bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of rabbit aortic Ca(2+)-activated K+ channels by pinacidil, cromakalim, and glibenclamide
Authors: Gelband G. H. , McCullough J. R.
Published: December 2017

Rabbit aortic smooth muscle microsomes were isolated and large-conductance Ca(2+)-activated K+ (BK) channels incorporated into planar lipid bilayers. The selectivity sequence and relative permeability ratios for monovalent cations was K+ (1.0) > Rb+ (0.68) > NH4+ (0.14) >> Na+, Cs+ (< 0.05). Application of pinacidil or cromakalim (0.05-10 microM) shifted the probability of opening (Po)-voltage relationship in the hyperpolarizing direction. The concentrations of pinacidil and cromakalim required to increase Po 50% of the maximum value at -40 mV were 0.96 +/- 0.04 and 0.52 +/- 0.03 microM, respectively. Neither pinacidil nor cromakalim altered the voltage sensitivity of the channel (11-13 mV/e-fold change in Po). Kinetic analysis of data at -40 mV demonstrated that pinacidil (1 microM) decreased the length of time the channel dwelled in its long-closed state by 50% from 173 +/- 50 to 86 +/- 19 ms. No significant change was observed for the open time constant (20 ms). Glibenclamide (10 microM) had no effect on Po of BK channels. However, glibenclamide reversed the pinacidil- or cromakalim-stimulated increase in Po of BK channels. These data suggest that both cromakalim and pinacidil increased the probability of opening of single rabbit aortic large-conductance Ca(2+)-activated K+ channels and that this channel modulation may contribute to the vasorelaxant properties of these drugs.
Responses to sphingosine-1-phosphate in X. laevis oocytes: similarities with lysophosphatidic acid signaling
Authors: Durieux M. E. , Carlisle S. J. , Salafranca M. N. , Lynch K. R.
Published: December 2017

Sphingosine-1-phosphate (S1P, 50 microM) induces inward currents in Xenopus laevis oocytes voltage clamped at -70 mV. The currents are Ca(2+)-activated Cl- currents, as shown by a reversal potential of -20 mV and absence of the response after intracellular injection of ethylene glycolbis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA; 10 mM). The response is nearly indistinguishable from that to the related compound lysophosphatidic acid (LPA), and complete cross-desensitization occurs between LPA and S1P responses. Both the LPA and S1P responses are inhibited by suramin (2 mM) and dithiothreitol (5 mM). These responses appear mediated by a specific membrane receptor, since intracellular injection of S1P (5 microM) does not induce currents, and sphingosine and the related compounds sphingosylphosphorylcholine and N,N-dimethylsphingosine, all at 100 microM, neither induce currents nor block the response to S1P. HEK-293 and COS-1 cells respond with intracellular Ca2+ release to both 50 microM S1P and 10 microM LPA; K-562 cells do not. No cross-desensitization was noted in the responsive cells. Our findings indicate that S1P and LPA might act through the same mechanism, probably a membrane receptor.
Regulation of cardiac L-type Ca channels in planar lipid bilayers by G proteins and protein phosphorylation
Authors: Wang Y. , Townsend C. , Rosenberg R. L.
Published: December 2017

We have studied the effects of activated G proteins (Gs alpha and Gi1 alpha), adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA), and okadaic acid on L-type Ca channels incorporated from porcine ventricular sarcolemma into planar lipid bilayers. Channel activity evoked by membrane depolarizations diminished to extremely low levels within 2 min of incorporation (channel "rundown"). When Gs alpha [activated with guanosine 5'-O-(3-thiotriphosphate)] was present in the intracellular chamber, the initial level of channel activity was increased and rundown was delayed, so that channel activity was sustained for longer times after incorporation. The effect was specific for activated Gs alpha; activated Gi1 alpha, heat-denatured, activated Gs alpha, and unactivated Gs alpha did not augment channel activity. Activated Gi1 alpha inhibited the stimulation of Ca channel activity by Gs alpha. Treatment of the sarcolemmal membranes with PKA and Mg-ATP also increased the initial channel open probability and delayed their rundown. Addition of intracellular Gs alpha to PKA-treated channels increased the initial level of activity above that seen with PKA or Gs alpha alone, suggesting different nonocclusive pathways for the channel stimulation. This was also supported by the observation that activated Gi1 alpha had no effect on PKA-treated channels. Okadaic acid (100 nM) increased the level of Ca channel activity, suggesting that dephosphorylation by endogenous phosphatases participated in the downregulation of the channels in cell-free membranes.(ABSTRACT TRUNCATED AT 250 WORDS)
Novobiocin forms cation-permeable ion channels in rat fetal distal lung epithelium
Authors: O'Brodovich H. , Wang X. , Li C. , Rafii B. , Correa J. , Bear C.
Published: December 2017

The antibiotic novobiocin has been previously reported to increase Na+ transport in frog skin, presumably by attenuation of Na+ self-inhibition of Na+ channels. To determine whether novobiocin had similar effects and utilized a similar mechanism in mammalian Na(+)-transporting tissues, we studied its effect on ion transport by primary cultures of fetal distal lung epithelium (FDLE) cultured from 20-day gestationally aged rats (term = 22 days). Novobiocin (10 mM) increased short-circuit current and markedly decreased the resistance in FDLE monolayers mounted in Ussing chambers. Fura-2 single-cell studies showed that novobiocin increased intracellular Ca2+ concentration and that this resulted from extracellular sources. Nystatin-perforated patch-clamp techniques demonstrated that novobiocin increased nonrectifying cation whole cell currents without inducing detectable anion currents. Novobiocin created nonrectifying monovalent cation-selective channels in lipid bilayers. These studies demonstrated that novobiocin affects the bioelectric properties of Na+ transporting lung epithelium and that this likely occurs by the formation of ion-permeant channels in their lipid membranes.
Whole cell Cl- currents in human neutrophils induced by cell swelling
Authors: Stoddard J. S. , Steinbach J. H. , Simchowitz L.
Published: December 2017

The properties of the conductive Cl- transport pathway underlying regulatory volume decrease (RVD) in human neutrophils were investigated using the whole cell patch-clamp technique. Cell swelling was induced during whole cell recordings by making the patch pipette solution hyperosmotic (approximately 20%) relative to the bath by addition of sucrose. Immediately after establishment of the whole cell configuration, no measurable Cl- currents were evident. Over a period of several minutes the outwardly rectifying Cl- current that developed displayed no apparent voltage dependence of activation and did not inactivate with time during voltage steps over the range of -80 to +80 mV. Reduction of Cl- currents by application of suction to the interior of the pipette implied that the swelling-induced Cl- channels are activated by membrane stretch. Based on reversal potential measurements, the volume-induced Cl- conductance was found to discriminate poorly among Cl-, Br-, I-, and NO3-, to possess a finite permeability to glucuronate (Pglucuronate/PCl approximately 0.1) and to be impermeable to cations. Single-channel conductance was estimated to be 1.5 pS from analysis of the variance of membrane current fluctuations. The activated Cl- currents were blocked by 100 microM of the compound MK-447 analogue A (inhibitor constant Ki = 37 microM) and by 200 microM 3,5-diiodosalicylate, 500 microM 4-acetamido-4'-iodothiocyanostilbene-2,2'-disulfonic acid, and 200 microM UK-5099. These results suggest that the initial event triggering RVD in neutrophils may be activation of stretch sensitive Cl- channels in the plasma membrane.
Regulation of human neutrophil degranulation by LY-83583 and L-arginine: role of cGMP-dependent protein kinase
Authors: Wyatt T. A. , Lincoln T. M. , Pryzwansky K. B.
Published: December 2017

The effects of guanosine 3',5'-cyclic monophosphate (cGMP) on the secretory response of activated human neutrophils were investigated using LY-83583, an inhibitor of soluble guanylate cyclase, and L-arginine, the precursor of nitric oxide formation. A 30% release of myeloperoxidase (MPO) and lactoferrin (LF) from the primary and specific granules, respectively, was detected by enzyme-linked immunosorbent assay in adhered neutrophils stimulated with 0.1 microM N-formyl-methionyl-leucyl-phenylalanine (FMLP) or 20 microM A-23187. LY-83583 (100 microM) inhibited the release of both LF and MPO after stimulation with FMLP or A-23187. Conversely, preincubation of neutrophils with 0.5 mM L-arginine augmented the release of LF and MPO in FMLP- and A-23187-stimulated cells. Concurrent with the increase in the degranulation response was an elevation of cGMP levels in L-arginine-treated cells, while stimulated cGMP levels were reduced in LY-83583-treated cells. Furthermore, cGMP-dependent protein kinase (G-kinase) activity was reduced in LY-83583-treated cells, as determined by the delay in G-kinase translocation to intermediate filaments and the inhibition of vimentin phosphorylation. Degranulation, elevation of cGMP levels, and targeting of G-kinase were also dependent on the concentration of A-23187 or FMLP. These data suggest that activators of neutrophil degranulation mediate this response through a cGMP-dependent protein kinase mechanism.
Differentiation-dependent expression of carbonic anhydrase II and III in 3T3 adipocytes
Authors: Lynch C. J. , Hazen S. A. , Horetsky R. L. , Carter N. D. , Dodgson S. J.
Published: December 2017

Carbonic anhydrase (CA) was examined in two adipocyte cell lines, 3T3-L1 and 3T3-F442A. Both CA III and non-CA III activities, measured by 18O mass spectrometry, were present in 3T3-L1 and 3T3-F442A adipocytes; however, no CA activity was detected in 3T3 preadipocytes of either line. These observations were supported by immunoblot experiments employing CA III and CA II isoform-specific antisera. CA III, a major protein in rodent and murine adipocytes, and CA II, another isoform known to be present in adipose tissue, were observed only in the differentiated 3T3 adipocytes. The differentiation-dependent expression of these isozymes may imply an adipocyte-related role for CA. Compared with cultures maintained in the absence of insulin, 3T3 adipocytes maintained in the presence of insulin exhibited 65-90% lower concentrations of CA III. CA II was unaffected. This negative effect of insulin on CA III may explain the metabolic regulation of adipose CA III observed in vivo. After media changes, 3T3 adipocyte cultures rapidly lower media pH, which in turn lowers the bicarbonate/CO2 of bicarbonate/CO2-buffered media. Cultures maintained at low pH displayed 50-90% lower concentrations of CA II and CA III. Similarly, cultures maintained in a low bicarbonate/CO2 media (GibCO2-I medium containing 1 mM bicarbonate under an atmosphere of 100% humidified air) displayed 30-50% lower CA II and CA III concentrations. Thus CA II and CA III concentrations are influenced by pH and bicarbonate/CO2. Neither effect, the pH or the GibCO2-I media effect, was associated with changes in the concentration of pyruvate carboxylase or ATP citrate lyase (2 markers of adipocyte differentiation). Because the regulation by pH and bicarbonate/CO2 may be relatively selective for CA in adipocytes, a simple method for reducing the concentration/activity of CA in 3T3 adipocytes is described that may be a useful tool for studies on the physiological role of the enzyme.
Nonselective cation and Cl channels in luminal membrane of the marginal cell
Authors: Sunose H. , Ikeda K. , Saito Y. , Nishiyama A. , Takasaka T.
Published: December 2017

Single-channel currents of the luminal membrane of marginal cells dissected from the guinea pig cochlea were investigated using the patch-clamp technique. Nonselective cation channels having a linear conductance of 27 pS were activated by depolarization, cytoplasmic Ca2+, and cytoplasmic acidification. Cytoplasmic ATP inactivated the channel. A mixture of 3-isobutyl-1-methylxanthine and forskolin activated a small-conductance Cl channel in the cell-attached mode. On excision in the inside-out mode, the Cl channel was inactivated, but it was reactivated by a cytoplasmic catalytic subunit of protein kinase A with ATP. This Cl channel had a linear conductance of 12 pS, and its activity was little affected by voltage. The sequence of permeation by anions was Br- > Cl > I-. The Cl channel blocker diphenylamine-2-carboxylic acid (3 mM) completely blocked the channel, but 5-nitro-2-(3-phenylpropylamino)-benzoic acid (50 microM) blocked it only partially. The above-mentioned characteristics are similar to those of the well-demonstrated Cl- channel, cystic fibrosis transmembrane regulator.
A myogenic regulatory gene, qmf1, is expressed by adult myonuclei after injury
Authors: Eppley Z. A. , Kim J. , Russell B.
Published: December 2017

Myogenic regulatory factors (MRFs) induce differentiation in developing muscle. We examined the role of MRFs in the repair of adult muscle using a model of stretch-induced injury in 5-wk-old chickens. The anterior latissimus dorsi muscle was stretched by loading the wing with 10% of body weight, while the contralateral muscle served as a control. At various intervals (0.5-72 h), chickens were killed by CO2 asphyxiation and the muscles were frozen. Slot hybridizations showed that the onset of high qmf1 expression occurred as early as 0.5 h, which was before regenerative processes involving satellite cell proliferation were observed. Maximal qmf1 expression varied among animals from 3 to 16 h and returned to control levels by 72 h. Within a muscle, in situ hybridization showed that maximal qmf1 expression varied spatially with > 60% of the nuclei within active fascicles being positive. We interpret this high percentage to mean that many of the nuclei of preexisting muscle fibers must be expressing qmf1. The expression of the protooncogene c-myc (presumably by proliferating cells such as satellite cells, fibroblasts, and capillary epithelial cells) and the MRF qmf1 (by myoblasts and adult muscle nuclei) are among the early molecular responses of injured muscle. We conclude that myogenic regulatory factors are not permanently repressed after embryonic development and that derepression plays a role in the repair of terminally differentiated myofibers.
Interrelationship between cell pH and cell calcium in rat inner medullary collecting duct cells
Authors: Slotki I. , Schwartz J. H. , Alexander E. A.
Published: December 2017

In this study we investigated the interrelationship between cell pH (pHi) and cell calcium (Cai) in cultured inner medullary collecting duct cells of the rat. Confluent monolayers were made quiescent by incubation for 24 h in Dulbecco's modified Eagle's medium supplemented with 0.1% serum before study. Changes in pHi and Cai were measured with the fluorescent probes 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein and fura 2. In nominally bicarbonate-free media containing 110 mM Na+ and 1 mM Cai, cell acidification to pH 6.70 increased Cai from 122 +/- 24 to 243 +/- 33 nM. In the absence of bath calcium, acidification increased Cai from 90 +/- 7 to 144 +/- 13 nM. An increase of pHi to 7.6 reduced Cai almost to baseline. Cell acidification increased inositol trisphosphate (IP3) production, and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester, an IP3 antagonist, partially inhibited the rise in Cai. Elevation of Cai resulted in a sustained cell alkalinization from 7.32 +/- 0.02 to 7.58 +/- 0.04. When Cai was reduced, pHi fell to 7.25 +/- 0.01. We conclude that Cai and pHi participate in a feedback loop that modulates changes in each respective parameter.
Contraction history modulates isotonic shortening velocity in smooth muscle
Authors: Gunst S. J. , Wu M. F. , Smith D. D.
Published: December 2017

The effect of contraction history on the isotonic shortening velocity of canine tracheal smooth muscle was investigated. Muscles were contracted isometrically for 20 s at initial lengths of L(o) (length of maximal active force), 85% L(o), or 70% L(o) using electrical field stimulation. Muscles were then allowed to shorten isotonically under different afterloads either with or without first being subjected to a step decrease in length to 70% L(o). Instantaneous velocities were plotted against instantaneous muscle length during isotonic shortening. Regardless of protocol, the velocity at any muscle length during shortening was lower when the muscle was initially activated at a longer length. The isotonic shortening velocity decreased progressively during shortening at a nearly linear rate with respect to instantaneous muscle length under all conditions. Results suggest that a longer muscle length at the time of activation leads to the development of higher loads on the contractile element during subsequent shortening, resulting in a slower shortening velocity. This plasticity of the force-velocity relationship may result from cytostructural reorganization of the smooth muscle cells in response to contractile activation at different muscle lengths.
Sarcoplasmic reticulum Ca(2+)-ATPase and ryanodine receptor in cold-acclimated ducklings and thermogenesis
Authors: Dumonteil E. , Barre H. , Meissner G.
Published: December 2017

In birds, prolonged cold exposure induces the development of a nonshivering thermogenesis (NST) of muscular origin. NST is characterized by an increased heat production, which may be achieved by an increased ATP-dependent cycling of Ca2+ between the sarcoplasmic reticulum (SR) and cytosolic compartments in muscle. In this study, the effects of prolonged cold exposure on SR function were assessed by determining the contents of the SR Ca(2+)-ATPase and Ca2+ release channel (ryanodine receptor) in the gastrocnemius muscle of ducklings (Cairina moschata) kept at thermoneutrality (25 degrees C) or cold acclimated (4 degrees C, 5 wk). Measurement of oxalate-supported 45Ca2+ uptake by whole muscle homogenates revealed that the SR Ca(2+)-ATPase activity, and fraction of vesicles containing a ryanodine-sensitive Ca2+ release channel were increased by 30-50% in response to prolonged cold exposure. Sodium dodecyl sulfate-polyacrylamide gel and immunoblot analysis, 45Ca2+ uptake, Ca(2+)-ATPase activity and [3H]ryanodine binding measurements with unfractionated and "heavy" SR membrane fractions also indicated an elevated Ca(2+)-ATPase and Ca2+ release channel content in cold-acclimated ducklings. These results showed that the contents of two components directly involved in Ca2+ cycling by the SR are increased by cold acclimation, and we suggest that this is related to NST.
Functional expression of cAMP-dependent and independent urea transporters in Xenopus oocytes
Authors: Hasegawa H. , Verkman A. S.
Published: December 2017

Facilitated transport of urea by the inner medullary collecting duct in kidney is important for the urinary concentrating mechanism. To examine the nature and tissue distribution of urea transporters, mRNA was isolated from different tissues and expressed in Xenopus oocytes. [14C]urea and [3H]methylglucose uptake were measured at 21 degrees C at 64 h after microinjection of mRNA. Relative urea uptake in oocytes injected with 50 ng of unfractionated mRNA was (n = 6-42): 1.0 (water-injected control), 1.0 +/- 0.3 (human kidney cortex), 2.9 +/- 0.5 (rat kidney papilla), 2.5 +/- 0.5 (human kidney papilla), 2.7 +/- 0.3 (rat liver), 1.1 +/- 0.3 (rat brain), 1.2 +/- 0.3 (rat muscle), and 2.6 +/- 0.3 (rabbit reticulocyte). Urea uptake was inhibited to near control values by 0.2 mM phloretin and 0.2 mM p-chloromercuribenzenesulfonate (pCMBS) in oocytes injected with mRNA from kidney medulla, liver, and reticulocyte; phloretin and pCMBS had no effect in control oocytes and oocytes injected with mRNA from kidney cortex, brain, and muscle. Urea uptake was strongly increased in oocytes injected with kidney medulla mRNA (4.4-fold over control) by a 5-min preincubation with the adenosine 3',5'-cyclic monophosphate (cAMP) agonist adenosine-3',5'-cyclic monophosphorothioate (Sp-cAMPS) or a mixture of CPT-cAMP, forskolin, and 3-isobutyl-1-methylxanthine; cAMP agonists did not affect urea uptake in oocytes expressing the reticulocyte and liver urea transporters. As an internal control, (phloretin inhibitable) glucose uptake was enhanced in all oocytes (up to 5-fold greater than control), and was not affected by pCMBS and the cAMP agonists.(ABSTRACT TRUNCATED AT 250 WORDS)
Physiological programmed cell death in thymocytes is induced by physical stress (exercise)
Authors: Concordet J. P. , Ferry A.
Published: December 2017

Thymic involution occurs in young adult male Wistar rats that have performed two runs to exhaustion (RTE) on a treadmill, separated by a 24-h rest period, but not after a single RTE. We were interested in determining whether programmed cell death (or apoptosis) is responsible for the corresponding decrease in T-cell numbers in the thymus. DNA fragmentation, which is an early feature of apoptosis and easily detected by agarose gel electrophoresis, was found in rat thymocytes after the second RTE (the duration of 1 RTE was approximately 5 h). It was also detected after a single RTE or after 2.5 h of running only, and the levels of DNA fragmentation were always roughly similar. In addition, DNA fragmentation was decreased in RU-486 vs. vehicle-treated rats that had run for 2.5 h. These results indicate that physical stress induces glucocorticoid receptor-mediated apoptosis of rat thymocytes. Because apoptosis is induced to similar levels during mild and severe physical stresses, some additional events must be associated to provoke thymic involution.
Nitric oxide synthesis is impaired in glutathione-depleted human umbilical vein endothelial cells
Authors: Ghigo D. , Alessio P. , Foco A. , Bussolino F. , Costamagna C. , Heller R. , Garbarino G. , Pescarmona G. P. , Bosia A.
Published: December 2017

Human endothelial cells cultured from umbilical vein (HUVEC) were tested for their ability to synthesize nitric oxide (NO), which has been identified as an endothelium-derived relaxing factor. The synthesis of this free radical (detected as citrulline, which is produced stoichiometrically with NO from arginine) in HUVEC is Ca2+ dependent, is increased sevenfold by the calcium ionophore ionomycin, and accounts for most basal and ionomycin-induced guanosine 3',5'-cyclic monophosphate (cGMP) production. Loading of cells with reduced glutathione (GSH), but not with N-(2-mercaptopropionyl)- glycine (MPG), led to increased citrulline production, both basally and after ionomycin stimulation. When the cells were depleted of GSH by incubation with 1-chloro-2,4-dinitrobenzene (CDNB), citrulline synthesis and cGMP production were inhibited in a concentration-dependent way. CDNB was not cytotoxic and did not inhibit cGMP increase elicited by sodium nitroprusside; cell loading with GSH (but not with MPG) relieved the block of citrulline synthesis. These results suggest that GSH is necessary in HUVEC for NO synthesis rather than for the NO effect on guanylate cyclase.
Autocrine control of wound repair by insulin-like growth factor I in cultured endothelial cells
Authors: Taylor W. R. , Alexander R. W.
Published: December 2017

The repair process of the vascular endothelium is modulated by growth factors from both endogenous (within the vessel wall) and exogenous (blood borne) sources. We utilized a tissue culture model of endothelial wounding to gain further insight into the potential autocrine control of proliferation during wound repair. Cultured porcine aortic endothelial monolayers were mechanically wounded by passing a 7-mm sterile glass rod over the surface of the culture. Proliferation at the wound edge was quantified using [3H]thymidine autoradiography. In wounded cultures incubated in media supplemented with 10% fetal calf serum, 81 +/- 2% of the nuclei at the wound edge were labeled. When the cultures were incubated in serum-free media, proliferation at the wound edge was only slightly diminished with 65 +/- 3% (P < 0.05) of the cells labeled. These findings raise the possibility that there is a significant contribution from autocrine growth factors to endothelial wound repair. To evaluate the potential role of insulin-like growth factor I (IGF-I) in the wound repair process, we used a radioimmunoassay to measure IGF-I secretion. Wounded cultures exhibited a 187 +/- 58% increase in IGF-I production when compared with nonwounded cultures (P < 0.05). To determine the extent to which endogenous IGF-I mediates the proliferative response of endothelial cell monolayers to wounding, wounded cultures were incubated with inactivating concentrations of IGF-I antibody. When IGF-I antibody was present in the culture media, only 26 +/- 3% of the nuclei at the wound edge were labeled with [3H]thymidine (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
Biphasic effects of carbachol on stimulated cAMP accumulation in mouse parotid acini
Authors: Watson E. L. , Jacobson K. L. , DiJulio D. H. , Dowd F. J.
Published: December 2017

Carbachol (0.1-10 microM) augmented the isoproterenol-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation by approximately 50% in mouse parotid acini; at carbachol concentrations > 10 microM the stimulatory trend was reduced. These effects were time dependent. In the presence of 3-isobutyl-1-methylxanthine (IBMX), the overall response to carbachol was an inhibition of the isoproterenol response. Pretreatment of acini with pertussis toxin failed to reverse this inhibition, suggesting that the effects of carbachol were not related to effects on the GTP binding protein, Gi. A-23187 mimicked the effects of carbachol on isoproterenol-stimulated cAMP accumulation in the presence and absence of IBMX. In the presence of IBMX, carbachol failed to inhibit isoproterenol-stimulated cAMP accumulation when calcium was absent from the extracellular media and depleted from intracellular stores by thapsigargin. By contrast, in the absence of IBMX, removal of calcium abolished augmentation of isoproterenol responses by low concentrations of carbachol, whereas at higher carbachol concentrations isoproterenol responses were significantly inhibited; the time to maximal cAMP accumulation was decreased approximately eightfold. The results show that the mechanisms underlying the effects of carbachol on cAMP metabolism involve both the enzymes that synthesize and degrade cAMP.
Glycosylphosphatidylinositol-specific phospholipase D is localized in keratinocytes
Authors: Xie M. , Sesko A. M. , Low M. G.
Published: December 2017

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is abundant in mammalian plasma, but little is known of its cellular and tissue distribution. In this study frozen sections of perfused tissues from adult rats were stained with monoclonal antibodies against GPI-PLD. The most intense staining was observed in the stratified squamous epithelium of the forestomach. Staining was also observed in the esophagus, the tongue, the hard palate, and the skin but not in most other tissues including the columnar epithelium of the stomach or the lower gastrointestinal tract. GPI-PLD expression was also detected in several keratinocyte cell lines. Biochemical assays of glycosylphosphatidylinositol-degrading activity using [3H]myristate-labeled variant surface glycoprotein as substrate provided independent evidence for the presence of GPI-PLD. Expression of GPI-PLD by keratinocytes was not affected by culture in serum-free media, indicating that it does not originate by uptake of serum GPI-PLD in the media. These data suggest that keratinocytes are an important site of action of GPI-PLD and possibly a contributor to the plasma GPI-PLD pool.
Positive and negative regulation of chloride secretion in T84 cells
Authors: Barrett K. E.
Published: December 2017

This review discusses recent findings regarding the mechanisms and regulation of chloride secretion in the T84 cell line, a widely used model for the study of transepithelial chloride transport, the properties of the cystic fibrosis gene product (cystic fibrosis transmembrane conductance regulator), and the interactions of inflammatory cell types with the intestinal epithelium. First, I review the features of the chloride secretory mechanism in this cell line, both as originally described by Dharmsathaphorn and co-workers and as modified by more recent findings. Second, I cover what is known of the intracellular regulation of the secretory process. Third, I review the ways in which the cell line has been utilized to dissect pathways of immune-epithelial interactions that may contribute to the pathogenesis of inflammatory diarrhea. Finally, I suggest areas of investigation with this cell line that may prove ripe for further study. The physiological and pathophysiological implications of findings obtained with the cell line are also discussed.
Influence of cloned voltage-gated K+ channel expression on alanine transport, Rb+ uptake, and cell volume
Authors: Felipe A. , Snyders D. J. , Deal K. K. , Tamkun M. M.
Published: December 2017

Voltage-gated K+ channels are involved in regulation of action potential duration and in setting the resting membrane potential in nerve and muscle. To determine the effects of voltage-gated K+ channel expression on processes not associated with electrically excitable cells, we studied cell volume, membrane potential, Na(+)-K(+)-ATPase activity, and alanine transport after the stable expression of the Kv1.4 and Kv1.5 human K+ channels in Ltk- mouse fibroblasts (L-cells). The fast-activating noninactivating Kv1.5 channel, but not the rapidly inactivating Kv1.4 channel, prevented dexamethasone-induced increases in intracellular volume and inhibited Na(+)-K(+)-ATPase activity by 25%, as measured by 86Rb+ uptake. Alanine transport, measured separately by systems A and ASC, was lower in Kv1.5-expressing cells, indicating that the expression of this channel modified the Na(+)-dependent amino acid transport of both systems. Expression of the Kv1.4 channel did not alter alanine transport relative to wild-type or sham-transfected cells. The changes specific to Kv1.5 expression may be related to the resting membrane potential induced by this channel (-30 mV) in contrast to that measured in wild-type sham-transfected, or Kv1.4-transfected cells (-2 to 0 mV). Blocking of the Kv1.5 channel by 60 microM quinidine negated the effects of Kv1.5 expression on intracellular volume, Na(+)-K(+)-ATPase, and Na(+)-dependent alanine transport. These results indicate that delayed rectifier channels such as Kv1.5 can play a key role in the control of cell membrane potential, cell volume, Na(+)-K(+)-ATPase activity, and electrogenic alanine transport across the plasma membrane of electrically unexcitable cells.
cAMP and protein synthesis in isolated adult rat heart preparations
Authors: Bogoyevitch M. A. , Fuller S. J. , Sugden P. H.
Published: December 2017

The involvement of adenosine 3',5'-cyclic monophosphate (cAMP) in the stimulation of ventricular protein synthesis by aortic hypertension or adrenergic agonists in the adult rat heart was investigated. In either the retrogradely or anterogradely perfused heart, aortic hypertension increased protein synthesis rates by up to 19%. However, no changes in cAMP concentrations or in cAMP-dependent protein kinase activity ratios could be detected either at early (< 5 min) or late (90 min) time points. Although isoproterenol, 3-isobutyl-1-methylxanthine, or forskolin raised cAMP concentrations (by up to 4.5-fold) and cAMP-dependent protein kinase ratios (by up to 4-fold), protein synthesis rates were not increased; however, under some perfusion conditions, glucagon did stimulate protein synthesis by 25%. Epinephrine stimulated protein synthesis by up to 32%, an effect that was not prevented by propranolol. Phenylephrine also stimulated protein synthesis, an effect that was prevented by prazosin but was unaffected by yohimbine. These findings implicate the alpha 1-adrenoceptor in the regulation of cardiac protein synthesis. Because changes in adenine nucleotide concentrations were similar in hearts perfused with epinephrine or with the agents that raised cAMP, it is unlikely that adenine nucleotide depletion is responsible for the failure to observe effects of the latter group of agents on protein synthesis. Although isoproterenol or forskolin raised cAMP concentrations in isolated ventricular cardiomyocytes where ATP depletion was minimal, neither stimulated protein synthesis. alpha 1-Adrenergic agonists stimulate phosphoinositide hydrolysis in the heart (Brown, J. H., I. L. Buxton, and L. L. Brunton. Circ. Res. 57:532-537, 1985). Aortic hypertension doubled the rate of phosphoinositide hydrolysis in the perfused heart. We suggest that the phosphoinositide-linked signal transduction pathway is more likely to be involved in stimulation of cardiac protein synthesis by hypertension or adrenergic agonism than the adenylyl cyclase/cAMP-linked pathway.
Time-dependent increase in Ca2+ influx in rabbit abdominal aorta: role of Na-Ca exchange
Authors: Khoyi M. A. , Bjur R. A. , Westfall D. P.
Published: December 2017

Exposure of the rabbit abdominal aorta to the combination of high K+ and norepinephrine resulted in a time-dependent increase in the rate of 45Ca influx and 45Ca and 22Na content over that observed after stimulation with either K+ or norepinephrine alone. The increase in 45Ca influx, but not the increase in 22Na content, was extracellular Ca2+ (Cao2+) dependent. This time-dependent increase in 45Ca influx was prevented by incubating the tissue in Na(+)-free medium. Nifedipine inhibited both the initial depolarization-induced 45Ca influx and time-dependent increase in 45Ca influx and 22Na content. The effect of nifedipine on time-dependent fluxes was prevented by ouabain. Phorbol dibutyrate mimicked the effects of norepinephrine on 22Na retention and 45Ca fluxes. The effects of phorbol dibutyrate and norepinephrine were not additive. It is concluded that, in rabbit abdominal aorta, norepinephrine plus K+ causes 22Na retention (possibly through inhibition of the sodium pump) and a Cao(2+)- and intracellular Na+ (Nai+)-dependent increase in 45Ca influx. This latter effect is possibly the result of increased Nai(+)-Cao2+ exchange.
Inward rectifier K+ currents in smooth muscle cells from rat resistance-sized cerebral arteries
Authors: Quayle J. M. , McCarron J. G. , Brayden J. E. , Nelson M. T.
Published: December 2017

Inward rectifier K+ channels have been implicated in the control of membrane potential and external K(+)-induced dilations of small cerebral arteries. In the present study, whole cell K+ currents through the inward rectifier K+ channel were measured in single smooth muscle cells isolated from the posterior cerebral artery of Wistar-Kyoto rats. The whole cell K+ current-voltage relationship showed inward rectification. Inward currents were recorded negative to the K+ equilibrium potential, whereas outward currents were small. When extracellular K+ was elevated, the zero current potential shifted to the new K+ equilibrium potential, and the conductance of the inward current increased. Inward currents were reduced by external barium or cesium. Inhibition by barium and cesium increased with membrane hyperpolarization. The half-inhibition constant for barium was 2.2 microM at -60 mV, increasing e-fold for a 23-mV depolarization. We provide the first direct measurements of inward rectifier K+ currents in single smooth muscle cells and show that external barium ions are effective blockers of these currents.
Determination of volume and water permeability of plated cells from measurements of light scattering
Authors: Fischbarg J. , Li J. , Kuang K. , Echevarria M. , Iserovich P.
Published: December 2017

Measurements of cell membrane water osmotic permeabilities can be inaccurate because of the technical difficulties inherent to cell volume measurements and because of the presence of an unstirred water layer in contact with the cells. We detail here a method we have developed to quantify transient changes in cell volumes from the intensity of light scattered by cells. For this, we theorize how an unstirred layer originates in a perfusing chamber, and we calculate values for both cell membrane water osmotic permeability and unstirred layer thickness from time transient changes in scattered light. We apply a computer algorithm that finds the best correspondence between experimental data and estimated values. This is done by solving a differential equation governing cell volume changes by numerical integration (Runge-Kutta) and iterating the procedure varying the test values of osmotic permeability and unstirred layer thickness until the best fit is achieved. We exemplify this procedure with experimental results obtained in adherent cultured cells.
Ionic basis of the action potential of guinea pig gallbladder smooth muscle cells
Authors: Zhang L. , Bonev A. D. , Nelson M. T. , Mawe G. M.
Published: December 2017

Smooth muscle cells in the intact guinea pig gallbladder had a resting membrane potential of about -45 mV and had spontaneous action potentials that consisted of a rapid depolarization, a transient repolarization, a plateau phase, and a complete repolarization. These action potentials lasted approximately 570 ms and occurred at a frequency of approximately 0.4 Hz. Action potentials were abolished by the dihydropyridine (DHP)-sensitive Ca2+ channel blocker nifedipine (1.0 microM) and were enhanced by the DHP-sensitive Ca2+ channel agonist BAY K 8644 (0.5 microM). The K+ channel blockers tetraethylammonium chloride (5.0 mM) and 4-aminopyridine (4-AP; 2.0 mM) prolonged the action potential, whereas charybdotoxin (100 nM), a blocker of calcium-activated potassium channels, had no effect. Whole cell currents were characterized in enzymatically isolated smooth muscle cells from the same preparation. 4-AP, a blocker of voltage-dependent K+ channels, suppressed 70% of the outward current at 0 mV. Charybdotoxin (100 nM) reduced an additional 15% of the current at 0 mV. Single calcium-activated potassium channels were identified. The potential for half-activation of these channels, at a cytosolic Ca2+ concentration of 100 nM, was 66.8 mV. A fivefold increase in cytosolic Ca2+ resulted in a shift of the activation curve by -53 mV. External tetraethylammonium chloride (200 microM) reduced the mean single channel current by 48% at 0 mV. The whole cell outward current was abolished by replacement of intracellular K+ for Cs+. Ca2+ currents were inhibited by nifedipine and were increased by BAY K 8644. We conclude that DHP-sensitive voltage-dependent Ca2+ channels are responsible for the depolarization of the action potentials and that the repolarization is due to primarily 4-AP-sensitive K+ current.
Characterization of the beta-subunit of the H(+)-K(+)-ATPase using an inhibitory monoclonal antibody
Authors: Chow D. C. , Forte J. G.
Published: December 2017

The gastric proton pump, H(+)-K(+)-ATPase, is composed of alpha- and beta-subunits. The 95-kDa alpha-subunit has been referred to as the catalytic subunit containing sites for ATP binding and phosphorylation. The beta-subunit is a glycoprotein with a 34-kDa core peptide that has a single transmembrane segment, a small cytoplasmic NH2-terminal, and a large extracellular COOH-terminal domain with seven potential N-linked glycosylation sites. To further study the beta-subunit, we developed monoclonal antibodies that identified a 52-kDa mannose-rich glycoprotein that was deglycosylated by endoglycosidase H such that six transient intermediates were identified, as well as a 34-kDa beta-subunit core peptide. These observations suggest that the beta-subunit is synthesized as a 52-kDa glycoprotein with seven N-linked precursor high-mannose oligosaccharides that mature into complex oligosaccharides. One antibody, 2G11, inhibits the K(+)-stimulated ATP hydrolysis as well as K(+)-stimulated p-nitrophenyl phosphatase (pNPPase) activity of the H(+)-K(+)-ATPase. Kinetic studies revealed that 2G11 inhibited maximum velocity (Vmax) of ATP hydrolysis by approximately 50% with no change in the Km for K+, whereas, for pNPPase both Vmax and Km were altered. These studies demonstrate a functional role for the beta-subunit in the H(+)-K(+)-ATPase activity, especially the K(+)-induced conformational states.
Association of the hepatic IP3 receptor with the plasma membrane: relevance to mode of action
Authors: Feng L. , Kraus-Friedmann N.
Published: December 2017

Studies were carried out to characterize the interaction between inositol 1,4,5-trisphosphate (IP3) receptors and the plasma membrane fraction. Extraction of the membranes with the nonionic detergents Nonidet P-40 and Triton X-100, followed by centrifugation at 100,000 g, resulted in the doubling of the IP3 receptor in the pellets, whereas no detectable binding was found in the supernatants. These data indicate that the detergents did not solubilize the receptor, that it remained associated with membrane particles, and that it is likely to be associated with the cytoskeleton. The cytoskeleton proteins actin, ankyrin, and spectrin were identified in the plasma membrane fraction. However, comparison of the amount of these proteins in different fractions of the detergent, or otherwise treated plasma membrane fractions, showed no direct correlation between the presence of any of these proteins in the plasma membrane fraction and their ability to bind [3H]IP3. This is in contrast to the brain and T-lymphoma cells in which the IP3 receptor is attached to ankyrin (L. Y. W. Bourguigon, H. Jin, N. Iida, N. R. Brandt, and S. H. Zhang. J. Biol. Chem. 268: 6477-6486, 1993; and S. K. Joseph and S. Samanta. J. Biol. Chem 268: 6477-6486, 1993). Thus the hepatic IP3 receptor, which is different from the brain receptor, might attach to the cytoskeleton by anchoring to a different protein. Because cytochalasin D treatment of livers diminishes the ability of IP3 to raise cytosolic free Ca2+ levels, the attachment of the IP3 receptor to the cytoskeleton seems to involve an association with microfilaments.
Effects of ATP and bradykinin on endothelial cell Ca2+ homeostasis and formation of cGMP and prostacyclin
Authors: Gosink E. C. , Forsberg E. J.
Published: December 2017

ATP and bradykinin are known to activate Ca2+ release from intracellular Ca2+ pools as well as induce the influx of Ca2+ in many cell types. In adrenal medulla endothelial cells, we found that ATP and bradykinin could activate Ca2+ influx, although Ca2+ influx did not appear to be due to depletion of intracellular Ca2+ pools per se, since depletion of intracellular Ca2+ pools with thapsigargin reduced rather than enhanced both unidirectional and steady-state 45Ca2+ uptake. In addition, Ca2+ influx, activated by ATP but not bradykinin, was mostly abolished after agonist removal in cells in which intracellular Ca2+ pools had not been allowed to refill, suggesting that continued receptor occupancy was necessary for ATP to activate Ca2+ influx. The role of Ca2+ in activating guanosine 3',5'-cyclic monophosphate (cGMP) formation [a marker for nitric oxide (NO) secretion] and prostacyclin (PGI2) secretion was also studied. Bradykinin-induced cGMP and PGI2 formation and ATP-induced PGI2 formation each required Ca2+ release from intracellular Ca2+ pools, since depletion of these pools with thapsigargin inhibited their formation. In contrast, ATP-induced cGMP formation, particularly at early time points, did not appear to require either Ca2+ release or Ca2+ influx. This suggests that ATP, but not bradykinin, either induces Ca(2+)-independent NO formation or that ATP stimulates the generation of cGMP independently of NO. The latter supposition is supported by our observation that NO synthase inhibitors inhibited ATP-induced cGMP formation by at most 50%.
Histamine H2 receptor mediates keratan sulfate secretion in rabbit chondrocytes: role of cAMP
Authors: Fukuda K. , Matsumura F. , Tanaka S.
Published: December 2017

We obtained evidence for the presence of a single class of histamine H2 receptor on rabbit chondrocytes. Stimulation of these receptors with specific H2 agonists led to an inhibition of keratan sulfate secretion and rapid (15 min) accumulation of intracellular adenosine 3',5'-cyclic monophosphate (cAMP). Factors such as prostaglandin E2 and parathyroid hormone, which stimulate short-term increases in cAMP, also caused a reduction in keratan sulfate secretion. Conversely, cholera toxin and forskolin, which enhance cAMP accumulation over 48 and 4 h, respectively, as well as a continuous exposure to dibutyryl cAMP, stimulated keratan sulfate secretion. These data suggest that intracellular cAMP must be kept above a certain level for a prolonged period to stimulate keratan sulfate secretion. We conclude that inhibition of keratan sulfate secretion is coupled with activation of the H2 histamine receptor.
ATP- and UTP-induced currents in macrophages and macrophage polykaryons
Authors: Albuquerque C. , Oliveira S. M. , Coutinho-Silva R. , Oliveira-Castro G. M. , Persechini P. M.
Published: December 2017

We have investigated the currents induced by extracellular ATP (ATPo), extra-cellular UTP, and other related compounds in macrophages. At potentials of -20 to -60 mV, a typical response to ATPo puffs consists of a fast-activating inward current followed by a transient outward current. The phenomenon lasts 5-20 s, but for sustained exposure to ATP the inward current persists for up to 10 min (our longest recording time). Both currents are inhibited by Mg2+, suggesting that the phenomenon is mediated by ATP4-. The outward current can be ascribed to a Ca(2+)-dependent K+ conductance, and release of Ca2+ from intracellular stores is at least in part responsible for this current. The inward current has a reversal potential of approximately 0 mV, and it is nonspecific for monovalent cations. UTP, a nucleotide that induces an increase in the cytoplasmic concentration of free Ca2+ but does not permeabilize macrophages, and ATP-gamma-S can also induce inward and outward current similar to those described for ATP, but higher doses are required. Adenosine and AMP produce no detectable effect, whereas ADP induces a small outward current. The implications of these results to the phenomenon of ATPo-induced permeabilization of macrophage membranes to large molecules are discussed.
Specific decrease of mitochondrial thermogenic capacity in brown adipose tissue of obese SHR/N-cp rats
Authors: Atgie C. , Marette A. , Desautels M. , Tulp O. , Bukowiecki L. J.
Published: December 2017

The metabolic properties of brown adipose tissue (BAT), liver, and skeletal muscles were compared in lean and obese diabetic SHR/N-cp rats (a new model of type II diabetes) to test whether the severe insulin resistance of obese animals is specifically associated with a thermogenic defect in BAT. The respiratory response of brown adipocytes to norepinephrine and to agents bypassing the adenylate cyclase complex (dibutyryl cyclic AMP and palmitate) was decreased by two-thirds in obese rats, thereby indicating the presence of a major postreceptor defect. Significantly, total BAT cytochrome oxidase activity, uncoupling protein content, and mitochondrial guanosine 5'-diphosphate binding (3 indexes of BAT thermogenic capacity) were also decreased by two-thirds. The specific activities of these parameters expressed per total BAT mitochondrial protein were not altered either. This indicates that the total number of mitochondria per cell is decreased in BAT of obese rats. In contrast, total tissue cytochrome oxidase activity, protein content, and DNA content all increased by two to three times in the liver of obese SHR/N-cp rats, but these parameters remained unchanged in skeletal muscles (vastus lateralis and soleus). Such a remarkable liver hypertrophy may have occurred as a consequence of the persistent hyperphagia-hyperinsulinemia of obese rats that induced a hyperplasia and/or a hepatocyte polyploidization. This observation together with the fact that daily energy expenditure associated with food intake was markedly increased in obese rats (representing as much as 25% of the total energy expenditure) strongly suggests that the liver plays a major role in energy balance in these animals.(ABSTRACT TRUNCATED AT 250 WORDS)