bluuebunny/crossref_metadata_2025 huggingface dataset:
Cl- secretion by cultured shark rectal gland cells. II. Effects of forskolin on cellular electrophysiology
Authors: Moran W. M. , Valentich J. D.
Published: December 2017

Employing microelectrode techniques we have assessed the cellular electrophysiological properties of shark rectal gland (SRG) cells in primary culture. In the absence of secretagogues a 10-fold reduction in the Cl- concentration of the apical superfusate shark Ringer solution had little effect on either apical membrane electrical potential difference (Va) or fractional resistance (fRa), indicating little, if any, apical membrane Cl- conductance. Superfusing the basolateral surface with high-K+ shark Ringer solution (K+ increased 10-fold) depolarized the basolateral membrane electrical potential difference (Vb) by 43 mV, indicating that this barrier is largely K+ conductive. In addition, basolateral Ba2+ (5 mM) depolarized Vb by 12 mV and reduced fRa from 0.92 to 0.58, results consistent with a K(+)-conductive basolateral membrane in unstimulated SRG cells. Basolateral forskolin (10(-6) M) depolarized Va by 25 mV and caused a dramatic reduction in fRa from 0.97 to approximately 0.10. Under these conditions, a 10-fold decrease in apical superfusate Cl- concentration depolarized Va by 37 mV, revealing an adenosine 3',5'-cyclic monophosphate-induced apical membrane Cl- conductance. The time course of the forskolin-induced changes in Va and Vb suggests that the basolateral membrane K+ conductance increased and maintained the driving force for apical Cl- exit, as in other Cl(-)-secreting epithelia. These electrophysiological properties compare favorably with those of the perfused SRG tubule and indicate that SRG primary cultures are a suitable model for Cl(-)-secreting epithelia.
Relationship between calcium current and cytosolic calcium in canine gastric smooth muscle cells
Authors: Vogalis F. , Publicover N. G. , Hume J. R. , Sanders K. M.
Published: December 2017

We measured free intracellular Ca2+ concentration ([Ca2+]i) and Ca2+ current (ICa) simultaneously in voltage-clamped, indo-1-loaded smooth muscle cells isolated from the circular layer of the canine antrum. Resting [Ca2+]i averaged 144 +/- 20 nM in cells held at -70 mV. Depolarization positive to -50 mV elicited ICa and increased [Ca2+]i. Peak [Ca2+]i occurred between 0 and +10 mV and averaged 372 +/- 48 nM. On repolarization, [Ca2+]i decreased slowly (time constant 2-3 s) and the rate depended on the magnitude of [Ca2+]i. Cells were also voltage clamped with protocols that mimicked the upstroke and plateau phases of slow waves. With simulated plateau potentials of -55 to -45 mV, [Ca2+]i increased transiently as a result of the small transient ICa elicited by the upstroke depolarization. Sustained ICa was of sufficient magnitude with plateau depolarizations positive to -40 mV to cause a secondary rise in [Ca2+]i throughout the plateau phase. These data suggest that at the plateau potential of slow waves in situ, ICa is sufficient to cause a sustained increase in [Ca2+]i. The resulting accumulation of Ca2+ may couple the slow wave plateau to contraction and may increase the open probability of Ca(2+)-activated K channels. The latter may provide the outward current necessary to initiate repolarization.
Whole cell current analyses of pancreatic acinar AR42J cells. II. CCK and receptor-activated membrane currents
Authors: Kusano K. , Gainer H.
Published: December 2017

Receptor currents generated in response to the application of sulfated octa-cholecystokinin (CCK) and various pancreatic secretagogues were studied in AR42J cells, a rat pancreatic acinar cell line. A whole cell configuration of the patch electrode voltage-clamp technique was utilized in these studies. Over 60% of the cells examined responded to 10(-6) M CCK by generating inward current at a membrane holding potential of -70 mV, accompanied by an increased membrane conductance. The CCK-induced receptor current (ICCK) was typically inactivated within 30 s in the presence of the ligand. The reversal potential (Erev) of the ICCK was approximately 0 mV when recorded in control bathing solution with a pipette containing 154 mM Cl-. When Cl- concentration in the bathing solution or in the recording pipette was modified, the Erev of the ICCK shifted toward the predicted Cl- potential. Na+ and K+ did not show significant contributions to the Erev of ICCK. Hence, the ICCK involves an increased Cl- conductance. This increased Cl- conductance appears to be due to an increase in intracellular Ca2+, since the ICCK could not be recorded from cells using pipettes containing 5 mM EGTA. Application of a Ca2+ ionophore A23187 (5 microM) to cells in control saline induced a similar inward current flow. The source of the Ca2+ involved in the ICCK appears to be mainly intracellular, since the ICCK could be recorded under conditions designed to completely block the voltage-dependent Ca2+ entry. These included exposure of the cells to a Ca2(+)-free saline for a brief period or the presence of 500 microM Cd2+ in the control bathing solution.
Miniglucagon [glucagon-(19-29)] is a component of the positive inotropic effect of glucagon
Authors: Pavoine C. , Brechler V. , Kervran A. , Blache P. , Le-Nguyen D. , Laurent S. , Bataille D. , Pecker F.
Published: December 2017

Glucagon is well known for its cardiotonic effect, but its mechanism of action remains undetermined. In the present study, we showed that glucagon, under minimal degradation conditions, had no effect on the amplitude of contractility of beating chick embryo ventricular cells. This raised the question of the contribution of the active metabolite of glucagon, glucagon-(19-29), referred to as miniglucagon, to the positive inotropic effect of glucagon. Incubation of glucagon with heart cells led to its rapid conversion into miniglucagon, as measured by radioimmunoassay. Accumulation of the metabolite was maximal after 8 min and remained stable until 15 min. reaching 6% of the initial glucagon concentration. Bacitracin inhibited this processing of glucagon into miniglucagon. Miniglucagon, from 0.1 pM to 1 nM, exerted a potent negative inotropic action. The most striking observation was a 45% increase in the amplitude of cell contractility elicited by the combination of 30 nM glucagon with 1 nM miniglucagon. A similar effect was obtained when glucagon was replaced by a low concentration (75 microM) of 8-bromoadenosine 3',5'-cyclic monophosphate. We conclude that glucagon processing into miniglucagon may be essential for the positive inotropic effect of glucagon on heart contraction.
Multiple calcium currents in a thyroid C-cell line: biophysical properties and pharmacology
Authors: Biagi B. A. , Enyeart J. J.
Published: December 2017

The whole cell version of the patch-clamp technique was used to characterize voltage-gated Ca2+ channels in the calcitonin-secreting rat thyroid C-cell line 6-23 (clone 6). Three types of Ca2+ channels could be distinguished based on differences in voltage dependence, kinetics, and pharmacological sensitivity. T-type current was half-maximal at -31 mV, showed steady-state voltage-dependent inactivation that was half-maximal at -57 mV, inactivated with a voltage-dependent time constant that reached a minimum of 20 ms at potentials positive to -20 mV, and deactivated with a single time constant of approximately 2 ms at -80 mV. Reactivation of inactivated channels occurred with a time constant of 1.26 s at -90 mV. T current was selectively blocked by Ni2+ at concentrations between 5 and 50 microM. La3+ and Y3+ blocked the T current at 10- to 20-fold lower concentrations. Dihydropyridine-sensitive L-type current was half-maximal at a test potential of -3 mV and was approximately doubled in size when Ba2+ replaced Ca2+ as the charge carrier. Unlike L-type Ca2+ current in many cells, this current in C-cells displayed little Ca(2+)-dependent inactivation. N-type current was composed of inactivating and sustained components that were inhibited by omega-conotoxin. The inactivating component was half-maximal at +9 mV and could be fitted by two exponentials with time constants of 22 and 142 ms. A slow inactivation of N current with a time constant of 24.9 s was observed upon switching the holding potential from -80 to -40 mV. These results demonstrate that, similar to other neural crest derived cells, thyroid C-cells express multiple Ca2+ channels, including one previously observed only in neurons.
Quantitation and topography of membrane proteins in highly water-permeable vesicles from ADH-stimulated toad bladder
Authors: Harris H. W. , Zeidel M. L. , Hosselet C.
Published: December 2017

Antidiuretic hormone (ADH) stimulation of toad bladder granular cells rapidly increases the osmotic water permeability (Pf) of their apical membranes by insertion of highly selective water channels. Before ADH stimulation, these water channels are stored in large cytoplasmic vesicles called aggrephores. ADH causes aggrephores to fuse with the apical membrane. Termination of ADH stimulation results in prompt endocytosis of water channel-containing membranes via retrieval of these specialized regions of apical membrane. Protein components of the ADH water channel contained within these retrieved vesicles would be expected to be integral membrane protein(s) that span the vesicle's lipid bilayer to create narrow aqueous channels. Our previous work has identified proteins of 55 (actually a 55/53-kDa doublet), 17, 15, and 7 kDa as candidate ADH water channel components. We now have investigated these candidate ADH water channel proteins in purified retrieved vesicles. These vesicles do not contain a functional proton pump as assayed by Western blots of purified vesicle protein probed with anti-H(+)-ATPase antisera. Approximately 60% of vesicle protein is accounted for by three protein bands of 55, 53, and 46 kDa. Smaller contributions to vesicle protein are made by the 17- and 15-kDa proteins. Triton X-114-partitioning analysis shows that the 55, 53, 46, and 17 kDa are integral membrane proteins. Vectorial labeling analysis with two membrane-impermeant reagents shows that the 55-, 53-, and 46-kDa protein species span the lipid bilayer of these vesicles. Thus the 55-, 53-, and 46-kDa proteins possess characteristics expected for ADH water channel components. These data show that the 55- and 53- and perhaps the 46-, 17-, and 15-kDa proteins are likely components of aqueous transmembrane pores that constitute ADH water channels contained within these vesicles.
Reduced glycolytic metabolism in regenerated fast-twitch skeletal muscle
Authors: Wineinger M. A. , Gorin F. , Tait R. , Froman B. , Carlsen R. C.
Published: December 2017

Freely grafted rat extensor digitorum longus (EDL) muscles were subjected to low-frequency stimulation in an anaerobic environment to determine whether regenerating fast-twitch muscles regain normal glycolytic metabolic capacity. Regenerating muscles were tested at 28, 42, and 76 days after the graft procedure. Stabilized grafts (76 days) produced approximately 60% of the lactate generated by intact, control EDL subjected to the same stimulus paradigm and developed half the estimated increase in H+. The grafts exhibited the same relative decline in force after 5 min of anaerobic stimulation as control EDL but maintained relatively constant levels of ATP while consuming phosphocreatine. This study indicates that regenerating fast-twitch skeletal muscle has a reduced ability to initiate glycolytic activity during exercise. The data also indicate that a small population of regenerating fast-twitch fibers express the slow isoform of myosin heavy chain (beta-MHC) with maximum expression occurring at 56 days postsurgery.
Depression of delayed outward K+ current by Co2+ in guinea pig ventricular myocytes
Authors: Fan Z. , Hiraoka M.
Published: December 2017

Effects of Co2+ on the delayed outward K+ current (IK) in guinea pig ventricular myocytes were studied using the whole cell patch-clamp technique. IK was activated by depolarizing voltage pulses positive to -30 mV and reached half-maximal activation at +24 mV. Co2+ shifted the activation curve to a more depolarized voltage range in a concentration-dependent manner, with a Co2+ concentration at which half-maximal response occurs (IC50) of 8 mM and a saturation value of +38 mV. The voltage dependency of IK gatings showed a shift similar to that of activation. In both cases the shift could be explained by screening of surface potential. The density of total negative surface charges sensed by Co2+ was estimated to be 1 e/225 A2. Co2+ also reduced the fully activated IK [IK(full)], and the dose-response curve had a Hill coefficient of 0.5 and an IC50 of 1 mM at 0 mV. Depression of IK(full) was mainly voltage independent. The single-channel unitary current estimated by fluctuation analysis was approximately 0.1 pA at -30 mV either in the absence or presence of Co2+. Therefore, the depression of IK(full) is due to an equivalent reduction in the number of functional channels. It is concluded that Co2+ depressed IK through multiple mechanisms.
Mechanism of insulin-induced activation of Na(+)-K(+)-ATPase in isolated rat soleus muscle
Authors: Weil E. , Sasson S. , Gutman Y.
Published: December 2017

Insulin augments Na(+)-K(+)-ATPase activity in skeletal muscles. It has been proposed that the sequence of events is activation of Na(+)-H+ antiporter, increased intracellular Na+ concentration ( [Na+]i), and stimulation of Na(+)-K+ pump. We have used isolated rat soleus muscles to test this hypothesis. Insulin increased the ouabain-suppressible K+ uptake in a dose- and time-dependent manner. The maximal effect was observed at 50-100 mU/ml insulin. Stimulation of K+ uptake was accompanied by increased specific [3H]ouabain binding and lowered [Na+]i. The ionophore monensin, which promotes Na(+)-H+ exchange, also increased the rate of ouabain-suppressible K+ uptake in soleus muscle, with a maximal effect obtained at 10-100 microM ionophore. However, this increase was accompanied by an elevation of [Na+]i. In the presence of 10-100 microM monensin, addition of 100 mU/ml insulin further increased K+ uptake but reduced [Na+]i. The effect on K+ uptake was additive. Ouabain (10(-3) M) completely suppressed the effect of insulin on [Na+]i. Insulin had no effect on the magnitude or the time course of insulin stimulation of K+ uptake. Thus equal stimulation of Na(+)-K(+)-ATPase by insulin was observed when [Na+]i was elevated (under monensin) or lowered (under amiloride). These data suggest that activation of Na(+)-K(+)-ATPase in soleus muscle by insulin is not secondary to stimulation of Na(+)-H+ antiporter.
Potassium currents in freshly dispersed myometrial cells
Authors: Piedras-Renteria E. , Stefani E. , Toro L.
Published: December 2017

K+ currents in freshly dispersed cells from rat myometrium at estrus were studied with the patch-clamp technique (whole cell). Three types of K+ currents were identified: 1) a fast-activating current (IKf), 2) a slowly activating current (IKs), and 3) a transient current (IKt). IKf had a half-activation voltage of 12 mV and a time constant of activation (tau on) of approximately 3 ms at +50 mV. IKs had a tau on of approximately 9 ms at +50 mV and a half-activation potential of 33 mV. Both IKf and IKs were sustained and became potentiated by the entrance of Ca2+ from the patch pipette. These two Ca(2+)-activated K+ currents were inhibited by 100 nM external charybdotoxin and were blocked by external tetraethylammonium (TEA, 2-20 mM). The third current (IKt) was transient, had a faster tau on (approximately 1 ms), and a decay phase with a time constant of approximately 8 ms at +50 mV. This current had a half-activation potential of 22 mV. IKt was not potentiated by intracellular Ca2+, was sensitive to 4-aminopyridine (1 mM), was insensitive to external charybdotoxin (100 nM) and TEA (2 mM), and spontaneously decreased with time.
Estrogen modulates phospholipid acylation in red blood cells: relationship to cell aging
Authors: Le Petit-Thevenin J. , Lerique B. , Nobili O. , Boyer J.
Published: December 2017

Ethinyl estradiol administered in vivo to female rats resulted in a mild anemia with a 120% increase in reticulocytosis. Consistent with a previous study, the red blood cell cholesterol-to-phospholipid molar ratio was decreased by 25%, whereas fatty acyl incorporation was significantly increased into phosphatidylethanolamine (PE) and not into phosphatidylcholine (PC), the major acyl acceptor in red blood cells. Analysis of this estrogen-dependent acylation increase as a function of cell age indicated that it was not expressed in reticulocytes but in erythrocytes and was associated with cell aging. Estrogen was further shown to increase the red blood cell susceptibility to peroxidation generated by incubation with H2O2. Altogether, the results suggest that estrogen indirectly increases phospholipid acylation in red blood cells by decreasing protection against oxidative damage, thereby favoring the action of endogenous phospholipases against oxidized substrates. This occurs predominantly in PE of oldest cells because 1) PE, being more unsaturated than PC, is more sensitive to oxidation, and 2) susceptibility to oxidation increases with cell age.
Mn and Cd transport by the Na-Ca exchanger of ferret red blood cells
Authors: Frame M. D. , Milanick M. A.
Published: December 2017

Ca influx via the Na-Ca exchanger into ferret red blood cells is easily measured from a Na-free solution; the intracellular Na concentration is normally approximately 150 mM in ferret red blood cells. We have found that Mn and Cd competitively inhibit Ca influx. Mn influx and Cd influx were a saturable function of the divalent cation concentration, consistent with a carrier mechanism. Indeed, the Km (approximately 10 microM) and the Vmax (usually 1-3 mmol.l packed cells-1.h-1) were similar for Ca, Cd, and Mn. Extracellular Na inhibited divalent cation influx, and intracellular Na stimulated influx. These results are consistent with Na-Cd and Na-Mn influx pathways in ferret red blood cells. Ca (1 mM) almost completely inhibited Mn influx and Cd influx, whereas 1 mM Mg inhibited 5-15%. These results strongly support the notion that Mn and Cd are alternative substrates for Ca on the ferret red cell Na-Ca exchanger. The similarity in the behavior of all three divalent cation places important constraints on kinetic and structural models of the exchanger.
Modulation of leukotriene B4 and platelet-activating factor binding to neutrophils
Authors: Yamazaki M. , Molski T. F. , Stevens T. , Huang C. K. , Becker E. L. , Sha'afi R. I.
Published: December 2017

Preincubation of human neutrophils with the human hormone granulocyte-macrophage colony-stimulating factor (GM-CSF) inhibits the specific binding of leukotriene B4 ([3H]LTB4) but not the nonmetabolizable bioactive platelet-activating factor ([3H]C-PAF) to intact cells. This inhibition requires that the GM-CSF interacts with intact cells. The action of GM-CSF is not prevented by pertussis toxin. Moreover, the rise in calcium produced by LTB4 but not by PAF is also inhibited in human neutrophils pretreated with GM-CSF. Interestingly, neither the inhibitory action of GM-CSF on [3H]LTB4 binding or LTB4-induced calcium rise nor the potentiation of superoxide production by GM-CSF is reduced by inhibitors of arachidonic acid metabolism by the lipoxygenase pathway. In contrast, preincubation of human neutrophils with either the chemotactic factor formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) or the active phorbol ester, phorbol 12-myristate 13-acetate (PMA), inhibits the binding of both [3H]LTB4 and [3H]C-PAF to intact cells. The inhibitory actions of GM-CSF, PMA, and fMet-Leu-Phe require that they interact with the intact cells; their actions cannot be reproduced in plasma membrane preparations. The effects of both GM-CSF and fMet-Leu-Phe cannot be prevented by the protein kinase C inhibitor staurosporine. The mechanisms of fMet-Leu-Phe and GM-CSF actions are probably not mediated through the release of LTB4 by the cells. Interestingly, this new action, unlike other reported effects of GM-CSF, is not mediated through a pertussis toxin-sensitive G protein (Gi alpha 2). This indicates that not all GM-CSF receptors are coupled to Gi alpha 2.
Nitric oxide synthesis in endothelial cells: evidence for a pathway inducible by TNF-alpha
Authors: Lamas S. , Michel T. , Brenner B. M. , Marsden P. A.
Published: December 2017

Nitric oxide (NO) release accounts for the biological activity of endothelium-derived relaxing factor. Given that tumor necrosis factor-alpha (TNF-alpha) has been implicated as an important mediator in septic shock, we explored whether TNF-alpha enhances L-arginine-dependent synthesis of NO and L-citrulline in endothelial cells. The release of NO was detected in a coincubation bioassay where measurement of guanosine 3',5'-cyclic monophosphate (cGMP) production in reporter monolayers, namely glomerular mesangial cells or fetal lung fibroblasts, reflected activation of soluble guanylate cyclase. Reporter monolayer cGMP content was greater in the presence of TNF-alpha-treated bovine aortic and renal artery endothelial cells than in the presence of vehicle-treated endothelial cells. TNF-alpha-stimulated endothelium-dependent increases in reporter monolayer cGMP content were first evident at 8 h and maximal at 16-24 h. In addition, TNF-alpha-stimulated endothelium-dependent increases in reporter monolayer cGMP content were abrogated by hemoglobin and methylene blue, blunted by N omega-nitro-L-arginine and augmented by superoxide dismutase and the calcium agonist bradykinin. These observations suggested that TNF-alpha enhanced release of NO. Furthermore, the formation of L-[14C]citrulline from L-[14C]arginine, as determined by quantitative cation-exchange chromatography and thin-layer chromatography, was enhanced by TNF-alpha in a time- and concentration-dependent manner. Thus it is evident that endothelial cells release NO for a prolonged period in response to TNF-alpha and transiently when stimulated with calcium agonists. The prolonged release of NO from TNF-alpha-stimulated endothelial cells may be implicated in the pathogenesis of septic shock.
Identification of a low-threshold T-type calcium channel in bovine ciliary epithelial cells
Authors: Jacob T. J.
Published: December 2017

The characteristics of an inward current in single pigmented cells from the bovine ciliary body were examined using the whole cell recording technique. The inward current appeared to activate at potentials 30 mV positive to a holding potential of -80 mV, although activation studies revealed that from more negative holding potentials the current activated at about -75 mV, peaked at around -10 mV, and reversed at a potential of +40 mV. The current was small, peak value -72 +/- 24 pA (n = 13), and exhibited rapid activation and slower inactivation kinetics, both processes being voltage sensitive. Inactivation occurred at holding potentials more positive than -120 mV and was complete at -50 mV with a half-maximal membrane potential of -88 mV. The overlap of the inactivation and activation curves between -75 and -50 mV means that, in the steady state, entry of calcium will be greatest in this range and calcium will thus enter the cell at the resting membrane potential. The current was resistant to tetrodotoxin and nifedipine. It was partially blocked by Cd2+ and almost completely blocked by Ni2+. It is concluded that this current is a low-threshold T-type calcium current.
Effects of pHi on Na(+)-H+, Na(+)-dependent, and Na(+)-independent C1(-)-HCO3-exchangers in vascular smooth muscle
Authors: Kahn A. M. , Cragoe E. J. , Allen J. C. , Seidel C. L. , Shelat H.
Published: December 2017

The mechanisms that control intracellular pH (pHi) in vascular smooth muscle are not fully understood. We reported that pHi in primary cultured vascular smooth muscle cells from canine femoral artery is 7.26, a value maintained via HCO3- influx by the Na(+)-dependent C1(-)-HCO3-exchanger but not via H+ efflux by the Na(+)-H+ exchanger [A. M. Kahn, E. J. Cragoe, Jr., J. C. Allen, R. D. Halligan, and H. Shelat. Am. J. Physiol 259 (Cell Physiol. 28): C134-C143, 1990]. To explain these findings, in the present study, we determined the pHi activity profile of these two transport systems. Although both were active at acidic pHi, Na(+)-H+ exchange activity was very low at and above pHi 7.0, while Na(+)-dependent C1(-)-HCO3-exchange activity maintained near-maximal activity up to pHi 7.26 but fell to undetectable levels by pHi 7.4. A Na(+)-independent C1(-)-HCO3-exchanger was present, which mediated HCO3-efflux after an acute alkaline load. The activity of this system was negligible at pHi 7.2 and was stimulated at alkaline pHi. In conclusion, the pHi of these vascular smooth muscle cells at rest is maintained via HCO3-influx by the Na(+)-dependent C1(-)-HCO3-exchanger. After acute acidic or alkaline loads, correction of pHi is mediated by activation of the normally quiescent Na(+)-H+ and Na(+)-independent C1(-)-HCO3-exchangers, respectively. All three acid-base transport systems have pHi set points that protect the cell from overcorrecting pHi after a disturbance in either direction.
Mechanism of phospholipase C-induced release of EDRF from pulmonary artery endothelium
Authors: Tracey W. R. , Johns R. A. , Romero G. , Peach M. J.
Published: December 2017

The mechanism of phospholipase (PL) C-induced release of endothelium-derived relaxing factor (EDRF) was investigated. Bovine pulmonary artery endothelial cells (BPAEC) were treated with phosphatidylinositol (PI)-selective PLCs, nonselective PLCs, or a nonselective PLD. PI-PLCs elicited PI-glycan anchor hydrolysis but did not alter either intracellular Ca2+ ([Ca2+]i) in fura-2-loaded BPAEC or EDRF production in BPAEC-vascular smooth muscle cocultures. In contrast, non-selective PLCs increased [Ca2+]i, an effect prevented by prior exposure to the PLCs, and EDRF production in a time- and concentration-dependent manner. Antibodies raised against PI-glycan anchors did not alter, while heat denaturation abolished, the PLC-dependent effects. Removal of extracellular Ca2+ with [ethylene-bis(oxyethylenenitrilo)]tetraacetic acid both prevented and reversed PLC-stimulated increases in [Ca2+]i and inhibited EDRF production. Although Mn2+ quenched PLC-induced increases in fura-2 fluorescence, high PLC concentrations elicited significant dye loss from fura-2-loaded BPAEC. We conclude that the effects of exogenous PLC on EDRF production are not dependent on release of a membrane PI-glycan-linked moiety. Rather, the PLC actions are mediated by a graded increase in cell membrane permeability, probably related to pore formation by the hemolytic activity of the enzyme, followed by an influx of extracellular Ca2+.
A compact and stable hydraulic micromanipulator patterned after a Huxley-style approach
Authors: Krueger J. W.
Published: December 2017

I describe a remote-controlled micromanipulator platform that is stable, durable, precise, and easy to construct. Small metallic bellows are used for hydraulic control, where all fluid connections are made by standard 1/16-in. high-performance liquid chromatography fittings. Inspired by the parallelogram suspension utilized in the larger Huxley-style micromanipulator (A. F. Huxley. J. Physiol. Lond. 157: 6-5P, 1961), the device is a compact cradle suspension of folded lever arms that creates vertical motions which have minimal cross-coupled horizontal error. A simple arrangement for securing the bellows in the remote controller counteracts the vertical cross-coupling error that arises in the parallelogram suspension so that the position of the microtool more faithfully corresponds to the micrometer settings. Being compact, the micromanipulator can be mounted on a microscope stage to eliminate the microscope's resonance as a source of vibration. This feature also reduces the cantilevering of the microtool that 1) is a source of parasitic vibrations and 2) limits the load bearing in larger devices which can only be placed alongside the microscope. The device has a good dynamic response, and one design suits both right- and left-handed use.
Pentose phosphate pathway in cellular trophoblasts from full-term human placentas
Authors: Moe A. J. , Farmer D. R. , Nelson D. M. , Smith C. H.
Published: December 2017

Glucose metabolism was investigated in cellular trophoblasts isolated from full-term human placentas. The specific yields of 14CO2 from D-[1-14C]glucose and D-[6-14C]glucose were used to determine glucose metabolism via the pentose cycle for cells freshly isolated or cells grown in culture for 1 and 3 days. Cells were mononucleated on day 1 but fused to form multinucleated syncytiotrophoblasts by day 3. The principal product of glucose metabolism under all conditions was lactate, accounting for approximately three-fourths of recovered 14C in products. Pentose cycle activity contributed 0.57 +/- 0.01, 0.39 +/- 0.06, and 0.21 +/- 0.05% of the glucose metabolized by cells freshly isolated, cultured for 1 day, and cultured for 3 days, respectively. In the presence of the electron acceptor methylene blue, pentose cycle activity increased to 16.5 +/- 2.1, 13.8 +/- 1.5, and 18.2 +/- 1.7% for cells freshly isolated, cultured for 1 day, and cultured for 3 days, respectively. Trace amounts of 14C were recovered in other products including amino acids and glycogen. These data suggest that pentose cycle activity in cellular trophoblasts from full-term placenta, like those in full-term villous tissue, is a minor component of glucose metabolism. However, these cultured cells maintain a capacity to oxidize glucose via the pentose cycle at relatively high rates.
Regulation of functional muscarinic receptor expression in tracheal smooth muscle cells
Authors: Yang C. M. , Chou S. P. , Sung T. C. , Chien H. J.
Published: December 2017

Recent studies have shown that cultured tracheal smooth muscle cells (TSMCs) do not respond to muscarinic agonists with a significant increase in intracellular Ca2+ concentration. This may be due to a downregulation of muscarinic receptors (mAChRs) in TSMCs. We report here that the individual component of growth factors or hormones at the concentration used is not sufficient to stimulate growth of TSMCs in the primary culture with 1% fetal bovine serum (FBS). In the presence of 1% FBS, TSMCs withdraw from the cell cycle and express high levels of cell surface mAChRs. Furthermore, insulin-like growth factor I (IGF-I) and insulin (Ins), alone or in combination, could stimulate the expression of mAChRs on the cultured TSMCs in 1% FBS without changing the affinity of receptors. Heparin could inhibit these stimulatory effects on mAChR expression. The pharmacological response of functional mAChRs, determined as accumulation of inositol phosphates induced by carbachol, is greater in the medium containing IGF-I and Ins than those cultured in 1% FBS. This action may be partially mediated through a cholera toxin-sensitive protein. The results conclude that IGF-I and Ins are necessary for TSMCs to express functional mAChRs.
Inhibition of atrial peptide secretion at different stages of the secretory process: Ca2+ dependence
Authors: Page E. , Upshaw-Earley J. , Goings G. E. , Hanck D. A.
Published: December 2017

We have used a noncontracting in vitro preparation of stretched and unstretched rat atria to estimate contributions of constitutive and regulated pathways to the rates of stretch-augmented and basal secretion of immunoreactive atrial natriuretic peptide (ANP) and to examine effects of inhibition of the secretory sequence by 1) protein synthesis inhibitors, 2) disruption of forward vesicular traffic between endoplasmic reticulum and Golgi with brefeldin A (BFA, and 3) cellular ATP depletion. Protein synthesis inhibition with cycloheximide for 44 min slowed neither basal nor stretch-augmented ANP secretion but instead accelerated stretch-augmented secretion at low (but not at physiological) external Ca2+ concentration, suggesting that the constitutive component does not contribute substantially to either basal or stretch-augmented secretion. BFA, which disassembled Golgi cisternae, increased the stretch-augmented secretory rate via the regulated pathway and prevented Ca(2+)-dependent inactivation with time. Cellular ATP depletion rapidly and completely inhibited stretch-augmented secretion. We conclude that both basal and stretch-augmented utilize the energy-dependent regulated pathway, drawing on a large reservoir of concentrated prohormone stored in granules that is not detectably depleted during 44 min of stretch-augmented secretion at 37 degrees C.
Synergism between cAMP and ATP in signal transduction in cardiac myocytes
Authors: Zheng J. S. , Christie A. , De Young M. B. , Levy M. N. , Scarpa A.
Published: December 2017

ATP transiently increases the intracellular Ca2+ concentration in cardiac myocyte suspensions. Pretreatment with norepinephrine (NE) greatly potentiates the ATP response. We performed experiments on adult rat myocyte suspensions loaded with fura-2 to investigate the mechanism of NE potentiation. We found that forskolin (an activator of adenylate cyclase), 3-isobutyl-1-methylxanthine (an inhibitor of phosphodiesterase), and permeative adenosine 3',5'-cyclic monophosphate (cAMP) analogues potentiate the increase in cytosolic Ca2+ concentration induced by ATP. NE, forskolin, and 8-(4-chlorophenylthio)-cAMP all increase Vmax of the Ca2+ response curve of ATP. Measurement of cAMP by radioimmunoassay confirmed that the changes in the ATP response were accompanied by an increase in cAMP. These results suggest that the noradrenergic potentiation of the ATP-induced Ca2+ mobilization involves cAMP as a second messenger. Patch-clamp studies of isolated myocytes showed that neither NE nor forskolin alters the inward current elicited by ATP, but rather they increase the duration of secondary slow action potentials elicited by ATP. NE also increases the Ca2+ current through L-type Ca2+ channels in the myocytes. We conclude that NE potentiates the ATP-induced Ca2+ transient by increasing cAMP levels and that one of the early events is the increase of the inward Ca2+ current during the action potential.
Regulation of extracellular calcium entry in endothelial cells: role of intracellular calcium pool
Authors: Dolor R. J. , Hurwitz L. M. , Mirza Z. , Strauss H. C. , Whorton A. R.
Published: December 2017

We have investigated the role of the intracellular Ca2+ pool in regulating Ca2+ entry into vascular endothelial cells. The intracellular Ca2+ pool was mobilized using either thapsigargin (TG) or 2',5'-di(tert-butyl)-1,4-benzohydroquinone (BHQ), inhibitors of the endoplasmic reticulum Ca(2+)-adenosinetriphosphatase (ATPase). Mobilization of intracellular Ca2+ stores with either inhibitor depleted intracellular Ca2+ and greatly reduced subsequent mobilization of the inositol 1,4,5-trisphosphate (IP3)-sensitive intracellular Ca2+ pool by bradykinin. However, bradykinin-induced mobilization of the IP3-sensitive intracellular Ca2+ pool only partially reduced the subsequent response of cells to TG and BHQ. Mobilization of the intracellular Ca2+ pool by either TG or BHQ led to a concentration-dependent elevation of cytosolic Ca2+ concentrations ([Ca2+]i) without initiating inositol polyphosphate formation. In contrast to the rapidly developing, transient rise in Ca2+ concentration initiated by bradykinin, maximal concentrations of TG and BHQ stimulated a slowly developing, prolonged elevation of [Ca2+]i that required extracellular Ca2+ and could be blocked by extracellular Ni2+. Extracellular Ca2+ entered the cell through an activated cation entry pathway, since bradykinin, TG, and BHQ stimulated Mn2+ and 45Ca2+ entry. Bradykinin-stimulated 45Ca2+ uptake reached a peak within 2 min, whereas 45Ca2+ influx initiated by TG or BHQ continued for at least 8 min. Importantly, the [Ca2+]i response after low concentrations of BHQ was more transient than that seen after TG. The return of [Ca2+]i to basal values after low concentrations of BHQ was associated with reversal of Ca(2+)-ATPase inhibition and refilling of the IP3-sensitive Ca2+ pool. The continued elevation of [Ca2+]i and prolonged Ca2+ entry seen with TG was associated with continued Ca(2+)-ATPase inhibition and an empty IP3-sensitive Ca2+ pool. We conclude that mobilization of intracellular Ca2+ stores induces Ca2+ entry in endothelial cells which continues until the intracellular Ca2+ pool is refilled.
Reversed anion selectivity in cultured cystic fibrosis sweat duct cells
Authors: Bell C. L. , Reddy M. M. , Quinton P. M.
Published: December 2017

The human genetic disease cystic fibrosis (CF) is characterized by defective epithelial Cl- conductance (GCl). To distinguish the CF-affected GCl from other Cl- channels, we have studied the properties of GCl in normal and CF cells grown from explanted reabsorptive sweat ducts (RD). The cultured cells from normal subjects retained some of the typical duct cell properties. The Na+ conductance inhibitor amiloride hyperpolarized intracellular potentials (Vm) by 10.4 +/- 1.6 mV (n = 12). Substitution of gluconate for Cl- depolarized Vm by 15.5 +/- 1.1 mV (n = 33). The apparent GCl (G'Cl) of normal cells was sensitive to adenosine 3',5'-cyclic monophosphate (forskolin, 10(-6) M), as evidenced by a significant increase (63%, n = 9) in the Cl- gradient induced depolarization, and more selective for Cl- than I- (substitution of Cl- by I- depolarized Vm by 6.3 +/- 0.3 mV, n = 49). Although the cells from CF subjects were statistically indistinguishable from normal cells based on Vm (-18.5 +/- 1.2 mV, n = 49 vs. -20.1 +/- 1.8 mV, n = 28), CF cells expressed differences in G'Cl, responses to forskolin, and anion selectivity. CF cells had a significantly reduced G'Cl as indicated by blunted responses to imposed Cl- gradients (26% of normal, n = 28). In contrast to our observations in normal cells, the G'Cl of CF cells was insensitive to forskolin.(ABSTRACT TRUNCATED AT 250 WORDS)
Modulation of neutrophil activation by okadaic acid, a protein phosphatase inhibitor
Authors: Lu D. J. , Takai A. , Leto T. L. , Grinstein S.
Published: December 2017

We determined the effects of okadaic acid (OA), a specific inhibitor of protein phosphatases 1 (PP1) and 2A (PP2A), on protein phosphorylation and on the activation of the NADPH oxidase in human neutrophils. In otherwise unstimulated cells, OA induced phosphoprotein accumulation, revealing the presence of constitutively active protein kinases. Pulse-chase experiments in electropermeabilized cells confirmed that this effect was due, at least in part, to inhibition of dephosphorylation. OA potentiated phosphoprotein accumulation induced by phorbol esters and by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). In phorbol ester-stimulated cells, OA prolonged the respiratory response after inhibition of protein kinase C (PKC) with staurosporine, consistent with a reduced rate of dephosphorylation of active phosphorylated components. Similarly, OA delayed the inactivation of the burst after displacement of FMLP from its receptor by a competitive antagonist. This suggests that the substrates of the protein kinases activated by FMLP are dephosphorylated by PP1 and/or PP2A. That phosphatases control the intensity and duration of the respiratory response is suggested by the finding that OA magnified and prolonged the oxidative burst elicited by FMLP. In contrast, pretreatment with OA produced a time-dependent inhibition of the phorbol ester-induced respiratory burst. Under conditions where inhibition of the phorbol ester response was nearly complete, activation by the chemoattractant peptide not only persisted but was in fact accentuated. These findings provide strong evidence that receptor-mediated stimulation of the NADPH oxidase can occur by pathways not involving PKC.
Fluid transport across cultured bovine corneal endothelial cell monolayers
Authors: Narula P. , Xu M. , Kuang K. Y. , Akiyama R. , Fischbarg J.
Published: December 2017

The mammalian corneal endothelium is known to transport fluid from the stromal compartment to the aqueous humor, thereby maintaining corneal transparency. Corneal endothelial cells have been cultured for some years now, but whether they preserve their in vivo ability to actively transport fluid is not known. We have now grown bovine corneal endothelial cell monolayers (BCECM) on permeable substrates (Transwell) and report that, just like their counterparts in vivo, these cultured cells pump fluid from the basal to the apical compartment and display measurable electrical resistance and potential difference across the monolayer. BCECM were grown on collagen-treated permeable supports using Dulbecco's modified Eagle's medium (DMEM)/20% fetal bovine serum with antibiotics. Cells grew to confluence in 5-7 days and displayed polygonal shape. Only cells from passages 1-3 were utilized. Inserts were fitted directly into Lucite chambers specially built. The rate of fluid pumping by BCECM was 3.96 +/- 0.49 (SE) microliter.h-1.cm-2 (n = 13) and could be measured continuously for several hours; fluid pumping was inhibited by 0.2 mM amiloride. The specific electrical resistance of the monolayers was 180 +/- 22 omega.cm2 (n = 11). A mean electrical potential difference of 63.8 +/- 3.7 microV (n = 15, range 40-100 microV, apical side negative) was recorded across the monolayers in DMEM. The availability of the commercial inserts makes this procedure practical; as a consequence, the rate of fluid transport by cultured corneal endothelium has been quantitated for the first time. This method can now be extended to other cultured layers.(ABSTRACT TRUNCATED AT 250 WORDS)
Relationship between intracellular pH and tension development in resting ventricular muscle and myocytes
Authors: Spitzer K. W. , Bridge J. H.
Published: December 2017

Simultaneous measurements of intracellular pH (pHi) and tension development were made in resting cat papillary muscles and resting ventricular myocytes (cat, guinea pig). pH microelectrodes and the fluorescent indicator carboxy-seminaphthorhodafluor-1 (SNARF-1) were used to measure pHi in muscles and myocytes, respectively. pHi-induced changes in isometric muscle tension and myocyte length were elicited by variations in PCO2, HCO3-, and [NH4Cl]. Increased pHi elevated resting tension and decreased resting cell length, whereas decreased pHi decreased tension and increased cell length. The tension-pHi and cell length-pHi relationships were nonlinear and displayed hysteresis. A reduction in extracellular [Ca2+] from 2.7 to 0.5 mM caused little or no change in the tension and cell length responses to elevated pHi. Ca2+ uptake and/or release by the sarcoplasmic reticulum (SR) does not appear to be involved in the tension response to intracellular alkalosis because the response was unaffected by a postpacing rest period and was not inhibited by ryanodine (5 microM), which depletes SR Ca2+ stores. The cross-bridge inhibitor 2,3-butanedione monoxime (15 mM), but not internal dialysis with 14 mM ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid, inhibited myocyte contractures elicited by elevated pHi. The latter finding suggests that factors other than pHi-induced increase in myofilament Ca2+ sensitivity may contribute to the resting contractile response to elevated pHi.
Discriminatory effects of protein kinase inhibitors and calcium ionophore on endothelial ICAM-1 induction
Authors: Myers C. L. , Desai S. N. , Schembri-King J. , Letts G. L. , Wallace R. W.
Published: December 2017

Intercellular adhesion molecule 1 (ICAM-1) is a proinflammatory adhesion glycoprotein induced by cytokines such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) as well as lipopolysaccharide (LPS). Little is known, however, concerning the intracellular regulatory mechanisms that modulate ICAM-1 expression in endothelial cells. We probed the involvement of protein kinase function and intracellular calcium ion upon ICAM-1 expression of human umbilical vein endothelial cells activated alternatively by TNF-alpha, IL-1 beta, LPS, or phorbol 12-myristate 13-acetate (PMA). Methodologies for the detection of ICAM-1 included both enzyme-linked immunosorbent assay and immunoprecipitation from biosynthetically labeled cells. The protein kinase inhibitor H-7 blocked induction of ICAM-1 by all of the activators; nonlinear regression analysis revealed 50% inhibitory concentration (IC50) values of 6-10 microM. Another kinase inhibitor, HA1004, did not block expression of the adhesion molecule at concentrations up to 50 microM. In contrast, the kinase inhibitor staurosporine dose dependently inhibited ICAM-1 expression triggered by PMA (IC50 67 +/- 4 nM) but, at similar concentrations, did not inhibit ICAM-1 expression induced by the other inflammatory stimuli. The divalent cation ionophore ionomycin (0.5 microM) interacted synergistically with PMA but not with cytokines or LPS in upregulating ICAM-1. We conclude from these data that although PMA-induced ICAM-1 expression may be triggered through activation of protein kinase C, ICAM-1 induction by IL-1 beta, TNF-alpha, or LPS may involve distinct regulatory pathway(s).
Substance P: a late-acting B lymphocyte differentiation cofactor
Authors: Bost K. L. , Pascual D. W.
Published: December 2017

The peptide substance P has been recognized for years as having dramatic effects on such diverse physiological responses as blood pressure regulation, peristalsis of the gut, and salivation. More recently, demonstration of substance P receptors on leukocytes and modulation of leukocyte functions by this peptide suggested that it might also have a role in immune regulation. This review focuses on the growing body of evidence that demonstrates substance P-induced effects on one population of leukocytes, namely B lymphocytes. Despite the diversity of experimental techniques used, there is surprisingly good agreement as to the role substance P has in modulating B lymphocyte responses. In vivo treatments of rodents, which increase substance P concentrations in the periphery, increase the number of immunoglobulin-secreting cells in these animals. Conversely, infusion of substance P antagonists or depletion of substance P-containing neurons in rodents substantially reduces the animals' ability to synthesize immunoglobulins. With the use of cultures of B lymphocytes it was possible to demonstrate similar results. In the presence of polyclonal B cell activators, substance P augmented immunoglobulin secretion in cultures of purified B lymphocytes or B cell clones. The absence of accessory cells in these cultures suggested that substance P could act directly on activated B lymphocytes, and in fact these B cells were shown to express specific receptors for this peptide. It appears that the substance P receptors expressed by leukocytes are similar or identical to those expressed by neurons as evidenced by radioreceptor binding assays and detection of the gene encoding the substance P receptor.(ABSTRACT TRUNCATED AT 250 WORDS)