bluuebunny/crossref_metadata_2025 huggingface dataset:
Roles of outward potassium currents in the action potentials of guinea pig ureteral myocytes
Authors: Sui J. L. , Kao C. Y.
Published: December 2017

Outward currents of freshly dissociated ureteral myocytes consist mainly of Ca(2+)-activated K+ current (IKCa) and a transient outward current (ITO). No delayed rectifier current was apparent. IKCa is small and nondecaying and fluctuates actively and irregularly. Blocking IKCa decreased resting membrane conductance and prolonged action potential plateaus, showing its roles in maintaining the resting potential and in repolarizing action potentials. It is also responsible for the membrane potential fluctuations on action potential plateaus. Neither 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride nor caffeine reduced the fluctuations in the outward current or in the action potentials, indicating that internal Ca2+ storage contributes little to the fluctuations. ITO has fast activation and inactivation kinetics with inactivation time constants of approximately 15 and 150 ms, respectively. Its highly negative voltage-availability relationship (V0.5 = -70.5 mV) suggests a low availability (< 5%) at normal resting potentials. It has only trivial effects on action potentials.
Stage-specific expression of P2Y receptors, ecto-apyrase, and ecto-5'-nucleotidase in myeloid leukocytes
Authors: Clifford E. E. , Martin K. A. , Dalal P. , Thomas R. , Dubyak G. R.
Published: December 2017

The expression of P2 purinergic receptor subtypes in leukocytes varies with both lineage and developmental stage. Given the recent identification and cloning of at least seven distinct G protein-coupled ATP receptor subtypes (P2Y family), we investigated P2Y receptor subtype expression during myeloid cell differentiation. We observed that KG-1 myeloblasts express P2Y1 but not P2Y2 receptors (previously termed P2U receptors), whereas later myeloid progenitors, including HL-60 promyelocytes and THP-1 monocytes, expressed P2Y2 but not P2Y1 receptors. In KG-1 cells, significant activation of Ca2+ mobilization by P2Y1 receptors was only observed after preincubation with potato apyrase, an exogenous ATPase. This indicated that P2Y1 receptors are desensitized in KG-1 cells by autocrine mechanisms that may involve enhanced release of endogenous nucleotides and/or decreased expression of cell-surface ecto-nucleotidases. We compared the levels of ecto-apyrase activity and expression in KG-1 myeloblasts and HL-60 promyelocytes. Extracellular ATP was rapidly metabolized by HL-60 but not by KG-1 cells. Reverse transcription-polymerase chain reaction analysis indicated that mRNA for CD39 (cluster of differentiation), an identified ecto-apyrase, was present in HL-60 but not KG-1 cells. Ecto-apyrase activity was modestly increased with differentiation of myeloid progenitors with either phorbol 12-myristate 13-acetate (PMA) or dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP). Differentiation of HL-60 cells with PMA, but not DBcAMP, strongly induced ecto-5'-nucleotidase activity and CD73 mRNA expression. These observations indicate that signal transduction by extracellular ATP in myeloid leukocytes can be regulated by developmentally programmed changes in the expression of P2Y receptor subtypes and multiple ecto-nucleotidases.
Genistein potentiates wild-type and delta F508-CFTR channel activity
Authors: Hwang T. C. , Wang F. , Yang I. C. , Reenstra W. W.
Published: December 2017

Effects of genistein on wild-type (wt) and delta F508-cystic fibrosis transmembrane conductance regulator (CFTR) were studied in NIH/3T3 cells stably transfected with wt or mutant CFTR cDNA. As measured by I- efflux, half-maximal concentration of agonist (K1/2) for forskolin-dependent activation was greater for delta F508-CFTR than wt-CFTR. Genistein decreased the K1/2 for both forms of the channel and increased the maximal activity of delta F508-CFTR by 3.7-fold. In cell-attached patches, 10 microM forskolin induced minimal delta F508-CFTR activity with characteristic prolonged closed times (estimated time constant, > 30 s). Genistein increased the forskolin-induced macroscopic currents of wt-CFTR and delta F508-CFTR by 3- and 19-fold, respectively. Variance analysis suggested that in the presence of forskolin and genistein the open probabilities (Po) of wt- and delta F508-CFTR were identical. In single-channel studies, at maximal adenosine 3',5'-cyclic monophosphate (cAMP) stimulation, genistein increased the Po of wt-CFTR by prolonging the open time, but, at submaximal cAMP stimulation, the Po was increased by prolonging the open time and shortening the closed time. In excised patches with CFTR channels preactivated in the cell-attached mode, genistein increased ATP-dependent wt- and delta F508-CFTR current about twofold by prolonging the open time. Our results thus suggest that phosphorylation-dependent activation of delta F508-CFTR is defective and that genistein corrects this defect at least in part by binding to the CFTR protein.
Activation of NF-κB in intestinal epithelial cells by enteropathogenic Escherichia coli
Authors: Savkovic Suzana D. , Koutsouris Athanasia , Hecht Gail
Published: December 2017

The initial response to infection is recruitment of acute inflammatory cells to the involved site. Interleukin (IL)-8 is the prototypical effector molecule for this process. Transcription of the IL-8 gene is primarily governed by the nuclear transcription factor (NF)-κB. Intestinal epithelial cells produce IL-8 in response to infection by enteric pathogens yet remain quiescent in a milieu where they are literally bathed in normal bacterial flora. We therefore sought to investigate NF-κB activation in response to enteropathogenic Escherichia coli (EPEC), nonpathogenic E. coli, and bacterial lipopolysaccharide in an intestinal epithelial cell (T84) model and to determine whether EPEC-induced activation of NF-κB factor is causally linked to IL-8 production. We report herein that NF-κB is activated by EPEC, yet such a response is not extended to nonpathogenic organisms or purified E. coli lipopolysaccharide. Transcription factor decoys significantly diminished IL-8 production in response to EPEC, demonstrating a causal relationship. Furthermore, deletion of specific EPEC virulence genes abrogates the NF-κB-activating property of this pathogen, suggesting that specific bacterial factors are crucial for inducing this response. These studies show for the first time that infection of intestinal epithelial cells with EPEC activates NF-κB, which in turn initiates IL-8 transcription, and highlight the differential response of these cells to bacterial pathogens vs. nonpathogens.
Cloning and functional expression of a ClC Cl− channel from the renal cell line A6
Authors: Lindenthal Sabine , Schmieder Sandra , Ehrenfeld Jordi , Wills Nancy K.
Published: December 2017

Cl− channels are important for ion transport and cell volume regulation in A6 renal cells. In the present study, we used reverse transcriptase (RT)-polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) to identify proteins homologous to ClC Cl− channel proteins in A6 cells. Using degenerate primers designed on consensus sequences for members of the ClC family, we amplified an RT-PCR product that had significant homology to the ClC sequences. RACE-PCR was then used to isolate several full-length clones that had total lengths from 2,764 to 3,016 base pairs. Although the coding regions were identical, sequence differences occurred in the 5′ noncoding regions. The amino acid sequences of the clones had high homologies to rat and human ClC-5 (85 and 84%, respectively, if the 5th methionine of the open reading frame represents the start codon). Three parts of the protein (53, 80, and 63 amino acids in length) were 97–100% homologous to the mammalian sequences. Ribonuclease protection assay analysis revealed mRNA for this protein in oocytes, kidney, intestine, liver, brain, and blood, with lower amounts in stomach, muscle, and skin. Expression of the clones in Xenopus laevis oocytes resulted in an outwardly rectifying Cl−current that was inhibited by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid and possessed an anion selectivity of I− > Cl− >> gluconate.
Protein synthesis-dependent potentiation by thyroxine of antiviral activity of interferon-γ
Authors: Lin Hung-Yun , Yen Paul M. , Davis Faith B. , Davis Paul J.
Published: December 2017

We have studied the prenuclear signal transduction pathway by which thyroid hormone potentiates the antiviral activity of human interferon-γ (IFN-γ) in HeLa cells, which are deficient in thyroid hormone receptor (TR). The action of thyroid hormone was compared with that of milrinone, which has structural homologies with thyroid hormone.l-Thyroxine (T4), 3,5,3′-l-triiodothyronine (T3), and milrinone enhanced the antiviral activity of IFN-γ up to 100-fold, a potentiation blocked by cycloheximide. The 5′-deiodinase inhibitor 6- n-propyl-2-thiouracil did not block the T4 effect. 3,3′,5,5′-Tetraiodothyroacetic acid prevented the effect of T4 but not of milrinone. The effects of T4 and milrinone were blocked by inhibitors of protein kinases C (PKC) and A (PKA) and restored by PKC and PKA agonists; only the effect of T4 was blocked by genistein, a tyrosine kinase inhibitor. In separate models, milrinone was shown not to interact with nuclear TR-β. T4 potentiation of the antiviral activity of IFN-γ requires PKC, PKA, and tyrosine kinase activities but not traditional TR.
Human umbilical vein and dermal microvascular endothelial cells show heterogeneity in response to PKC activation
Authors: Mason Justin C. , Yarwood Helen , Sugars Katharine , Haskard Dorian O.
Published: December 2017

Changes in endothelial cell (EC) phenotype are central to the function of endothelium in inflammation. Although these events mainly occur in the microvasculature, previous studies have predominantly used large-vessel EC. Using enzyme-linked immunosorbent and flow cytometric assays, we compared the responses of human umbilical vein endothelial cells (HUVEC) and dermal microvascular endothelial cells (DMEC) to the activation of protein kinase C (PKC). Stimulation with phorbol 12,13-dibutyrate and more selective PKC agonists, including 12-deoxyphorbol 13-phenylacetate 20-acetate (dPPA), induced morphological changes and proliferation in both EC types. PKC activation induced a marked increase in Thy-1 expression on DMEC and only a moderate rise on HUVEC. Furthermore, heterogeneity in the induction of the adhesion molecules intercellular adhesion molecule 1, vascular cell adhesion molecule 1 (VCAM-1), and E-selectin between the two EC types following activation of PKC was demonstrated. In particular, E-selectin and VCAM-1 were significantly upregulated on HUVEC but not DMEC. The data indicate that the PKC pathway is unlikely to be important for E-selectin and VCAM-1 expression in the microvasculature but are consistent with a role for PKC in angiogenesis. This diversity in signaling in response to PKC activation may depend on differential utilization of PKC isozymes and may facilitate specialized endothelial responses.
Regulation of vascular angiotensin II receptors by EGF
Authors: Ullian Michael E. , Raymond John R. , Willingham Mark C. , Paul Richard V.
Published: December 2017

After vascular endothelial injury, angiotensin II (ANG II) plays a role in the resulting hypertrophic response, and expression of epidermal growth factor (EGF) is enhanced. Therefore, we tested the possibility that EGF regulates vascular ANG II action and receptor expression. Incubation of cultured aortic vascular smooth muscle cells (VSMC) with EGF (or basic fibroblast growth factor but not platelet-derived growth factor isoforms) resulted in concentration-dependent (1–50 ng/ml EGF), time-dependent (>8 h), and reversible decreases in ANG II surface receptor density. For example, a 50% reduction was observed after exposure to 50 ng/ml EGF for 24 h. Incubation of cultured VSMC with 50 ng/ml EGF for 24 h resulted in a 77% reduction in ANG II-stimulated inositol phosphate formation. EGF not only prevented but also reversed ANG II receptor upregulation by 100 nM corticosterone. The specific tyrosine kinase inhibitor tyrphostin A48 (50 μM) reduced EGF-stimulated thymidine incorporation and EGF-stimulated phosphorylation of mitogen-activated protein kinase but did not prevent EGF from reducing ANG II receptor density. Neither pertussis toxin (100 ng/ml) nor downregulation of protein kinase C by phorbol myristate acetate (100 nM for 24 h) prevented EGF from reducing ANG II receptor density. In summary, EGF is a potent negative regulator of vascular ANG II surface receptor density and ANG II action by mechanisms that do not appear to include tyrosine phorphorylation, pertussis toxin-sensitive G proteins, or phorbol ester-sensitive protein kinase C. The possibility that EGF shifts the cell culture phenotype to one that exhibits reduced surface ANG II density cannot be eliminated by the present studies.
Tunicamycin increases intracellular calcium levels in bovine aortic endothelial cells
Authors: Buckley Barbara J. , Whorton A. R.
Published: December 2017

Tunicamycin is a nucleoside antibiotic that inhibits protein glycosylation and palmitoylation. The therapeutic use of tunicamycin is limited in animals because of its toxic effects, particularly in cerebral vasculature. Tunicamycin decreases palmitoylation of the endothelial isoform of nitric oxide synthase, stimulates nitric oxide synthesis, and increases the concentration of intracellular calcium ([Ca2+]i) in bovine aortic endothelial cells (B. J. Buckley and A. R. Whorton. FASEB J. 11: A110, 1997). In the present study, we investigated the mechanism by which tunicamycin alters [Ca2+]iusing the Ca2+-sensitive dye fura 2. We found that tunicamycin increased [Ca2+]iwithout increasing levels of inositol phosphates. When cells were incubated in the absence of extracellular Ca2+, [Ca2+]irapidly rose in response to tunicamycin, although a full response was not achieved. The pool of intracellular Ca2+ mobilized by tunicamycin overlapped with that mobilized by thapsigargin. Extracellular nickel blocked a full response to tunicamycin when cells were incubated in the presence of extracellular Ca2+. The effects of tunicamycin on [Ca2+]iwere partially reversed by washing out the drug, and the remainder of the response was inhibited by removing extracellular Ca2+. These results indicate that tunicamycin mobilizes Ca2+ from intracellular stores in a manner independent of phospholipase C activation and increases the influx of Ca2+ across the plasma membrane.
Male sex steroids are responsible for depressing macrophage immune function after trauma-hemorrhage
Authors: Wichmann Matthias W. , Ayala Alfred , Chaudry Irshad H.
Published: December 2017

Recent studies suggest beneficial effects of castration before soft tissue trauma and hemorrhagic shock on splenocyte immune functions. Nonetheless, it remains unknown whether this effect of testosterone depletion is limited to splenocytes or is a generalized effect on immune function. The present study was therefore carried out to determine whether androgen depletion before trauma-hemorrhage also has salutary effects on splenic and peritoneal macrophage as well as on Kupffer cell function, as indicated by interleukin (IL)-1 and IL-6 release. Male C3H/HeN mice were castrated or sham-castrated 2 wk before the experiment and were killed at 24 h after trauma-hemorrhage and resuscitation. Significant depression of macrophage IL-1 and IL-6 release was only observed in sham-castrated mice, as opposed to normal levels of cytokine release from castrated animals after trauma-hemorrhage. In addition, only sham-castrated animals showed significantly increased levels of IL-6 release from Kupffer cells, which is believed to contribute to the systemic inflammatory response to trauma-hemorrhage. These observations suggest that the beneficial effects of androgen depletion before trauma-hemorrhage are not limited to splenocyte immune functions but are more global in nature. These results in surgically castrated animals suggest that androgen-blocking agents should be studied for their potential to reverse the immunodepression associated with trauma-hemorrhage.
Parathyroid hormone stimulates calcium influx and the cAMP messenger system in rat enterocytes
Authors: Picotto Gabriela , Massheimer Virginia , Boland Ricardo
Published: December 2017

Direct effects of parathyroid hormone (PTH) on calcium uptake by isolated rat duodenal cell preparations enriched in enterocytes were investigated. PTH significantly stimulated enterocyte45Ca2+influx in a time-dependent (1–10 min) manner and at all doses tested (2 × 10−13 to 10−7 M). The Ca2+ channel antagonists verapamil (10 μM) and nitrendipine (1 μM) completely blocked the stimulation of Ca2+ influx by the hormone (10−8 M). PTH markedly increased cAMP levels in rat duodenal cells (88, 167, and 67%, after 1, 2, and 3 min, respectively). In agreement with these observations, forskolin (adenylate cyclase activator), dibutyryl adenosine 3′,5′-cyclic monophosphate (DBcAMP), and Sp-cAMPS (cAMP analogs) mimicked, whereas Rp-cAMPS (cAMP antagonist) suppressed PTH and DBcAMP activation of enterocyte calcium uptake. Furthermore, the effects of DBcAMP were abolished by nitrendipine. These results show direct rapid effects of PTH on duodenal cells’ Ca2+ influx, which involve the activation of a dihydropyridine-sensitive Ca2+ influx pathway and the cAMP second messenger system.
Adenosine stimulates Cl− channels of nonpigmented ciliary epithelial cells
Authors: Carré David A. , Mitchell Claire H. , Peterson-Yantorno Kim , Coca-Prados Miguel , Civan Mortimer M.
Published: December 2017

Ciliary epithelial cells possess multiple purinergic receptors, and occupancy of A1 and A2 adenosine receptors is associated with opposing effects on intraocular pressure. Aqueous adenosine produced increases in short-circuit current across rabbit ciliary epithelium, blocked by removing Cl− and enhanced by aqueous Ba2+. Adenosine’s actions were further studied with nonpigmented ciliary epithelial (NPE) cells from continuous human HCE and ODM lines and freshly dissected bovine cells. With gramicidin present, adenosine (≥3 μM) triggered isosmotic shrinkage of the human NPE cells, which was inhibited by the Cl− channel blockers 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) and niflumic acid. At 10 μM, the nonmetabolizable analog 2-chloroadenosine and AMP also produced shrinkage, but not inosine, UTP, or ATP. 2-Chloroadenosine (≥1 μM) triggered increases of whole cell currents in HCE cells, which were partially reversible, Cl− dependent, and reversibly inhibited by NPPB. Adenosine (≥10 μM) also stimulated whole cell currents in bovine NPE cells. We conclude that occupancy of adenosine receptors stimulates Cl− secretion in mammalian NPE cells.
Tissue distribution of Na+/H+exchanger isoforms NHE2 and NHE4 in rat intestine and kidney
Authors: Bookstein Crescence , Xie Yue , Rabenau Karen , Musch Mark W. , McSwine Rebbecca L. , Rao Mrinalini C. , Chang Eugene B.
Published: December 2017

We present evidence that tissue distribution of two highly conserved Na+/H+exchanger isoforms, NHE2 and NHE4, differs significantly from previously published reports. Riboprobes unique to each of these antiporters, from 5′ (noncoding and coding) and 3′ coding regions, were used to analyze mRNA from adult rat kidney and intestine by ribonuclease protection assay and in situ hybridization. In contrast to earlier work that concluded that both NHE2 and NHE4 were expressed throughout the intestine and in the kidney, our data show that there is no NHE2 message in the kidney and NHE4 is not expressed in small or large intestine. Analyses of intestinal epithelial and kidney membrane proteins by an NHE2-specific antibody identified a doublet at <90 kDa in intestine but not in kidney. NHE2 is highly expressed in the Na+-absorptive epithelium of jejunum, ileum, and ascending and descending colon. NHE4 mRNA message is found in the inner medulla of the kidney as previously reported (C. Bookstein, M. W. Musch, A. DePaoli, Y. Xie, M. Villereal, M. C. Rao, and E. B. Chang. J. Biol. Chem. 269: 29704–29709, 1994) and not in the intestine. From these data, we speculate that neither NHE2 nor NHE4 has a role in renal Na+ absorption. NHE2 is likely involved in gut Na+ absorption, whereas NHE4 may have a specialized role in cell volume rectification of inner medullary collecting duct cells. Knowledge of the correct tissue and cell-specific distribution of these two antiporters should help significantly in understanding their physiological roles.
Functional characterization of the neuronal-specific K-Cl cotransporter: implications for [K+]oregulation
Authors: Payne John A.
Published: December 2017

The neuronal K-Cl cotransporter isoform (KCC2) was functionally expressed in human embryonic kidney (HEK-293) cell lines. Two stably transfected HEK-293 cell lines were prepared: one expressing an epitope-tagged KCC2 (KCC2–22T) and another expressing the unaltered KCC2 (KCC2–9). The KCC2–22T cells produced a glycoprotein of ∼150 kDa that was absent from HEK-293 control cells. The 86Rb influx in both cell lines was significantly greater than untransfected control HEK-293 cells. The KCC2–9 cells displayed a constitutively active86Rb influx that could be increased further by 1 mM N-ethylmaleimide (NEM) but not by cell swelling. Both furosemide [inhibition constant ( K i) ∼25 μM] and bumetanide (Ki∼55 μM) inhibited the NEM-stimulated86Rb influx in the KCC2–9 cells. This diuretic-sensitive86Rb influx in the KCC2–9 cells, operationally defined as KCC2 mediated, required external Cl−but not external Na+ and exhibited a high apparent affinity for external Rb+(K+) [Michaelis constant ( K m) = 5.2 ± 0.9 (SE) mM; n = 5] but a low apparent affinity for external Cl−( K m >50 mM). On the basis of thermodynamic considerations as well as the unique kinetic properties of the KCC2 isoform, it is hypothesized that KCC2 may serve a dual function in neurons: 1) the maintenance of low intracellular Cl− concentration so as to allow Cl− influx via ligand-gated Cl− channels and 2) the buffering of external K+ concentration ([K+]o) in the brain.
Hormone-induced rise in cytosolic Ca2+ in axolotl hepatocytes: properties of the Ca2+ influx channel
Authors: Lenz Thomas , Kleineke Jochen W.
Published: December 2017

Calcium entry in nonexcitable cells occurs through Ca2+-selective channels activated secondarily to store depletion and/or through receptor- or second messenger-operated channels. In amphibian liver, hormones that stimulate the production of adenosine 3′,5′-cyclic monophosphate (cAMP) also regulate the opening of an ion gate in the plasma membrane, which allows a noncapacitative inflow of Ca2+. To characterize this Ca2+ channel, we studied the effects of inhibitors of voltage-dependent Ca2+ channels and of nonselective cation channels on 8-bromoadenosine 3′,5′-cyclic monophosphate (8-BrcAMP)-dependent Ca2+ entry in single axolotl hepatocytes. Ca2+ entry provoked by 8-BrcAMP in the presence of physiological Ca2+ followed first-order kinetics (apparent Michaelis constant = 43 μM at the cell surface). Maximal values of cytosolic Ca2+ (increment ∼300%) were reached within 15 s, and the effect was transient (half time of 56 s). We report a strong inhibition of cAMP-dependent Ca2+ entry by nifedipine [half-maximal inhibitory concentration (IC50) = 0.8 μM], by verapamil (IC50 = 22 μM), and by SK&F-96365 (IC50 = 1.8 μM). Depolarizing concentrations of K+were without effect. Gadolinium and the anti-inflammatory compound niflumate, both inhibitors of nonselective cation channels, suppressed Ca2+ influx. This “profile” indicates a novel mechanism of Ca2+ entry in nonexcitable cells.
Niflumic acid differentially modulates two types of skeletal ryanodine-sensitive Ca2+-release channels
Authors: Oba Toshiharu
Published: December 2017

The effects of niflumic acid on ryanodine receptors (RyRs) of frog skeletal muscle were studied by incorporating sarcoplasmic reticulum (SR) vesicles into planar lipid bilayers. Frog muscle had two distinct types of RyRs in the SR: one showed a bell-shaped channel activation curve against cytoplasmic Ca2+ or niflumic acid, and its mean open probability ( P o) was increased by perchlorate at 20–30 mM (termed “α-like” RyR); the other showed a sigmoidal activation curve against Ca2+ or niflumic acid, with no effect on perchlorate (termed “β-like” RyR). The unitary conductance and reversal potential of both channel types were unaffected after exposure to niflumic acid when clamped at 0 mV. When clamped at more positive potentials, the β-like RyR channel rectified this, increasing the unitary current. Treatment with niflumic acid did not inhibit the response of both channels to Ca2+ release channel modulators such as caffeine, ryanodine, and ruthenium red. The different effects of niflumic acid on P o and the unitary current amplitude in both types of channels may be attributable to the lack or the presence of inactivation sites and/or distinct responses to agonists.
Oxygen sensitivity of mitochondrial metabolic state in isolated skeletal and cardiac myocytes
Authors: Richmond Keith Neu , Burnite Steven , Lynch Ronald M.
Published: December 2017

In striated muscle the coupling of blood flow to changes in tissue metabolism is hypothesized to be dependent in part on release of vasodilating metabolic by-products generated when mitochondrial metabolism becomes O2 limited. Cytochrome oxidase, the terminal step in oxidative phosphorylation, is half-maximally saturated at <1 mmHg [Formula: see text] in isolated mitochondria. However, blood flow is regulated at tissue[Formula: see text] of ∼20 mmHg. If the affinity of mitochondrial respiration for O2were higher in vivo than in vitro, O2 limitation of mitochondrial metabolism near mean tissue levels could occur. In the present study the [Formula: see text] at which mitochondrial metabolism becomes inhibited (critical[Formula: see text]) was measured for cardiac myocytes in suspension (1.1 ± 0.15 mmHg) and single cells (1.0 ± 0.22 and 1.25 ± 0.22 mmHg in cardiac myocytes and rat spinotrapezius cells, respectively). These measurements are consistent with those from isolated mitochondria, indicating that vasodilators produced when oxidative phosphorylation becomes inhibited may be important for regulating blood flow only in highly glycolytic muscles or under conditions of severe O2limitation.
Dipeptide-induced Cl−secretion in proximal tubule cells
Authors: Jin Wenwu , Hopfer Ulrich
Published: December 2017

During a survey of dipeptides that might be transported by the renal PEPT2 transporter in proximal tubule cells, we discovered that acidic dipeptides could stimulate transient secretory anion current and conductance increases in intact cell monolayers. The stimulatory effect of acidic dipeptides was observed in several proximal tubule cell lines that have been recently developed by immortalization of early proximal tubule primary cultures from the Wistar-Kyoto and spontaneously hypertensive rat strains and humans, suggesting that this phenomenon is a characteristic of proximal tubule cells. The electrical current induced in intact monolayers by Ala-Asp, a representative of these acidic dipeptides, must represent Cl− secretion rather than Na+ or H+ absorption, because 1) it was Na+ independent, 2) it showed a pH dependence different from that of the PEPT2 cotransporter, and 3) it correlated with an Ala-Asp-induced increase in Cl− conductance of the apical membrane in basolaterally amphotericin B-permeabilized monolayers. The secretory current could be inhibited by stilbene disulfonates, but not diphenylamine-2-carboxylates, suggesting a non-cystic fibrosis transmembrane conductance regulator type of Cl− conductance. The effect of Ala-Asp was dose dependent, with an apparent 50% effective concentration of ∼1 mM. Ala-Asp also produced intracellular acidification, suggesting that acidic dipeptides are also substrates for an H+-peptide cotransporter.
Steroid hormone-dependent expression of blockersensitive ENaCs in apical membranes of A6 epithelia
Authors: Baxendale-Cox Lynn M. , Duncan Randall L. , Liu Xuehong , Baldwin Kieron , Els Willem J. , Helman Sandy I.
Published: December 2017

Weak channel blocker-induced noise analysis was used to determine the way in which the steroids aldosterone and corticosterone stimulated apical membrane Na+ entry into the cells of tissue-cultured A6 epithelia. Among groups of tissues grown on a variety of substrates, in a variety of growth media, and with cells at passages 73–112, the steroids stimulated both amiloride-sensitive and amiloride-insensitive Na+ transport as measured by short-circuit currents in chambers perfused with either growth medium or a Ringer solution. From baseline rates of blocker-sensitive short-circuit current between 2 and 7 μA/cm2, transport was stimulated about threefold in all groups of experiments. Single channel currents averaged near 0.3 pA (growth medium) and 0.5 pA (Ringer) and were decreased 6–20% from controls by steroid due to the expected decreases of fractional transcellular resistance. Irrespective of baseline transport rates, the steroids in all groups of tissues stimulated transport by increase of the density of blocker-sensitive epithelial Na+ channels (ENaCs). Channel open probability was the same in control and stimulated tissues, averaging ∼0.3 in all groups of tissues. Accordingly, steroid-mediated increases of open channel density responsible for stimulation of Na+ transport are due to increases of the apical membrane pool of functional channels and not their open probability.
The mineralocorticoid aldosterone activates a proton conductance in cultured kidney cells
Authors: Gekle Michael , Silbernagl Stefan , Oberleithner Hans
Published: December 2017

The mineralocorticoid aldosterone is the most important hormone for the regulation of Na+ and K+ homeostasis in mammals and is thereby involved in the regulation of extracellular volume and blood pressure. Because aldosterone is a steroid hormone, the classical way of action involves transcription, translation, and protein synthesis. We previously reported a rapid, nongenomic, and Zn2+-sensitive action of aldosterone on Na+/H+exchange in renal epithelial [Madin-Darby canine kidney (MDCK)] cells (M. Gekle, N. Golenhofen, H. Oberleithner, and S. Silbernagl. Proc. Natl. Acad. Sci. 93: 10500–10504, 1996). Here we show that, in the absence of Na+ (i.e., with inactive Na+/H+exchange), aldosterone induces a membrane potential-dependent and Zn2+-sensitive cytoplasmic acidification in MDCK cells within 2–4 min. This aldosterone-induced activation of a proton conductance is insensitive to the inhibitor of the classical genomic pathway, spironolactone. Furthermore, the inhibitor of serine/threonine kinases and staurosporine, as well as the specific inhibitor of protein kinase C (PKC), calphostin C, prevented proton conductance activation. Activation of PKC by phorbol esters mimicked the effect of aldosterone. Furthermore, preincubation of the cells with pertussis toxin reduced the effect of aldosterone significantly. We propose a new nongenomic mechanism of action for aldosterone, independently of the intracellular type 1 mineralocorticoid receptor: G protein-dependent stimulation of PKC by aldosterone leads to the activation of a plasma membrane proton conductance that enhances the activity of Na+/H+exchange. This rapid nongenomic effect could explain the observation that aldosterone may alter renal Na+ and K+ excretion within 5–10 min.
Long-term regulation of contractility and calcium current in smooth muscle
Authors: Gomez Maria , Swärd Karl
Published: December 2017

Longitudinal smooth muscle strips from guinea pig ileum were cultured in vitro for 5 days, and the relationship between extracellular Ca2+ and force in high-K+ medium was evaluated. In strips cultured with 10% fetal calf serum (FCS), this relationship was shifted to the right (50% effective concentration changed by 2–3 mM) compared with strips cultured without FCS. The shift was prevented by inclusion of verapamil (1 μM) during culture and mimicked by ionomycin in the absence of FCS. The intracellular Ca2+ concentration ([Ca2+]i) during stimulation with high-K+solution or carbachol was reduced after culture with FCS, whereas the [Ca2+]i-force relationship was unaffected. Cells were isolated from cultured strips, and whole cell voltage-clamp experiments were performed. Maximum inward Ca2+ current (10 mM Ba2+), normalized to cell capacitance, was almost three times smaller in cells isolated from strips cultured with FCS. Culture with 1 μM verapamil prevented this reduction. These results suggest that increased [Ca2+]iduring culture downregulates Ca2+current density, with associated effects on contractility.
Human cytomegalovirus infection enhances osmotic stimulation of Na+/H+exchange in human fibroblasts
Authors: Crowe William E. , Altamirano Aníbal A. , Russell John M.
Published: December 2017

Infection with human cytomegalovirus (HCMV) causes an enlargement (cytomegaly) of human fibroblasts (MRC-5). As a first step toward determining whether solute uptake, mediated in part by Na+/H+exchange, is responsible for the development of cytomegaly, we studied the effects of HCMV infection on intracellular pH (pHi) regulation (nominal CO2/[Formula: see text]concn = 0) by comparing cytomegalic cells with mock-infected cells. Seventy-two hours after HCMV infection of MRC-5 cells we observed the following changes relative to mock-infected cells: resting pHi is 0.1–0.2 pH unit more alkaline; the intrinsic buffering power of the cytoplasm was reduced by ∼40–50%; acid-loading H+-equivalent fluxes were reduced; and there were alterations of Na+/H+exchanger (NHE) properties, including an alkaline shift of the pHi dependence of activity, a reduction of the apparent affinity for extracellular Na+, and an increase of the apparent maximum velocity and a large increase in stimulation by a hyperosmotic challenge. These results indicate that HCMV infection exerts a profound effect on functional properties of the NHE, on acid-loading mechanisms, and on intrinsic cellular buffering power. These effects are consistent with a role for the NHE in the development of cytomegaly.
Elementary events of agonist-induced Ca2+ release in vascular endothelial cells
Authors: Hüser Jörg , Blatter Lothar A.
Published: December 2017

The subcellular spatial and temporal organization of agonist-induced Ca2+ signals was investigated in single cultured vascular endothelial cells. Extracellular application of ATP initiated a rapid increase of intracellular Ca2+ concentration ([Ca2+]i) in peripheral cytoplasmic processes from where activation propagated as a [Ca2+]iwave toward the central regions of the cell. The average propagation velocity of the [Ca2+]iwave in the peripheral processes was 20–60 μm/s, whereas in the central region the wave propagated at <10 μm/s. The time course of the recovery of [Ca2+]idepended on the cell geometry. In the peripheral processes (i.e., regions with a high surface-to-volume ratio) [Ca2+]ideclined monotonically, whereas in the central region [Ca2+]idecreased in an oscillatory fashion. Propagating [Ca2+]iwaves were preceded by small, highly localized [Ca2+]itransients originating from 1- to 3-μm-wide regions. The average amplitude of these elementary events of Ca2+ release was 23 nM, and the underlying flux of Ca2+ amounted to ∼1–2 × 10−18mol/s or ∼0.3 pA, consistent with a Ca2+ flux through a single or small number of endoplasmic reticulum Ca2+-release channels.
Spontaneous acetylcholine release in mammalian neuromuscular junctions
Authors: Losavio Adriana , Muchnik S.
Published: December 2017

Spontaneous secretion of the neurotransmitter acetylcholine in mammalian neuromuscular synapsis depends on the Ca2+ content of nerve terminals. The Ca2+ electrochemical gradient favors the entry of this cation. We investigated the possible involvement of three voltage-dependent Ca2+ channels (VDCC) (L-, N-, and P/Q-types) on spontaneous transmitter release at the rat neuromuscular junction. Miniature end-plate potential (MEPP) frequency was clearly reduced by 5 μM nifedipine, a blocker of the L-type VDCC, and to a lesser extent by the N-type VDCC blocker, ω-conotoxin GVIA (ω-CgTx, 5 μM). On the other hand, nifedipine and ω-CgTx had no effect on K+-induced transmitter secretion. ω-Agatoxin IVA (100 nM), a P/Q-type VDCC blocker, prevents acetylcholine release induced by K+ depolarization but failed to affect MEPP frequency in basal conditions. These results suggest that in the mammalian neuromuscular junction Ca2+ enters nerve terminals through at least three different channels, two of them (L- and N-types) mainly related to spontaneous acetylcholine release and the other (P/Q-type) mostly involved in depolarization-induced neurotransmitter release. Ca2+-binding molecule-related spontaneous release apparently binds Ca2+ very rapidly and would probably be located very close to Ca2+ channels, since the fast Ca2+ chelator (BAPTA-AM) significantly reduced MEPP frequency, whereas EGTA-AM, exhibiting slower kinetics, had a lower effect. The increase in MEPP frequency induced by exposing the preparation to hypertonic solutions was affected by neither external Ca2+concentration nor L-, N-, and P/Q-type VDCC blockers, indicating that extracellular Ca2+ is not necessary to produce hyperosmotic neurosecretion. On the other hand, MEPP frequency was diminished by BAPTA-AM and EGTA-AM to the same extent, supporting the view that hypertonic response is promoted by “bulk” intracellular Ca2+concentration increases.
Na+-K+-ATPase in frog esophagus mucociliary cell membranes: inhibition by protein kinase C activation
Authors: Gertsberg Irena , Brodsky Irena , Priel Zvi , Danilenko Michael
Published: December 2017

We examined protein kinase C (PKC)-dependent regulation of Na+-K+-ATPase in frog mucociliary cells. Activation of PKC by 12- O-tetradecanoylphorbol-13-acetate (TPA) or 1,2-dioctanoyl- sn-glycerol (diC8) either in intact cells or isolated membranes resulted in a specific inhibition of Na+-K+-ATPase activity by ∼25–45%. The inhibitory effects in membranes exhibited time dependence and dose dependence [half-maximal inhibition concentration (IC50) = 0.5 ± 0.1 nM and 2.4 ± 0.2 μM, respectively, for TPA and diC8] and were not influenced by Ca2+. Analysis of the ouabain inhibition pattern revealed the presence of two Na+-K+-ATPase isoforms with IC50 values for cardiac glycoside of 2.6 ± 0.8 nM and 409 ± 65 nM, respectively. Most importantly, the isoform possessing a higher affinity for ouabain was almost completely inhibited by TPA, whereas its counterpart was hardly sensitive to the PKC activator. The results suggest that, in frog mucociliary cells, PKC regulates Na+-K+-ATPase and that this action is related to the specific Na+-K+-ATPase isoform.
Changes in intracellular Ca2+concentration induced by L-type Ca2+ channel current in guinea pig gastric myocytes
Authors: Kim Sung Joon , Ahn Seung Cheol , Kim Jin Kyung , Kim Young Chul , So Insuk , Kim Ki Whan
Published: December 2017

We investigated the relationship between voltage-operated Ca2+ channel current and the corresponding intracellular Ca2+concentration ([Ca2+]i) change (Ca2+ transient) in guinea pig gastric myocytes. Fluorescence microspectroscopy was combined with conventional whole cell patch-clamp technique, and fura 2 (80 μM) was added to CsCl-rich pipette solution. Step depolarization to 0 mV induced inward Ca2+ current ( I Ca) and concomitantly raised [Ca2+]i. Both responses were suppressed by nicardipine, an L-type Ca2+ channel blocker, and the voltage dependence of Ca2+transient was similar to the current-voltage relation of I Ca. When pulse duration was increased by up to 900 ms, peak Ca2+ transient increased and reached a steady state when stimulation was for longer. The calculated fast Ca2+ buffering capacity ( B value), determined as the ratio of the time integral of I Ca divided by the amplitude of Ca2+ transient, was not significantly increased after depletion of Ca2+ stores by the cyclic application of caffeine (10 mM) in the presence of ryanodine (4 μM). The addition of cyclopiazonic acid (CPA, 10 μM), a sarco(endo)plasmic reticulum Ca2+-ATPase inhibitor, decreased B value by ∼20% in a reversible manner. When KCl pipette solution was used, Ca2+-activated K+ current [ I K(Ca)] was also recorded during step depolarization. CPA sensitively suppressed the initial peak and oscillations of I K(Ca) with irregular effects on Ca2+transients. The above results suggest that, in guinea pig gastric myocyte, Ca2+ transient is tightly coupled to I Caduring depolarization, and global [Ca2+]iis not significantly affected by Ca2+-induced Ca2+ release from sarcoplasmic reticulum during depolarization.
P2U purinergic receptor inhibits apical IsK/KvLQT1 channel via protein kinase C in vestibular dark cells
Authors: Marcus Daniel C. , Sunose Hiroshi , Liu Jianzhong , Shen Zhijun , Scofield Margaret A.
Published: December 2017

Vestibular dark cells (VDC) are known to electrogenically secrete K+ via slowly activating K+(IsK) channels, consisting of IsK regulatory and KvLQT1 channel subunits, and the associated short-circuit current ( I sc) is inhibited by agonists of the apical P2U(P2Y2) receptor (J. Liu, K. Kozakura, and D. C. Marcus. Audit. Neurosci. 2: 331–340, 1995). Measurements of relative K+ flux ( J K) with a self-referencing K+-selective probe demonstrated a decrease in J K after apical perfusion of 100 μM ATP. On-cell macropatch recordings from gerbil VDC showed a decrease of the IsKchannel current ( I IsK) by 83 ± 7% during pipette perfusion of 10 μM ATP. The magnitude of the decrease of I scby ATP was diminished in the presence of inhibitors of phospholipase C (PLC) and protein kinase C (PKC), U-73122 and GF109203X. Activation of PKC by phorbol 12-myristate 13-acetate (PMA, 20 nM) decreased I IsK by 79 ± 3% in perforated-patch whole cell recordings, whereas the inactive analog, 4α-PMA, had no effect. In contrast, elevation of cytosolic Ca2+ concentration by A-23187 increased the whole cell I IsK . The expression of the isk gene transcript was confirmed, and the serine responsible for the species-specific response to PKC was found to be present in the gerbil IsKsequence. These data provide evidence consistent with a direct effect of the PKC branch of the PLC pathway on the IsK channel of VDC in response to activation of the apical P2Ureceptor and predict that the secretion of endolymph in the human vestibular system may be controlled by PKC in the same way as in our animal model.
Pacing rate, halothane, and BDM affect fura 2 reporting of [Ca2+]iin intact rat trabeculae
Authors: Jiang Yandong , Julian Fred J.
Published: December 2017

Experiments were done on intact trabeculae from rats. Fura 2 in the salt form was microinjected directly into the myoplasm. The experiments were conducted at 30°C, with 2 mM extracellular Ca2+ concentration and pacing at either 0.5 or 5 Hz. The aims were to establish a new method for in vivo calibration of fura 2 and to determine the effect of autofluorescence changes on intracellular Ca2+ concentration ([Ca2+]i) reported by fura 2. Autofluorescence was recorded under optimal conditions for fura 2 fluorescence (emission at 510 nm). By alteration of the oxidation-reduction state, it was shown that NADH is the main component of autofluorescence in heart. An increase in pacing frequency caused a decrease in autofluorescence. Both halothane and 2,3-butanedione monoxime (BDM) at 5-Hz pacing produced a substantial rise in autofluorescence, approaching the levels observed at 0.5-Hz pacing. The values for the dissociation constant (678 nM) and maximum fluorescence ratio of fura 2 for Ca2+ for the in vivo calibration are 3.4 times larger and 2.6 times smaller, respectively, than those found in vitro. Using the parameters obtained in vivo, we found that the diastolic and systolic [Ca2+]iof a twitch at 30°C were 0.2 and 2.4 μM, respectively. Proper correction of the autofluorescence change unmasks the [Ca2+]ielevation caused by 5-Hz pacing. It was concluded that autofluorescence is not constant and that interventions affecting autofluorescence need correction if fura 2 is used to report [Ca2+]i.
A century of (epithelial) transport physiology: from vitalism to molecular cloning
Authors: Schultz Stanley G.
Published: December 2017

During the past century, the generally accepted view of the function of biological membranes has evolved from that of a static, inert lipoprotein envelope that simply separates the intracellular machinery from the outside milieu, to that of a dynamic structure that is, in fact, an integral part of that machinery actively involved in homeostasis and bioenergetics. This brief review traces that paradigm shift with special emphasis on the evolution of central concepts in epithelial transport physiology.
Modulation of K+ channels by arachidonic acid in T84 cells. I. Inhibition of the Ca2+-dependent K+ channel
Authors: Devor Daniel C. , Frizzell Raymond A.
Published: December 2017

The Cl− secretory response of colonic cells to Ca2+-mediated agonists is transient despite a sustained elevation of intracellular Ca2+. We evaluated the effects of second messengers proposed to limit Ca2+-mediated Cl− secretion on the basolateral membrane, Ca2+-dependent K+ channel (KCa) in colonic secretory cells, T84. Neither protein kinase C (PKC) nor inositol tetrakisphosphate (1,3,4,5 or 3,4,5,6 form) affected KCa in excised inside-out patches. In contrast, arachidonic acid (AA; 3 μM) potently inhibited KCa, reducing NP o, the product of number of channels and channel open probability, by 95%. The apparent inhibition constant for this AA effect was 425 nM. AA inhibited KCa in the presence of both indomethacin and nordihydroguaiaretic acid, blockers of the cyclooxygenase and lipoxygenase pathways. In the presence of albumin, the effect of AA on KCa was reversed. A similar effect of AA was observed on KCa during outside-out recording. We determined also the effect of the cis-unsaturated fatty acid linoleate, the trans-unsaturated fatty acid elaidate, and the saturated fatty acid myristate. At 3 μM, all of these fatty acids inhibited KCa, reducing NP o by 72–86%. Finally, the effect of the cytosolic phospholipase A2 inhibitor arachidonyltrifluoromethyl ketone (AACOCF3) on the carbachol-induced short-circuit current ( I sc) response was determined. In the presence of AACOCF3, the peak carbachol-induced I sc response was increased ∼2.5-fold. Our results suggest that AA generation induced by Ca2+-mediated agonists may contribute to the dissociation observed between the rise in intracellular Ca2+ evoked by these agonists and the associated Cl− secretory response.
Use of DNA injection for identification of slow nerve-dependent regions of the MLC2sgene
Authors: Lupa-Kimball Valerie A. , Esser Karyn A.
Published: December 2017

It has been well established that expression of slow contractile protein genes in skeletal muscle is regulated, in part, by activity from slow motoneurons. However, very little is understood about the mechanism by which neural activity regulates transcription of slow isoform genes. The purpose of this investigation was first to more fully define the in vivo DNA injection technique for use in both fast-twitch and slow-twitch muscles and second to use the injection technique for the identification of slow nerve-dependent regions of the myosin light chain 2 slow ( MLC2s) gene. Initial experiments determined that the same amount of plasmid DNA was taken up by both the slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles and that injection of from 0.5 to 10 μg DNA/muscle is ideal for analysis of promoter activity during regeneration. This technique was subsequently used to identify that the region from −800 to +12 base pairs of MLC2s gene directed ∼100 times higher activity in the innervated soleus than in innervated EDL, denervated soleus, or denervated EDL muscles. Placing the introns upstream of either the MLC2s or SV40 promoter increased expression 5- and 2.7-fold, respectively, in innervated soleus but not in innervated EDL, denervated soleus, or denervated EDL muscles. These results demonstrate that 1) in vivo DNA injection is a sensitive assay for promoter analysis in both fast-twitch and slow-twitch skeletal muscles and 2) both 5′ flanking and intronic regions of the MLC2s gene can independently and synergistically direct slow nerve-dependent transcription in vivo.