bluuebunny/crossref_metadata_2025 huggingface dataset:
A role for CFTR in human autosomal dominant polycystic kidney disease
Authors: Hanaoka K. , Devuyst O. , Schwiebert E. M. , Wilson P. D. , Guggino W. B.
Published: December 2017

Human autosomal dominant polycystic kidney disease (ADPKD) is the most common lethal dominant hereditary disorder characterized by enormous renal enlargement and the development of multiple cysts originating from nephrons. We investigated the pathogenesis of cyst formation in ADPKD by using patch-clamp and immunocytochemical techniques. Adenosine 3',5'-cyclic monophosphate-activated Cl- currents are present in primary cultures of ADPKD cells and have characteristics such as a linear current-voltage relation, insensitivity to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, sensitivity to glibenclamide and diphenylamine carboxylic acid, and an anion selectivity sequence of Br- > Cl- > I- > glutamate, all of which are identical to cystic fibrosis transmembrane conductance regulator (CFTR). With the use of CFTR antibodies raised against the regulatory and first nucleotide-binding domains, CFTR was detected in primary cultures of ADPKD cells. Similar results were obtained in vivo in cyst-lining epithelial cells in ADPKD kidneys, where staining was seen associated with the apical membrane regions. These data indicate that the CFTR Cl- channel exists in apical membranes of ADPKD cells and may play an important role in cyst formation or enlargement.
Continuous measurement of 13C16O2 production from [13C]pyruvate by intact liver mitochondria: effect of HCO3-
Authors: Ono Y. , Lin L. , Storey B. T. , Taguchi Y. , Dodgson S. J. , Forster R. E.
Published: December 2017

We have measured continuously the production of mass 45 CO2(13C16O2) from 13C-labeled pyruvate in a guinea pig liver mitochondrial suspension and simultaneously the O2 consumption at 37 degrees C and pH 7.4. The reactions took place in a closed 3-ml volume, stirred, thermoregulated chamber separated from the ion source of a mass spectrometer by a gas-permeable membrane that permitted recording the mass peaks of any gas dissolved in the reaction mixture with a response time as fast as 3 s. If the pyruvate was labeled on C-2, no 13C16O2 was formed, even after 1 h, indicating that C-2 and C-3 were not metabolized in the citric acid cycle. We found that production of 13C16O2 was five times greater in the presence of 25 mM HCO3- than in its absence. A probable mechanism of this CO2/HCO3- effect is carboxylation of pyruvate to oxaloacetate, which would react with acetyl CoA to form citrate and with NADH to form malate, thus removing two major inhibitors of pyruvate dehydrogenase. We conclude that CO2/HCO3- has a potent and hitherto unappreciated regulatory effect on liver pyruvate dehydrogenase.
Modulation of pump function by mutations in the first transmembrane region of Na(+)-K(+)-ATPase alpha 1-subunit
Authors: Yamamoto S. , Askew G. R. , Heiny J. , Masaki H. , Yatani A.
Published: December 2017

The Cys in the first transmembrane region of the Na(+)-K(+)-adenosinetriphosphatase (ATPase) alpha 1-subunit has been shown to be a critical determinant of cardiac glycoside binding. To study the role of this Cys on ion transport activity, we measured pump currents in HeLa cells expressing wild-type or mutant alpha 1-subunit cDNAs. The endogenous ouabainsensitive Na(+)-K(+)-ATPase was selectively inhibited by growing the cells in 0.1 microM ouabain. A Cys-to-Tyr substituted mutant exhibited decreased sensitivity to digitoxin but not digoxin compared with wild type. The decreased affinity for digitoxin was due to a faster dissociation rate. In contrast, the Cys-to-Ala substitution did not significantly alter the sensitivity to digitoxin or digoxin. Both wild-type and mutant cells displayed marked external K(+)-dependent pump currents; however, the affinity for K+ was reduced by the mutations. The decrease in K+ affinity was due to a slower association rate. The results show that the Cys that interacts with cardiac glycosides also participates in the sensitivity of the pump to external K+.
Effects of calponin on isometric force and shortening velocity in permeabilized taenia coli smooth muscle
Authors: Obara K. , Szymanski P. T. , Tao T. , Paul R. J.
Published: December 2017

Calponin, a thin filament-associated protein, inhibits actomyosin adenosinetriphosphatase in solution and has been suggested to modulate smooth muscle contractility. We used permeabilized guinea pig taenia coli smooth muscle to investigate whether calponin can modulate actin-myosin interaction in a more organized contractile system. Fibers were permeabilized with Triton X-100 and glycerol, which permit access of large macromolecules to the contractile apparatus. For contractures elicited by Ca2+ (6.6 microM + 0.1 microM calmodulin), the recombinant alpha-isoform of chicken gizzard calponin (CaP) decreased isometric force (Fo) and unloaded shortening velocity (Vus) in a dose-dependent manner; 1 microM CaP had minimal effects on force (< 10%) but reduced Vus by approximately 50% and 10 microM CaP reduced Fo to 27% of control and Vus to near zero levels. To eliminate any effects of the binding of calmodulin by CaP and consequent inhibition of myosin light chain kinase activity, we also studied fibers activated by thiophosphorylation of the myosin regulatory light chain. Fo was only moderately inhibited, remaining at approximately 75% of control in the presence of CaP (10 microM), whereas Vus was reduced to 32% of control. A similar inhibition was obtained with a mutant (CaPcys175) that retains the ability to bind to actin. CaP phosphorylated by protein kinase C and CaPcys175 mutant labeled with 1,5-IAEDANS, which bind actin poorly, were not effective inhibitors. Our results indicate that 1) CaP more strongly inhibits Vus (approximately cross-bridge cycle rate) than Fo (approximately number of activated cross bridges) and 2) the effects of CaP are related to its binding to actin. Thus the function of CaP in regulation of smooth muscle contractility may be more strongly related to its function as a modulator of velocity, as related to the "latch state," than as an "on-off" switch.
Betaine transporter cDNA cloning and effect of osmolytes on its mRNA induction
Authors: Ferraris J. D. , Burg M. B. , Williams C. K. , Peters E. M. , Garcia-Perez A.
Published: December 2017

Cells generally adapt to long-term hyperosmolality by accumulating compatible organic osmolytes, thereby helping to normalize both volume and intracellular inorganic ion concentration. When organic osmolytes are accumulated, as in renal inner medullary cells, it is the sum of their concentrations that is theoretically important. In effect, when one organic osmolyte rises, the others generally fall to maintain their sum approximately constant. The present study addresses the mechanism controlling betaine accumulation. Hypertonicity induces accumulation of betaine, sorbitol, inositol, and other organic osmolytes in PAP-HT25 cells, a line derived from rabbit renal papilla. Hypertonicity increases the betaine transporter expression in these cells. To obtain a specific probe for betaine transporter mRNA, we cloned from PAP-HT25 cells a cDNA that encodes the full protein. We then examined the effect of betaine, sorbitol, and inositol on betaine transporter mRNA abundance. Increased accumulation of any of these three organic osmolytes reduces betaine transporter mRNA. We previously observed similar results for aldose reductase, the enzyme responsible for osmotically regulated sorbitol accumulation. We conclude that the accumulation of organic osmolytes regulates betaine transporter gene expression. Because the aldose reductase gene is controlled in a similar fashion, we surmise that the two genes share a common signal for induction.
Mechanisms of electrolyte transport across the endometrium. I. Regulation by PGF2 alpha and cAMP
Authors: Vetter A. E. , O'Grady S. M.
Published: December 2017

The purpose of this study was to characterize the transport mechanisms in endometrial epithelial cells that are responsible for regulation of Na and K concentrations in uterine luminal fluid. Porcine endometrial tissues were mounted in Ussing chambers and bathed in plasmalike Ringer solution. The mean basal short-circuit current (Isc) was 40 microA/cm2, and the mean tissue conductance was 3.6 mS/cm2. Addition of amiloride to the luminal solution inhibited 86% of the basal Isc. Concentration-response experiments using amiloride analogues showed a rank order of potency of benzamil > amiloride > 5-(N-methyl-N-isobutyl)-amiloride in blocking the Isc, with no response to ethylisopropylamiloride. Na channel immunoreactivity was localized to the apical membrane of surface epithelial cells. The Na-to-K selectivity ratio of the amiloride-sensitive Na channel was calculated to be 6.4:1. Prostaglandin (PG) F2 alpha or 8-(chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP) added to the luminal solution stimulated a twofold increase in Isc that was inhibited by pretreatment with amiloride. Experiments using both amphotericin B-permeabilized tissues and intact tissues showed that PGF2 alpha and cAMP increased Na absorption by activation of basolateral K channels. Treatment of the luminal solution with 4-aminopyridine produced an effect on Isc that was consistent with block of K secretion and a subsequent decrease in Na absorption. These experiments showed that Na and K transport are tightly coupled processes occurring under basal conditions in surface endometrial epithelial cells and that these processes are regulated by PGF2 alpha and cAMP.
Differential regulation of putative K(+)-ATPase by low-K+ diet and corticosteroids in rat distal colon and kidney
Authors: Jaisser F. , Escoubet B. , Coutry N. , Eugene E. , Bonvalet J. P. , Farman N.
Published: December 2017

K+ homeostasis depends on K+ absorption in digestive and renal epithelia. Recently, a cDNA encoding for a putative K(+)-adenosinetriphosphatase (ATPase) alpha-subunit has been characterized. We studied its expression by ribonuclease protection assay and in situ hybridization in the distal colon and the kidney of rats in various physiological states. In the distal colon of control rats, high expression of the colonic putative K(+)-ATPase mRNA was restricted to the surface epithelial cells. A low-K+ diet did not modify this expression, adrenalectomy decreased it, and aldosterone or dexamethasone treatment for 2 days restored normal levels. In the kidney of control rats, levels of K(+)-ATPase mRNA were very low. A low-K+ diet revealed a clear mRNA expression, which is consistent with a recent report [J.A. Kraut, F. Starr, G. Sachs, and M. Reuben. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F581-F587, 1995]. This expression was restricted to the outer medullary collecting duct, presumably in principal cells. Changes in corticosteroid status did not influence the renal expression. Our results, together with previous studies on K+ absorption and K(+)-ATPase activity, suggest that more than a single molecular form of K(+)-ATPase is likely to be responsible for the regulation of K+ absorption in the colon and distal nephron.
Xenobiotic transport differences in mouse mesangial cell clones expressing mdr1 and mdr3
Authors: Ernest S. , Bello-Reuss E.
Published: December 2017

P-glycoprotein (PGP), which confers multidrug resistance to cancer cells, is expressed in mouse kidney proximal tubule and mesangium. We report on the expression of PGP and its xenobiotic transport function in mesangial cells. Studies were performed in a mouse mesangial cell line (TKGM) and two cell clones. Ribonuclease protection assay and Western blot analysis demonstrated that TKGM cells expressed mdr1 and mdr3, the isoforms responsible for multidrug resistance. TKGM-F12 cells coexpressed mdr1 and mdr3 whereas TKGM-G2 cells expressed only mdr1. The drug transport function, measured by rhodamine 123 (R-123) efflux, was smaller in TKGM-F12 than in TKGM-G2 cells. The PGP substrates adriamycin, cyclosporin A, vinblastine, and verapamil inhibited R-123 transport in TKGM and TKGM-G2 cells. In the cells studied, PGP conferred some resistance to adriamycin; concomitant exposure to adriamycin with another PGP substrate impaired cell growth. The differential expression of mdr1 and mdr3 in mouse mesangial cell clones, the ability of mdr1 PGP to transport R-123, and the impairment of PGP-mediated transport in TKGM-F12 cells, coexpressing mdr1 and mdr3 products, are demonstrated. PGP may play a physiological role in mesangial cells.
Desmin gene expression in cardiac myocytes is responsive to contractile activity and stretch
Authors: Watson P. A. , Hannan R. , Carl L. L. , Giger K. E.
Published: December 2017

Experiments were performed to assess the ability of mechanical stimuli, experienced by ventricular cardiac myocytes during the progression of hypertrophic and dilated pathology, to increase the expression of desmin in cultured neonatal rat cardiac myocytes. Results indicate that both contractile activity and load due to passive stretch increase desmin content in neonatal rat cardiac myocytes through increased desmin gene transcription. Western blot analysis demonstrated that contraction induced a selective increase in desmin protein content in neonatal rat cardiac myocytes above increases observed in the content of total cellular protein. Northern blot analysis indicated that desmin mRNA content increased in response to contraction as well as to alpha-adrenergic stimulation. Desmin mRNA content also increased in cultured neonatal myocytes in response to stretch. Angiotensin II (ANG II) treatment of contracting neonatal cardiac myocytes further increased desmin mRNA content, whereas similar treatment in arrested neonatal cardiac myocytes further increased desmin mRNA content, whereas similar treatment in arrested neonatal cardiac myocytes failed to increase desmin mRNA. This contraction-dependent responsiveness to ANG II is not a function of increases in the density or relative subtype composition of ANG II receptors. Treatment of contracting neonatal rat cardiac myocytes with actinomycin D prevented increases in desmin mRNA content, suggesting regulation of transcription of the desmin gene by contraction. Nuclear run-on experiments indicate that contraction. Nuclear run-on experiments indicate that contraction increases transcription of the desmin gene in cardiac myocytes. These results are consistent with the modulation of desmin gene expression secondarily to changes in the mechanical environment that occur in cardiac tissue undergoing dilation or hypertrophy.
Contractile and metabolic effects of increased creatine kinase activity in mouse skeletal muscle
Authors: Roman B. B. , Foley J. M. , Meyer R. A. , Koretsky A. P.
Published: December 2017

The effects of increased expression of creatine kinase (CK) in skeletal muscle were studied in control and transgenic animals homozygous for expression of the B subunit of CK. CK activity was 47% higher in transgenic gastrocnemius muscle. The CK activity was distributed as follows: 45 +/- 1% MM dinner, 31 +/- 4% MB dimer, and 22 +/- 5% BB dimer. No significant differences in metabolic or contractile proteins were detected except for a 22% decrease in lactate dehydrogenase activity and a 9% decrease in adenylate kinase activity. The only significant effect in contractile activity was that the rise time of a 5-s isometric contraction was 28% faster in the transgenic muscle. 31P nuclear magnetic resonance (NMR) spectra were obtained from control and transgenic muscles during mechanical activation, and there were no NMR measurable differences detected. These results indicate that a 50% increase in CK activity due to expression of the B subunit does not have large effects on skeletal muscle metabolism or contractile function. Therefore, control muscle has sufficient CK activity to keep up with changes in cellular high-energy phosphates except during the early phase of intense contractile activity.
The spectrin-based membrane skeleton as a membrane protein-sorting machine
Authors: Beck K. A. , Nelson W. J.
Published: December 2017

Normal cell function is dependent on the existence of membrane compartments that have unique populations of membrane proteins. Sorting of membrane proteins forms the basis for the biogenesis of distinct membrane compartments. There are many examples of membrane protein-sorting events in cells, but the molecular machinery involved is poorly understood. We discuss characteristics of a putative membrane protein-sorting machine and show that the spectrin-based membrane skeleton conforms to these characteristics. The spectrin-based membrane skeleton is a submembranous, spatially limited, two-dimensional lattice that binds a subset of membrane proteins. These properties allow the membrane skeleton to facilitate the formation of distinct membrane domains and thus reveal its potential as a membrane protein-sorting machine.
Nitric oxide regulates the low-conductance K+ channel in basolateral membrane of cortical collecting duct
Authors: Lu M. , Wang W. H.
Published: December 2017

Two types of K+ channels, low conductance (28 pS) and intermediate conductance (85 pS), have been previously identified in the basolateral membrane of the cortical collecting duct (CCD) of the rat kidney (31, 32). In the present study, we used the patch-clamp technique to explore further the mechanism by which the low-conductance K+ channel is regulated. The conductance of the low-conductance K+ channel is inward rectifying, with an inward slope conductance of 30 pS between 0 and -20 mV and an outward slope conductance of 16 pS between 0 and 50 mV in symmetrical 140 mM KCl in the bath and in the pipette. This K+ channel was not sensitive to ATP (10 mM), tetraethylammonium chloride (5 mM), and quinidine (1 mM). Addition of 100 microM N omega-nitro-L-arginine methyl ester (L-NAME) or N omega-(imonoethyl)-L-ornithine (L-NIO), an inhibitor of nitric oxide synthase (NOS), completely blocked channel activity in cell-attached patches. In contrast, addition of 200 microM-D-NAME, which does not block NOS, had no effect on channel activity. The inhibitory effect of L-NAME or L-NIO was fully reversible and completely overcome by addition of exogenous nitric oxide (NO) donors, such as 10 microM S-nitroso-N-acetyl-penicillamine or sodium nitroprusside. Furthermore, addition of 100 microM 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) restored the activity of the channel when it had been inhibited by either L-NAME or L-NIO, indicating that the effect of NO on the channel activity was mediated by a cGMP-dependent pathway. In conclusion, NO plays a key role in the regulation of the basolateral 30-pS K+ channel and the effect of NO on channel activity is mediated by a cGMP-dependent pathway.
Prostaglandin production in cultured cerebral microvascular smooth muscle is serum dependent
Authors: Rich G. , Yoder E. J. , Prokuski L. , Moore S. A.
Published: December 2017

To expand the understanding of cerebrovascular eicosanoid metabolism, the ability of smooth muscle isolated from murine cerebral microvessels to produce prostaglandins (PGs) was studied in vitro. Cultures from SJL and BALB/c mice produced primarily prostaglandin E2 (PGE2) and I2 (PGI2) in response to exogenous arachidonate and calcium ionophore as well as the agonists acetylcholine and epinephrine. Subconfluent smooth muscle cultures demonstrated a two- to threefold increased capacity to produce PG compared with confluent cultures. In contrast, serum deprivation of smooth muscle caused an 80-90% diminution in both PGE2 and PGI2 production but had no effect on PG release in cerebromicrovascular endothelium. Reintroduction of serum to smooth muscle restored PG production within 6h, and the restoration was inhibited by 1 microM dexamethasone. Message for both prostaglandin H synthase (PGHS)-1 and -2 was detectable in smooth muscle grown in the presence of serum, but PGHS-2 message was not present in serum-deprived cultures. Furthermore, readdition of serum induced a massive increase in PGHS-2 mRNA with only a small increase in PGHS-1 message. The serum induction of PGHS-2 was corroborated by immunohistochemistry and Western blotting. Thus cerebromicrovascular smooth muscle may contribute significantly to the formation of PG under circumstances likely to be present during central nervous system pathologies. The induction of PGHS, particularly PGHS-2, may play a key role in this process.
Endothelin and PDGF enhance arachidonic acid release and DNA synthesis in vascular smooth muscle cells
Authors: Irons C. E. , Flynn M. A. , Mok L. M. , Reynolds E. E.
Published: December 2017

Intracellular signaling mechanisms affected by endothelin (ET), a hypertrophic agonist, and platelet-derived growth factor (PDGF)-BB, a proliferative agonist, in vascular smooth muscle cells were examined. PDGF-BB was a potent mitogen compared with untreated cultures, stimulating both [3H]thymidine incorporation and cell number. In contrast, ET was a poor mitogen, enhancing [3H]thymidine incorporation but not cell number. Simultaneous ET and PDGF-BB treatment was significantly more effective than either agonist alone at stimulating both [3H]thymidine uptake and cell number. Although either ET or PDGF-BB alone stimulated arachidonic acid release, phosphoinositide hydrolysis, protein kinase C activation, PDGF receptor phosphorylation, and mitogen-activated protein kinase activity, of these effectors, only arachidonic acid release was further enhanced by simultaneous ET and PDGF-BB treatment. These results link proliferative and hypertrophic signal transduction pathways in these cells and suggest that arachidonic acid or its metabolites mediate the observed effects of ET on PDGF-BB-stimulated vascular smooth muscle cell proliferation.
Telokin expression is mediated by a smooth muscle cell-specific promoter
Authors: Herring B. P. , Smith A. F.
Published: December 2017

The carboxy terminus of the smooth muscle myosin light chain kinase (smMLCK) is expressed as an independent protein, telokin. Western and Northern blotting analyses demonstrated that telokin protein and mRNA are expressed at high levels only in adult and embryonic smooth muscle tissues and cells. In vitro transfection assays in A10 smooth muscle cells identified a functional promoter located in an intron in the 3' region of the smMLCK gene that directs the smooth muscle cell-specific transcription of telokin. To test the cell specificity of the telokin promoter in vivo, transgenic mice were generated in which the telokin promoter was used to drive expression of SV40 large T-antigen. Expression of T-antigen in the transgenic mice paralleled that of the endogenous telokin gene. High levels of T-antigen expression were observed in smooth muscle tissues of the digestive, urinary, and reproductive tracts, with lower levels of expression in airway and vascular smooth muscle. Expression was restricted to smooth muscle cells, with no expression detected in any other cell type.
Heterogeneity of smooth muscle myosin heavy chain expression at the single cell level
Authors: Meer D. P. , Eddinger T. J.
Published: December 2017

Expression of the smooth muscle myosin heavy chain (SM-MHC) isoforms was examined in individual rabbit arterial smooth muscle cells with the use of reverse transcription-polymerase chain reactions (RT-PCR). The RT-PCR amplification protocol used oligonucleotide primers complementary to regions that flank the alternative exon that encodes the nine unique amino acids found in the carboxy terminal domain of SM2. RT-PCR products of SM1 and SM2 mRNA differ in length and electrophoretic mobility. Partial DNA sequencing of the PCR products confirmed SM1 and SM2 identity. Densitometric analyses of adjacent samples extracted from the same tissues and processed for SM2-to-SM1 protein and PCR-amplified SM2-to-SM1 RNA ratios exhibited a high correlation (R = 0.92). RT-PCR-amplified SM2-to-SM1 mRNA ratios of individual adult rabbit arterial cells ranged from 0.0 to 1.8 (n = 59), whereas multicellular vascular samples varied much less (0.4-0.6, n = 5). These results indicate that individual cells within a blood vessel differ significantly in SM-MHC expression. This difference may be important for the regulation of contraction in these vessels.
Coincident expression of Menkes gene with copper efflux in human placental cells
Authors: Qian Y. , Majumdar S. , Reddy M. C. , Harris E. D.
Published: December 2017

BeWo cells, a human choriocarcinoma cell line, have a high-affinity system for transporting copper ions into the cell (Km = 0.21 microM) but are sluggish in releasing copper back into the medium from preloaded cells. The slow efflux rate has recently been shown to correlate with a failure of BeWo cells to express the Menkes transcript [Y. Qian, E. Tiffany-Castiglioni, and E. D. Harris. Am. J. Physiol. 271 (Cell Physiol. 40). In press]. We have now determined that only when BeWo cells were grown on plastic surfaces such as petri dishes or flasks did they display negligible release and enhanced retention of 67Cu. Reverse transcriptase-polymerase chain reaction with the use of primers selective for the Menkes gene failed to show any evidence of a Menkes transcript in cells cultured on plastic surfaces. In contrast, cells grown on porous filters previously shown to allow apical and basolateral surfaces to develop did display the transcript and showed significant copper release with normal retention. Release of copper from filter-grown cells was blocked with p-chloromercuribenzoate, thus confirming sulfhydryl group involvement. Absorption of the 67Cu, either as a free ion or bound to ceruloplasmin, was unaffected by the different culture conditions. The data link the Menkes gene product with the ability of cells to release copper ions. They also suggest that the expression of the Menkes gene may be regulated by the development of polarized cell membranes.
Preservation of structural and functional polarity in isolated epithelial cells
Authors: Torres R. J. , Altenberg G. A. , Copello J. A. , Zampighi G. , Reuss L.
Published: December 2017

Pages C1864-C1874: R. J. Torres, G. A. Altenberg, J. A. Copello, G. Zampighi, and L. Reuss, “Preservation of structural and functional polarity in isolated epithelial cells.” Pages C1866 (Fig. 2), C1869 (Fig. 51, and C1873 (Fig. 9): due to a printer processing error at the final publication stage, Figs. 2, 5, and 9 were poorly reproduced. The figures should have appeared as follows:

(See PDF)
William Hewson's studies of red blood corpuscles and the evolving concept of a cell membrane
Authors: Kleinzeller A.
Published: December 2017

The botanist Carl Nageli is generally considered to have laid the basis for the cell membrane concept by his 1855 study of the osmotic properties of plant cells. It is shown here that William Hewson in 1773 presented cogent experimental evidence for the concept of a cell membrane in red blood corpuscles. Although his work was largely confirmed in subsequent studies, and a cell membrane became an attribute of the cell in T. Schwann's cell theory, the idea of a cell membrane was rejected by anatomists in 1861 essentially on theoretical grounds, and plant physiologists did not mention Hewson's pioneering endeavour. As a consequence, Hewson's work has been ignored to this day. A broad cell membrane concept then had to await the ingenious work of Overton, started in 1895. The possible reasons for these lapses in scientific recognition are analyzed.
Aldosterone regulation of sodium channel gamma-subunit mRNA in cortical collecting duct cells
Authors: Denault D. L. , Fejes-Toth G. , Naray-Fejes-Toth A.
Published: December 2017

Specific regulatory mechanisms of aldosterone-stimulated Na+ reabsorption through the apical amiloride-sensitive channel are unknown. In this study, we examined the effects of aldosterone on Na+ channel gamma-subunit mRNA levels in cultured rabbit cortical collecting duct cells. With the use of reverse transcriptase-polymerase chain reaction (RT-PCR) with RNA isolated from aldosterone-treated cells and degenerate primers, a 446-base pair (bp) PCR product was amplified and further characterized by nested PCR and sequencing. The nested PCR yielded a predicted 164-bp product. Sequencing of the 446-bp PCR product revealed 83% nucleotide and 91% amino acid identity to the rat colonic Na+ channel gamma-subunit. The relative abundance of Na+ channel mRNA was determined by quantitative PCR after a 24-h aldosterone treatment. The results demonstrate that Na+ channel gamma-subunit mRNA levels were significantly higher (2.6 +/- 0.42) in aldosterone-treated cultures vs. the controls. This increase, however, is less than the aldosterone-induced increase (3.2 +/- 2.0) in the amiloride-sensitive short-circuit current. These results indicate that Na+ channel gamma-subunit mRNA levels are increased by aldosterone and that this increase is likely to be responsible, at least in part, for the aldosterone-induced Na+ current in the kidney.
Characteristics and physiological role of the Ca(2+)-activated Cl- conductance in smooth muscle
Authors: Large W. A. , Wang Q.
Published: December 2017

In this review we discuss the properties of the Ca(2+)-activated Cl- current [ICl(Ca)] recorded in isolated smooth muscle cells with electrophysiological techniques and speculate on the possible physiological role(s) of ICl(Ca) in smooth muscle function. In particular, we concentrate on 1) the Ca2+ dependence of ICl(Ca), 2) the mechanisms that link pharmacological receptor stimulation on the cell surface membrane to activation of ICl(Ca), 3) the biophysical properties of ICl(Ca), and 4) the pharmacology of ICl(Ca). It is evident that a diverse array of pharmacological agonists can evoke ICl(Ca) in many types of smooth muscle, and it seems that the well-established G protein-phosphoinositide metabolism (inositol 1,4,5-trisphosphate)-intracellular Ca2+ store pathway couples the receptor to the membrane channels. Also, the results indicate that the biophysical and pharmacological properties of ICl(Ca) are not only similar in the various smooth muscle types studied so far but, possibly, are also similar to ICl(Ca) in non-smooth muscle tissue. Evidence is presented that the Ca(2+)-activated Cl- channel exists in two states, open and closed, with a relatively long mean open time and that some of the agents that inhibit ICl(Ca) interact directly with the open channel. It is suggested that the most likely role of ICl(Ca) in smooth muscle is to produce membrane depolarization and contraction to neurotransmitters and local mediators.
Distinct cellular mechanisms of cholinergic and beta-adrenergic sweat secretion
Authors: Reddy M. M. , Bell C. L.
Published: December 2017

The cholinergic and beta-adrenergic sweat secretions from human sweat glands differ with respect to secretory rates and their susceptibility to cystic fibrosis (CF). Using the cultured beta-adrenergic-sensitive sweat secretory cell, we sought to determine the intracellular electrophysiological mechanisms underlying these functional differences. We found that the cholinergic agonist methacholine (10(-6) M) induced a Ca(2+)-dependent biphasic membrane potential (Vm) response: an initial hyperpolarization and a secondary depolarization. The initial hyperpolarization was independent of bath Cl- and dependent on transmembrane K+ gradient. However, the secondary depolarization of Vm was dependent on bath Cl-. In contrast, the beta-adrenergic agonist isoproterenol (10(-5) M) induced a monophasic depolarization of Vm. This depolarization was 1) dependent on bath Cl-, 2) independent of K+ conductance (GK) blocker Ba2+ (5mM), 3) unaffected by the methacholine-induced secondary depolarization of Vm, and 4) absent in cells derived from CF subjects. These results indicated that the cholinergic agonist-induced secretion mainly involves the activation of Ca(2+)-dependent GK and Cl- conductance (GCl), whereas the beta-adrenergic secretion seems to mainly depend on the activation of cystic fibrosis transmembrane conductance regulator-GCl.
Copper deficiency increases hepatic apolipoprotein B secretion and mRNA editing in rats
Authors: Reaves S. K. , Hoogeveen R. C. , Wang Y. R. , Wu J. Y. , Lei K. Y.
Published: December 2017

Male weanling Sprague-Dawley rats were assigned to copper-deficient (9.0 mumol Cu/kg diet) or copper-adequate (102 mumol Cu/kg diet) dietary treatments for 6 wk. Pulse-chase studies using freshly isolated rat liver parenchymal cells demonstrated that apolipoprotein B (apoB)-48 and apoB-100 syntheses were not altered, but secretion was increased twofold in hepatocytes derived from copper-deficient rats. Both plasma apoB-48 and apoB-100 levels were increased by copper deficiency, but only the apoB-48 increase was significant. Hepatic apoB mRNA editing, expressed as a ratio of apoB-48 mRNA to apoB-48 plus apoB-100 mRNA, was significantly increased from 60.8% in copper-adequate to 70.2% in copper-deficient rats. Moreover, hepatic apoB mRNA abundance was not significantly altered by copper deficiency. Thus the increased amount of nascent apoB-48 secreted into the medium as well as the enhanced apoB mRNA editing may have contributed to the differential increase in plasma apoB-48 over apoB-100 level in copper-deficient rats.
Membrane depolarization in PC-12 cells during hypoxia is regulated by an O2-sensitive K+ current
Authors: Zhu W. H. , Conforti L. , Czyzyk-Krzeska M. F. , Millhorn D. E.
Published: December 2017

The effects of hypoxia on K+ current (IK), resting membrane potential, and cytosolic free Ca2+ in rat pheochromocytoma (PC-12) cells were studied. Whole cell voltage- and current-clamp experiments were performed to measure IK and membrane potential, respectively. Cytosolic free Ca2+ level was measured using the Ca(2+)-sensitive fluorescent dye fura 2. Depolarizing voltage steps to +50 mV from a holding potential of -90 mV elicited a slowly inactivating, tetraethylammonium chloride-sensitive, and Ca(2+)-insensitive IK that was reversibly inhibited by reduced O2 tension. Graded reduction in PO2 (from 150 to 0 mmHg) induced a graded inhibition of O2-sensitive IK [IK(O2)] up to 46% at 0 mmHg. Moreover, hypoxia induced a 19-mV membrane depolarization and a twofold increase in cytosolic free Ca2+. In Ca(2+)-free condition, inhibition of IK(O2) induced an 8-mV depolarization, suggesting that inhibition of IK(O2) was responsible for initiating depolarization. The effect of reduced PO2 on the current-voltage relationship showed a reduction of outward current and a 14-mV shift in the reversal potential comparable with the amount of depolarization measured in current clamp experiments. Neither Ca(2+)-activated IK nor inwardly rectifying IK are responsible for the hypoxia-induced depolarization. In conclusion, PC-12 cells express an IK(O2), inhibition of which leads to membrane depolarization and increased intracellular Ca2+, making the PC-12 clonal cell line a useful model for studying the molecular and biophysical mechanisms that mediate O2 chemosensitivity.
Distribution of Ca2+ channels on cochlear outer hair cells revealed by fluorescent dihydropyridines
Authors: Oshima T. , Ikeda K. , Furukawa M. , Ueda N. , Suzuki H. , Takasaka T.
Published: December 2017

Physiological evidence has shown that cochlear outer hair cells (OHC) possess L-type voltage-dependent Ca2+ channels through which Ca2+ enters the OHC during depolarization. Their subcellular distribution has, however, remained unclear. In this study, the distribution of L-type Ca2+ channels on the basolateral plasma membrane of OHC has been demonstrated by the use of a laser scanning confocal microscope (LSCM) and a fluorescent probe DMBODIPY-DHP. The fluorescent staining pattern on the basolateral wall is nonuniform, suggesting a heterogeneous distribution of the channels in the plasma membrane. Direct imaging of intracellular Ca2+ visualized in real time by means of the LSCM and the fluorescent Ca2+ probe fluo 3 revealed temporal and spatial integration of Ca2+ movements and Ca2+ channel distribution. Exposure to high-K+ solution induced heterogeneity in the subcellular increase in the intracellular Ca2+ concentration. These results suggest that the heterogeneous distribution of L-type Ca2+ channels on the basolateral membrane might induce heterogeneous intracellular Ca2+ distribution during electrical activity in the OHC.
Fluid shear stress induces the phosphorylation of small heat shock proteins in vascular endothelial cells
Authors: Li S. , Piotrowicz R. S. , Levin E. G. , Shyy Y. J. , Chien S.
Published: December 2017

The small molecular mass heat shock protein of 27 kDa (HSP27) has been shown to influence actin filament dynamics and endothelial cell behavior in ways similar to those observed during laminar flow. We have employed human umbilical vein endothelial cells to determine whether fluid shear stress affects HSP27 expression or phosphorylation. After a shear stress of 16 dyn/cm2, HSP27 became more highly phosphorylated, with maximum increase in phosphorylation levels (3-fold) attained by 30 min and sustained for at least 20 h. HSP27 antigen levels did not change; however, HSP27 mRNA levels decreased by 20% after 16 h. In bovine aortic endothelial cells stably transfected with the wild-type human HSP27 gene, shear stress induced the phosphorylation of both the exogenous human HSP27 and the endogenous bovine HSP25. The product of a transfected mutant HSP27 gene in which the putative phosphorylation sites Ser-15, Ser-78, and Ser-82 had been replaced with Gly was not phosphorylated. Thus the modulation of HSP27 and its activity by shear stress is mediated through a posttranslational mechanism and differs from the shear stress induction of immediate early genes at the level of transcription.
Mechanisms of ANG II-induced mitogenic responses: role of 12-lipoxygenase and biphasic MAP kinase
Authors: Wen Y. , Nadler J. L. , Gonzales N. , Scott S. , Clauser E. , Natarajan R.
Published: December 2017

The potential mechanisms of angiotensin II (ANG II)-induced mitogenesis were studied in a Chinese hamster ovary fibroblast cell line overexpressing the rat vascular type 1a ANG II receptor (CHO-AT1a). ANG II had potent mitogenic effects in these CHO-AT1a cells, leading to a sustained increase in cell number as well as a dose-dependent increase in DNA synthesis. ANG II treatment also induced a biphasic elevation of mitogen-activated protein (MAP) kinase activity of both p42MAPK and p44MAPK with a rapid early peak at 5 min (2- to 6-fold) followed by a second sustained increase that reached a peak at 3 h (1.5- to 3-fold). We have previously shown that the 12-lipoxygenase (12-LO) pathway of arachidonate metabolism plays a key role in ANG II-induced growth of vascular smooth muscle and adrenal cells. In the present study, ANG II (10(-7) M) increased the formation of the 12-LO product, 12-hydroxyeicosatetraenoic acid (12-HETE). ANG II-induced DNA synthesis was inhibited by a specific LO inhibitor, cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (CDC, 10 microM). In contrast, a cyclooxygenase blocker of arachidonate metabolism such as ibuprofen had no effect on ANG II-induced DNA synthesis. ANG II-induced DNA synthesis was also partially (32%) blocked by pertussis toxin (PTX). CDC and PTX also selectively blocked only the late (3 h) peak of ANG II-induced MAP kinase activity, suggesting that the late sustained peak of MAP kinase activity may be linked to the mitogenic effect of ANG II. Direct addition of 12-HETE (10(-7) M) led to a sustained increase in cell number similar to the effect of ANG II. 12-HETE also caused an increase in MAP kinase activity, and 12-HETE effects were blocked by PTX. These results suggest that ANG II-induced mitogenic response is associated with sustained MAP kinase activation and that LO activation may play a key role in this process.
Maturation effects on the NMR microimaging characteristics of single neurons
Authors: Aiken N. R. , Hsu E. W. , Horsman A. , Blackband S. J.
Published: December 2017

We have used an isolated neuronal preparation of single Aplysia californica L7 cells to study the effects of development on the nuclear magnetic resonance (NMR) characteristics, T2 spin-spin relaxation rate (RT2) and apparent diffusion coefficient (D), of intracellular water within the nuclear and cytoplasmic compartments. These studies demonstrate a significant correlation of animal weight, but not age, with RT2. On the other hand, D was not significantly different as a function of either age or weight in single L7 neurons. Demonstration of maturation dependence in single cells is important in understanding the cellular origins of developmental effects on NMR characteristics in cell assemblies such as brain.
Developmental shift of myosin heavy chain mRNA expression due to neural factor(s) and muscle activity
Authors: Camoretti-Mercado B. , Qin Y. , Jakovcic S. , Salazar-Grueso E. , Zak R.
Published: December 2017

The adult ventricular isoform of chicken myosin heavy chain (MHC-V) is transiently expressed in all skeletal muscle primordia analyzed and is completely repressed around embryonic days 10-12, when functional innervation is established. By ribonuclease protection assay, we demonstrated that denervation of the adult anterior latissimus dorsi muscle resulted in reexpression of MHC-V mRNA. In contrast, treatment of primary cultures of fetal breast or leg muscles with embryonic brain extract or conditioned media from glial or neuroblastoma cell lines, but not from a myogenic cell line or primary muscle cell cultures, led to inhibition of MHC-V expression. This inhibitory activity was abolished by heating and increased with protein concentration. The acquisition of both brain inhibitory activity and the competence of myogenic cells to downregulate MHC-V mRNA expression were age dependent. Furthermore, either paralysis of muscle in ovo by curare or contraction arrest of cultured myotubes resulted in persistent expression of MHC-V mRNA. Thus a putative soluble factor(s) of nerve origin as well as muscle activity are involved in the developmental downregulation of MHC-V expression in muscle primordia.
ATP activates K and Cl channels via purinoceptor-mediated release of Ca2+ in human coronary artery smooth muscle
Authors: Strobaek D. , Christophersen P. , Dissing S. , Olesen S. P.
Published: December 2017

Coronary artery smooth muscle cells express G protein-coupled purinoceptors, and we report here for the first time how receptor activation by extracellular ATP influences cell membrane currents and membrane potential in human cells. ATP (100 microM) stimulated a triphasic change in membrane potential lasting several seconds, which was caused by sequential opening of transient inward and outward conductances. The inward current was carried by Cl- and the outward current by K+, as shown by ion substitution and changes in holding potential. Both currents were independent of the presence of external Ca2+ but were blocked by strong buffering of Ca2+ in the internal solution. The P2u- and P2y-purinoceptor agonists UTP and 2-methylthioadenosine 5'-triphosphate activated similar currents, whereas the P2x-receptor agonist alpha, beta-methyleneadenosine 5'-triphosphate and the P1-receptor agonist adenosine failed to stimulate any whole cell currents. The ATP-activated K+ current was inhibited by iberiotoxin (200 nM), and it was potentiated by the BK channel activator NS-1619 (30 microM). In cell-attached recordings, ATP activated a 230-pS BK channel. In conclusion, ATP acting via P2 purinoceptors stimulated release of Ca2+ from internal stores and transiently activated depolarizing Cl- and hyperpolarizing BK channels in human coronary artery smooth muscle cells.
Expression of Drosophila melanogaster P-glycoproteins is associated with ATP channel activity
Authors: Bosch I. , Jackson G. R. , Croop J. M. , Cantiello H. F.
Published: December 2017

Two distinct Drosophila melanogaster P-glycoprotein (Pgp) gene homologues of different chromosomal origin, MDR49 and MDR65, have been previously identified (38). Most Pgps are implicated in the development of the multidrug-resistance phenotype. Despite intense efforts to identify the molecular mechanism(s) associated with Pgp function, the endogenous substrate(s) of these transport molecules is largely unknown. Recent studies from our laboratory indicate that a murine Pgp homologue (E. H. Abraham, A. G. Prat, L. Gerweck, T. Seneveratne, R. J. Arceci, R. Kramer, G. Guidotti, and H. F. Cantiello. Proc. Natl. Acad. Sci. USA 90: 312-316, 1993) and a related protein, the cystic fibrosis transmembrane conductance regulator (CFTR; I. L. Reisin, A. Prat, E. H. Abraham, J. F. Amara, R. J. Gregory, D. A. Ausiello, and H. F. Cantiello. J. Biol. Chem. 269: 20584-20591, 1994), are novel ATP-permeable ion channels. The common feature of these two proteins is the conserved ATP-binding cassettes (ABC); thus molecules structurally linked to the ABC transporter family may be also functionally associated with ATP channel activity. In this study, MDR65 and MDR49 Pgps were functionally expressed in Sf9 cells, and patch-clamp techniques were applied to assess the role of these proteins in the electrodiffusional movement of ATP. In the presence of intracellular ATP and external NaCl, expression of MDR65 was associated with a linear electrodiffusional pathway that was permeable to both ATP and Cl-. Under symmetrical ATP conditions, only voltage depolarization activated a MDR65-mediated ATP-conductive pathway. Expression of MDR49 was also associated with a voltage-activated ATP conductance in symmetrical ATP, but no apparent permeability to either Cl- or ATP was observed under asymmetrical conditions. The different functional properties of MDR65 and MDR49 may be indicative of distinct physiological roles in this organism. The study indicates, however, that the two Drosophila Pgp homologues share strong functional similarities with their mammalian relatives Pgp and CFTR.
Angiotensin II activation of Ca(2+)-permeant nonselective cation channels in rat adrenal glomerulosa cells
Authors: Lotshaw D. P. , Li F.
Published: December 2017

A Ca(2+)-permeant, nonselective cation channel was observed in cell-attached and inside-out membrane patches from rat adrenal glomerulosa cells maintained in primary cell culture. In cell-attached patches under near physiological ionic conditions, single-channel currents exhibited a reversal potential near -10 mV, inward rectification, a nearly linear slope conductance between 0 and -80 mV of 17.4 pS, and voltage-dependent block at potentials more negative than -80 mV. Channels exhibiting identical conductance and gating properties were observed in inside-out patches; however, channel gating ran down within minutes in this configuation. In the inside-out configuration, channel gating did not require cytosolic Ca2+ (Ca2+ < 10(-9) M), and inward rectification was relieved by removal of intracellular Mg2+. Relative ionic permeability was calculated using reversal potential measurements from inside-out patches under bi-ionic conditions. The channel discriminated poorly among monovalent cations (PLi > PK > PCs > PNa) and was not significantly permeable to anions. The channel was permeable to Ca2+, exhibiting a relative permeability ratio of 0.29 PCa/PNa) when measured with 110 mM Ca2+ on the intracellular face and a permeability ratio of 4.38 (PCa/PNa) with 110 mM Ca2+ on the extracellular face. Channel gating behavior was episodic with open times ranging from milliseconds to tens of seconds and closed times lasting up to several minutes or longer. Channel gating appeared to be relatively voltage independent except that mean channel open time and open probability were reduced by membrane hyperpolarization. In cell-attached patches, bath application of 1 nM angiotensin II (ANG II) increased the channel open probability, primarily affecting channels exhibiting a low open probability, primarily affecting channels exhibiting a low open probability before stimulation. With the use of nystatin perforated-patch current clamp to measure membrane potential, ANG II was observed to induce large transient membrane depolarizations, consistent with activation of an inward current. We hypothesize that this channel is an important component of ANG II-induced membrane depolarization and Ca2+ influx during stimulation of aldosterone secretion.