bluuebunny/crossref_metadata_2025 huggingface dataset:
Ca2+ excitability of the ER membrane: an explanation for IP3-induced Ca2+ oscillations
Authors: Li Y. X. , Keizer J. , Stojilkovic S. S. , Rinzel J.
Published: December 2017

Recent research dealing with experiments and theoretical models of Ca2+ excitability of the endoplasmic reticulum (ER) membrane induced by inositol 1,4,5-trisphosphate (IP3) is reviewed. Ca2+ excitability refers to the ability of a small increment of cytoplasmic Ca2+ concentration ([Ca2+]i) to trigger a large [Ca2+]i pulse or oscillations. Such nonlinear regenerative behavior is conferred by the existence of IP3 channels and Ca(2+)-ATPase transporters on the ER membrane, which extends throughout the cytoplasm. Ca2+ excitability resembles the plasma membrane electrical excitability of neurons and other cells: it is driven by the ionic concentration gradient across the ER membrane (higher Ca2+ concentration inside the ER); each [Ca2+]i spike partially consumes the prestored energy that is reestablished through ATP-dependent active transport; and [Ca2+]i, the excitation variable, controls the nonlinear dynamic release rate of ER Ca2+. This review focuses on the kinetic models based on these features and on experiments dealing with the kinetic properties of [Ca2+]i-dependent gating of the IP3 receptor channel. We summarize evidence in favor of two roles for [Ca2+]i in gating the channel's opening: activation at a rapid time scale and inactivation on a slower time scale. Exploiting an analogy to the well-known Hodgkin-Huxley model for neuronal electrical excitability, we show how Ca2+ excitability of the ER membrane can be explained by these gating properties combined with the ER Ca2+ pump activity. The theory's ability to predict is illustrated by comparing calculated with experimental [Ca2+]i responses for pituitary gonadotrophs under various stimulus conditions.
Lysophosphatidic acid, serum, and hyposmolarity activate Cl- currents in corneal keratocytes
Authors: Watsky M. A.
Published: December 2017

The influence of serum, lysophosphatidic acid (LPA), and hyposmotic stress on the ion channel activity of normal and cryo-injured rabbit corneal keratocytes was investigated. Whole cell currents were examined using the amphotericin perforated-patch technique. In cells from wounded corneas, fetal bovine serum activated large, holding voltage-insensitive, fast-activating, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-, flufenamic acid-, and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB)-blockable outward currents showing inactivation at depolarized voltages. LPA activated identical currents, also only in cells from wounded corneas. Blocker and reversal potential experiments characterized the current as a Cl- currents (Icl). Lysophosphatidylcholine (10 microM) failed to activate the current. An identical current was activated by hyposmotic stimulation in cells from control and wounded corneas. Hyposmotic stimulation also activated Icl in cells from wounded corneas that were unresponsive to LPA. We conclude that serum, LPA, and hypotonic stress activate Icl in keratocytes from wounded corneas. We also conclude that LPA is a serum factor that can activate Icl and that hyposmotic activation may work through a signaling pathway separate from that of LPA.
Calcium sparks and [Ca2+]i waves in cardiac myocytes
Authors: Cheng H. , Lederer M. R. , Lederer W. J. , Cannell M. B.
Published: December 2017

Local elevations in intracellular calcium ("Ca2+ sparks") in heart muscle are elementary sarcoplasmic reticulum (SR) Ca(2+)-release events. Ca2+ sparks occur at a low rate in quiescent cells but can also be evoked by electrical stimulation of the cell to produce the cell-wide Ca2+ transient. In this study we investigate how Ca2+ sparks are related to propagating waves of elevated cytosolic Ca2+ induced by "Ca2+ overload." Single ventricular myocytes from rat were loaded with the Ca(2+)-sensitive indicator fluo 3 and imaged with a confocal microscope. After extracellular Ca2+ concentration was increased from 1 to 10 mM to produce Ca2+ overload, the frequency of spontaneous Ca2+ sparks, which occur at the t tubule/SR junction, increased approximately 4-fold, whereas the spark amplitude and spatial size increased 4.1-and 1.7-fold, respectively. In addition, a spectrum of larger subcellular events, including propagating Ca2+ waves, was observed. Ca2+ sparks were seen to occur at the majority (65%) of the sites of wave initiation. For slowly propagating Ca2+ waves, discrete Ca(2+)-release events, similar to Ca2+ sparks, were detected in the wave front. These Ca2+ sparks appeared to recruit other sparks along the wave front so that the wave progressed in a saltatory manner. We conclude that Ca2+ sparks are elementary events that can explain both the initiation and propagation of Ca2+ waves. In addition, we show that Ca2+ waves and electrically evoked Ca2+ transients have the same time course and interact with each other in a manner that is consistent with both phenomena having the same underlying mechanism(s). These results suggest that SR Ca2+ release during Ca2+ waves, like that during normal excitation-contraction coupling, results from the spatial and temporal summation of Ca2+ sparks.
Stretch modulates amiloride sensitivity and cation selectivity of sodium channels in human B lymphocytes
Authors: Achard J. M. , Bubien J. K. , Benos D. J. , Warnock D. G.
Published: December 2017

Stretch-mediated regulation of amiloride-sensitive Na+ channels was examined in Epstein-Barr virus-transformed human B lymphocytes. Cation conductances were measured using whole cell patch-clamp techniques. Stretch activation, induced by increasing the hydrostatic pressure of the bath solution, immediately and reversibly increased both inward and outward ionic conductances once a threshold of 2-5 mmH2O was reached. Ionic substitutions confirmed that stretch enhanced membrane conductivity for both Na+ and K+. Amiloride (2 microM) completely prevented the response to elevated hydrostatic pressure; however, when amiloride was applied after stretch-induced activation, the sensitivity to amiloride was dramatically decreased (inhibitor concentration that reduces whole cell current by 50% of approximately 20 microM). Evidence that the currents induced by stretch were mediated by Na+ channels was provided by the lack of response to stretch in lymphocytes from patients with Liddle's syndrome, which is caused by expression of a truncated mutant of the beta-subunit of the amiloride-sensitive Na+ channel. Pretreatment with colchicine (0.5 mM, 30 min) prevented stretch-induced activation, which shows evidence of the involvement of the cytoskeleton. These data indicate that stretch regulates the conductance of amiloride-sensitive Na+ channels in immortalized human B lymphocytes and also alters its cationic selectivity and its sensitivity to amiloride.
Parvalbumin relaxes frog skeletal muscle when sarcoplasmic reticulum Ca(2+)-ATPase is inhibited
Authors: Jiang Y. , Johnson J. D. , Rall J. A.
Published: December 2017

Inhibition of sarcoplasmic reticulum (SR) Ca(2+)-adenosinetriphosphatase (ATPase) with 2,5-di-(tert-butyl)-1,4-benzohydroquinone (TBQ) in frog skeletal muscle fibers at 10 degrees C prolonged the half time of the fall of the Ca2+ transient by 62% and twitch force by 100% and increased peak force by 120% without increasing the amplitude of the Ca2+ signal. In the presence of TBQ the rate of relaxation and the rate of fall of Ca2+ became progressively slower in a series of twitches until relaxation failed. Relaxation rate decreased with a time course (approximately 2 s-1) similar to the Mg2+ off rate from purified parvalbumin (PA; 3.6 s-1). TBQ slowed the rate of fall of Ca2+ (5-fold) and force (8-fold) in a 0.3-s tetanus so that the rate of fall of Ca2+ (approximately 2.5 s-1) was similar to the Mg2+ off rate from PA. TBQ caused a near total failure of both Ca2+ sequestration and relaxation in a 1.1-s tetanus, during which PA would be saturated with Ca2+ and could not contribute to relaxation. Thus, when the SR Ca(2+)-ATPase is inhibited, Mg(2+)-PA can sequester Ca2+ and produce relaxation at a rate that is defined by the Mg2+ off rate from PA.
Stretch overload-induced satellite cell activation in slow tonic muscle from adult and aged Japanese quail
Authors: Carson J. A. , Alway S. E.
Published: December 2017

Stretch overload-induced activation of satellite cells in the anterior latissimus dorsi (ALD) muscle was examined in full-grown adult (12 wk old) and aged (90 wk old) Japanese quail. 5'-Bromo-2'-deoxyuridine (BrdU) constant-release pellets (0.22 mg BrdU.g body wt-1.day-1) were implanted subcutaneously before weighting the left wing of each bird. Nuclei that incorporated BrdU were localized by immunohistochemistry after 1 or 2 wk of stretch overload. Total fiber number was quantified by counting all fibers in a histological cross section from the midbelly of the ALD. Aging reduced increases in ALD mass and fiber number during 2 wk of stretch overload. Fiber proliferation in the ALD of aged birds also demonstrated an altered time course. The percentage of BrdU-positive nuclei associated with muscle fibers and the percentage of fibers associated with BrdU-positive nuclei did not differ between age groups. The altered time course of new fiber formation in the ALD of aged birds during 2 wk of stretch overload does not appear to be related to the capability to activate satellite cells.
Increased synthesis of high-molecular-weight cPLA2 mediates early UV-induced PGE2 in human skin
Authors: Gresham A. , Masferrer J. , Chen X. , Leal-Khouri S. , Pentland A. P.
Published: December 2017

Ultraviolet light (UV) B-induced inflammation is characterized by dramatic increases in prostaglandin E2 (PGE2) synthesis due to enhanced arachidonate deacylation from the membrane. Therefore, the effect of UV on sythesis, mass, and distribution of the high-molecular-weight phospholipase A2 (cPLA2) in cultured human keratinocytes and human skin was studied. The 105-kDa cPLA2 was demonstrated to be the critical enzyme in UV-induced PGE2 synthesis and erythema in the first 6 h postirradiation. Immunoprecipitation of 35S-labeled protein showed cPLA2 synthesis increased three- to fourfold 6 h after irradiation. Immunoprecipitated 32P-labeled cPLA2 demonstrated phosphorylation of cPLA2 was concurrently induced, suggesting that UV also activates cPLA2. This increase in cPLA2 synthesis and activation also closely correlated with increased PGE2 synthesis and [3H]arachidonic acid release and was effectively blocked by both an S-oligonucleotide antisense to cPLA2 and methyl arachidonate fluorophosphate, a specific inhibitor of cPLA2. Biopsy and histochemical examination of erythematous sites expressed increased amounts of cPLA2 whereas nonerythematous irradiated sites did not. In contrast, cyclooxygenase-1 and -2 in cultures and skin explants were unaffected 6 h post-UV, and no change in cyclooxygenase activity was observed at this time. These results suggest that increased cPLA2 synthesis occurs only when skin is exposed to UV doses that are sufficient to cause erythema and indicate expression of cPLA2 participates in acute UV inflammation.
Cell volume in vascular smooth muscle is regulated by bumetanide-sensitive ion transport
Authors: Orlov S. N. , Tremblay J. , Hamet P.
Published: December 2017

Vascular smooth muscle cells (VSMC) exhibit shrinkage-induced bumetanide-inhibited 86Rb influx and ethylisopropylamiloride (EIPA)-inhibited 22Na influx. In this study, we examined the role of these transport pathways in volume adjustment of VSMC after isosmotic and hyperosmotic shrinkage. Cell volume was assessed by measurement of [14C]urea distribution. An initial 18-20% cell volume decrease in isosmotically shrunken VSMC was followed by a regulatory volume increase (RVI). RVI was completely abolished by bumetanide but not by EIPA. No RVI was noted in hyperosmotically shrunken VSMC. The initial rate of bumetanide-inhibited 86Rb influx was two- to threefold higher in isosmotically shrunken VSMC than with hyperosmotic shrinkage. Hyperosmotic shrinkage of VSMC was accompanied by a three- to fourfold increase in the rate of bumetanide-inhibited 86Rb efflux, whereas isosmotic shrinkage augmented this component by only 20-30%. In contrast to bumetanide-inhibited 86Rb influx, isosmotic shrinkage slightly increased the rate of EIPA-sensitive 22Na influx. Hyperosmotic shrinkage led to transient activation of EIPA-inhibited 22Na influx, which was completely abolished in 15 min. Activation of adenosine 3',5'-cyclic monophosphate (cAMP) signaling with isoproterenol arborized VSMC and decreased their volume by approximately 15%. A similar volume decrease was seen in VSMC treated with the microfilament-disrupting compound, cytochalasin B. The isoproterenol-induced cell volume decrease was prolonged by the addition of bumetanide. Unlike isoproterenol, agents that raise intracellular Ca2+ (A-23187 and angiotensin II) did not modify VSMC volume. Thus our data demonstrate involvement of cAMP signaling in the regulating of VSMC volume and a key role of bumetanide-inhibited ion transport in the RVI after isosmotically induced shrinkage.
Activation of a novel metabolic gene regulatory pathway by chronic stimulation of skeletal muscle
Authors: Cresci S. , Wright L. D. , Spratt J. A. , Briggs F. N. , Kelly D. P.
Published: December 2017

To determine whether expression of a nuclear gene encoding a mitochondrial fatty acid oxidation enzyme is regulated in parallel with skeletal muscle fibre-type-specific energy substrate preference, expression of the gene encoding medium-chain acyl-CoA dehydrogenase (MCAD) was delineated in canine latissimus dorsi muscle subjected to chronic motor nerve stimulation. In predominantly fast-twitch canine latissimus dorsi muscle, MCAD mRNA levels were regulated by chronic stimulation in a biphasic pattern. During the 1st wk of stimulation, steady-state MCAD mRNA levels decreased to 50% of unstimulated levels. MCAD mRNA levels began to increase during the 3rd wk of stimulation to reach a level 3.0-fold higher than levels in unstimulated contralateral control muscle by day 70. Immunodetectable MCAD mRNA levels throughout the stimulation period. The temporal pattern and magnitude of MCAD mRNA accumulation in response to muscle stimulation was distinct from that of mRNAs encoding other enzymes known to be regulated by this stimulus, including glyceraldehyde phosphate dehydrogenase, citrate synthase, and sarcoplasmic reticulum Ca-ATPase, but paralleled the protein levels of the peroxisome proliferator-activated receptor (PPAR), an orphan member of the nuclear hormone receptor superfamily known to regulate genes encoding fatty acid oxidation enzymes in liver. The skeletal muscle expression pattern of PPAR was also similar to that of MCAD in unstimulated rat skeletal muscles with distinct fiber-type compositions. These results demonstrate that a nuclear gene encoding a mitochondrial beta-oxidation enzyme is dynamically regulated in a pattern that parallels skeletal muscle fiber-type-specific energy substrate utilization and implicate an orphan nuclear receptor transcription factor as a candidate transducer of this response.
cAMP motifs regulating transcription in the aquaporin 2 gene
Authors: Hozawa S. , Holtzman E. J. , Ausiello D. A.
Published: December 2017

Genomic clones including the 5' flanking regions of the AQP2 (aquaporin 2) gene were isolated, and the promoter region was examined by transiently transfecting a promoter-luciferase reporter fusion gene into renal cultured epithelial cells. An orientation specific promoter for the AQP2 gene was found within the proximal 3 kb of 5'-flanking region. Minimal basal promoter activity of the AQP2 gene was found within 198 bp upstream from the transcription start site by deletion analysis. Sequencing the transcriptionally active region revealed a typical TATA box, adenosine 3',5'-cyclic monophosphate (cAMP) responsive element (CRE) and three putative CCAAT boxes in the proximal 1.2-kb region. Significantly, a GATA motif, AP1, AP2, and SP1 transcriptional factor consensus sites were also found in this region. Exposure to cAMP-enhancing agents (1 nM vasopressin or 20 mM forskolin and 250 mM 3-isobutyl-1-methylxanthine) showed that these agents increased luciferase activity in a parallel fashion, suggesting that vasopressin-induced AQP2 gene transcription is mediated through increases in intracellular cAMP in at least one renal cell type, the LLC-PK1 cells. The mechanism of cAMP responsiveness of AQP2 gene transcription was further studied using a series of deletion mutants in renal epithelial cells and other cell types. The cAMP regulatory motifs were shown to exist in a 50-bp sequence between -340 and -290 (containing CRE) and a 65-bp sequence (containing an AP2 site) between -150 and the ATG start site in LLC-PK1 cells. In rat inner medullary collecting duct (IMCD) cells, the cAMP regulatory motifs also exist in a 50-bp sequence between -340 and -290 (containing CRE) and in a 10-bp sequence between -160 and -150 (containing an SP1 site). These separate regions may cooperate to confer full cAMP inducibility to the AQP2 gene in a cell-specific manner
Cytochrome P-450 metabolites mediate extracellular Ca(2+)-induced inhibition of apical K+ channels in the TAL
Authors: Wang W. H. , Lu M. , Hebert S. C.
Published: December 2017

We used the patch-clamp technique to study the effect of extracellular Ca2+ (Ca2+o) on the activity of the apical 70-pS K+ channel in the isolated split-open thick ascending limb (TAL) of the rat kidney. Raising Ca2+o from 1.1 to 5 mM reversibly reduced the activity of the 70-pS K+ channel in cell-attached patches to 16 +/- 2% of the control value within 300 s. In addition, 50 microM neomycin mimicked the effect of an increase in Ca2+o on channel activity in cell-attached patches and completely inhibited channel activity. The effect of neomycin on the channel activity in cell-attached patches is an indirect effect, since addition of 50 microM neomycin on the 70-pS K+ channel in inside-out patches reduced only the apparent amplitude of the channel current without changing channel open probability. We examined further the role of protein kinase C (PKC) and the cytochrome P-450-dependent metabolites of arachidonic acid in mediating the Ca2+o -induced inhibition of channel activity. Addition of phorbol 12-myristate 13-acetate (2 microM) reversibly blocked channel activity in cell-attached patches to 4 +/- 1% of the control value, whereas 75 nM calphostin C increased the channel activity by 115 +/- 10%. Moreover, addition of 1 nM exogenous PKC reversibly and completely inhibited the 70-pS K+ channel. However, inhibition of PKC with calphostin C (75 nM) only slightly prolonged the time course of the effect of Ca2+o on channel activity (370 +/- 40 s) and failed to abolish the inhibitory effect of 5 mM Ca2+o on channel activity in cell-attached patches, indicating that PKC was not mainly responsible for the effect of Ca2+o on channel activity. In contrast, the effect of 5 mM Ca2+o on the apical 70-pS K+ channel was completely abolished when TAL tubules were first incubated in the 17-octadecynoic acid (5 microM)-containing solution, an agent that specifically blocks cytochrome P-450 monooxygenase. In conclusion, these data indicate that Ca2+o is an important regulator of the apical 70-pS K+ channel and that a cytochrome P-450-dependent metabolite of arachidonic acid is involved in mediating this inhibitory effect.
Regulation of HSP70 by PTH: a model of gene regulation not mediated by changes in cAMP levels
Authors: Fukayama S. , Lanske B. , Guo J. , Kronenberg H. M. , Bringhurst F. R.
Published: December 2017

Parathyroid hormone (PTH) activates both adenylate cyclase and phospholipase C in target cells, and cloned PTH/PTH-related protein (PTHrP) receptor can mediate both responses when expressed in host cells such as LLC-PK1 renal epithelial cells. Because calcitonin (CT) is known to augment 70-kDa heat shock protein (HSP70) mRNA by an adenosine 3',5'-cyclic monophosphate (cAMP)-independent mechanism in LLC-PK1 cells, we examined regulation of HSP70 transcription by PTH in these cells. Like CT, human PTH-(1-34) [hPTH-(1-34); 10(-10) to 10(-7) M)] increased porcine HSP70 mRNA and human HSP70 promoter-chloramphenicol acetyltransferase (CAT) expression within 4 h in LLC-PK1 cells that stably express > or = 100,000 PTH/PTHrP receptors per cell. The effect of PTH on HSP70 mRNA was not mimicked by cAMP analogues, forskolin, phorbol esters, Ca2+ ionophores, or alpha-thrombin; was insensitive to pertussis toxin; and was not due to increased mRNA stability. The upregulation of HSP70 gene transcription by hPTH (and CT) was clearly observed even after deletion of the functional heat shock consensus element in the promoter region of the human HSP70/CAT reporter. Upregulation of HSP70 transcription via endogenous PTH receptors also was observed in the osteoblastic cell lines SaOS-2 and ROS 17/2.8. Regulation of HSP70 gene transcription by PTH may be a common cellular response to the hormone, which, in some cells, may not be mediated by activation of adenylate cyclase or protein kinase C.
Increased sensitivity to 1,25(OH)2D3 in bone from genetic hypercalciuric rats
Authors: Krieger N. S. , Stathopoulos V. M. , Bushinsky D. A.
Published: December 2017

As a model of human hypercalciuria, we have selectively inbred genetic hypercalciuric stone-forming (GHS) Sprague-Dawley rats whose mean urine calcium excretion is eight to nine times greater than that of controls. A large component of this excess urine calcium excretion is secondary to increased intestinal calcium absorption, which is not due to an elevation in serum 1,25(OH)2D3, but appears to result from an increased number of intestinal 1,25(OH)2D3 receptors (VDR). When GHS rats are fed a low-calcium diet, the hypercalciuria is only partially decreased and urine calcium excretion exceeds intake, suggesting that an additional mechanism contributing to the hypercalciuria is enhanced bone demineralization. To determine if GHS rat bones are more sensitive to exogenous 1,25(OH)2D3, we cultured calvariae from neonatal (2- to 3-day-old) GHS and control rats with or without 1,25(OH)2D3 or parathyroid hormone (PTH) for 48 h at 37 degrees C. There was significant stimulation of calcium efflux from GHS calvariae at 1 and 10 nM 1,25(OH)2D3, whereas control calvariae showed no significant response to 1,25(OH)2D3 at any concentration tested. In contrast, PTH induced similar bone resorption in control and GHS calvariae. Immunoblot analysis demonstrated a fourfold increase in the level of VDR in GHS calvariae compared with control calvariae, similar to the increased intestinal receptors described previously. There was no comparable change in VDR RNA levels as measured by slot blot analysis, suggesting the altered regulation of the VDR occurs posttranscriptionally. That both bone and intestine display an increased amount of VDR suggests that this may be a systemic disorder in the GHS rat and that enhanced bone resorption may be responsible, in part, for the hypercalciuria in the GHS rat.
Dephosphorylation of activated protein kinase C contributes to downregulation by bryostatin
Authors: Lee H. W. , Smith L. , Pettit G. R. , Bingham Smith J.
Published: December 2017

We show that bryostatin 1 (Bryo) rapidly produces an inactive, incompetent 76-kDa form of protein kinase C-alpha (PKC-alpha) in the LLC-MK2 line of renal epithelial cells. Bryo, like phorbol 12-myristate 13-acetate (PMA), acutely activated PKC, as indicated by autophosphorylation and translocation of PKC-alpha, the predominant PMA-sensitive isoform expressed by the cells. Bryo concomitantly increased the 32P labeling of 80-kDa PKC-alpha by autophosphorylation and produced a 76-kDa form of PKC-alpha that lacked detectable 32P. The 76-kDa form was in the particulate rather than the cytosolic fraction, which suggests that it was produced from activated kinase. Alkaline phosphatase treatment of immunoprecipitated PKC-alpha converted the 80-kDa form to 76 kDa, but it had no effect on the mobility of the 76-kDa form, suggesting that it was not phosphorylated. Pulse-chase labeling of PKC-alpha with [35S]Met/Cys indicated that there is a precursor-product relationship between the 80- and 76-kDa forms, respectively. Inhibition of protein synthesis had no effect on the production of 76-kDa PKC-alpha by Bryo. PMA also produced 76-kDa PKC-alpha but was less potent and efficacious than Bryo. Bryo produced a more rapid loss of 80-kDa PKC-alpha protein and total Ca(2+)- and phospholipid-dependent PKC activity than PMA. The 76-kDa form is inactive and incompetent because it lacked detectable 32P under conditions that strongly autophosphorylated the 80-kDa form. We suggest that dephosphorylation predisposes PKC to proteolysis, and greater production of the 76-kDa form explains the more efficient downregulation of the kinase by Bryo vs. PMA.
Hydrolytic and nonhydrolytic interactions in the ATP regulation of CFTR Cl- conductance
Authors: Reddy M. M. , Quinton P. M.
Published: December 2017

Previously, we showed in the native sweat duct that, in the presence of 0.1-0.5 mM ATP, nonhydrolyzable ATP analogue adenosine 5'-adenylylimidodiphosphate (AMP-PNP) can activate cystic fibrosis transmembrane conductance regulator Cl- conductance (CFTR GCl) (15). The objective of this study is to determine if 1) nonhydrolytic ATP binding alone can activate CFTR GCl after stable phosphorylation [in the presence of adenosine 5'-O-(3-thiotriphosphate) and phosphatase inhibition cocktail] of CFTR or 2) an ATP hydrolysis (in addition to phosphorylation) is required to support subsequent nonhydrolytic ATP regulation of CFTR GCl. We show that stably phosphorylated CFTR could only be activated by AMP-PNP in the presence of a small background ATP concentration. However, AMP-PNP can sustain previously activated CFTR GCl in the absence of ATP, even though Mg2+ is required for phosphorylation activation of CFTR GCl. However, once stably phosphorylated, ATP activation of CFTR GCl is independent of Mg2+. Our results show that both hydrolytic and nonhydrolytic interactions regulate CFTR GCl in vivo. Nonhydrolytic ATP interaction plays a significant role in both activation and deactivation of CFTR GCl.
Plasmin-platelet interaction involves cleavage of functional thrombin receptor
Authors: Kimura M. , Andersen T. T. , Fenton J. W. , Bahou W. F. , Aviv A.
Published: December 2017

We tested the hypothesis that the inhibition of thrombin-induced platelet activation by plasmin is mediated via the enzymatic action of plasmin on the functional thrombin receptor. We monitored the binding of the anti-thrombin receptor antibody [anti-TR-(34-46)] to platelets; this binding is sensitive to the cleavage of the thrombin receptor at amino acid residues Arg-41 to Ser-42. Plasmin inhibited anti-TR-(34-46) binding in dose- and time-dependent manners. The inactive synthetic peptide with the amino acid sequence 40-55 of the thrombin receptor (D-FPRSFLLRNPNDKYEPF) was similarly cleaved by thrombin and plasmin to an active peptide (SFLLRNPNDKYEPF) that produced robust cytosolic Ca2+ responses. At high concentrations, plasmin itself can activate platelets. We explored this effect with the use of anti-TR-(1-160). This antibody abolished the cytosolic Ca2+ responses to thrombin and to the thrombin receptor-activating peptide SFLLRN but did not attenuate the plasmin-induced cytosolic Ca2+ response. Thus plasmin inhibits thrombin-evoked platelet activation by cleaving the thrombin receptor, but the plasmin-induced cytosolic Ca2+ response is not due to the generation of the tethered peptide of the thrombin receptor.
Role of tyrosine kinase pathways in ETB receptor activation of NHE3
Authors: Chu T. S. , Tsuganezawa H. , Peng Y. , Cano A. , Yanagisawa M. , Alpern R. J.
Published: December 2017

Endothelin-1 (ET-1) binding to ETB receptors increases the activity of the apical membrane Na+/H+ antiporter (NHE3) of renal proximal tubule and cultured OKP cells. In OKPETB6 cells, a clonal cell line of OKP cells that overexpresses ETB receptors, ET-1-induced increases in Na+/H+ antiporter activity are mediated 50% by Ca2(+)-dependent pathways and 50% by tyrosine kinase pathways. ET-1 induces tyrosine phosphorylation of proteins of 68, 110, 125, 130, and 210 kDa. ET-1-induced tyrosine phosphorylation is mediated by the ETB receptor and is not dependent on increases in cell Ca2+ or protein kinase C. The 68-, 110-, 125-, and 130-kDa phosphoproteins are cytosolic, whereas the 210-kDa phosphoprotein is an integral membrane protein. Immunoprecipitation studies showed that the 68-kDa protein is paxillin and the 125-kDa protein is p125FAK (focal adhesion kinase). Cytochalasin D, which disrupts focal adhesions, prevented ET-1-induced tyrosine phosphorylation of paxillin, p110, p125FAK, and p130 but did not prevent tyrosine phosphorylation of p210 and did not prevent ET-1-induced increases in Na+/H+ antiporter activity. Thus 50% of ETB receptor-induced Na+/H+ antiporter activation is mediated by tyrosine kinase pathways, possibly involving p210. ETB receptor activation also induces tyrosine phosphorylation of focal adhesion proteins, but this is not required for antiporter activation.
Identification of muscarinic receptor subtypes in mouse parotid gland
Authors: Watson E. L. , Abel P. W. , DiJulio D. , Zeng W. , Makoid M. , Jacobson K. L. , Potter L. T. , Dowd F. J.
Published: December 2017

Immunoprecipitation of muscarinic receptors from mouse parotid membranes by specific subtype antisera showed that M3 and M1 receptors represented 75 and 15% of the total number of precipitable receptors, respectively. [N-methyl-3H]methylscopolamine (NMS) labeled a single class of high-affinity binding sites in membranes from parotid glands with a dissociation constant of 0.67 +/- 0.02 nM and a maximum binding capacity of 176 +/- 15 fmol/mg protein. Competition curves for NMS, atropine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and para-fluoro-hexahydro-sila-difenidol fit best to a one-site binding model, whereas pirenzepine and methoctramine fit best to a two-site binding model, indicating 76-90% M3 receptors. Results from the use of pirenzepine indicated that the second mouse parotid receptor subtype, unlike that of the submandibular gland, has atypical characteristics for an M1 receptor. The rank order of potency of muscarinic antagonists in inhibiting phosphoinositide turnover and biphasic effects of carbachol on isoproterenol-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation was atropine > or = 4-DAMP >> pirenzepine > AF-DX 116. A specific M1 antagonist, m1-toxin, had no effect on carbachol augmentation or inhibition of isoproterenol responses. Results suggest that M3 receptors couple to both augmentation and inhibition of stimulated cAMP levels.
Effects of aging on sarcoplasmic reticulum function and contraction duration in skeletal muscles of the rat
Authors: Narayanan N. , Jones D. L. , Xu A. , Yu J. C.
Published: December 2017

The impact of aging on the Ca2+ pump function of skeletal muscle sarcoplasmic reticulum (SR) was investigated using SR-enriched membrane vesicles isolated from the slow-twitch soleus muscle (SM) and the relatively fast-twitch gastrocnemius muscle (GM) isolated from adult (6-8 mo old) and aged (26-28 mo old) Fischer 344 rats. In addition, isometric twitch characteristics of SM and GM were determined in situ in adult and aged rats under anesthesia. The rates of ATP-supported Ca2+ uptake by SM SR was markedly lower ( approximately 50%) in the aged compared with adult at varying Ca2+ (0.11-8.24 microM) concentrations. Kinetic analysis of the data revealed age-associated decrease in maximum activity reached (Vmax) and increase in the concentration of Ca2+ giving half of Vmax. In contrast, no significant age-related difference was observed in ATP-supported Ca2+ uptake activity of GM SR. The Ca(2+)-stimulated adenosinetriphosphatase (ATPase) activities and the amount of Ca(2+)-ATPase protein did not vary significantly with aging in SM or GM SR. Also, no significant age-related difference was observed in the content of the ryanodine receptor (Ca(2+)-release channel) or the Ca2+ binding protein, calsequestrin in SM and GM SR. In isometrically contracting SM, the time to peak force, half-relaxation time, and contraction duration were significantly prolonged in the aged compared with adult, whereas there was no age-related difference in maximum developed force. None of these isometric twitch parameters differed significantly with age in the GM. These results demonstrate that the effects of aging on skeletal muscle contractile properties and SR function are muscle specific. Furthermore, the data strongly suggest that impairment in SR Ca2+ pump function, apparently due to uncoupling of ATP hydrolysis from Ca2+ transport, contributes to the age-associated slowing of relaxation in the soleus muscle.
Nitric oxide donor SIN-1 inhibits insulin release
Authors: Sjoholm A.
Published: December 2017

Preceding the onset of insulin-dependent diabetes mellitus, pancreatic islets are infiltrated by macrophages secreting interleukin-1 beta, which exerts cytotoxic and inhibitory actions on islet beta-cell insulin secretion through induction of nitric oxide (NO) synthesis. The influence of the NO donor 3-morpholinosydnonimine (SIN-1) on insulin secretion from isolated pancreatic islets in response to various secretagogues was investigated. Stimulation of insulin release evoked by glucose, phospholipase C activation with carbachol, and protein kinase C activation with phorbol ester were obtained by SIN-1, whereas the response to adenylyl cyclase activation or K(+)-induced depolarization was not affected. It is concluded that enzymes involved in glucose catabolism, phospholipase C or protein kinase C, may be targeted by NO. Reversal of SIN-1 inhibition of glucose-stimulated insulin release by dithiothreitol suggests that NO may inhibit insulin secretion partly by S-nitrosylation of thiol residues in key proteins in the stimulus-secretion coupling. These adverse effects of NO on the beta-cell stimulus-secretion coupling may be of importance for the development of the impaired insulin secretion characterizing diabetes mellitus.
IL-1 stimulates intestinal myofibroblast COX gene expression and augments activation of Cl- secretion in T84 cells
Authors: Hinterleitner T. A. , Saada J. I. , Berschneider H. M. , Powell D. W. , Valentich J. D.
Published: December 2017

Because interleukin-1 (IL-1) is an important mediator in the inflamed intestine, its effects on enterocyte-subepithelial myofibroblast (SEMF) interaction were investigated in vitro. Acutely juxtaposing T84 cells with 18Co or P2JF SEMF preincubated with IL-1 alpha significantly enhanced T84 short-circuit current (Isc) responsiveness to secretagogues in comparison to SEMF not activated by IL-1 alpha. The sensitivity of T84 cell Isc to Ca(2+)-dependent, but not adenosine 3',5'-cyclic monophosphate-dependent, secretagogues was augmented by IL-1 alpha-treated SEMF. These effects of IL-1 alpha are directly correlated with SEMF prostaglandin E2 (PGE2) production. Both IL-1 alpha augmentation of Cl secretagogue responsiveness and PGE2 formation were inhibited by IL-1 receptor antagonist. Within 5 h, IL-1 alpha stimulated a 10-fold increase in cyclooxygenase (COX)-2 steady-state mRNA levels in 18Co cells. In contrast, COX-1 message levels increased more slowly to two- to threefold above control levels after 24 h incubation. These results demonstrate that the proinflammatory cytokine IL-1 alpha accentuates intestinal SEMF augmentation of enterocyte responsiveness to Ca(2+)-dependent CI-secretagogues. PGE2 is an important mediator of SEMF-enterocyte interaction. The effects of IL-1 alpha on SEMF PGE2 productions are, at least in part, due to stimulation of COX gene expression.
Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly
Authors: Beckman J. S. , Koppenol W. H.
Published: December 2017

Nitric oxide contrasts with most intercellular messengers because it diffuses rapidly and isotropically through most tissues with little reaction but cannot be transported through the vasculature due to rapid destruction by oxyhemoglobin. The rapid diffusion of nitric oxide between cells allows it to locally integrate the responses of blood vessels to turbulence, modulate synaptic plasticity in neurons, and control the oscillatory behavior of neuronal networks. Nitric oxide is not necessarily short lived and is intrinsically no more reactive than oxygen. The reactivity of nitric oxide per se has been greatly overestimated in vitro because no drain is provided to remove nitric oxide. Nitric oxide persists in solution for several minutes in micromolar concentrations before it reacts with oxygen to form much stronger oxidants like nitrogen dioxide. Nitric oxide is removed within seconds in vivo by diffusion over 100 microns through tissues to enter red blood cells and react with oxyhemoglobin. The direct toxicity of nitric oxide is modest but is greatly enhanced by reacting with superoxide to form peroxynitrite (ONOO-). Nitric oxide is the only biological molecule produced in high enough concentrations to out-compete superoxide dismutase for superoxide. Peroxynitrite reacts relatively slowly with most biological molecules, making peroxynitrite a selective oxidant. Peroxynitrite modifies tyrosine in proteins to create nitrotyrosines, leaving a footprint detectable in vivo. Nitration of structural proteins, including neurofilaments and actin, can disrupt filament assembly with major pathological consequences. Antibodies to nitrotyrosine have revealed nitration in human atherosclerosis, myocardial ischemia, septic and distressed lung, inflammatory bowel disease, and amyotrophic lateral sclerosis.
Expression of the catalytic domain of myosin light chain kinase increases paracellular permeability
Authors: Hecht G. , Pestic L. , Nikcevic G. , Koutsouris A. , Tripuraneni J. , Lorimer D. D. , Nowak G. , Guerriero V. , Elson E. L. , Lanerolle P. D.
Published: December 2017

Contractile events resulting from phosphorylation of the 20-kDa myosin light chain (MLC20) have been implicated in the regulation of epithelial tight junction permeability. To address this question, Madin-Darby canine kidney cells were transfected with a murine leukemia retroviral vector containing DNA encoding either the catalytic domain of myosin light chain kinase (tMK) or the beta-galactosidase gene (beta-gal). Autoradiograms of sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of myosin immunoprecipitated from 32Pi-labeled transfected cells demonstrated that MLC20 phosphorylation was increased 3.1 +/- 0.9-fold in cells expressing tMK compared with cells expressing beta-gal. Phosphopeptide mapping confirmed that myosin light chain kinase was responsible for the increased MLC20 phosphorylation. Transepithelial electrical resistance, a measurement of barrier function, of tMK cell monolayers was consistently < 10% (123 +/- 20 omega.cm2) of that of monolayers comprised of wild-type cells (1,456 +/- 178 omega.cm2) or cells expressing beta-gal (1,452 +/- 174 omega.cm2). Dual 22Na+ and [3H]mannitol flux studies indicated that the decrease in resistance in tMK cells was attributable to increased paracellular flow. These data support the idea that MLC20 phosphorylation by myosin light chain kinase is involved in regulating epithelial tight junction permeability.
Continuous contractile activity induces fiber type specific expression of HSP70 in skeletal muscle
Authors: Neufer P. D. , Ordway G. A. , Hand G. A. , Shelton J. M. , Richardson J. A. , Benjamin I. J. , Williams R. S.
Published: December 2017

Continuous contractile activity of skeletal muscle elicits an early and dramatic increase in ribosomal RNA, suggesting that translational efficiency and/or capacity is enhanced during the adaptive response to increased metabolic demand. In view of the important role heat shock or stress proteins (HSPs) play as molecular chaperones during protein synthesis, we examined whether expression of the inducible 70-kDa HSP (HSP70) and/or mitochondrial 60-kDa HSP (HSP60) is altered in rabbit tibialis anterior muscle during continuous low-frequency motor nerve stimulation. Induction of the HSP70 gene was evident within 24 h after the onset of stimulation as reflected by increases in HSP70 transcription (> 20-fold) and mRNA (> 50-fold). HSP70 protein levels were significantly elevated (10- to 12-fold) after 14 and 21 days of stimulation. Mitochondrial HSP60 mRNA and protein also increased during stimulation (> 18- and > 5-fold after 21 days, respectively). In situ hybridization and immunohistochemistry coupled with myosin ATPase staining revealed that expression of HSP70 was restricted to oxidative type I and IIa fibers during the first 3 days of stimulation but shifted to primarily type II fibers after 21 days of stimulation. These findings demonstrate that induction of HSP70 during the adaptive response to chronic motor nerve stimulation proceeds from type I/IIa to type IId(x)/b fibers, suggesting that the expression of HSPs may be required to support the folding and compartmentalization of nascent proteins during the transformation process.
Sensitivity of volume-sensitive Cl- conductance in human epithelial cells to extracellular nucleotides
Authors: Tsumura T. , Oiki S. , Ueda S. , Okuma M. , Okada Y.
Published: December 2017

The sensitivity to extracellular nucleotides of volume-sensitive Cl- channel activity was investigated by whole cell and single-channel patch-clamp recordings in a human small intestinal epithelial cell line (Intestine 407) during steady osmotic swelling. Adenine nucleotides added to the bathing solution suppressed whole cell volume-sensitive Cl- currents with the potency sequence of ATP > ADP > AMP. In contrast, extracellular adenosine 3',5'-cyclic monophosphate (cAMP) at over 0.1 mM increased volume-sensitive Cl- currents in the entire voltage range examined, whereas guanosine 3',5'-cyclic monophosphate was without effect. Neither the single-channel conductance nor the open probability was affected by extracellular cAMP. Extracellular ATP (at over 30 microM), in the Mg(2+)-free form, inhibited the whole cell volume-sensitive Cl- current, preferentially in the outward direction. By exposure to extracellular ATP, the single Cl- channel current became flickery at positive potentials. These results indicate that the volume-sensitive Cl- channel in the human epithelial cell is stimulated voltage independently by extracellular cAMP but blocked voltage dependently by the Mg(2+)-free form of extracellular ATP.
Changes of actin cytoskeleton during swelling and regulatory volume decrease in cultured astrocytes
Authors: Moran J. , Sabanero M. , Meza I. , Pasantes-Morales H.
Published: December 2017

Swelling of cultured astrocytes exposed to hyposmotic medium modified the organization of the filamentous actin (F-actin) cytoskeleton, making the actin network diffuse in the cell body but concentrated at foci corresponding to the tips of the cell projections retracted by swelling. This change was reversible, and, after 2 h, the actin cytoskeleton tended to recover, and cells regained their flat and stellate shape. Cytochalasins B and D (CB and CD, respectively), which disrupt the actin cytoskeleton, did not affect regulatory volume decrease (RVD) or the swelling-activated efflux of Cl- and inositol, although 10 microM CD increased the basal efflux of taurine. The mercurial p-chloromercuribenzenesulfonate (0.5-1 mM), known to disrupt the membrane cytoskeleton in isosmotic conditions, induced a 46, 50, and 38% release of [3H]taurine, 125I, and [3H]inositol, respectively, causing cell shrinkage and retraction of the cytoskeleton. Coincidently, the swelling-stimulated release of [3H]taurine and 125I was reduced by 60 and 30%, respectively. Results of this study do not exclude the possibility that changes in the actin cytoskeleton elicited by swelling are involved in mechanisms of RVD and only indicate that the disruption caused by cytochalasins is unrelated to that process.
Induction of human myeloblastic ML-1 cell G1 arrest by suppression of K+ channel activity
Authors: Xu B. , Wilson B. A. , Lu L.
Published: December 2017

Our previous studies have shown that a voltage-gated K+ channel is highly expressed in proliferating human myeloblastic ML-1 cells and is suppressed in the early stages of 12-O-tetradecanoylphorbol-13-acetate-induced ML-1 cell differentiation. In the present study, we report that inhibition of the K+ channel activity by 4-aminopyridine (4-AP) suppressed ML-1 cell proliferation, as measured by DNA synthesis. Cell cycle mapping indicated that ML-1 cells were arrested in G1 phase after 24-h treatment with 4-AP. Blockade of ML-1 cells at the G1/S boundary of the cell cycle with aphidicolin revealed that ML-1 cells past the G1 checkpoint were capable of entering S phase and synthesizing DNA independently of the channel blockade. ML-1 cell differentiation, measured by CD14 marker protein expression, revealed that the effect of 4-AP was to cause growth arrest and that it did not cause differentiation. Dephosphorylation of retinoblastoma protein accompanied inhibition of ML-1 cell proliferation and suggested that suppression of K+ channel activity by 4-AP is associated with retinoblastoma protein-mediated G1 arrest in ML-1 cells. Moreover, we found that ML-1 cell volume increased 35 +/- 7% after 4-AP treatment, which could be an early event triggering inhibition of ML-1 cell proliferation. These findings suggest that a 4-AP-sensitive K+ channel may play an important role in the transduction of mitogenic signals in ML-1 cells.
Pathways of hepatic oxalate synthesis and their regulation
Authors: Poore R. E. , Hurst C. H. , Assimos D. G. , Holmes R. P.
Published: December 2017

Important features of hepatic oxalate synthesis remain uncertain despite its clinical significance. To clarify the terminal steps of the biosynthetic pathway and their modulation, we have examined oxalate and glyoxylate synthesis in vitro using isolated guinea pig peroxisomes and purified lactate dehydrogenase (LDH). Glycolate was rapidly oxidized to glyoxylate by isolated peroxisomes followed by a slower conversion of glyoxylate to oxalate. The glycolate oxidase (GO)-catalyzed conversion of glyoxylate to oxalate was strongly inhibited by physiological concentrations of glycolate and lactate. In contrast, the LDH-catalyzed conversion of glyoxylate to oxalate was only marginally affected by physiological concentrations of lactate and unaffected by physiological glycolate concentrations. This inhibition pattern suggests that LDH, not GO, catalyzes this conversion in vivo. Alanine inhibited oxalate synthesis by converting the bulk of the glyoxylate to glycine. On exposure to high alanine concentrations, however, inhibition was not complete and peroxisomes were able to convert sufficient glycolate to oxalate to account for daily endogenous oxalate production. NADH was a potent inhibitor of oxalate production by LDH by increasing glycolate formation from glyoxylate. Glycine was an ineffective source of glyoxylate, and an alkaline pH, a high-glycine concentration, and a prolonged incubation time were required to obtain a detectable synthesis. These results suggest that oxalate synthesis will be modulated by the metabolic state of the liver and resultant changes in NADH, lactate, and alanine levels.