyte secretion
Authors: Tamai H. , Kachur J. F. , Grisham M. B. , Musch M. W. , Chang E. B. , Gaginella T. S.
Published: December 2017

The granulocyte-derived oxidant, monochloramine (NH2Cl), is known to stimulate chloride ion secretion in rat distal colonic mucosa mounted in Ussing chambers, through mechanisms that are sensitive and insensitive to tetrodotoxin (TTX). The possible role of intracellular thiols, in the mechanism of action of NH2Cl as a secretagogue, was evaluated with the thiol-oxidizing agent diamide and by measuring tissue sulfhydryl levels in response to NH2Cl. Serosal exposure to the antioxidant glutathione (0.25 mM), 5 min before NH2Cl (50 microM) addition, decreased the maximal effect of 50 microM NH2Cl on short-circuit current (Isc). The NH2Cl-stimulated increase in Isc was not affected by mucosal amiloride (5 microM). Pretreatment with 0.1 mM diamide shortened the lag period before the increase in Isc in response to NH2Cl, but it did not affect the maximal increase in Isc. Although TTX (0.5 microM) increased the lag time for achievement of the maximal Isc response to NH2Cl, the neurotoxin did not inhibit the effect of diamide, suggesting that diamide acts primarily on the nonneural component of NH2Cl-stimulated secretion. Incubation of colonic mucosa with NH2Cl, with or without diamide, decreased cellular acid-soluble sulfhydryl concentrations. Taken together, the results support a role for epithelial cell thiols in NH2Cl-stimulated electrolyte secretion by the rat colon.
Multiple modulation of Na-dependent Pi uptake by cellular Ca in MDCK cells
Authors: Escoubet B. , Garestier M. C. , Le Grimellec C. , Amiel C.
Published: December 2017

The events accounting for the adaptation of the sodium-dependent phosphate cotransport (Na-Pi) to phosphate deprivation other than genomic regulation remain unknown. The involvement of changes in intracellular calcium concentration was investigated in Madin-Darby canine kidney (MDCK) cells. Calcium concentration was decreased by 15 h of phosphate deprivation (-24 to -35%) or low-calcium medium (calcium deprivation) (-45%), or 8-(N,N'-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB8) (-32%). Calcium deprivation stimulated Na-Pi (2-fold at 1 h and up to 15 h) by increasing the affinity for phosphate. Combined calcium and phosphate deprivation had more than additive effects on phosphate uptake. The effect of a 15-h calcium deprivation, but not of a 2-h one, was dependent on gene transcription and protein synthesis. TMB8 stimulated phosphate uptake similarly to phosphate deprivation (increase in maximum velocity dependent on gene transcription). The ionophore A23187 decreased basal Na-Pi as well as its stimulation by phosphate or calcium deprivation or by TMB8. Calcium deprivation stimulated (3.2-fold increase) the sodium-coupled alanine transport, whereas phosphate deprivation and TMB8 did not. We conclude that 1) phosphate deprivation decreases intracellular calcium concentration, 2) low intracellular calcium concentration is instrumental in the stimulation by prolonged calcium or phosphate deprivation of Na-Pi, and 3) phosphate or calcium deprivation modulates Na-Pi through different cellular pathways.
Vasopressin and protein kinase A activate G protein-sensitive epithelial Na+ channels
Authors: Prat A. G. , Ausiello D. A. , Cantiello H. F.
Published: December 2017

To determine the molecular steps involved in the vasopressin-induced renal Na+ reabsorption, the patch-clamp technique was utilized to study the role of this hormone in the regulation of apical Na+ channels in renal epithelial A6 cells. Addition of arginine vasopressin (AVP) induced and/or enhanced Na+ channel activity within 5 min of addition under cell-attached conditions. The AVP-induced channel activity was a reflection of both an increase in the average apparent channel number (0.2-1.7) and the percent open time (2-56%). Addition of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, the adenosine 3',5'-cyclic monophosphate (cAMP) analogues, 8-(4-chlorophenylthio)-cAMP and 8-bromo-cAMP, or forskolin elicited a comparable effect to that of AVP. The induced channels had similar properties to Na+ channels previously reported, including a channel conductance of 9 pS, Na(+)-to-K+ selectivity of 3-5:1, and high amiloride sensitivity. The cAMP-dependent protein kinase A (PKA) in the presence of ATP induced and/or enhanced Na+ channel activity in excised inside-out patches with a change in average apparent channel number and percent open probability similar to those observed with either AVP or cAMP analogues in intact cells. Addition of activated pertussis toxin (100 ng/ml) completely blocked the AVP- or PKA-induced Na+ channel activity in excised inside-out patches, whereas incubation of intact cells with the toxin completely prevented the effect of both activators. The data indicate that AVP mediates its effect through a cAMP-dependent pathway involving PKA activation whose target is the G protein pathway that regulates apical epithelial Na+ channel activity.
Conditionally immortalized intestinal epithelial cells: novel approach for study of differentiated enterocytes
Authors: Paul E. C. , Hochman J. , Quaroni A.
Published: December 2017

Clonal cell lines have been established from primary fetal rat intestinal epithelial cells by stable transfection with plasmids containing either the simian virus 40 (SV40) large T-antigen gene under the control of the heavy metal inducible metallothionein promoter (pMTWt) or the thermolabile SV40 T-antigen gene under the control of the SV40 early promoter (pZipSVtsa58). pMTWt-transfected cells produced sufficient T-antigen to allow them to proliferate both when the metallothionein promoter was induced and uninduced. No differences were observed in the pattern of intestinal epithelial markers expressed when the cells were cultured in the presence or absence of inducing agent (zinc). In contrast, fetal rat intestinal epithelial cells transfected with pZipSVtsa58 were immortalized conditionally; cells proliferated at 32 degrees C but ceased to proliferate between 48 and 72 h of culture at 39 degrees C. Four of these cell lines were characterized in detail; they showed microvilli and tight junctions as well as dome formation and expressed functional and biochemical markers of intestinal epithelial cells, including keratins 8, 19, and 21, aminopeptidase N, and dipeptidyl peptidase IV. One cell line, 2/4/A1, expressed in addition a low level of lactase and sucrase-isomaltase. The amount and/or activity of some of these markers changed during the switch from the proliferative to the nonproliferative state (switch from culture at 32 to 39 degrees C), resulting in a more differentiated phenotype and mimicking similar changes taking place during intestinal epithelial cell differentiation in vivo.
cGMP-dependent protein kinase activates Ca-activated K channels in cerebral artery smooth muscle cells
Authors: Robertson B. E. , Schubert R. , Hescheler J. , Nelson M. T.
Published: December 2017

Guanosine-3',5'-cyclic monophosphate (cGMP)-dependent protein kinase (cGMP-PK) plays a central role in the mediation of the vasodilator response to nitric oxide (NO) and other nitrovasodilators. It is unclear whether cGMP-PK affects calcium-activated potassium channels (KCa channels) or any other type of ion channel in smooth muscle. We provide here the first direct evidence that cGMP-PK can activate KCa channels in arterial smooth muscle cells. We demonstrate that NO and a membrane-permeable analogue of cGMP can activate KCa channels in on-cell patches approximately twofold. Furthermore, cGMP-PK, in the presence of ATP and cGMP added directly to the intracellular surface of inside-out patches, increases channel activity by approximately eightfold. These results suggest that cGMP-PK-mediated activation of KCa channels may contribute to the actions of NO and other nitrovasodilators.
Ezrin-calpain I interactions in gastric parietal cells
Authors: Yao X. , Thibodeau A. , Forte J. G.
Published: December 2017

Gastric ezrin, a membrane-cytoskeletal linker with sequence homology to talin and erythrocyte band 4.1, has been associated with the remodeling of parietal cell apical membrane that occurs with adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase stimulation. Here we examine the interrelationship between parietal cell ezrin and Ca(2+)-dependent protease activity. Addition of Ca2+ to sonicated gastric gland preparations rendered a relatively selective proteolysis of the 80-kDa ezrin, accompanied by the appearance of a 55-kDa breakdown product. Ca(2+)-dependent proteolysis of ezrin was blocked by E64, a cysteine protease inhibitor, or calpastatin, indicating calpain as the responsible protease. Degradation of ezrin in intact gastric glands was achieved by varying extracellular [Ca2+] and [ionomycin]. Ezrin degradation in situ was rapid and relatively selective, although Ca(2+)-dependent degradation of some spectrin-like bands was also observed. The effect of activated calpain I on parietal cell function was assessed by probing the secretory response to histamine stimulation using [14C]aminopyrine uptake, along with parallel measurements of calpain activity, over a wide range of ionomycin. Activation of calpain, as evidenced by loss of parietal cell ezrin, was correlated with decreased AP uptake by stimulated gastric glands, supporting a role for ezrin in the oxyntic secretory process. The calpain-ezrin interaction established here, and the similarities of calpain with talin and erythrocyte band 4.1, suggest a common feature to this family of ezrin/band 4.1/talin proteins that have been implicated in membrane-cytoskeletal association.
Stimulation of the slow calcium current in bullfrog skeletal muscle fibers by cAMP and cGMP
Authors: Kokate T. G. , Heiny J. A. , Sperelakis N.
Published: December 2017

The effects of adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) on slow calcium currents (ICa) were investigated using the Vaseline-gap voltage-clamp technique in bullfrog skeletal muscle cut fibers. Both cAMP and cGMP induced a pronounced increase in the amplitude of ICa when applied to the cut ends of fibers. Both cyclic nucleotides also decreased time to peak current at all membrane potentials. The current-voltage relationship was shifted toward more negative potentials by cAMP as well as cGMP. The potentiating effects of cAMP and cGMP on ICa were additive. 8-Bromo analogues of both nucleotides had similar effects on ICa. The beta-adrenergic agonist isoproterenol, applied extracellularly, also produced an increase in the amplitude of ICa and produced a leftward shift in the current-voltage relationship. These results suggest that both cAMP and cGMP modulate calcium slow channels in bullfrog skeletal muscle fibers, causing stimulation of the ICa. The effect of cyclic nucleotides on ICa in bullfrog skeletal muscle contrasts with that in mammalian cardiac muscle, in which the same nucleotides produce opposite effects on the slow ICa, i.e., in cardiac muscle cAMP stimulates, and cGMP inhibits, the slow ICa.
Alpha-, beta-MHC mRNA quantification in adult cardiomyocytes by in situhybridization: effect of thyroid hormone
Authors: Dubus I. , Mercadier A. , Lucas O. , Contard F. , Nallet O. , Oliviero P. , Rappaport L. , Samuel J. L.
Published: December 2017

Cardiac myocytes isolated from adult rats and cultured for up to 5 days in a defined serum- and 3,5,3'-triiodothyronine-(T3) free medium were processed for in situ hybridization using [35S]cRNA probes specific for alpha- or beta-myosin heavy chain (MHC) mRNAs. A computer-assisted image analysis system was used to quantitate the hybridization signals within individual myocytes (100 cells/experimental point). The method was validated by comparison with dot-blot quantitation. The mean alpha-MHC mRNA density per cell decreased by 50% (P < 0.01) after 2 days in culture and remained stable thereafter, whereas the relative amount of beta-MHC mRNA did not increase until day 5. Addition of 10(-12) M T3 to the culture medium for 2 or 3 days was sufficient to maintain alpha-MHC mRNA levels similar to the day 0 values, whereas 10(-9) M T3 was necessary to completely inhibit beta-MHC mRNA expression. The independent analysis of myocytes exhibiting different morphological phenotypes with time in culture demonstrated that rounded myocytes contain relatively more alpha-MHC mRNA and were as sensitive to T3 as their rod-shaped counterparts. Their beta-MHC RNA content was similar to that found in rod-shaped cells and was still depressed by T3. In conclusion, we show that 1) physiological doses of T3 are sufficient to maintain in vitro a MHC phenotype close to that observed in vivo in adult, 2) the dose responsiveness of adult myocytes to T3 differs from that reported in neonatal myocytes, and 3) the alpha-MHC mRNA content and the T3 sensitivity of spheroidal myocytes imply that there is no alteration in their state of maturation.
Regulation by phosphorylation of purified epithelial Na+ channels in planar lipid bilayers
Authors: Oh Y. , Smith P. R. , Bradford A. L. , Keeton D. , Benos D. J.
Published: December 2017

To determine the mechanism by which vasopressin increases apical membrane Na+ entry, we evaluated whether or not this hormone could recruit Na+ channels from a subapical membrane pool using specific polyclonal antibodies raised against high amiloride affinity bovine renal papillary Na+ channels. We also studied the effect of protein kinase A (PKA)-mediated phosphorylation on single-channel activity of highly purified Na+ channels incorporated into planar lipid bilayer membranes. PKA induced a significant increase in open-channel probability (Po) with no change in single-channel conductance. As shown previously and reconfirmed in the present work, PKA catalyzed the phosphorylation of a single subunit of this Na+ channel protein, namely, a 300-kDa polypeptide. On the other hand, protein kinase C, in combination with diacylglycerol, Ca2+, and phosphatidylserine, phosphorylated both the 130- and 55-kDa subunits of this purified Na+ channel, with a concomitant decrease in Po of both untreated and previously PKA-treated channels. We also found, in expression studies conducted in confluent monolayers of amphibian renal A6 cells, that vasopressin did not induce the apical insertion of new channel proteins. These observations support the hypothesis that vasopressin increases the apical Na+ permeability by activating Na+ channels already resident in the apical membrane by a direct phosphorylation mechanism rather than by cytoplasmic recruitment of latent Na+ channels.
Effects of atrial natriuretic peptide and cGMP on uptake of IgG complexes by macrophages
Authors: Mattana J. , Singhal P. C.
Published: December 2017

Macromolecular handling by macrophages and glomerular mesangial cells may be important in the development of renal injury. We undertook the present study to determine whether atrial natriuretic peptide (ANP), a particulate guanylate cyclase stimulator, plays a direct role in uptake of immunoglobulin G (IgG) complexes by macrophages. Macrophages incubated with ANP at 10(-5) and 10(-6) M showed significantly suppressed uptake of IgG complexes compared with control. Macrophage uptake of IgG complexes was also significantly suppressed by the soluble guanylate cyclase stimulator sodium nitroprusside. Dibutyryl guanosine 3',5'-cyclic monophosphate and dibutyryl adenosine 3',5'-cyclic monophosphate both significantly suppressed IgG complex uptake as well. ANP was found to significantly enhance macrophage guanosine 3',5'-cyclic monophosphate (cGMP) levels compared with control cells, and this effect was antagonized by angiotensin II. Angiotensin II significantly enhanced uptake of IgG complexes and suppressed macrophage adenosine 3',5'-cyclic monophosphate synthesis, and both effects were antagonized by coincubation with ANP. These results suggest that ANP modulates uptake of IgG complexes by macrophages and that this effect may be mediated via cGMP.
Differentiation of Na(+)-K+ pumps of low-K+ sheep red blood cells is promoted by Lp membrane antigens
Authors: Xu Z. C. , Dunham P. B. , Dyer B. , Blostein R.
Published: December 2017

Na(+)-K+ pumps of red blood cells from sheep of the low-K+ (LK) phenotype undergo differentiation during circulation, manifested in part by a striking increase in sensitivity to inhibition by intracellular K+ (Ki). Pumps of red blood cells from sheep from the allelic phenotype, high K+ (HK), do not undergo this type of maturation. The hypothesis was tested that the Lp antigen, found on LK but not HK cells, is responsible for the maturation of LK pumps. Lp antigens have been shown to inhibit LK pumps because anti-Lp antibody stimulates the pumps by relieving inhibition by the antigen. Lp antigens were recently shown to be molecular entities separate from Na(+)-K+ pumps [Xu, Z.-C., P. Dunham, J. Munzer, J. Silvius, and R. Blostein. Am. J. Physiol. 263 (Cell Physiol. 32): C1007-C1014, 1992]. The test of the hypothesis was to modify the Lp antigens of immature LK red blood cells with two kinds of treatments, anti-Lp antibody and trypsinization (which cleaves Lp), and to observe the effects of these treatments on maturation of pumps during culture of the cells in vitro. Both of these treatments prevented the maturation of the kinetics of the pumps to the Ki-sensitive pattern, supporting the hypothesis that interaction of the pumps with Lp antigens is responsible for the maturation of the pumps. Strong supportive evidence came from experiments on Na(+)-K+ pumps from rat kidney delivered into immature LK sheep red blood cells by microsome fusion.(ABSTRACT TRUNCATED AT 250 WORDS)
Change in K+ current of HeLa cells with progression of the cell cycle studied by patch-clamp technique
Authors: Takahashi A. , Yamaguchi H. , Miyamoto H.
Published: December 2017

The K+ channel of HeLa S3 cells in metaphase was analyzed by inside-out and whole cell patch-clamp techniques. The channel had the characteristics of strong inward rectification, small conductance (22 pS at -100 mV), and dependence on intracellular Ca2+. We investigated the cell cycle dependency of the channel, using cells synchronized by harvesting them at the mitotic stage. The cell capacitance increased gradually with increases in the cell volume toward the S phase. The inward K+ currents through the channel at fixed membrane potentials were highest in early G1 and then decreased with time to a minimum in the S phase, increasing again in the M phase. The permeabilities at fixed membrane potentials were also highest in early G1, decreased to minima in the S phase, and increased again toward the next mitosis. In contrast, mean amplitude and the open probability of the single channel at a fixed membrane potential (-60 mV) did not change significantly during the cell cycle. Therefore the capacitance increases with progression of the cell cycle, whereas the permeability decreases from early G1 to an apparent minimum in the S phase. These changes may be caused by cell cycle-dependent changes in the number of channels.
Perinatal enhancement of cardiac myofibrillar creatine kinase activity without change in enzyme Km
Authors: Dowell R. T. , Fu M. C.
Published: December 2017

Myofibrillar creatine kinase (CK) serves as one microcompartment of the phosphorylcreatine shuttle by providing ATP as substrate for adenosinetriphosphatase (ATPase). During perinatal heart development, augmentations of myofibrillar ATPase and CK occur in concert with increased contractile performance. The maximal reaction velocity (Vmax) for CK doubles during development in both intact native myofibril and enzyme extracted from myofibril. The absence of alterations in ADP and creatine phosphate substrate Michaelis constants (Km), isoenzyme composition, or total number of -SH groups suggests active site function (Vmax) is influenced indirectly via a subunit domain effect on enzyme conformation.
Polylysine activates smooth muscle myosin ATPase activity via induction of a 10S to 6S transition
Authors: Szymanski P. T. , Ferguson D. G. , Paul R. J.
Published: December 2017

Polylysine (10-13 kDa) stimulates contraction in smooth muscle skinned fibers and activates actomyosin adenosinetriphosphatase (ATPase) activity in the absence of myosin light chain phosphorylation [P. T. Szymanski and R. J. Paul. Adv. Exp. Med. 304: 363-368, 1991; P. T. Szymanski, J. D. Strauss, G. Doerman, J. DiSalvo, and R. J. Paul. Am J. Physiol. 262 (Cell Physiol. 31): C1445-C1455, 1992]. To provide further information on the mechanism of polylysine action on contractility in smooth muscle, we investigated its effect on ATPase activity and conformation of purified gizzard myosin. We report here that polylysine directly stimulates myosin ATPase activity in a concentration-dependent manner. This stimulation could be completely abolished with the addition of heparin, a negatively charged heteropolysaccharide. Polylysine (10 microM) increases myosin ATPase activity to a level similar to that of myosin phosphorylation. Addition of 10 microM polylysine to phosphorylated myosin [with myosin light chain kinase and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), to approximately 1.9 mol P/mol myosin], however, did not further stimulate ATPase activity. At 0.2 M KCl (the salt concentration at which myosin exists primary in the 10S form), the addition of polylysine increases myosin ATPase activity to a level comparable to that of untreated myosin in 0.3 M KCl. These changes parallel the increase in solution viscosity elicited by polylysine. These results suggest that polylysine induces a transition in myosin conformation from the 10S to the 6S form, and this was confirmed by electron microscopy.(ABSTRACT TRUNCATED AT 250 WORDS)
Hyperosmotic stress stimulates tissue plasminogen activator expression by a PKC-independent pathway
Authors: Levin E. G. , Santell L. , Saljooque F.
Published: December 2017

Shear, stretch, and the generation of oxygen radicals stimulate increases in tissue plasminogen activator (t-PA) mRNA levels and antigen production, suggesting that environmental stress may regulate t-PA gene expression. We have examined whether t-PA production is also responsive to a hyperosmotic environment. Endothelial and HeLa cells were treated with hyperosmotic medium, and t-PA mRNA and antigen secretion were measured. Endothelial cells incubated in hyperosmotic medium showed a dose-dependent decrease in cell volume and a 1.9 +/- 0.3- and 3.7 +/- 0.9-fold increase in t-PA secretion at 425 and 485 mosmol/kgH2O, respectively. HeLa cells showed a 3.3 +/- 0.6- and 5.1 +/- 1.2-fold increase at the same osmolalities. Increased secretion began between 8 and 16 h and continued through 24 h. Cultures returned to isosmotic medium after 8 h of treatment continued to release 98.1 +/- 7% of the maximum levels of t-PA for the next 16 h, despite the reversal of other responses to hyperosmotic environment. t-PA mRNA levels also increased between 8 and 16 h to five times control levels but returned to baseline by 24 h. No change in intracellular Ca2+ concentration, inositol 1,4,5-trisphosphate, or diacylglycerol content was detected, suggesting that a different intracellular signal pathway may be involved in the response to hyperosmolar stimulus. Thus environmental stress may be a general stimulatory signal through which t-PA production can be induced.