bluuebunny/crossref_metadata_2025 huggingface dataset:
Occurrence and putative hormone regulatory function of a constitutive heme oxygenase in rat pancreatic islets
Authors: Henningsson R. , Alm P. , Lundquist I.
Published: December 2017

Recent observations suggest that carbon monoxide (CO) may serve as a neuroendocrine modulator in hypothalamus. Here we provide evidence, for the first time, that the islets of Langerhans contain the constitutive isoform of the CO-producing enzyme heme oxygenase (HO-2), the activity of which was found to modulate islet hormone release. Most insulin and glucagon cells in the rat endocrine pancreas expressed strong immunoreactivity for HO-2. In the exocrine parenchyma, scattered HO-2-positive ganglionic cell bodies were occasionally observed. Furthermore, Western blot analysis revealed the presence of HO-2 in isolated islets but not in acinar cells. Islet homogenates displayed a comparatively high HO-2 enzymatic activity measured as CO formation (approximately 600 pmol CO.min-1.mg islet protein-1). This HO-2 enzymatic activity was greatly suppressed by zincprotoporphyrin-IX (ZnPP-IX), a recognized inhibitor of HO activity. Neither ZnPP-IX nor the HO activator, hemin, influenced basal insulin release from isolated rat islets at low (1 mM) glucose. However, glucagon release at 1 mM glucose was increased by hemin and inhibited by ZnPP-IX. The hemin-induced increase in glucagon secretion was abolished by ZnPP-IX. Furthermore, a series of experiments at high glucose (16.7 mM) revealed that hemin induced a dose-dependent potentiation of glucose-stimulated insulin release. Moreover, glucose-induced insulin release was dose-dependently suppressed by ZnPP-IX but unaffected by protoporphyrin-IX, a compound known not to influence HO-2 activity in other tissues. Similarly, glucagon release at high glucose was dose-dependently increased by hemin and suppressed by ZnPP-IX. Finally, the hemin-induced increase in islet hormone release at high glucose was totally abolished by ZnPP-IX. The data strongly suggest that CO production positively modulates both glucagon and insulin secretion. We propose that CO may serve as a novel messenger molecule within the islets of Langerhans.
Genistein activates CFTR Cl- channels via a tyrosine kinase- and protein phosphatase-independent mechanism
Authors: French P. J. , Bijman J. , Bot A. G. , Boomaars W. E. , Scholte B. J. , de Jonge H. R.
Published: December 2017

Previous studies have revealed an adenosine 3',5'-cyclic monophosphate (cAMP)-independent activation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels by the tyrosine kinase inhibitor genistein. To further explore its mechanism of action, we have reconstituted genistein activation of CFTR in excised inside-out membrane patches. In the presence or absence of ATP, genistein appeared unable to open silent CFTR Cl- channels. However, on CFTR prephosphorylation by cAMP-dependent protein kinase (cAK), genistein enhanced CFTR activity by twofold, resulting from a prolonged burst duration. Genistein could also hyperactivate partially phosphorylated CFTR in the absence of cAK and therefore is different from 5'-adenylylimidodiphosphate, which required fully phosphorylated CFTR. Phosphatase-resistant thiophosphorylation likewise primed the CFTR Cl- channel for hyperactivation by genistein in the absence of cAK. Replacement of ATP by GTP as a hydrolyzable nucleotide triphosphate for CFTR did not impair the ability of genistein to activate thiophosphorylated CFTR, despite the fact that GTP is a poor substrate for tyrosine kinases. These findings argue against a role of protein phosphatases or tyrosine kinases but suggest a more direct interaction of genistein with CFTR, possibly at the level of the second nucleotide-binding domain.
Optimal shortening velocities for in situ power production of rat soleus and plantaris muscles
Authors: Swoap S. J. , Caiozzo V. J. , Baldwin K. M.
Published: December 2017

Force-velocity (FV) relationships have been used previously to calculate maximal power production and to identify an optimal velocity of shortening (V(opt)-fv) to produce such power in skeletal muscle. The cyclical nature of muscle position during locomotion for muscles such as the soleus and plantaris is such that either constant force or velocity is rarely attained. In the present study, the work loop technique, a technique developed to measure maximal attainable power output from muscles undergoing cyclic length changes, was undertaken to determine whether simulating in vivo function alters the power-velocity relationship of the soleus and plantaris and, in particular, the velocity of shortening that produces maximal power (V(opt)-wl). FV relationships were determined for both soleus (n = 4) and plantaris (n = 4) muscles in situ from adult female Sprague-Dawley rats by measuring shortening velocities during afterloaded isotonic contractions. The velocity that produced maximal power using FV relationships, V(opt)-fv, was 54.6 +/- 0.7 mm/s for the plantaris vs. 20.2 +/- 1.2 mm/s for the soleus. Then, the work loop technique was employed to measure net power from these same muscles at multiple cycling frequencies (1.5 to 4.0 Hz for the soleus; 4.0 to 8.0 Hz for the plantaris). Multiple power-velocity curves were generated (one at each cycle frequency) by varying the strain (1-8 mm). Thus, at each cycle frequency, V(opt)-wl could be identified. For both the plantaris and soleus, V(opt)-wl at each cycle frequency was not different from their respective V(opt)-fv value. Thus both fast and slow skeletal muscles have inherent optimal shortening velocities, identifiable with FV relationships, that dictate their respective maximal attainable mechanical power production using the work loop technique.
Evidence for an amiloride-insensitive Na+/H+ exchanger in rat renal cortical tubules
Authors: Chambrey R. , Achard J. M. , St John P. L. , Abrahamson D. R. , Warnock D. G.
Published: December 2017

We have characterized the Na+/H+ exchanger (NHE) isoforms expressed in rat renal cortical tubule fragments. Amiloride sensitivity of the Na(+)-dependent intracellular pH (pHi) recovery in suspended tubules that had been acid loaded by an NH4+ prepulse was determined in nominally CO2/HCO3(-)-free solution, using the fluorescent pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. In the presence of 140 mM extracellular Na+, 800 microM amiloride inhibited the rate of Na(+)-dependent pHi recovery by only 65%, demonstrating the presence of a Na(+)-dependent amiloride-insensitive H+ extrusion system. This system was not affected by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid but was activated by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Lowering extracellular Na+ concentration permitted 300 microM amiloride to completely inhibit Na(+)-dependent pHi recovery. These results can be explained by the expression of a Na+/H+ exchange with the pharmacological properties of NHE4. Using reverse transcriptase-polymerase chain reaction, we found specific mRNA for NHE1, NHE2, NHE3, and NHE4 isoforms in the renal cortex. Immunohistochemical studies using polyclonal antibodies against rat NHE4 peptide demonstrated that NHE4 is heterogeneously expressed on basolateral membrane domains of cortical tubules. These results strongly suggest that amiloride-insensitive Na+/H+ exchange expressed in renal cortical tubule suspensions is mediated by NHE4.
Isolated bovine retinal pigment epithelial cells express delayed rectifier type and M-type K+ currents
Authors: Takahira M. , Hughes B. A.
Published: December 2017

Outwardly rectifying K+ currents in freshly isolated bovine retinal pigment epithelial (RPE) cells were characterized using the whole cell and perforated-patch configurations of the patch-clamp technique. All cells exhibited a delayed rectifier type K+ current. This current had an activation threshold voltage of approximately -40 mV, activated with a sigmoidal trajectory, and inactivated completely over a period of several seconds. External tetraethylammonium (TEA) was an effective blocker of the delayed rectifier current [apparent dissociation constant (Kd) = 5.1 mM], but external Ba2+ was relatively ineffective. Approximately 24% of the cells also exhibited a sustained outwardly rectifying K+ current that became activated at voltages positive to approximately -80 mV. This current resembled the neuronal M-current. External Ba2+ was a potent blocker of this current (apparent Kd = 1.1 mM), but external TEA and Cs+ were relatively ineffective. These results indicate that freshly isolated bovine RPE cells express K+ currents of both the delayed rectifier and M types. The latter may contribute to the resting K+ conductances of the apical and basolateral membranes.
Ecto-enzymatic hydrolysis of diadenosine polyphosphates by cultured adrenomedullary vascular endothelial cells
Authors: Mateo J. , Miras-Portugal M. T. , Rotllan P.
Published: December 2017

We investigated the extracellular degradation of diadenosine polyphosphates (ApnA) by cultured adrenomedullary endothelial cells using fluorogenic analogs of ApnA, the di(1,N6-ethenoadenosine) 5',5"'-P1,Pn-polyphosphates [epsilon-(ApnA)]. Kinetic parameters of epsilon-(ApnA) cleavage and effects of pH, ions, and inhibitors were determined by continuous fluorometric assays, using suspensions of endothelial cells grown on Cytodex-1 microspheres. Ecto-enzyme kinetic parameters for epsilon-(Ap3A), epsilon-(Ap4A), and epsilon-(Ap5A) hydrolysis are as follows: Michaelis-Menten constants of 0.39 +/- 0.07, 0.42 +/- 0.09, and 0.37 +/- 0.05 microM respectively, and maximal velocities of 26.1 +/- 6.8, 74.2 +/- 16.4, and 24.4 +/- 3.4 pmol.min-1.10(6) cells-1, respectively. ApnA and guanosine 5',5"'-P1,P4-tetraphosphate behave as competitor substrates of epsilon-(Ap4A) hydrolysis. The ectoenzyme is activated by Mg2+ and Mn2+ and inhibited by Ca2+, F-, adenosine 5'-tetraphosphate, adenosine 5'-O-(3-thiotriphosphate), and suramin. Optimum pH is around 9.0. High-performance liquid chromatography analysis reveals that the ecto-enzyme hydrolyzes epsilon-(ApnA) to give epsilon-adenosine-5'(n-1)-phosphate and epsilon-AMP, which are then further catabolized up to epsilon-adenosine via the membrane-bound nucleotidase system ecto-ATPase, ecto-ADPase (or apyrase), and ecto-5'-nucleotidase. The endothelial ecto-diadenosine polyphosphate hydrolase studied here exhibits different kinetic parameters and sensitivity to ions with respect to the enzyme from the tissue-related neurochromaffin cells. These different properties may be important in the extracellular signaling by ApnA.
Role of water and electrolyte influxes in anoxic plasma membrane disruption
Authors: Chen Jing , Mandel Lazaro J.
Published: December 2017

The role of water and electrolyte influxes in anoxia-induced plasma membrane disruption was investigated using rabbit proximal tubule suspension. The results indicated that normal proximal tubule (PT) cells have a great capacity for expanding cell volume in response to water influx, whereas anoxia increases the susceptibility to water influx-induced disruption, and this was attenuated by glycine. However, resistance of anoxic plasma membranes to water influx-induced stress is not lost, although their mechanical strength was diminished, compared with normoxic membranes. Anoxic membranes did not disrupt under an intra-to-extracellular osmotic difference as great as 150 mosM. Potentiating or attenuating water influx by incubating PT cells in hypotonic or hypertonic medium, respectively, during anoxia, did not affect anoxia-induced membrane disruption. After the transmembrane electrolyte concentration gradient was eliminated by a “intracellular” buffer or by permeabilizing the plasma membrane to molecules <4 kDa using α-toxin, anoxia still caused further membrane disruption that was prevented by glycine or low pH. These results demonstrate that 1) water or net electrolyte influxes are probably not a primary cause for anoxia-induced membrane disruption and 2) glycine could prevent the plasma membrane disruption during anoxia independently from its effect on transmembrane electrolyte or water influxes. The present data support a biochemical rather than a mechanical alteration of the plasma membrane as the underlying cause of membrane disruption during anoxia.
Letters to the Editor
Authors: Cuthbert A. W.
Published: December 2017

The following is the abstract of the article discussed in the subsequent letter:

Blazer-Yost, Bonnie L., and Sandy I. Helman.The amiloride-sensitive epithelial Na+ channel: binding sites and channel densities. Am. J. Physiol. 272 ( Cell Physiol. 41): C761–C769, 1997.—The amiloride-sensitive Na+ channel found in many transporting epithelia plays a key role in regulating salt and water homeostasis. Both biochemical and biophysical approaches have been used to identify, characterize, and quantitate this important channel. Among biophysical methods, there is agreement as to the single-channel conductance and gating kinetics of the highly selective Na+ channel found in native epithelia. Amiloride and its analogs inhibit transport through the channel by binding to high-affinity ligand-binding sites. This characteristic of high-affinity binding has been used biochemically to quantitate channel densities and to isolate presumptive channel proteins. Although the goals of biophysical and biochemical experiments are the same in elucidating mechanisms underlying regulation of Na+transport, our review highlights a major quantitative discrepancy between methods in estimation of channel densities involved in transport. Because the density of binding sites measured biochemically is three to four orders of magnitude in excess of channel densities measured biophysically, it is unlikely that high-affinity ligand binding can be used physiologically to quantitate channel densities and characterize the channel proteins.
Antisense oligodeoxynucleotide to PKC-δ blocks α1-adrenergic activation of Na-K-2Cl cotransport
Authors: Liedtke Carole M. , Cole Thomas
Published: December 2017

A role for protein kinase C (PKC)-δ and -ζ isotypes in α1-adrenergic regulation of human tracheal epithelial Na-K-2Cl cotransport was studied with the use of isotype-specific PKC inhibitors and antisense oligodeoxynucleotides to PKC-δ or -ζ mRNA. Rottlerin, a PKC-δ inhibitor, blocked 72% of basolateral-to-apical, bumetanide-sensitive36Cl flux in nystatin-permeabilized cell monolayers stimulated with methoxamine, an α1-adrenergic agonist, with a 50% inhibitory concentration of 2.3 μM. Methoxamine increased PKC activity in cytosol and a particulate fraction; the response was insensitive to PKC-α and -βIIisotype-specific inhibitors, but was blocked by general PKC inhibitors and rottlerin. Rottlerin also inhibited methoxamine-induced PKC activity in immune complexes of PKC-δ, but not PKC-ζ. At the subcellular level, methoxamine selectively elevated cytosolic PKC-δ activity and particulate PKC-ζ activity. Pretreatment of cell monolayers with antisense oligodeoxynucleotide to PKC-δ for 48 h reduced the amount of whole cell and cytosolic PKC-δ, diminished whole cell and cytosolic PKC-δ activity, and blocked methoxamine-stimulated Na-K-2Cl cotransport. Sense oligodeoxynucleotide to PKC-δ and antisense oligodeoxynucleotide to PKC-ζ did not alter methoxamine-induced cotransport activity. These results demonstrate the selective activation of Na-K-2Cl cotransport by cytosolic PKC-δ.
Stretch-dependent activation and desensitization of mitogen-activated protein kinase in carotid arteries
Authors: Franklin Michael T. , Wang C. L.-Albert , Adam Leonard P.
Published: December 2017

Arterial smooth muscle stretch is an important physiological modulator of vascular function. To identify intracellular processes altered during muscle stretch, we found previously that extracellular signal-regulated kinase-mitogen-activated protein kinase (MAPK) activity increased in response to the application of mechanical loads. In the present study, stretch-dependent activation of MAPK in porcine carotid arteries was investigated as was the phosphorylation of the thin filament-binding protein caldesmon, which is known to be a substrate for the kinase in fully differentiated smooth muscle. MAPK activity was 67 pmol ⋅ min−1 ⋅ mg protein−1 in unloaded muscle strips immediately after attachment to force transducers and 139 pmol ⋅ min−1 ⋅ mg protein−1 within 30 s of muscle stretch. When muscle strips were continually stretched, MAPK activity remained elevated for ∼2 h and then decreased over 16 h to 16 pmol ⋅ min−1 ⋅ mg protein−1. When muscle strips were stretched and then unloaded, MAPK activity decreased within 1 h to the level present in the muscle before the stretch. These effects of muscle stretch on MAPK activity were additive to the effects of KCl or phorbol ester stimulation and were partially inhibited by reducing extracellular Ca2+. Eliminating extracellular Ca2+ had no effect on phorbol 12,13-dibutyrate (PDBu)-dependent contractions or MAPK activity; however, KCl-dependent contractions and MAPK activity were completely abolished by this procedure. An antibody specific for detecting caldesmon phosphorylated by MAPK, vs. protein kinase C (PKC), was developed and used to assess relative caldesmon phosphorylation in unstimulated and PDBu-stimulated muscle strips. In all cases investigated, the level of MAPK activity correlated with phosphocaldesmon immunoreactivity. Because arterial MAPK activity is regulated by PKC- and stretch-dependent mechanisms, these data are consistent with a role for MAPK and the subsequent phosphorylation of caldesmon as mediators in the stretch activation of vascular smooth muscle.
Contribution of the NH2terminus of Kv2.1 to channel activation
Authors: Pascual Juan M. , Shieh Char-Chang , Kirsch Glenn E. , Brown Arthur M.
Published: December 2017

Opening and closing of voltage-operated channels requires the interaction of diverse structural elements. One approach to the identification of channel domains that participate in gating is to locate the sites of action of modifiers. Covalent reaction of Kv2.1 channels with the neutral, sulfhydryl-specific methylmethanethiosulfonate (MMTS) caused a slowing of channel gating with a predominant effect on the kinetics of activation. These effects were also obtained after intracellular, but not extracellular, application of a charged MMTS analog. Single channel analysis revealed that MMTS acted primarily by prolonging the latency to first opening without substantially affecting gating transitions after the channel first opens and until it inactivates. To localize the channel cysteine(s) with which MMTS reacts, we generated NH2- and COOH-terminal deletion mutants and a construct in which all three cysteines in transmembrane regions were substituted. Only the NH2-terminal deletion construct gave rise to currents that activated slowly and displayed MMTS-insensitive kinetics. These results show that the NH2-terminal tail of Kv2.1 participates in transitions leading to activation through interactions involving reduced cysteine(s) that can be modulated from the cytoplasmic phase.
Activation of the human histamine H2 receptor is linked to cell proliferation and c-fos gene transcription
Authors: Wang L.-D. , Hoeltzel M. , Butler K. , Hare B. , Todisco A. , Wang M. , Del Valle J.
Published: December 2017

We examined whether histamine could regulate cell proliferation and expression of the early response gene c- fos in HEK-293 cells stably transfected with the human H2receptor (HEK-H2). Histamine stimulated [3H]thymidine incorporation [50% effective concentration (EC50) = 3.6 × 10−6 M] in HEK-H2 cells in a cimetidine-sensitive manner and increased c- fos mRNA in a time-dependent fashion, reaching maximal induction after 30 min. Histamine induced luciferase activity in HEK-H2cells transiently transfected with a construct containing the luciferase reporter gene ( Luc) coupled to the serum response element (SRE) of the c- fos gene promoter (EC50 = 1.5 × 10−6 M) or a plasmid containing the SRE core fragment (bases −320 to −298). The protein kinase C (PKC) inhibitor staurosporine and long-term pretreatment of HEK cells with phorbol ester inhibited the effect of histamine on PKC activation, SRE- Lucactivity, and [3H]thymidine incorporation. We have demonstrated that activation of the human H2 receptor can lead to induction of c- fos gene transcription and cell proliferation through a PKC-dependent mechanism.
Protein kinase Cβ regulates heterologous desensitization of thrombin receptor (PAR-1) in endothelial cells
Authors: Yan Weihong , Tiruppathi Chinnaswamy , Lum Hazel , Qiao Renli , Malik Asrar B.
Published: December 2017

We studied the effects of protein kinase C (PKC) activation on endothelial cell surface expression and function of the proteolytically activated thrombin receptor 1 (PAR-1). Cell surface PAR-1 expression was assessed by immunofluorescence (using anti-PAR-1 monoclonal antibody), and receptor activation was assessed by measuring increases in cytosolic Ca2+ concentration in human dermal microvascular endothelial cells (HMEC) exposed to α-thrombin or phorbol ester, 12- O-tetradecanoylphorbol-13-acetate (TPA). Immunofluorescence showed that thrombin and TPA reduced the cell surface expression of PAR-1. Prior exposure of HMEC to thrombin for 5 min desensitized the cells to thrombin, indicating homologous PAR-1 desensitization. In contrast, prior activation of PKC with TPA produced desensitization to thrombin and histamine, indicating heterologous PAR-1 desensitization. Treatment of cells with staurosporine, a PKC inhibitor, fully prevented heterologous desensitization, whereas thrombin-induced homologous desensitization persisted. Depletion of PKCβ isozymes (PKCβI and PKCβII) by transducing cells with antisense cDNA of PKCβIprevented the TPA-induced decrease in cell surface PAR-1 expression and restored ∼60% of the cytosolic Ca2+ signal in response to thrombin. In contrast, depletion of PKCβ isozymes did not affect the loss of cell surface PAR-1 and induction of homologous PAR-1 desensitization by thrombin. Therefore, homologous PAR-1 desensitization by thrombin occurs independently of PKCβ isozymes, whereas the PKCβ-activated pathway is important in signaling heterologous PAR-1 desensitization in endothelial cells.
Stimulation of membrane serine-threonine phosphatase in erythrocytes by hydrogen peroxide and staurosporine
Authors: Bize Isabel , Muñoz Patricia , Canessa Mitzy , Dunham Philip B.
Published: December 2017

Indirect evidence has suggested that K-Cl cotransport in human and sheep erythrocytes is activated physiologically by a serine-threonine phosphatase. It is activated experimentally by H2O2and by staurosporine, a kinase inhibitor. Activation by H2O2and staurosporine is inhibited by serine-threonine phosphatase inhibitors, suggesting that the activators stimulate the phosphatase. The present study shows that sheep and human erythrocytes contain membrane-associated as well as cytosolic serine-threonine phosphatases, assayed from the dephosphorylation of32P-labeled glycogen phosphorylase. In cells from both species, the relatively low sensitivity of the membrane enzyme to okadaic acid suggests it is type 1 protein phosphatase. The cytosolic phosphatase was much more sensitive to okadaic acid. Membrane-associated phosphatase was stimulated by both H2O2and staurosporine. The results support earlier conclusions that the membrane-associated type 1 phosphatase identified here is regulated by phosphorylation and oxidation. The results are consistent with the phosphatase, or a portion of it, being responsible for activating K-Cl cotransport.
Activity and protein localization of multiple glutamate transporters in gestation day 14 vs. day 20 rat placenta
Authors: Matthews James C. , Beveridge Mark J. , Malandro Marc S. , Rothstein Jeffrey D. , Campbell-Thompson Martha , Verlander Jill W. , Kilberg Michael S. , Novak Donald A.
Published: December 2017

Concentrative absorption of glutamate by the developing placenta is critical for proper fetal development. The expression of GLAST1, GLT1, EAAC1, and EAAT4, known to be capable ofd-aspartate-inhibitable and Na+-coupled glutamate transport (system [Formula: see text]), was evaluated in day 14 vs. day 20 rat chorioallantoic placenta. Steady-state mRNA levels were greater at day 20 for all transporters. Immunohistochemistry determined that the expression of GLAST1, GLT1, and EAAC1 was greater throughout the day 20 placenta and was asymmetric with respect to cellular localization. EAAT4 protein was not detected. System[Formula: see text] activity was responsible for most of the Na+-dependent glutamate uptake and was greater in day 20 than in day 14apical and basal membrane subdomains of the labyrinth syncytiotrophoblast. Greater quantities of EAAC1 and GLAST1 protein were identified on day 20, and quantities were greater in basal than in apical membranes. GLT1 expression, unchanged in apical membranes, was decreased in basal membranes. These data correlate transporter mRNA and protein content with transport activity and demonstrate an increasing capacity for glutamate absorption by the developing placenta.
Cellular differentiation and I-FABP protein expression modulate fatty acid uptake and diffusion
Authors: Atshaves Barbara P. , Foxworth William B. , Frolov Andrey , Roths John B. , Kier Ann B. , Oetama Betty K. , Piedrahita Jorge A. , Schroeder Friedhelm
Published: December 2017

The effect of cellular differentiation on fatty acid uptake and intracellular diffusion was examined in transfected pluripotent mouse embryonic stem (ES) cells stably expressing intestinal fatty acid binding protein (I-FABP). Control ES cells, whether differentiated or undifferentiated, did not express I-FABP. The initial rate and maximal uptake of the fluorescent fatty acid, 12-( N-methyl)- N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-octadecanoic acid (NBD-stearic acid), was measured in single cells by kinetic digital fluorescence imaging. I-FABP expression in undifferentiated ES cells increased the initial rate and maximal uptake of NBD-stearic acid 1.7- and 1.6-fold, respectively, as well as increased its effective intracellular diffusion constant ( D eff) 1.8-fold as measured by the fluorescence recovery after photobleaching technique. In contrast, ES cell differentiation decreased I-FABP expression up to 3-fold and decreased the NBD-stearic acid initial rate of uptake, maximal uptake, and D eff by 10-, 4.7-, and 2-fold, respectively. There were no significant differences in these parameters between the differentiated control and differentiated I-FABP-expressing ES cell lines. In summary, differentiation and expression of I-FABP oppositely modulated NBD-stearic acid uptake parameters and intracellular diffusion in ES cells.
Endotoxin-induced skeletal muscle contractile dysfunction: contribution of nitric oxide synthases
Authors: El-Dwairi Q. , Comtois A. , Guo Y. , Hussain S. N. A.
Published: December 2017

The aims of this study were to assess the role of nitric oxide (NO) and the contribution of different NO synthase (NOS) isoforms in skeletal muscle contractile dysfunction in septic shock. Four groups of conscious rats were examined. Group 1 served as control; groups 2, 3, and 4 were injected with Escherichia coli endotoxin [lipopolysaccharide (LPS), 20 mg/kg ip] and killed after 6, 12, and 24 h, respectively. Protein expression was assessed by immunoblotting and immunostaining. LPS injection elicited a transient expression of the inducible NOS isoform, which peaked 12 h after LPS injection and disappeared within 24 h. This expression coincided with a significant increase in nitrotyrosine formation (peroxynitrite footprint). Muscle expression of the endothelial and neuronal NOS isoforms, by comparison, rose significantly and remained higher than control levels 24 h after LPS injection. In vitro measurement of muscle contractility 24 h after LPS injection showed that incubation with NOS inhibitor ( S-methyliosothiourea) restored the decline in submaximal force generation, whereas maximal muscle force remained unaffected. We conclude that NO plays a significant role in muscle contractile dysfunction in septic animals and that increased NO production is due to induction of the inducible NOS isoform and upregulation of constitutive NOS isoforms.
Growth factor-like phospholipids generated after corneal injury
Authors: Liliom Károly , Guan Zhiwei , Tseng Jih-Lie , Desiderio Dominic M. , Tigyi Gábor , Watsky Mitchell A.
Published: December 2017

The present study provides evidence that growth factor-like glycerophosphate mediators of the lysophosphatidic acid (LPA) family are present in the aqueous humor and the lacrimal gland fluid of the rabbit eye. By use of a combination of HPLC, two-dimensional TLC, mass spectrometry, and the Xenopus oocyte bioassay, the LPA-like phospholipids LPA, cyclic PA, alkenyl-glycerophosphate (GP), lysophosphatidylserine, and phosphatidic acid were detected as physiological constituents of the fluids bathing the cornea. Corneal injury resulted in an increased production of some of these mediators. Alkenyl-GP, a novel member of the LPA family, has been identified in postinjury aqueous humor, establishing that it is generated endogenously. LPA and its homologues were found to be mitogenic in freshly dissociated keratocytes from uninjured corneas. There appears to be a link between the occurrence of LPA responsiveness in keratocytes activated by injury and the increase in LPA-like activity in aqueous humor. These data suggest that LPA and its homologues are involved in maintaining the integrity of the normal cornea and in promoting cellular regeneration of the injured cornea.
Inhibition of proximal tubular fluid absorption by nitric oxide and atrial natriuretic peptide in rat kidney
Authors: Eitle Eveline , Hiranyachattada Siriphun , Wang Hui , Harris Peter J.
Published: December 2017

Atrial natriuretic factor (ANF) and nitric oxide (NO) stimulate production of guanosine 3′,5′-cyclic monophosphate (cGMP) and are natriuretic. Split-drop micropuncture was performed on anesthetized rats to determine the effects of ANF and the NO donor sodium nitroprusside (SNP) on proximal tubular fluid absorption rate ( J va). Compared with control solutions, SNP (10−4 M) decreased J va by 23% when administered luminally and by 35% when added to the peritubular perfusate. Stimulation of fluid uptake by luminal angiotensin II (ANG II; 10−9 M) was abolished by SNP (10−4 and 10−6 M). In proximal tubule suspensions, ANF (10−6 M) increased cGMP concentration to 143%, whereas SNP (10−6, 10−5, 10−4, 10−3 M) raised cGMP to 231, 594, 687, and 880%, respectively. S-nitroso- N-acetylpenicillamine (SNAP) also raised cGMP concentrations with similar dose-response relations. These studies demonstrate inhibition by luminal and peritubular NO of basal and ANG II-stimulated proximal fluid absorption in vivo. The ability of SNP to inhibit basal fluid uptake whereas ANF only affected ANG II-stimulated transport may be because of production of higher concentrations of cGMP by SNP.
Norepinephrine stimulates arachidonic acid release from vascular smooth muscle via activation of cPLA2
Authors: LaBelle Edward F. , Polyak Erzsebet
Published: December 2017

The mechanism of agonist-activated arachidonate release was studied in segments of rat tail artery. Tail artery segments were prelabeled with [3H]arachidonate and then stimulated with norepinephrine (NE), and the radioactivity of the extracellular medium was determined. NE stimulated arachidonate release from the tissue without increasing arachidonic acid levels within cellular cytosol or crude membranes. About 90% of the extracellular radioactivity was shown to be unmetabolized arachidonate by TLC. Arachidonic acid release was not inhibited by the removal of the endothelium from the artery. NE exerted a half-maximal effect at a concentration of 0.2 μM. NE-stimulated arachidonate release was not inhibited by blockers of phospholipase C (U-73122), diacylglycerol lipase (RHC-80267), secretory phospholipase A2 (manoalide), calcium-insensitive phospholipase A2 (HELSS), or β-adrenergic receptors (propranolol). NE-stimulated arachidonic acid release was inhibited by blockers of cytosolic phospholipase A2(cPLA2) (AACOCF3), α1-adrenergic receptors (prazosin), and specific G proteins (pertussis toxin). This indicated that NE stimulated arachidonate release from vascular smooth muscle via activation of α-adrenergic receptors, either Gi or Go, and cPLA2. NE-activated arachidonic acid release from vascular smooth muscle may play a role in force generation by the tissue. Perhaps arachidonic acid extends the effect of NE on one specific smooth muscle cell to its nearby neighbor cells.
Model of β-cell mitochondrial calcium handling and electrical activity. I. Cytoplasmic variables
Authors: Magnus Gerhard , Keizer Joel
Published: December 2017

We continue our development of a kinetic model of bursting electrical activity in the pancreatic β-cell ( J. Keizer and G. Magnus. Biophys. J. 56: 229–242, 1989), including the influence of Ca2+ handling by the mitochondria. Our minimal model of mitochondrial Ca2+handling [G. Magnus and J. Keizer. Am. J. Physiol. 273 ( Cell Physiol. 42): C717–C733, 1997] is expanded to include the d-glucose dependence of the rate of production of mitochondrial reducing equivalents. The Ca2+ dependence of the mitochondrial dehydrogenases, which is also included in the model, plays only a small role in the simulations, since the dehydrogenases appear to be maximally activated whend-glucose concentrations are sufficient to produce bursting. A previous model of ionic currents in the plasma membrane is updated using a recent experimental characterization of the dependence of the conductance of the ATP-sensitive K+(KATP) current on adenine nucleotides. The resulting whole cell model is complex, involving 12 dynamic variables that couple Ca2+ handling in the cytoplasm and the mitochondria with electrical activity in the plasma and inner mitochondrial membranes. Simulations with the whole cell model give rise to bursting electrical activity similar to that seen in pancreatic islets and clusters of pancreatic β-cells. The full d-glucose dose response of electrical activity is obtained if the cytosolic rate of ATP hydrolysis is a sigmoidal function of glucose. The simulations give the correct shape, period, and phase of the associated oscillations in cytosolic Ca2+, predict that the conductance of the KATPcurrent oscillates out of phase with electrical activity [as recently observed in ob/ ob mice (O. Larsson, H. Kindmark, R. Bränstrom, B. Fredholm, and P.-O. Berggren. Proc. Natl. Acad. Sci. USA 93: 5161–5165, 1996)], and make other novel predictions. In this model, bursting results because Ca2+uptake into mitochondria during the active phase reduces the mitochondrial inner membrane potential, reducing the rate of production of ATP, which in turn activates the KATP current and repolarizes the plasma membrane.
Regulation of cardiac AMP-specific 5′-nucleotidase during ischemia mediates ATP resynthesis on reflow
Authors: Bak Marianna I. , Ingwall Joanne S.
Published: December 2017

The ability to resynthesize ATP during recovery from ischemia is limited to the size of endogenous pool of adenine nucleotides. Cytosolic AMP-specific 5′-nucleotidase (5′-NT) plays a key role in ATP degradation and hence the capacity for ATP resynthesis. We have suggested ( J. Clin. Invest. 93: 40–49, 1994) that intracellular acidosis [intracellular pH (pHi)] is a potent inhibitor of 5′-NT under in vivo conditions. To test this hypothesis further, we used the hyperthyroid rat heart because we could alter pHi during ischemia and determine the consequences of lower pHi on AMP accumulation (by chemical assay) and ATP resynthesis (by31P nuclear magnetic resonance spectroscopy) during reperfusion. Global no-flow ischemia caused pHi to decrease from 7.1 under well-oxygenated control perfusion to 6.7. We found that decreasing pHi further from pH 6.7 to 6.4 leads to increased accumulation (30%) of AMP during ischemia and to a 2.5-fold increase in ATP resynthesis during reperfusion. Analysis of all known substrates, products, activators, and inhibitors of the 5′-NT suggests that 5′-NT is activated primarily by Mg2+ and ADP and is inhibited by H+. Thus these observations provide evidence for a salutary effect of intracellular acidosis on preserving the AMP pool due to inhibition of 5′-NT and suggest a novel role of H+ in protecting ischemic tissue.
cGMP-mediated Ca2+ release from IP3-insensitive Ca2+ stores in smooth muscle
Authors: Murthy Karnam S. , Makhlouf Gabriel M.
Published: December 2017

Recent studies on the role of nitric oxide (NO) in gastrointestinal smooth muscle have raised the possibility that NO-stimulated cGMP could, in the absence of cGMP-dependent protein kinase (PKG) activity, act as a Ca2+-mobilizing messenger [K. S. Murthy, K.-M. Zhang, J.-G. Jin, J. T. Grider, and G. M. Makhlouf. Am. J. Physiol. 265 ( Gastrointest. Liver Physiol. 28): G660–G671, 1993]. This notion was examined in dispersed gastric smooth muscle cells with 8-bromo-cGMP (8-BrcGMP) and with NO and vasoactive intestinal peptide (VIP), which stimulate endogenous cGMP. In muscle cells treated with cAMP-dependent protein kinase (PKA) and PKG inhibitors (H-89 and KT-5823), 8-BrcGMP (10 μM), NO (1 μM), and VIP (1 μM) stimulated45Ca2+release (21 ± 3 to 30 ± 1% decrease in45Ca2+cell content); Ca2+ release stimulated by 8-BrcGMP was concentration dependent with an EC50 of 0.4 ± 0.1 μM and a threshold of 10 nM. 8-BrcGMP and NO increased cytosolic free Ca2+ concentration ([Ca2+]i) and induced contraction; both responses were abolished after Ca2+ stores were depleted with thapsigargin. With VIP, which normally increases [Ca2+]iby stimulating Ca2+ influx, treatment with PKA and PKG inhibitors caused a further increase in [Ca2+]ithat reverted to control levels in cells pretreated with thapsigargin. Neither Ca2+ release nor contraction induced by cGMP and NO in permeabilized muscle cells was affected by heparin or ruthenium red. Ca2+ release induced by maximally effective concentrations of cGMP and inositol 1,4,5-trisphosphate (IP3) was additive, independent of which agent was applied first. We conclude that, in the absence of PKA and PKG activity, cGMP stimulates Ca2+ release from an IP3-insensitive store and that its effect is additive to that of IP3.
Effect of phorbol esters on Ca2+ sensitivity and myosin light-chain phosphorylation in airway smooth muscle
Authors: Bremerich Dorothee H. , Kai Tetsuya , Warner David O. , Jones Keith A.
Published: December 2017

We studied in β-escin-permeabilized canine tracheal smooth muscle (CTSM) the effect of the protein kinase C (PKC) agonist phorbol 12,13-dibutyrate (PDBu) on isometric force at a constant submaximal Ca2+ concentration (i.e., the effect on Ca2+ sensitivity) and regulatory myosin light-chain (rMLC) phosphorylation. PDBu increased Ca2+sensitivity, an increase associated with a concentration-dependent, sustained increase in rMLC phosphorylation. PDBu altered the relationship between rMLC phosphorylation and isometric force such that the increase in isometric force was less than that expected for the increase in rMLC phosphorylation observed. The effect of four PKC inhibitors [calphostin C, chelerythrine chloride, a pseudosubstrate inhibitor for PKC, PKC peptide-(19—31) (PSSI), and staurosporine] on PDBu-induced Ca2+ sensitization as well as the effect of calphostin C and PSSI on rMLC phosphorylation were determined. Whereas none of these compounds prevented or reversed the PDBu-induced increase in Ca2+sensitivity, the PDBu-induced increase in rMLC phosphorylation was inhibited. We conclude that PDBu increases rMLC phosphorylation by activation of PKC but that the associated PDBu-induced increases in Ca2+ sensitivity are mediated by mechanisms other than activation of PKC in permeabilized airway smooth muscle.
5′ Heterogeneity in epithelial sodium channel α-subunit mRNA leads to distinct NH2-terminal variant proteins
Authors: Thomas Christie P. , Auerbach Scott , Stokes John B. , Volk Kenneth A.
Published: December 2017

The amiloride-sensitive epithelial sodium channel (ENaC) is composed of three subunits: α, β, and γ. The human α-ENaC subunit is expressed as at least two transcripts (N. Voilley, E. Lingueglia, G. Champigny, M. G. Mattei, R. Waldmann, M. Lazdunski, and P. Barbry. Proc. Natl. Acad. Sci. USA91: 247–251, 1994). To determine the origin of these transcripts, we characterized the 5′ end of the α-ENaC gene. Four transcripts that differ at their first exon were identified. Exon 1A splices to exon 2 to form the 5′ end of α-ENaC1, whereas exon 1B arises separately and continues into exon 2 to form α-ENaC2. Other variant mRNAs, α-ENaC3 and α-ENaC4, are formed by activating 5′ splice sites within exon 1B. Although α-ENaC3 and -4 did not change the open reading frame for α-ENaC, α-ENaC2 contains upstream ATGs that add 59 amino acids to the previous (α-ENaC1) protein. To address the significance of these isoforms, both proteins were expressed in Xenopus oocytes. The cRNA for each α-ENaC transcript when combined with β- and γ-ENaC cRNA reconstituted a low-conductance ion channel with amiloride-sensitive currents of similar characteristics. We have thus identified variant α-ENaC mRNAs that lead to functional ENaC peptides.
Aquaporins in complex tissues: distribution of aquaporins 1–5 in human and rat eye
Authors: Hamann Steffen , Zeuthen Thomas , Cour Morten La , Nagelhus Erlend A. , Ottersen Ole Petter , Agre Peter , Nielsen Søren
Published: December 2017

Multiple physiological fluid movements are involved in vision. Here we define the cellular and subcellular sites of aquaporin (AQP) water transport proteins in human and rat eyes by immunoblotting, high-resolution immunocytochemistry, and immunoelectron microscopy. AQP3 is abundant in bulbar conjunctival epithelium and glands but is only weakly present in corneal epithelium. In contrast, AQP5 is prominent in corneal epithelium and apical membranes of lacrimal acini. AQP1 is heavily expressed in scleral fibroblasts, corneal endothelium and keratocytes, and endothelium covering the trabecular meshwork and Schlemm’s canal. Although AQP1 is plentiful in ciliary nonpigmented epithelium, it is not present in ciliary pigmented epithelium. Posterior and anterior epithelium of the iris and anterior lens epithelium also contain significant amounts of AQP1, but AQP0 (major intrinsic protein of the lens) is expressed in lens fiber cells. Retinal Müller cells and astrocytes exhibit notable concentrations of AQP4, whereas neurons and retinal pigment epithelium do not display aquaporin immunolabeling. These studies demonstrate selective expression of AQP1, AQP3, AQP4, and AQP5 in distinct ocular epithelia, predicting specific roles for each in the complex network through which water movements occur in the eye.
Dying enterocytes downregulate signaling pathways converging on Ras: rescue by protease inhibition
Authors: Scheving Lawrence A. , Jin Wen-Hui , Chong Kang-Mei , Gardner Wendi , Cope Frederick O.
Published: December 2017

Organ and cell cultures of the small intestine serve as excellent in vitro models for programmed cell death (PCD). Cells cultured in serum-free, minimal medium rapidly died, as evidenced by histological changes, internucleosomal DNA cleavage, and TdT-mediated dUTP nick end labeling. Cell death was pervasive, although nonepithelial cells within the fibrovascular villus core were spared. PCD did not require a functional p53 gene. Serine and cysteine protease inhibitors, but not FCS, suppressed it. Relative to structural and functional proteins, dying enterocytes rapidly downregulated Ras-convergent proteins, including epidermal growth factor receptor, Erb-B2, and the son of sevenless guanine nucleotide exchangers. Reductions in the steady-state levels of both protein and mRNA were observed. These reductions were prevented by a combination of death-defying serine and caspase inhibitors, indicating a requirement for the initiation of death. Thus, during catastrophic PCD, intestinal epithelial cells delete cell surface signaling pathways responsible for Ras activation.
Peroxynitrite inhibits amiloride-sensitive Na+ currents inXenopus oocytes expressing αβγ-rENaC
Authors: DuVall M. D. , Zhu S. , Fuller C. M. , Matalon S.
Published: December 2017

We examined the effect of peroxynitrite (ONOO−) on the cloned rat epithelial Na+ channel (αβγ-rENaC) expressed in Xenopusoocytes. 3-Morpholinosydnonimine (SIN-1) was used to concurrently generate nitric oxide (⋅ NO) and superoxide ([Formula: see text] ⋅), which react to form ONOO−, a species known to promote protein nitration and oxidation. Under control conditions, oocytes displayed an amiloride-sensitive whole cell conductance of 7.4 ± 2.8 (SE) μS. When incubated at 18°C with SIN-1 (1 mM) for 2 h (final ONOO− concentration = 10 μM), the amiloride-sensitive conductance was reduced to 0.8 ± 0.5 μS. To evaluate whether the observed inhibition was due to ONOO−, as opposed to ⋅ NO, we also exposed oocytes to SIN-1 in the presence of urate (500 μM), a scavenger of ONOO− and superoxide dismutase, which scavenges[Formula: see text] ⋅, converting SIN-1 from an ONOO− to an ⋅ NO donor. Under these conditions, conductance values remained at control levels following SIN-1 treatment. Tetranitromethane, an agent that oxidizes sulfhydryl groups at pH 6, also inhibited the amiloride-sensitive conductance. These data suggest that oxidation of critical sulfhydryl groups within rENaC by ONOO− directly inhibits channel activity.
IP3 receptor blockade fails to prevent intracellular Ca2+release by ET-1 and α-thrombin
Authors: Mathias Robert S. , Mikoshiba Katsuhiko , Michikawa Takayuki , Miyawaki Atsushi , Ives Harlan E.
Published: December 2017

The effect of inositol 1,4,5-trisphosphate (IP3) receptor blockade on platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), endothelin-1 (ET-1), or α-thrombin receptor-mediated intracellular Ca2+([Formula: see text]) release was examined using fura 2 microspectrofluorometry in single Chinese hamster ovary cells and myoblasts. Blockade of the IP3receptor was achieved by microinjection of heparin or monoclonal antibody (MAb) 18A10 into the IP3type 1 receptor. Heparin completely inhibited[Formula: see text] release after flash photolysis with caged IP3 and after exposure to PDGF and FGF. In contrast, heparin failed to block[Formula: see text] release after α-thrombin and ET-1. After application of ligand, IP3 levels were five- to sevenfold higher for α-thrombin than for ET-1 or PDGF. IP3 levels after PDGF and ET-1 were comparable. Similar to heparin, MAb 18A10 blocked[Formula: see text] release after PDGF but failed to block [Formula: see text] release after ET-1 or α-thrombin. These data suggest that the mechanisms of[Formula: see text] release by tyrosine kinase and certain 7-transmembrane receptors may differ. Although both receptor types use the IP3-signaling system, the ET-1 and α-thrombin receptors may have a second, alternative mechanism for activating[Formula: see text] release.
Developmental regulation of ENaC subunit mRNA levels in rat kidney
Authors: Vehaskari V. Matti , Hempe James M. , Manning Jennifer , Aviles Diego H. , Carmichael Mary Catherine
Published: December 2017

To assess the role of distal nephron apical Na channel (ENaC) gene expression in Na wasting by the immature kidney, ENaC α-, β-, and γ-subunit mRNA levels were examined in the rat by RT-PCR. In microdissected nephron segments, all three ENaC subunit mRNAs were detected in the distal convoluted tubule, connecting tubule, cortical collecting duct, and outer medullary collecting duct. The inner medullary collecting duct and all other nephron segments were consistently negative. The mRNA levels were quantified in kidneys at different developmental stages by multiplex RT-PCR with “primer dropping,” with endoplasmic reticulum-specific cyclophilin mRNA as an internal standard. All three ENaC mRNA levels were low or undetectable on gestational day 16 and only slightly higher 3 days before birth. A sharp rise was observed between 3 days before and 1–3 days after birth; the levels at postnatal days 1–3 were already similar to those of adult kidneys. The results suggest that ENaC subunit gene expression is not a limiting factor in the full-term newborn rat kidney, but low levels of expression may limit distal Na absorption in more immature kidneys, such as those of very premature human infants.