bluuebunny/crossref_metadata_2025 huggingface dataset:
Nerve growth factor stimulates rapid metabolic responses in PC12 cells
Authors: Pitchford S. , De Moor K. , Glaeser B. S.
Published: December 2017

Research into the effects of nerve growth factor (NGF) has involved study of either the signal transduction process or the morphological result of growth factor treatment (cell proliferation and/or differentiation). The Cytosensor Microphysiometer, a silicon-based biosensor system that allows the continuous and real-time monitoring of extracellular acidification rate changes of cells, was used to study the response of PC12 cells to NGF. Stimulation resulted in a rapid increase in the acidification rate of cells in a concentration-dependent fashion (0.1-200 ng/ml NGF; mean effective concentration value of 153 +/- 54 pM). Inhibition of the NGF receptor-linked protein tyrosine kinase by either genistein or K252a attenuated the acidification rate response to NGF. In addition, the acidification response to NGF could be modified by inhibiting Na+/H+ exchange and, separately, glycolysis. This implicates these processes in the metabolic response of PC12 cells to NGF stimulation.
Circulating hyaluronan levels in the rodent: effects of age and diet
Authors: Yannariello-Brown J. , Chapman S. H. , Ward W. F. , Pappas T. C. , Weigel P. H.
Published: December 2017

Circulating hyaluronan (HA) levels were investigated as a function of age and diet in Fischer 344 male rats. A biphasic pattern of age-related changes was observed in rats fed ad libitum a diet in which the protein source was soya/fish meal. HA levels in 3- to 6- and 22- to 29-mo-old rats were not statistically different. However, HA levels in 12- to 20-mo-old rats were 10-29% of the levels in younger or aged adults. HA levels were also measured in rats fed ad libitum a semisynthetic diet in which the protein source was hydrolyzed casein. Whereas the two colonies exhibited similar biphasic age-related changes, HA levels differed 4- to 20-fold at every age examined. Caloric restriction affected HA levels in 19-mo-old casein-fed rats; HA levels were 2.3 times higher than age-matched controls and were not statistically different from young or aged animals. Serum and plasma HA levels were identical in the same individuals at all ages tested. These data suggest that HA turnover and metabolism in the rat are affected by age, dietary composition, and caloric intake.
Duality of plasmin effect on cytosolic free calcium in human platelets
Authors: Nakamura K. , Kimura M. , Fenton J. W. , Andersen T. T. , Aviv A.
Published: December 2017

Plasmin caused a modest and gradual increase in platelet cytosolic Ca2+, mediated through both Ca2+ mobilization and external Ca2+ entry. This response was associated with accelerated Ca2+ extrusion and protein tyrosine phosphorylation. Plasmin-enhanced external Ca2+ entry and Ca2+ extrusion (but not Ca2+ mobilization) were attenuated by the tyrosine kinase inhibitor, genistein. Plasmin inhibited the thrombin-evoked increase in cytosolic Ca2+ and also inhibited the Ca2+ response to the tethered peptide TRAP-6 of the thrombin receptor. Furthermore, plasmin inhibited the binding of 125I-labeled alpha-thrombin to platelets. The inhibitory effect of plasmin on the thrombin response shared some characteristics with the effect of protein kinase C stimulators but was not reversed by protein kinase C inhibitors. Plasmin did not change platelet cyclic nucleotides. These results suggest a dual effect of plasmin. Plasmin produces a small rise in platelet cytosolic Ca2+ and a tyrosine kinase-dependent enhancement of Ca2+ turnover (external Ca2+ influx and Ca2+ efflux). However, it also attenuates the thrombin-evoked cytosolic Ca2+ response by blocking Ca2+ mobilization and slowing the rate of external Ca2+ influx. The latter feature would result in a plasmin-induced inhibition of thrombogenesis.
Role of NO generation in beta-adrenoceptor-mediated stimulation of rabbit airway ciliary motility
Authors: Tamaoki J. , Chiyotani A. , Kondo M. , Konno K.
Published: December 2017

To determine possible contribution of nitric oxide (NO) to the stimulatory action of beta-adrenoceptor agonist on ciliary motility, we measured ciliary beat frequency (CBF) of rabbit cultured tracheal epithelial cells by photoelectric method and NO release by specific amperometric sensors for this molecule in vitro. Salbutamol increased CBF, an effect that was potentiated by superoxide dismutase. Pretreatment of cells with NG-nitro-L-arginine methyl ester (L-NAME) attenuated the salbutamol-induced increase in CBF, causing a rightward displacement of the concentration-response curve by 2-2.5 log units, whereas NG-nitro-D-arginine methyl ester had no effect. The inhibitory effect of L-NAME was reversed by L-arginine but not by D-arginine. Immersion of the NO-selective electrode in the medium containing epithelial cells detected baseline current of 4.6-14.5 pA, which was abolished by L-NAME. Salbutamol dose-dependently increased the concentration of NO in the medium, the maximal increase being 56.2 +/- 5.3 nM (mean +/- SE; P < 0.001). These results suggest that NO is spontaneously released by airway epithelium and that the enhanced release of this molecule may play a role in the beta-adrenoceptor-mediated stimulation of ciliary motility
Decreased polysomal HSP-70 may slow polypeptide elongation during skeletal muscle atrophy
Authors: Ku Z. , Yang J. , Menon V. , Thomason D. B.
Published: December 2017

Slowed elongation rate is the apparent cause of the rapid decrease in rat soleus muscle protein synthesis rate during non-weight bearing. We found that elongation factor 2 was not phosphorylated and thus could not explain the slowed elongation rate. However, we observed a 44 +/- 19 and 28 +/- 14% decrease in the chaperone protein 70-kDa heat-shock cognate/heat shock protein (HSC/HSP-70) associated with the polysomes after 12 and 18 h of non-weight bearing, respectively. Size-fractionated polysomes had less HSC/HSP-70 associated with the larger polysomes in 18-h non-weight-bearing soleus muscle. ATP concentration increased in the non-weight-bearing muscle, so, because ATP enhances HSC/HSP-70 dissociation, we tested the potential role of ATP. Digitonin-permeabilized myoblasts treated with increasing concentrations of ATP showed both a decreased association of HSC/HSP-70 with the polysomes and a shift toward heavier polysomes; these responses were blocked by adenosine 5'-O-(3-thiotriphosphate). These data are consistent with the role of HSC/HSP-70 as a chaperone of nascent protein. The absence of HSC/HSP-70 may slow ribosome translocation, thus slowing elongation rate.
Quasi-linear relationship between Gibbs free energy of ATP hydrolysis and power output in human forearm muscle
Authors: Jeneson J. A. , Westerhoff H. V. , Brown T. R. , Van Echteld C. J. , Berger R.
Published: December 2017

The postulated strictly linear descriptions of the rate dependence of oxidative phosphorylation in skeletal muscle on the free energy of ATP hydrolysis (delta GP) over the range of physiological steady states fail to harmonize with reported findings of identical basal respiration rates in mammalian muscles at different delta GP values. The relevance of an extension of the strictly linear description to a description deriving from enzyme kinetics that predicts a sigmoidal dependence was investigated in human finger flexor muscle using 31P-nuclear magnetic resonance spectroscopy. At constant pH 7.0, the experimental variation of adenine nucleotide concentrations with power output, which reflects the rate of oxidative phosphorylation, was compared with predictions by various formulations of adenine nucleotide control of respiration. The quasi-linear sigmoidal description was found to be statistically equivalent but physiologically superior to the strictly linear description. The predicted maximal oxidatively sustained steady-state power output and rate-dependent sensitivity of respiration to changes in delta GP were in agreement both with theoretical considerations and with experimental observations in the present study and other studies of intact mammalian skeletal muscle.
Microtubule disruption stimulates system A transport in cultured vascular smooth muscle cells
Authors: Chen J. G. , Strawbridge A. B. , Kempson S. A.
Published: December 2017

This study has focused on the possible influence of microtubules for the regulation of Na(+)-dependent system A neutral amino acid transport in A10 cells, a cultured cell line derived from rat aortic vascular smooth muscle. When microtubules were disrupted by incubating cells for 5 h in serum-free medium containing colchicine, nocodazole, or vinblastine, there was a twofold increase in system A transport (Vmax change). The dose for the disruption of microtubules by colchicine was similar to the dose required for the stimulation of system A. The time course showed that system A stimulation did not occur until widespread disruption of microtubules was established. The stimulation was specific for system A; there were no changes in glucose transport and Na(+)-dependent transport of phosphate and glutamate. Serum refeeding of quiescent cells from 2 days of serum starvation led to stimulation of system A, glucose, and phosphate transport. However, only system A was activated when colchicine was added to the serum-free medium. Addition of colchicine during serum refeeding had no additive effect for the stimulation of system A. The stimulation by both colchicine and serum was blocked by cycloheximide and actinomycin D. These findings suggest that microtubule disruption may activate system A gene expression.
Protein kinase C isoforms in rat kidney proximal tubule: acute effect of angiotensin II
Authors: Karim Z. , Defontaine N. , Paillard M. , Poggioli J.
Published: December 2017

The present study examined the effect of phorbol esters, Ca2+, and angiotensin II (ANG II) on protein kinase C (PKC) isoforms in the rat proximal tubule. The immunoblot analysis of PKC isoforms of particulate and cytosolic fractions of proximal tubules revealed immunoreactive proteins when antibodies against PKC-alpha, -delta, -epsilon, and -zeta, but not -beta and -gamma were used. Phorbol dibutyrate (PDBU) induced the translocation of PKC-alpha, -delta, and -epsilon, whereas an inactive phorbol ester had no effect. PDBU and ionomycin increased particulate PKC specific activity from 0.67 +/- 0.09 to 1.56 +/- 0.18 and 0.96 +/- 0.04 pmol.microgram protein-1.2 min-1, respectively. ANG II (10(-7) M) induced a time-dependent increase in particulate PKC-alpha immunoreactivity observed after 2 min and maintained for 12 min. Particulate PKC-epsilon immunoreactivity increased after 4 min. Meanwhile, PKC-delta and -zeta were not modified by ANG II. Accordingly, ANG II elicited a rise in the specific activity of the particulate PKC, which increased to 0.89 +/- 0.09 pmol.micrograms protein-1.2 min-1 after 2 min. This was inhibited by a preincubation in the presence of 10(-5) M losartan, specific inhibitor of angiotensin subtype 1 receptors. These data indicate that PKC-alpha and -epsilon are potential candidates to regulate the activity of Na+/H+ and Na(+)-HCO3- transporters because they are translocated with a time course fitting with that of the reported effect of ANG II on those transporters.
Ca(2+)-induced mitochondrial membrane permeabilization: role of coenzyme Q redox state
Authors: Kowaltowski A. J. , Castilho R. F. , Vercesi A. E.
Published: December 2017

Rotenone-poisoned rat liver mitochondria energized by succinate addition, after a 5-min period of preincubation in presence of 10 microM Ca2+, produce H2O2 at much faster rates, undergo extensive swelling, and are not able to retain the membrane potential and accumulated Ca2+. Similar results were obtained when a suspension of rat liver mitochondria preincubated in anaerobic medium for 5 min was reoxygenated. The addition of either ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, ruthenium red, catalase, or dithiothreitol, just before succinate or O2 addition, prevented mitochondrial swelling, indicating the involvement of Ca2+, reactive oxygen species, and oxidation of membrane protein thiols in this process of membrane permeabilization. Inhibition of mitochondrial swelling by cyclosporin A suggests that the membrane alterations observed under these experimental conditions are related to opening of the permeability transition pore. The presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone, which prevents Ca2+ cycling across the membrane, did not inhibit mitochondrial swelling when Ca2+ influx into the mitochondrial matrix was driven by a high Ca2+ gradient. When rotenone plus antimycin A-poisoned mitochondria were energized by N,N,N',N'-tetramethyl-p-phenylenediamine, which reduces respiratory chain complex IV, mitochondrial swelling did not occur, unless succinate, which reduces coenzyme Q, was also added. It is concluded that reduced coenzyme Q is the electron source for oxygen radical production during Ca(2+)-stimulated oxidative damage of mitochondria.
Inhibition by polyunsaturated fatty acids of cell volume regulation and osmolyte fluxes in astrocytes
Authors: Sanchez-Olea R. , Morales-Mulia M. , Moran J. , Pasantes-Morales H.
Published: December 2017

The polyunsaturated fatty acids, arachidonic, linoleic, and linolenic acids, were potent blockers of regulatory volume decrease (RVD) and of the swelling-activated efflux of [3H]taurine, D-[3H]aspartate, [3H]inositol, and 125I (used as marker of Cl) from rat cerebellar astrocytes in culture. The monounsaturated oleic and ricinoleic acids and saturated fatty acids were ineffective. The amino acid and 125I fluxes were similarly inhibited by fatty acids, whereas inositol release was less sensitive. Polyunsaturated fatty acids appear to directly affect RVD in trypsinized astrocytes as the inhibition was immediate and fully reversible. Blockers of the arachidonic acid metabolic pathways, indomethacin (cyclooxygenase), esculetin (lipoxygenases), and metyrapone (P-450 monooxygenases), did not prevent the effect of arachidonic acid, suggesting that further metabolism is not required for displaying the effects of arachidonic acid on RVD and osmolyte fluxes. Some blockers of arachidonic acid metabolic pathways, such as nordihydroguaiaretic acid (lipoxygenases) and naphthoflavone (P-450 monooxygenases), also exhibited marked inhibitory effects on RVD and on osmolyte fluxes. The predominant arachidonic acid metabolite in astrocytes, 12-hydroxyeicosatetraenoic acid, did not affect RVD or osmolyte fluxes. These results suggest that arachidonic acid and other polyunsaturated fatty acids directly inhibit the permeability pathways correcting cell volume after swelling in cultured astrocytes.
Calcium responses of endothelial cell monolayers subjected to pulsatile and steady laminar flow differ
Authors: Helmlinger G. , Berk B. C. , Nerem R. M.
Published: December 2017

The vascular endothelium is the primary transducer of hemodynamically imposed mechanochemical events. In this study, we measured the intracellular free calcium concentration ([Ca2+]i) using the fluorescent probe fura 2 and ratiometric digital imaging in cultured bovine aortic endothelial cells (BAEC) subjected to various laminar flow patterns. These were steady shear stress (0.2-70 dyn/cm2) and three types of sinusoidal pulsatile shear stress (nonreversing: 40 +/- 20 dyn/cm2; reversing: 20 +/- 40 dyn/cm2; and purely oscillatory: 0 +/- 20 dyn/cm2; flow frequencies: 0.4, 1.0, and 2.0 Hz) in a serum-containing medium. The most dramatic finding was failure of a purely oscillatory flow to increase [Ca2+]i in BAEC monolayers. In contrast, steady flow, as well as nonreversing and reversing pulsatile flows, increased [Ca2+]i. The dynamics of the response were dependent on the flow pattern. Both internal Ca2+ release and extracellular Ca2+ entry were involved in these [Ca2+]i increases. Also, switching from either a steady nonreversing pulsatile or reversing pulsatile flow back to a static condition resulted in a [Ca2+]i increase. However, switching from an oscillatory flow to a static condition did not induce any changes in average [Ca2+]i. This study shows that endothelial cells are able to sense different flow environments in terms of [Ca2+]i signaling and is relevant to further studies of the influence of hemodynamic forces on vascular pathophysiology.
Deoxygenation-induced cation fluxes in sickle cells. IV. Modulation by external calcium
Authors: Joiner C. H. , Jiang M. , Franco R. S.
Published: December 2017

Net cation movements were measured in low-density sickle red blood cells (SS RBC) in the presence and absence of oxygen. External Ca2+ (Ca2+o) partially inhibited deoxygenation-induced fluxes of both Na+ and K+. Deoxygenation-induced Na+ influx was reduced by 2 mM Ca2+o to 0.71 +/- 0.04 (SE) of its value in Ca(2+)-free solutions, whereas this ratio was 0.90 +/- 0.05 for K+ efflux (P < 0.01 by paired t-test). Because Ca2+o inhibited Na+ influx more than K+ efflux, net cation loss in deoxygenated SS RBC was higher in the presence of Ca2+o. In separate experiments, Ca2+o reduced deoxygenation-induced Na+ influx to 0.66 +/- 0.03 of its Ca(2+)-free value compared with 0.77 +/- 0.03 for Rb+ influx (P < 0.001), indicating relative selectivity of this effect for Na+ over Rb+. However, this effect is not specific for Ca2+ because other divalent cations also inhibited deoxygenation-induced Na+ and K+ fluxes. Under the conditions of these experiments, no evidence for K+ channel activation was found, indicating that K+ loss measured in deoxygenated SS RBC was mediated by the deoxygenation-induced pathway. These studies show that in the presence of Ca2+o deoxygenation-induced Na+ influx and K+ efflux are unbalanced. This pathway can, therefore, mediate cation loss and contribute directly to cellular dehydration in SS RBC.
Lipid signal transduction pathways in angiotensin II type 1 receptor-transfected fibroblasts
Authors: Wen Y. , Cabot M. C. , Clauser E. , Bursten S. L. , Nadler J. L.
Published: December 2017

A stable Chinese hamster ovary fibroblast line expressing the rat vascular type 1a angiotensin II (ANG II) receptor was used to study the lipid-derived signal transduction pathways elicited by type 1a ANG II receptor activation. ANG II caused a biphasic and dose-dependent increase in diacylglycerol (DAG) accumulation with an initial peak at 15 s (181 +/- 11% of control, P < 0.02) and a second sustained peak at 5-10 min (214 +/- 10% of control, P < 0.02). The late DAG peak was derived from phosphatidylcholine (PC), and the formation was blocked by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. ANG II also increased phosphatidic acid (PA) production nearly fourfold by 7.5 min. In the presence of ethanol, ANG II markedly increased phosphatidylethanol (PEt) formation, indicating activation of phospholipase D (PLD). ANG II was shown to increase the mass of three separate PA species, one of which apparently originated from DAG kinase action on PC-phospholipase C (PLC)-produced DAG, providing evidence for PC-PLC activity. ANG II also formed a third PA species, which originated neither from PLD nor from DAG kinase. These results demonstrate that multiple lipid signals propagated via collateral stimulation of PLC and PLD are generated by specific activation of the vascular type 1a ANG II receptor.
Reconstitution of a KATP channel from basolateral membranes of Necturus enterocytes
Authors: Mayorga-Wark O. , Dubinsky W. P. , Schultz S. G.
Published: December 2017

We have previously reported that basolateral membrane vesicles isolated from Necturus maculosa small intestinal epithelial cells and incorporated into planar phospholipid bilayers display a highly selective "maxi"-conductance K+ channel whose open-time probability is affected by voltage. We now report that this channel is inhibited by MgATP in the solution bathing the intracellular face of the channel but not by Mg2+ or the Na+ or K+ salts of ATP; the effects of MgATP can be prevented or reversed by MgADP. The channel is also inhibited by the nonhydrolyzable ATP analogue magnesium adenosine 5'-O-(3-thiotriphosphate) and the sulfonylurea derivatives tolbutamide and glibenclamide; all of these agents are effective in the intracellular compartment but not when added to the extracellular compartment alone. Channel activity is stimulated by the "K+ channel opener," diazoxide, which also reverses the effect of glibenclamide but not of MgATP. The possible role of this channel as a mediator of the parallelism between basolateral membrane Na(+)-K+ pump activity and the macroscopic K+ conductance of that barrier is discussed.
Nitric oxide modulates signaling between cultured adult peripheral cardiac neurons and cardiomyocytes
Authors: Horackova M. , Armour J. A. , Hopkins D. A. , Huang M. H.
Published: December 2017

To determine whether nitric oxide (NO) modifies cardiomyocytes directly or indirectly via peripheral autonomic neurons, the effects of NO were studied in long-term (3-6 wk) cultures of adult guinea pig ventricular myocytes alone as well as in cocultures with adult extracardiac (stellate ganglion) or intrinsic cardiac neurons. NADPH diaphorase was associated histochemically with cultured intrinsic cardiac and, to a lesser extent, stellate ganglion neurons. The beating frequency of ventricular myocytes cocultured with intrinsic cardiac neurons (M-intrinsic) or stellate ganglion neurons (M-stellate) increased by 20-30% (P < 0.001) after administration of the NO donor S-nitroso-N-acetylpenicillamine (SNAP); this effect was abolished by the guanylate cyclase inhibitor LY-83583. The beating frequency of noninnervated myocyte cultures was not affected by SNAP. The precursor of NO, L-arginine, also increased the beating rate (approximately 20%; P < 0.05) of M-intrinsic cocultures, not affecting that of M-stellate cocultures or noninnervated myocyte cultures. Augmentor effects induced by SNAP were no longer elicited in the presence of tetrodotoxin and were unaffected by beta-adrenergic or muscarinic receptor blockade. It is concluded that 1) NO-sensitive neurons are present in stellate and intrinsic cardiac ganglia, and these neurons increase the beating rate of cardiomyocytes in the presence of NO; 2) more NO-synthesizing neurons are present in M-intrinsic than M-stellate cocultures, since L-arginine increased the beating frequency of myocytes significantly only in M-intrinsic cocultures; and 3) the beating rate of noninnervated myocyte cultures is not directly affected by NO.
Immunopurification and functional reconstitution of a Na+ channel complex from rat lymphocytes
Authors: Bradford A. L. , Ismailov I. I. , Achard J. M. , Warnock D. G. , Bubien J. K. , Benos D. J.
Published: December 2017

Patch-clamp experiments have demonstrated an amiloride-sensitive Na+ conductance in human B lymphoid cells. We measured whole cell currents in rat lymphocytes and observed a similar Na(+)-specific inward conductance. The presence of 400 microM 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate in the bath significantly increased the inward current, and this adenosine 3',5'-cyclic monophosphate activation was abolished by 2 microM amiloride. We immunopurified a protein complex from rat lymphocyte membranes using an anti-bovine kidney Na+ channel antibody. The complex consisted of five distinct polypeptides with apparent M(r) values of 110,000, 92,000, 59,000, 48,000, and 42,000. This putative channel complex was incorporated into planar lipid bilayers, where we observed single Na+ channel activity that was blocked by amiloride in a concentration-dependent manner. The addition of protein kinase A and ATP to the "intracellular" solution elicited a twofold increase in channel activity. Reverse transcription-polymerase chain reaction analysis was used to determine if the rat lymphocytes express the message for the recently cloned Na+ channel of the rat colon (rENaC). Primers for the alpha-subunit of rENaC identified no message in the lymphocyte RNA, while primers for the beta-subunit of the clone produced low levels of the expected product. Thus it appears that a rENaC-like beta-subunit may be an essential component of the lymphocyte Na+ channel that was isolated. At the same time, this channel is different from those recently cloned in that it does not include an alpha-subunit homologous to that of rENaC.
Whole cell sodium conductance of principal cells freshly isolated from rat cortical collecting duct
Authors: Bubien J. K.
Published: December 2017

Cortical collecting duct fragments were manually dissected from 6-wk-old Sprague-Dawley rats. The fragments were enzymatically digested (collagenase A) into single cells, washed, and resuspended in serum-free RPMI 1640. Individual cells were examined electrophysiologically using the whole cell patch-clamp technique. Two morphologically distinct cell types were present in the cell suspension. Small round cells that had a capacitance of 7 pF and larger oval cells with a capacitance of 29 pF were consistently observed. Whole cell electrophysiological examination revealed that the small round cells had virtually no plasma membrane ionic conductance, whereas both inward and outward currents were observed in the larger oval-type cells. Also, superfusion of 250 pM arginine vasopressin specifically increased the inward conductance of only the larger cells. The effect could be completely inhibited by 2 microM amiloride or 100 mumol of the Rp diastereomer of 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (a specific adenosine 3',5'-cyclic monophosphate inhibitor). These findings are consistent with the hypothesis that the larger cells are principal cells and the smaller cells are intercalated cells and directly demonstrate that an amiloride-sensitive whole cell conductance is readily observable in freshly isolated cortical collecting duct cells. Thus the whole cell configuration of the patch-clamp technique appears to be well suited for assessing cellular mechanisms that regulate the ionic conductances of cortical collecting duct cells.
G protein regulation of cardiac muscarinic potassium channel
Authors: Kurachi Y.
Published: December 2017

Several ion channels can be regulated by G proteins in a “membrane-delimited” manner. The cardiac muscarinic K+ (KACh) channel, which is responsible for the acetylcholine (ACh) or adenosine-induced deceleration of heart beat and atrioventricular conduction, is the prototype of this type of receptor-dependent regulation of ion channels. Because similar transduction mechanisms are utilized by various membrane receptors, such as somatostatin, 5-hydroxytryptamine-1, alpha 2-adrenergic, mu-and delta-opioid, D2-dopamine, and gamma-aminobutyric acid B receptors, in neuronal, hormone-secreting, renal, or smooth muscle cells, the G protein (GK)-KACh channel system illustrates the principles underlying one of the most important cell signaling mechanisms (B. Hille. Neuron 9: 187-195, 1992). It seems that both alpha- and beta gamma-subunits of GK may be involved in the regulation of the KACh channel of mammalian atrial muscle. A general consensus of opinion has emerged, after some years of controversy, to support the notion that physiological activation of the channel by GK is the responsibility of the beta gamma-subunits. Recent evidence suggests that the KACh channel interacts with the alpha-subunit in the terminating process of activation.
Human vascular smooth muscle cells produce an intracellular form of interleukin-1 receptor antagonist
Authors: Beasley D. , McGuiggin M. E. , Dinarello C. A.
Published: December 2017

Interleukin-1 (IL-1) is a proinflammatory monocyte- and macrophage-derived cytokine that has potent vasorelaxant effects on vascular smooth muscle cells (VSMC). VSMC themselves also express both IL-1 alpha- and beta-genes, suggesting that IL-1 may be an autocrine regulator of VSMC function. The present study demonstrates that human saphenous vein VSMC (HSVSMC) produce IL-1 receptor antagonist (IL-1Ra), a specific inhibitor of IL-1 action. IL-1Ra was produced constitutively in most experiments, and its production was upregulated by phorbol 12-myristate 13-acetate and by IL-1 beta. IL-1Ra produced by HSVSMC remained predominately cell associated and was not detectable extracellularly. Furthermore, reverse transcription-polymerase chain reaction analysis and cDNA sequencing indicated that HSVSMC express the alternatively spliced form of IL-1Ra which lacks the signal peptide present in secreted IL-1Ra. HSVSMC also produced IL-1 alpha and the precursor form but not the mature form of IL-1 beta. These results suggest that HSVSMC lack active IL-1 beta-converting enzyme. Like IL-1Ra, IL-1 beta precursor and IL-1 alpha remained cell associated, predominately in the cytosolic fraction. IL-1 beta induced production of both IL-1Ra and IL-1 alpha at each time point and concentration tested. In contrast, platelet-derived growth factor and transforming growth factor-beta augmented production of IL-1Ra, but not that of IL-1 alpha. These results are suggestive of an autocrine role for cell-associated IL-1Ra, as well as for IL-1 alpha and IL-1 beta, in the regulation of VSMC function.
Inhibition of MAP kinase by prostaglandin E2 and forskolin in rat renal mesangial cells
Authors: Li X. , Zarinetchi F. , Schrier R. W. , Nemenoff R. A.
Published: December 2017

Activation of the mitogen-activated protein (MAP) kinase pathway is believed to play a critical role in normal and pathophysiological proliferation of mesangial cells. Recent studies have shown that MAP kinase activation by growth factors in other cell types involves activation of the low-molecular-weight G protein Ras and the protooncogene serine kinase c-Raf-1. In this study, the role of this pathway in rat renal mesangial cells was assessed. Platelet-derived growth factor (PDGF), epidermal growth factor (EGF), as well as phorbol esters (PMA) rapidly activated MAP kinase three- to fourfold in these cells. PDGF and EGF, but not PMA, were able to activate c-Raf-1 and Ras activity. Stimulation of mesangial cells with the inflammatory mediator prostaglandin E2 (PGE2) or elevation of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) by treatment with forskolin markedly blunted activation of MAP kinase induced by PDGF and EGF, but not by PMA. Consistent with this observation, PGE2 abolished growth factor-induced activation of c-Raf-1. However, Ras activation induced by growth factors was not affected by PGE2 and forskolin. These results suggest that MAP kinase activation can occur by at least two separate pathways in mesangial cells. Tyrosine kinase receptors activate MAP kinase through activation of Ras and Raf. This pathway can be blocked by PGE2 and elevation of cAMP, presumably by interfering with the ability of Ras to activate Raf. In addition, activation of protein kinase C by phorbol esters can activate MAP kinase in a Ras/Raf-independent manner.(ABSTRACT TRUNCATED AT 250 WORDS)
Ca2+ influx into bovine retinal rod outer segments mediated by Na+/Ca2+/K+ exchange
Authors: Schnetkamp P. P. , Tucker J. E. , Szerencsei R. T.
Published: December 2017

Ca(2+)-depleted rod outer segments (ROS) were purified from bovine retinal rod photoreceptors, and factors influencing Ca2+ influx into ROS via the plasma membrane Na+/Ca2+/K+ exchanger were analyzed. Intracellular alkali cation concentrations were manipulated by 1) previous loading via the ionophore monensin followed by removal of monensin and 2) addition of the channel ionophore gramicidin during Ca(2+)-influx measurements. Ca2+ influx was measured as a rise in cytosolic free Ca2+ with the Ca(2+)-indicating dye fluo 3. An absolute requirement for intracellular Na+ was observed with a Na+ dissociation constant of 30-40 mM, whereas intracellular K+ was a potent inhibitor of Ca2+ influx, apparently by competing with Na+ for a common site on the Na+/Ca2+/K+ exchanger. Half-maximal Ca2+ influx was observed at an external free Ca2+ concentration of 0.9 microM when no external Na+ was present and 3.5 microM when 10 mM external Na+ was present. Our observations are discussed in the context of 1) a three-site model for the Na+/Ca2+/K+ exchanger and 2) earlier experiments on light adaptation in rods, which depended on minimizing Ca2+ fluxes across the ROS plasma membrane.
Activation of a selective pathway of lysosomal proteolysis in rat liver by prolonged starvation
Authors: Cuervo A. M. , Knecht E. , Terlecky S. R. , Dice J. F.
Published: December 2017

Lysosomal uptake and degradation of polypeptides such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribonuclease A (RNase A), and RNase S-peptide (residues 1-20 of RNase A) are progressively activated in rat liver by starvation before isolation of lysosomes. This pathway of proteolysis is selective, since it is stimulated by the heat shock cognate protein of 73 kDa (HSC73) and ATP-MgCl2, and lysosomal uptake of RNase A could be competed by GAPDH but not by ovalbumin. A portion of intracellular HSC73 is associated with certain lysosomes, and the amount of lysosomal HSC73 increases by 5- to 10-fold during prolonged starvation. The lysosome-associated HSC73 is primarily within the lysosomal lumen. Double immunogold labeling of lysosomes incubated in vitro with RNase A detects this protein substrate as well as HSC73 within lysosomes. More than two-thirds of the labeled lysosomes contain both RNase A and HSC73. The possible physiological significance of the activation of this selective pathway of lysosomal proteolysis in long-term starvation is discussed.
Induction of hypertrophic responsiveness to isoproterenol by TGF-beta in adult rat cardiomyocytes
Authors: Schluter K. D. , Zhou X. J. , Piper H. M.
Published: December 2017

In a previous publication we reported that hypertrophic responsiveness to beta-adrenoceptor stimulation can be induced in isolated cardiomyocytes when these are cultured for 6 days in presence of fetal calf serum (FCS; Pinson et al., J. Mol. Cell. Cardiol.. 25: 477-490, 1993). The role of transforming growth factor-beta (TGF-beta) in this induction process has now been investigated. Isolated cardiomyocytes from adult rats were cultured for 6 days in presence of 20% FCS. It was found that induction of hypertrophic responsiveness to beta-adrenoceptor stimulation was abolished when a neutralizing anti-TGF-beta 1 antibody was added to FCS-containing culture medium. In culture media with FCS contents (5%) too low to induce hypertrophic responsiveness to beta-adrenoceptor stimulation, addition of 1 ng/ml TGF-beta 1,2 induces this responsiveness. It was demonstrated that cardiomyocytes already release TGF-beta into culture media on day 1 of culture and that they continue to do so in presence of FCS supplements of > 5%. The results demonstrate that hypertrophic responsiveness to beta-adrenoceptor stimulation is induced in cardiomyocytes by an autocrine mechanism involving TGF-beta 1 as mediator.
Antisense inhibition of Na+/Ca2+ exchange in primary cultured arterial myocytes
Authors: Slodzinski M. K. , Juhaszova M. , Blaustein M. P.
Published: December 2017

The effects of chimeric phosphorothioated antisense oligodeoxynucleotides (AS-oligos) targeted against the Na+/Ca2+ exchanger (NCX) were tested in primary cultured rat mesenteric artery myocytes. In parallel cultures, myocytes proliferated and were morphologically normal in the presence of scrambled nonsense (NS-) or AS-oligos or no oligos (controls). NCX function was examined with digital imaging, using fura 2 to estimate the cytosolic free Ca2+ concentration ([Ca2+]cyt). Resting [Ca2+]cyt was higher (145 +/- 4 nM; P < 0.05) in AS-oligo-treated cells than in controls (125 +/- 5 nM) or NS-oligo-treated cells (131 +/- 4 nM). Lowering external Na+, to promote Ca2+ entry via NCX, increased [Ca2+]cyt transiently in controls and NS-oligo-treated cells but not in AS-oligo-treated cells. Raising the cytosolic free Na+ concentration with ouabain augmented the low-Na(+)-induced rise in [Ca2+]cyt in controls and NS-oligo-treated cells, but AS-oligo-treated cells still did not respond. Nevertheless, serotonin (5-HT) increased [Ca2+]cyt in all three groups. Thus AS-oligos selectively blocked NCX activity but not the 5-HT response. To determine the effect of NCX knockdown on the modulation of stored Ca2+, the 5-HT response was tested immediately after removal of external Ca2+. Ouabain augmented the 5-HT-induced rise in [Ca2+]cyt in control and NS-oligo-treated cells but not AS-oligo-treated cells. This indicates that the NCX can modulate intracellular Ca2+ stores. We conclude that AS-oligos are useful for investigating the physiological role of NCX in vascular smooth muscle.
Hyperglycemia alters cytoplasmic Ca2+ responses to capacitative Ca2+ influx in rat aortic smooth muscle cells
Authors: Rivera A. A. , White C. R. , Guest L. L. , Elton T. S. , Marchase R. B.
Published: December 2017

Concentrations of free cytoplasmic Ca2+ in rat aortic smooth muscle (RASM) cells were monitored using the ratiometric Ca2+ indicator fura 2-acetoxymethyl ester (AM). In RASM cells cultured in 5 mM Glc, incubation with angiotensin II, ATP, or thapsigargin [a selective inhibitor of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase] depleted SR Ca2+ stores and initiated a capacitative Ca2+ influx through the plasma membrane. This influx was resistant to verapamil, a selective inhibitor of L-type voltage-gated Ca2+ channels, but was sensitive to SKF-96365, an inhibitor of the receptor-operated Ca2+ entry pathway. RASM cells cultured in 25 mM Glc exhibited a significant decrease in cytoplasmic Ca2+ responses to agonist-induced Ca2+ release from SR stores and to subsequent capacitative Ca2+ entry. In addition, the cytoplasmic response to thapsigargin-induced release of Ca2+ from the SR in hyperglycemic cells peaked more sharply than in control cells and returned to baseline more rapidly. The effects of hyperglycemia were not overcome by myo-inositol supplementation.
Evidence for voltage-sensitive, calcium-conducting channels in airway epithelial cells
Authors: Boitano S. , Woodruff M. L. , Dirksen E. R.
Published: December 2017

In airways epithelial cultures, mechanical stimulation induces intracellular Ca2+ concentration ([Ca2+]i) changes by causing Ca2+ entry and intracellular Ca2+ release. Mechanically induced Ca2+ fluxes across the plasma membrane are blocked by Ni2+ (Boitano, S., M. J. Sanderson, and E. R. Dirksen. J. Cell. Sci. 107: 3037-3044, 1994). In this report we use fluorescence imaging microscopy with fura 2 and intracellular recording of the transmembrane potential to further characterize Ca2+ flux in the plasma membrane of these cells. Mechanically induced Ca2+ influx is blocked by nifedipine. Addition of the dihydropyridine agonist BAY K8644 (2 microM) leads to a delayed increase of [Ca2+]i that is dependent on extracellular Ca2+. Switching to high extracellular K+ concentration ([K+]o) causes depolarization of the plasma membrane and a transient increase in [Ca2+]i. The number of cells that respond to high [K+]o is significantly decreased by Ni2+ (1 mM) or nifedipine (10 microM). Mechanical stimulation causes a rapid depolarization of the stimulated cell that can be suppressed by the K+ ionophore valinomycin. Valinomycin treatment also blocks mechanically induced Ca2+ dux. These results suggest that voltage-sensitive Ca(2+)-conducting channels exist in airway epithelial cells, and these channels contribute to the [Ca2+]i changes observed after mechanical stimulation or depolarization of the plasma membrane.
Cell cycle arrest in the G2 phase induced by phorbol ester and diacylglycerol in vascular endothelial cells
Authors: Kosaka C. , Sasaguri T. , Ishida A. , Ogata J.
Published: December 2017

The role of protein kinase C (PKC) in vascular endothelial cell proliferation was investigated using human umbilical vein endothelial cells released from the G1/S border. Phorbol 12-myristate 13-acetate (PMA) caused G2 arrest because 1) when added to G2 cells, PMA inhibited subsequent cell division; 2) these growth-arrested cells did not show morphological features of mitotic cells; and 3) PMA did not interrupt mitosis in cells released from nocodazole-induced M phase arrest. 1-Oleoyl-2-acetyl-sn-glycerol (OAG) added repeatedly from G2 also inhibited mitosis. The activation of cdc2 kinase around the G2/M transition was suppressed by PMA and OAG. Although cdc2 was expressed in the presence of PMA, dephosphorylation of its tyrosine residue was inhibited by PMA. In parallel, the expression of cdc25B was suppressed by PMA. The total and the cdc2-associated amount of cyclin B were both reduced by PMA. These data suggested that the PKC pathway negatively regulates the G2/M transition and that the inhibition of cdc2 kinase by the reduction in the levels of cdc25B and cyclin B may contribute to this effect.
Steady-state twitch Ca2+ fluxes and cytosolic Ca2+ buffering in rabbit ventricular myocytes
Authors: Delbridge L. M. , Bassani J. W. , Bers D. M.
Published: December 2017

Intracellular Ca2+ ([Ca2+]i) transients and transsarcolemmal Ca2+ currents were measured in indo 1-loaded isolated rabbit ventricular myocytes during whole cell voltage clamp to quantitate the components of cytosolic Ca2+ influx and to describe the dynamic aspects of cytosolic Ca2+ buffering during steady-state contraction (0.5 Hz, 22 degrees C). Sarcolemmal Ca2+ influx was directly measured from the integrated Ca2+ current (Ica) recorded during the clamp (158 +/- 10 attomoles; amol). Sarcoplasmic reticulum (SR) Ca2+ content was determined from the integrated electrogenic Na+/Ca2+ exchange current (Ix) induced during rapid application and sustained exposure of cells to caffeine to elicit the release of the SR Ca2+ load (1,208 +/- 170 amol). The mean steady-state SR Ca2+ load was calculated to be 87 +/- 13 microM (mumol/l nonmitochondrial cytosolic volume). Ca2+ influx via Ica represented approximately 14% of the stored SR Ca2+ and 23% of the total cytosolic Ca2+ flux during a twitch (47 +/- 6 microM). Comparison of electrophysiologically measured Ca2+ fluxes with Ca2+ transients yields apparent buffering values of 60 for caffeine contractures and 110 for twitches (delta Ca2+ total/delta Ca2+ free). This is consistent with the occurrence of "active" buffering of cytosolic Ca2+ by SR Ca2+ uptake during the twitch.
Betaine and inositol reduce MDCK cell glycerophosphocholine by stimulating its degradation
Authors: Kwon E. D. , Zablocki K. , Peters E. M. , Jung K. Y. , Garcia-Perez A. , Burg M. B.
Published: December 2017

The amount of glycerophosphocholine (GPC) in renal medullary cells in vivo and in cultured renal [Madin-Darby canine kidney (MDCK)] cells varies with extracellular NaCl and urea. We previously showed that this is largely due to modulation of GPC degradation catalyzed by GPC:choline phosphodiesterase (GPC: PDE). GPC also varies inversely with the levels of other compatible osmolytes, the accumulation of which is induced by high tonicity. We tested whether GPC:PDE activity and GPC degradation are affected by accumulation of compatible osmolytes other than GPC. We find that MDCK cell GPC content decreases when the cells take up betaine and/or inositol from the medium. The effect is considerably greater for cells in isosmotic or high-NaCl medium than in high-urea medium. This difference is associated with suppression of betaine and inositol accumulation with high urea. We then measured GPC:PDE activity with a novel chemiluminescent assay. Addition of inositol and/or betaine to the medium greatly increases GPC:PDE activity in cells in isosmotic or high-NaCl media, but the increase is much less in high-urea medium. The increases in GPC:PDE activity, associated with the presence of betaine, are accompanied by commensurate increases in absolute rates of endogenous GPC degradation by cells in isosmotic or high-NaCl medium. We found previously that, in MDCK cells incubated for 2 days in high-NaCl medium, the rate of GPC synthesis from phosphatidylcholine is increased, correlated with an increase in phospholipase activity. However, in the present experiments, betaine accumulation has no effect on phospholipase activity under those conditions and, thus, presumably does not affect GPC synthesis. Collectively, these data support the conclusion that betaine and/or inositol reduces GPC by increasing GPC degradation catalyzed by GPC:PDE. This mechanism enables GPC to be reciprocally regulated relative to other compatible osmolytes, thus maintaining an appropriate total osmolyte content.
Potentiation of regulatory volume decrease by P2U purinoceptors in HSG-PA cells
Authors: Kim H. D. , Bowen J. W. , James-Kracke M. R. , Landon L. A. , Camden J. M. , Burnett J. E. , Turner J. T.
Published: December 2017

HSG-PA human salivary gland duct cells exhibit progressively increased regulatory volume decrease (RVD) in response to decreased medium osmolarity. The P2U purinoceptor agonist UTP causes a potentiation of RVD, the extent of which is most pronounced in 220 mosM medium and is least apparent in 180 mosM medium. We examined the underlying mechanisms for this effect. Exposure of HSG-PA cells to UTP promotes Ca2+ mobilization, hyperpolarization, and net K+ efflux, suggesting the participation of Ca(2+)-activated K+ channels in RVD. To delineate the anion counterpart of K+ movement during RVD, cell swelling in the presence of gramicidin, which abolishes the membrane potential, was measured. In response to a sudden dilution in hypotonic media, gramicidin-treated cells swelled immediately, followed by a "secondary swelling" in 180 but not in 220 mosM medium. The results suggest that in 180 mosM cells perform spontaneous RVD mediated by increased anion conductance. In 220 mosM medium in which RVD is minimal, the increase in anion conductance is marginal. In our model of RVD in which cells were challenged by UTP, the ensuing hyperpolarization provides the driving force for net Cl- efflux, which is confirmed by tracer flux studies during purinoceptor-activated RVD. Thus RVD, which has long been regarded as a self-sufficient cellular program, appears to be subject to extracellular control in HSG-PA cells through receptor-mediated processes.
A possible role for phospholipase A2 in the action of general anesthetics
Authors: Denson D. D. , Worrell R. T. , Eaton D. C.
Published: December 2017

General anesthetics inhibit Ca(2+)-activated potassium (BK) channels at clinically relevant concentrations. This study examined the possibility that general anesthetics produce their effect on BK channels by disrupting the phospholipase A2 (PLA2)-arachidonic acid signal transduction pathway. Treatment of excised patches with exogenous arachidonic acid (2.5 microM) resulted in a 3.6 +/- 1.3-fold increase in BK channel activity. Subsequent exposure of these patches to concentrations of halothane (0.6 mM), ketamine (100 microM), or etomidate (10 microM) that would normally block the channel by approximately 60-80% in the absence of arachidonic acid did not reduce the channel activity. Arachidonic acid resulted in a significant increase in the 50% effective concentration for the ketamine dose-response curve from 3.4 +/- 0.4 to 693 +/- 379 microM (P < 0.001) as well as a significant decrease in slope from 1.40 +/- 0.21 to 0.59 +/- 0.05 (P < 0.001). The PLA2 inhibitors quinacrine (1 microM), aristolochic acid (250 microM), and octadecylbenzoylacrylic acid (7 microM) inhibited BK channels by 61 +/- 6, 47 +/- 2, and 30 +/- 9%, respectively, and in a manner indistinguishable from general anesthetics inhibition. Aristolochic acid and ketamine significantly inhibit the PLA2-mediated production of arachidonic acid in GH3 cells.