bluuebunny/crossref_metadata_2025 huggingface dataset:
pHi dependence of Na-H exchange and H delivery in IEC-6 cells
Authors: Sjaastad M. D. , Wenzl E. , Machen T. E.
Published: December 2017

The intracellular pH (pHi) dependencies of the Na-H exchanger and also of unidentified acid-loading processes (“H delivery”) in IEC-6, a rat intestinal crypt cell line, were determined using microspectrofluorimetry of the pH-sensitive dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein on groups of cells grown on glass cover slips mounted in a perfusion chamber in HCO3-free solutions (to inactivate Cl-HCO3 exchange and Na-HCO3 cotransport). Na-H exchange and H delivery rates were measured using two methods. 1) Cells were acid loaded with NH4Cl, and rates of pHi recovery and acidification were measured just before and then just after adding amiloride at various times during the recovery. Multiplying rates of pHi recovery times the intrinsic buffer capacity (beta i) yielded net H efflux rates, rates of pHi decrease (during amiloride treatment) times beta i yielded H delivery rates, and Na-H exchange equals net H efflux plus H delivery. 2) In control cells, time courses of amiloride-induced pHi decreases (from 7.36 to 6.65) and of pHi recovery following amiloride removal (from 6.65 to 7.49) were curve fit by computer; rates of pHi decrease and recovery were calculated; and H delivery, net H efflux, and Na-H exchange were then calculated at the different pHi as above. H delivery and Na-H exchange were both pHi dependent, but in the opposite directions. H delivery was 2-7 mM/min at the control pHi, decreasing to zero around pHi 6.6. Na-H exchange was also equal to 2-7 mM/min at the control pHi, and this increased as pHi decreased; at pHi 6.6, Na-H exchange was 20-35 mM/min.(ABSTRACT TRUNCATED AT 250 WORDS)
Citrate alters Ca channel gating and selectivity in rabbit ventricular myocytes
Authors: Hryshko L. V. , Bers D. M.
Published: December 2017

Addition of 10 mM citrate at constant free extracellular Ca concentration [( Ca]o; 2 mM) reduced contraction in rabbit ventricular muscle and isolated myocytes. We have recently shown that extracellular citrate decreases contraction and Ca current (ICa) in cardiac muscle by a direct effect on Ca channels rather than by Ca buffering per se [D. M. Bers, L. V. Hryshko, S. M. Harrison, and D. Dawson. Am. J. Physiol. 260 (Cell Physiol. 29): C900-C909, 1991]. Citrate rapidly depressed peak ICa and shifted both the peak ICa and the apparent reversal potential (Erev) to more negative potentials. When the impermeant cations, tetraethylammonium or N-methylglucamine were used instead of intracellular Cs, the citrate-induced shift in Erev was reduced or eliminated but depression of ICa was still observed. Thus citrate appears to alter the selectivity (PCa/PCs) of the Ca channel and reduce ICa. We also studied the effects of citrate on Na current through the Ca channel, observed when the divalent cation concentration is submicromolar. This current, termed INS for nonspecific, also exhibited leftward shifts in peak INS and smaller changes in Erev in the presence of citrate. However, neither peak INS nor single-channel conductance were affected by citrate. Thus the reduced PCa/PCs is due primarily to alteration of Ca permeation rather than monovalent cation permeation. Activation and inactivation curves for both ICa and INS were shifted toward more negative potentials by citrate. The shifts in gating and peak current to more negative membrane potentials would be consistent with a surface charge effect. The much larger shift in Erev for ICa (than for INS) is consistent with a reduction in Ca selectivity.(ABSTRACT TRUNCATED AT 250 WORDS)
Exercise inhibits glucocorticoid-induced glutamine synthetase expression in red skeletal muscles
Authors: Falduto M. T. , Young A. P. , Hickson R. C.
Published: December 2017

One purpose of this study was to determine whether the suppression of glucocorticoid-induced glutamine synthetase (GS) gene expression by exercise is localized to fiber types that are known to be primarily recruited during endurance running. A second purpose examined whether denervation, which is associated with a reduction in contractile activity, would upregulate GS expression. Exercise consisted of treadmill running at 31 m/min for 12-16 wk. Glucocorticoid treatment (100 mg/kg body wt hydrocortisone 21-acetate) was administered during the last 11 days of the exercise program. Basal GS expression was lowest (GS enzyme activity, 43 +/- 3 nmol.h-1.mg protein-1; GS mRNA, 1.0 arbitrary units) in the slow-twitch red soleus, a muscle type that is known to resist glucocorticoid-induced muscle wasting, intermediate (74 +/- 10 and 1.7 +/- 0.2) in fast-twitch red quadriceps, a muscle type susceptible to atrophy, and highest (106 +/- 16 and 5.4 +/- 1.3) in fast-twitch white quadriceps, a muscle type known to be most susceptible to atrophy. Hormone treatment increased GS enzyme activity and mRNA by two- to fourfold in all muscle types. Exercise diminished GS enzyme activity and mRNA in the fast-twitch red fibers to 35-70% of sedentary control values in both basal and glucocorticoid-stimulated muscles. The running also reduced GS enzyme activity in hormone-treated slow-twitch fibers but did not alter basal or glucocorticoid-induced GS expression in fast-twitch white fibers. These results indicate that glucocorticoids induce similar relative GS expression across all muscle types, but the low absolute levels of expression in slow-twitch muscles are not related to any atrophy.(ABSTRACT TRUNCATED AT 250 WORDS)
Na current density at and away from end plates on rat fast- and slow-twitch skeletal muscle fibers
Authors: Ruff R. L.
Published: December 2017

Na current density and membrane capacitance were studied with the loose patch voltage clamp technique on rat fast- and slow-twitch skeletal muscle fibers at three different regions on the fibers: 1) the end plate border, 2) greater than 200 microns from the end plate (extrajunctional), and 3) on the end plate postsynaptic membrane. Fibers were treated with collagenase to improve visualization of the end plate and to enzymatically remove the nerve terminal. The capacitance of membrane patches was similar on fast- and slow-twitch fibers and patches of membrane on the end plate had twice the capacitance of patches elsewhere. For fast- and slow-twitch fibers, the sizes of the Na current normalized to the area of the patch were as follows: end plate greater than end plate border greater than extrajunctional. For both types of fibers, the amplitudes of the Na current normalized to the capacitance of the membrane patch were as follows: end plate approximately end plate border greater than extrajunctional. At each of the three regions, the Na current densities were larger on fast-twitch fibers and fast-twitch fibers had a larger increase in Na current density at the end plate border compared with extrajunctional membrane.
Hormonal regulation of Cl transport in polar airway epithelia measured by a fluorescent indicator
Authors: Verkman A. S. , Chao A. C. , Hartmann T.
Published: December 2017

Cl transport mechanisms in polarized cultures of canine tracheal epithelium were examined using an Ussing-type chamber with independent mucosal and serosal perfusion. Cl activity was monitored continuously from fluorescence of entrapped 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). When added to the serosal (but not mucosal) solution, isoproterenol increased Cl fluxes across the apical membrane Cl more than fourfold. Apical Cl transport was sensitive to diphenylamine-2-carboxylate (DPC) but not to furosemide, whereas basolateral membrane Cl transport was sensitive to furosemide but not to DPC. Based on a mathematical model of Cl transport, we developed a sensitive protocol to measure hormone-sensitive Cl transport. In Cl-loaded cells in which basolateral Cl transport was partially inhibited by furosemide, mucosal Cl removal caused no Cl efflux before but rapid efflux (0.25 mM/s) after addition of isoproterenol or chlorophenylthio-cAMP. In the presence of indomethacin to block prostaglandin production, elevation of intracellular Ca by bradykinin or 4-bromo-A23187 did not cause Cl efflux, nor did Ca buffering with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid affect stimulation by the cAMP pathway. Phorbol 12-myristate 13-acetate increased Cl efflux submaximally (0.09 mM/s) but did not affect maximal stimulation by cAMP agonists. Methoxamine did not alter apical or basolateral membrane Cl transport.(ABSTRACT TRUNCATED AT 250 WORDS)
1 alpha,25-(OH)2 vitamin D3 enhances expression of the genes encoding Ca(2+)-binding proteins MRP-8 and MRP-14
Authors: Sellmayer A. , Krane S. M. , Ouellette A. J. , Bonventre J. V.
Published: December 2017

Two closely related Ca(2+)-binding proteins, migration inhibitory factor-related protein (MRP)-8 and MRP-14, are synthesized under specific conditions of myeloid cell differentiation. Because 1 alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] induces myeloid cell differentiation and expression of other S-100 class calcium-binding proteins, we examined the effects of 1,25-(OH)2D3 on MRP mRNA levels in human U-937 histiocytic lymphoma cells. 1,25-(OH)2D3 increased MRP-8 and MRP-14 mRNA levels in a time- and dose-dependent manner. MRP mRNA levels were maximal at 24 h and remained elevated for at least 96 h after exposure of the cells to 1,25-(OH)2D3. MRP-8 mRNA accumulation required 100- to 1,000-fold higher concentrations of 25-(OH)D3, which binds to the 1,25-(OH)2D3 intracellular receptor with 100- to 1,000-fold lower affinity. Other differentiating agents, dimethyl sulfoxide, retinoic acid, and dexamethasone, also increased levels of MRP-8 and MRP-14 mRNA. Phorbol myristate acetate enhanced MRP-14 mRNA levels to a greater extent than MRP-8 mRNA levels, suggesting differential regulation of MRP gene expression by protein kinase C. The 1,25-(OH)2D3-induced relative increase in MRP mRNA levels was not changed by a 1,000-fold reduction in extracellular [Ca2+]. Thus 1,25-(OH)2D3 is potentially a physiological modulator of MRP gene expression. Expression of the MRP-8 and MRP-14 genes may be important for differentiation of myeloid cells.
Phorbol ester inhibits granulocyte-macrophage colony-stimulating factor binding and tyrosine phosphorylation
Authors: Gomez-Cambronero J. , Huang C. K. , Yamazaki M. , Wang E. , Molski T. F. , Becker E. L. , Sha'afi R. I.
Published: December 2017

Exposure of human polymorphonuclear neutrophils to phorbol 12-myristate 13-acetate (PMA) results in a 70-75% reduction in the specific binding of 125I-granulocyte-macrophage colony-stimulating factor (GM-CSF) to its receptors. The PMA-induced reduction in 125I-GM-CSF binding is due to a decrease in the number of available GM-CSF receptors, as derived from Scatchard analysis of the binding data. On the other hand, the phorbol ester 4-alpha-phorbol 12,13-didecanoate (4 alpha-PDD) fails to affect 125I-GM-CSF binding. PMA promotes phosphorylation on tyrosine residues of several proteins, as demonstrated by Western blotting analysis using antiphosphotyrosine antibodies. The molecular masses of those proteins are 41, 55, 66, 78, 85, 104, and 115 kDa. GM-CSF increases the levels of the tyrosine phosphorylation of several proteins, the majority of which have similar Mr to those found in PMA-stimulated neutrophils. This increase, on all but the 41-kDa protein, is partially prevented by treatment of the cells with PMA. The inhibition by PMA of GM-CSF binding to its receptors and its phosphorylated effects is partially prevented by the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine and, to a greater extent, by staurosporine. It is suggested that PMA, through the activation of protein kinase C, interrupts the excitation-response sequence initiated by GM-CSF, which includes tyrosine phosphorylation, and that the earliest altered step is the binding of GM-CSF to its receptor.
Electrophysiologically distinct cell types in human sweat gland secretory coil
Authors: Reddy M. M. , Quinton P. M.
Published: December 2017

The human sweat gland secretory coil consists of three histologically distinct cell types: myoepithelial (ME), light (or clear), and dark cells. The electrophysiological properties of all these cells are poorly defined. Employing electrophysiological techniques, we report distinct pharmacological responses of three different cell types from freshly isolated human sweat gland secretory coil. The superficial ME cells are characterized by 1) spontaneous depolarizing spikes (2 to 50 mV), 2) high cell membrane potentials [Vm = -68.6 +/- 3.9 (SE) mV; n = 21], 3) a K(+)-selective cell membrane (slope response = 54.2 +/- 6.7 mV per decade K+ concentration; n = 4), 4) depolarizing responses to cholinergic agonist mecholyl (delta Vm = 29.1 +/- 3.1 mV, n = 21), and 5) insensitivity to beta-adrenergic stimulation (n = 12). Two other types of cells, presumably secretory, were also observed. We arbitrarily labeled these cells as beta-adrenergic sensitive (beta-S) and beta-adrenergic insensitive (beta-I) cells based on their respective sensitivity to isoproterenol (IPR), a beta-adrenomimetic. Properties of the beta-S cells include 1) relatively higher basolateral membrane potentials (Vm = -57.3 +/- 3.1 mV; n = 13), 2) depolarizing responses to IPR (delta Vm = 16.8 +/- 2.6 mV; n = 9) inhibitable by the beta-adrenergic antagonist propranolol, and 3) hyperpolarizing responses to mecholyl (delta Vm = -21.8 +/- 2.0 mV; n = 13). The beta-I cells are characterized by 1) low basolateral membrane potentials (Vm = -23.6 +/- 2.1 mV; n = 16), 2) insensitivity to beta-adrenergic stimulation, and 3) hyperpolarizating responses to mecholyl (delta Vm = -16.1 +/- 2.1 mV; n = 16).
Electrogenic sodium-bicarbonate cotransport in human ciliary muscle cells
Authors: Stahl F. , Lepple-Wienhues A. , Kuppinger M. , Tamm E. , Wiederholt M.
Published: December 2017

We investigated membrane voltage and intracellular pH (pHi) in cultured human ciliary muscle cells using a cell line (H7CM) and primary-cultured human ciliary muscle cells. 1) Resting potential was 58.9 +/- 1.0 mV in H7CM cells and 61.9 +/- 1.4 mV in primary cultures. The following data are from H7CM cells, but results from primary cultures were basically similar. 2) In HCO3(-)-CO2-buffered solution, removal of extracellular sodium resulted in a depolarization [change in membrane resistance (delta V) = 31.3 +/- 2.8 mV] that was less marked in the absence of HCO3(-)-CO2 (delta V = 0.5 +/- 2.6 mV) and reduced by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) (delta V = 19.3 +/- 1.9 mV). 3) Removal of extracellular HCO3(-)-CO2 led to a depolarization (delta V = 13.2 +/- 0.8 mV) that was abolished in the absence of extracellular sodium and inhibited by DIDS. 4) Intracellular alkalinization led to a depolarization (delta V = 24.7 +/- 2.3 mV), and intracellular acidification resulted in a hyperpolarization (delta V = 9.4 +/- 1.1 mV) that was inhibited by DIDS and dependent on extracellular HCO3(-)-CO2 and sodium. 5) pHi backregulation after an acid load occurred in both the presence and absence of extracellular bicarbonate but not in the absence of extracellular sodium. Our data are consistent with an electrogenic Na(+)-HCO3- cotransport in human ciliary muscle cells, which is activated by intracellular acidification.
Regulation of vascular endothelial cell volume by Na-K-2Cl cotransport
Authors: O'Neill W. C. , Klein J. D.
Published: December 2017

The relationship between cell volume and Na-K-2Cl cotransport was studied in cultured bovine aortic endothelial cells. Hypertonic cell shrinkage increased bumetanide-sensitive, Na- or Cl-dependent K influx without altering bumetanide-insensitive influx. Greater stimulation of cotransport was observed in cells shrunken isosmotically either by preincubation in K-free and Na-free medium or by preincubation in hypotonic medium. Cell swelling, produced by preincubation in isotonic high-K medium, inhibited bumetanide-sensitive K influx. Simultaneous measurements of [3H]bumetanide binding and K influx revealed an increased number of binding sites without an increased influx per binding site in shrunken cells. Bumetanide did not alter the volume or ion content of cells in isotonic or hypertonic medium, indicating that no net influx of ions occurs through cotransport under these conditions. In isosmotically shrunken cells, there was greater stimulation of bumetanide-sensitive influx than of bumetanide-sensitive efflux, resulting in net bumetanide-sensitive influx. Rapid recovery of cell K, Na, and water occurred over 10-20 min and was inhibited by bumetanide or by the removal of external Na or Cl. These data demonstrate that Na-K-2Cl cotransport in aortic endothelial cells is regulated by cell volume, possibly through changes in the number of functional cotransporters, and mediates a brisk regulatory volume increase in isosmotically shrunken cells. Although thermodynamically favored, no net influx occurs through Na-K-2Cl cotransport in cells of normal volume or in hypertonically shrunken cells. This suggests additional regulation of cotransport, perhaps through trans-inhibition by intracellular Cl. Regulation of cell volume by Na-K-2Cl cotransport may be important in maintaining endothelial integrity.
Differential responses of Ca-activated K channels to bradykinin in sensory neurons and F-11 cells
Authors: Naruse K. , McGehee D. S. , Oxford G. S.
Published: December 2017

The nonapeptide bradykinin (BK) excites a subset of dorsal root ganglion (DRG) neurons with putative nociceptive functions by stimulating an inward cation current. In addition, BK stimulates various intracellular signaling pathways including an elevation of intracellular Ca2+. In a DRG neuron x neuroblastoma hybrid cell (F-11), BK stimulates similar increases in intracellular [Ca2+] and inward current but also elicits a large transient outward current through Ca(2+)-activated K channels. We have investigated the mechanisms underlying differential expression of outward current responses in the two cell types at the single channel level. Although K(Ca) channel activity appears in inside-out patches from both cells exposed to Ca2+, BK applied to the extrapatch membrane of cell-attached patches activates K(Ca) channels in F-11 but not DRG neurons. Whereas single K(Ca) channels are quantitatively similar in terms of conductance, voltage-dependence, and sensitivity to tetraethylammonium, they differ in sensitivity to intracellular Ca2+. Channel activation in both cells requires at least four Ca2+ ions, but half-maximal activation occurs at slightly higher [Ca2+] for DRG neurons. The shift in the Ca2+ dose-response curve combined with the steep [Ca2+] dependence of channel open probability makes it less likely that a BK-induced rise in internal [Ca2+] induced will trigger a transient outward current and resultant hyperpolarization in a DRG neuron.
Na+ binding to the Na(+)-glucose cotransporter is potential dependent
Authors: Bennett E. , Kimmich G. A.
Published: December 2017

Activity of the Na(+)-glucose cotransporter in LLC-PK1 epithelial cells was assayed by measuring sugar-induced currents (IAMG) using whole cell recording techniques. IAMG was compared among cells by standardizing the measured currents to cell size using cell capacitance measurements. IAMG at a given membrane potential was measured as a function of alpha-methylglucoside (AMG) concentration and can be fit to Michaelis-Menten kinetics. IAMG at varying Na+ concentrations can be described by the Hill equation with a Hill coefficient of 1.6 at all tested potentials. At high external Na+ levels (155 mM), Na+ is at least 90% saturating at all tested potentials. Maximal currents at a given membrane potential (Im) are calculated from the Michaelis-Menten equation fit to data measuring IAMG vs. AMG concentration at a constant Na+ concentration. Im showed potential dependence under all conditions. Potential-dependent Na+ binding rate(s) cannot alone explain the observed potential dependence of Im under saturating Na+ conditions. Therefore, because Im is potential dependent, at least one step of the transport cycle other than external Na+ binding must be potential dependent. Im was also calculated from data taken at 40 mM external Na+. At all potentials studied, Im at 155 mM Na+ is greater than Im calculated at 40 mM Na+. This implies that the rate of external Na+ binding to the transporter at 40 mM also affects the maximal transport rate. Furthermore, Im at 40 mM external Na+ increases with hyperpolarization faster than Im at 155 mM Na+. Together, these facts indicate that the rate at which Na+ binds to the transporter is also potential dependent.(ABSTRACT TRUNCATED AT 250 WORDS)
Role of G proteins in stimulation of Na-H exchange by cell shrinkage
Authors: Davis B. A. , Hogan E. M. , Boron W. F.
Published: December 2017

Many cells respond to shrinkage by stimulating specific ion transport processes (e.g., Na-H exchange). However, it is not known how the cell senses this volume change, nor how this signal is transduced to an ion transporter. We have studied the activation of Na-H exchange in internally dialyzed barnacle muscle fibers, measuring intracellular pH (pHi) with glass microelectrodes. When cells are dialyzed to a pHi of approximately 7.2, Na-H exchange is active only in shrunken cells. We found that the shrinkage-induced stimulation of Na-H exchange, elicited by increasing medium osmolality from 975 to 1,600 mosmol/kgH2O, is inhibited approximately 72% by including in the dialysis fluid 1 mM guanosine 5'-O-(2-thiodiphosphate). The latter is an antagonist of G protein activation. Even in unshrunken cells, Na-H exchange is activated by dialyzing the cell with 1 mM guanosine 5'-O-(3-thiotriphosphate), which causes the prolonged activation of G proteins. Activation of Na-H exchange is also elicited in unshrunken cells by injecting cholera toxin, which activates certain G proteins. Neither exposing cells to 100 nM phorbol 12-myristate 13-acetate nor dialyzing them with a solution containing 20 microM adenosine 3',5'-cyclic monophosphate (cAMP) (or 50 microM dibutyryl cAMP) plus 0.5 mM 3-isobutyl-1-methylxanthine substantially stimulates the exchanger. Thus our data suggest that a G protein plays a key role in the transduction of the shrinkage signal to the Na-H exchanger via a pathway that involves neither protein kinase C nor cAMP.
Calcium oscillations in freshly dispersed and cultured interstitial cells from canine colon
Authors: Publicover N. G. , Horowitz N. N. , Sanders K. M.
Published: December 2017

Spontaneous oscillations in intracellular Ca2+ concentration ([Ca2+]i) and membrane potential were used to monitor rhythmicity in freshly dispersed and cultured interstitial cells (IC) from the canine colon. The frequency of oscillations and responses to a number of channel blockers, agonists, ionic substitutions, and temperature were similar in freshly dispersed and cultured cells. An increase in the amplitude of Ca2+ oscillations after 3-6 days in culture and an increase in the rate of decline of [Ca2+]i in cultured IC were two differences noted between freshly dispersed and cultured cells. The frequency and amplitude of oscillations were a function of extracellular Ca2+ concentrations, and oscillations were abolished when the transmembrane flux of Ca2+ was reduced by nicardipine, La3+, or removal of Ca2+ from the extracellular medium. Oscillations persisted in the presence of ryanodine and ouabain. Lowered temperatures or a reduction in the concentration of Ca2+ in the medium reduced the frequency of spontaneous oscillations. Carbachol and substance P caused a transient increase in [Ca2+]i. Substance P then abolished spontaneous events. ATP and calcitonin gene-related peptide increased the frequency of spontaneous activity. Vasoactive intestinal peptide caused a temporary delay in spontaneous oscillations when added to the medium. Results indicate that freshly dispersed and cultured IC may be useful in studies of the mechanisms of rhythmicity in the gastrointestinal system.
Intracellular Ca2+ concentration and pHi during metabolic inhibition
Authors: Hayashi H. , Miyata H. , Noda N. , Kobayashi A. , Hirano M. , Kawai T. , Yamazaki N.
Published: December 2017

To study the changes in intracellular Ca2+ concentration ([Ca2+]i) and pH (pHi) during metabolic inhibition, rat ventricular myocytes were dual loaded with the acetoxymethyl esters of fura-2 (fura-2/AM) and 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF/AM) and perfused with 2 mM sodium cyanide (NaCN). The percent of rod-shaped cells was 30% of the control after 30 min NaCN in the absence of glucose. [Ca2+]i increased from 82 +/- 8 to 151 +/- 25 (SE) nM (P less than 0.05) when cells were shortened, and to 421 +/- 106 nM (P less than 0.05) when cells were rounded. There was a positive relationship between pCai and pHi (r = 0.425, P less than 0.01). When 50 mM glucose was added during NaCN, there were no significant changes in [Ca2+]i and the percent of rod-shaped cells after 30 min. The pHi of rod-shaped cells, however, decreased to 6.95 +/- 0.03 (P less than 0.01). Although the acidic solution (70% O2-30% CO2) decreased pHi to 6.90 +/- 0.05 (P less than 0.01), there were no changes in the cell shape or [Ca2+]i. The addition of NaCN for 30 min decreased the rod-shaped cells to 18% of the control. Mild acidosis did not cause the changes in cell shape or [Ca2+]i. There was also no protection of the NaCN-induced changes in cell shape by intracellular acidosis. It is likely that the changes in cell shape during metabolic inhibition were related to the depletion of metabolic energy and the increase in [Ca2+]i.
Potassium channel block does not affect metabolic responses of brown fat cells
Authors: Pappone P. A. , Lucero M. T.
Published: December 2017

Hormonally stimulated brown fat cells are capable of extremely high metabolic rates, making them an excellent system in which to examine the role of plasma membrane ion channels in cell metabolism. We have previously shown that brown fat cell membranes have both voltage-gated and calcium-activated potassium channels (Voltage-gated potassium channels in brown fat cells. J. Gen. Physiol. 93: 451-472, 1989; Membrane responses to norepinephrine in cultured brown fat cells. J. Gen. Physiol. 95: 523-544, 1990). Currents through both the voltage-activated potassium channels, IK,V, and the calcium-activated potassium channels, IK,Ca, can be blocked by the membrane-impermeant K channel blocker tetraethylammonium (TEA). We used microcalorimetric measurements from isolated neonatal rat brown fat cells to assess the role these potassium conductances play in the metabolic response of brown fat cells to adrenergic stimulation. Concentrations of TEA as high as 50 mM, sufficient to block approximately 95% of IK,V and 100% of IK,Ca, had no effect on norepinephrine-stimulated heat production. These results show that neither voltage-gated nor calcium-activated K channels are necessary for a maximal thermogenic response in brown fat cells and suggest that K channels are not involved in maintaining cellular homeostasis during periods of high metabolic activity.
Regulation of calcium current by voltage and cytoplasmic calcium in canine gastric smooth muscle
Authors: Vogalis F. , Publicover N. G. , Sanders K. M.
Published: December 2017

The regulation of Ca2+ current by intracellular Ca2+ was studied in isolated myocytes from the circular layer of canine gastric antrum. Ca2+ current was measured with the whole cell patch-clamp technique, and changes in cytoplasmic Ca2+ ([Ca2+]i) were simultaneously measured with indo-1 fluorescence. Ca2+ currents were activated by depolarization and inactivated despite maintained depolarization. Ca2+ current inactivation was fit with a double exponential function. Using Ba2+ or Na+ as charge carriers removed the fast component of inactivation, whereas enhanced intracellular buffering of Ca2+ did not remove the fast component. Ca2+ currents were associated with a rise in [Ca2+]i. The decrease in [Ca2+]i following repolarization was exponential, and during the relaxation of [Ca2+]i, Ca2+ current was inactivated. The inward current recovered with a similar time course as the decrease in [Ca2+]i, suggesting that [Ca2+]i regulates the basal availability of Ca2+ channels. These data support the hypothesis that, although [Ca2+]i may influence the resting level of inactivation, it is the "submembrane" compartment of [Ca2+]i that regulates the development of inactivation.
U46619, a thromboxane A2 agonist, inhibits KCa channel activity from pig coronary artery
Authors: Scornik F. S. , Toro L.
Published: December 2017

Thromboxane A2 (TxA2) is a potent vasoconstrictor derived from the metabolism of arachidonic acid. Because potassium channels are involved in the contraction of vascular smooth muscle, their blockade could contribute to the TxA2-induced contraction. To test this possibility, we studied the effect of the TxA2 stable analogue U46619 on calcium-activated potassium (KCa) channels from coronary artery reconstituted into lipid bilayers. Addition of U46619 (50-150 nM) to the external but not to the internal side of the channel decreased the channel open probability (Po) between 15 and 80% of the control value. The inhibitory effect of U46619 affected both the open and closed states of the channel and could be reversed by internal calcium. Thromboxane B2, the inactive hydrolysis derivative of TxA2, did not affect channel activity. SQ 29548, a TxA2 receptor antagonist, was able to prevent the inhibition by U46619. Furthermore, SQ 29548 added after U46619 could restore channel activity to near control values. These results suggest that TxA2 could be a regulatory factor of KCa channels from coronary smooth muscle and that this regulation could be related to its action as a vasoconstrictor.
Factors affecting movement of F-actin filaments propelled by skeletal muscle heavy meromyosin
Authors: Homsher E. , Wang F. , Sellers J. R.
Published: December 2017

The measurement of fluorescent-labeled actin filament movement driven by mechanoenzymes (e.g., myosin) is an important methodology for the study of molecular motors. It is assumed that the filament velocity (Vf) is analogous to the unloaded shortening velocity (Vu) seen in muscle fibers. Methods are described to reproducibly quantitate the movement of these filaments and to select uniformly moving filaments and specify their Vf. Use of these techniques allowed comparison of Vf to literature values for Vu with regard to [ATP], [ADP], [Pi], pH, ionic strength (10-150 mM), and temperature (15-30 degrees C). Vf and Vu are quantitatively similar with respect to the effects of substrate and product concentrations and temperatures greater than 20 degrees C. However, Vf is more sensitive to decreases in pH and temperatures less than 20 degrees C than Vu. At ionic strengths of 50-150 mM, Vf and Vu exhibit similar ionic strength dependencies (decreasing with ionic strength). At ionic strengths less than 50 mM, Vf is markedly reduced. Results of experiments using adenosine 5'-O-(3-thiotriphosphate) suggest that increasing the number of weakly bound cross bridges does not seriously affect Vf. Thus, although Vf is a good analogue for Vu under certain conditions (elevated ionic strength and temperatures greater than 20 degrees C), under others it is not. The results of motility assays must be cautiously interpreted.
Regulated endocytosis in a chloride secretory epithelial cell line
Authors: Bradbury N. A. , Jilling T. , Kirk K. L. , Bridges R. J.
Published: December 2017

The colonic epithelial cell line T84 has been shown to be a good model to investigate the regulation of Cl- secretion by the adenosine 3',5'-cyclic monophosphate (cAMP)-mediated second messenger cascade. Regulated exocytic insertion and endocytic retrieval of transport proteins, or proteins that regulate transport proteins, is one mechanism proposed to regulate plasma membrane solute permeabilities. The aims of our studies were to characterize endocytic processes in T84 cells and to investigate their regulation by known activators of Cl- secretion that are mediated by the cAMP second messenger cascade. Forskolin, an activator of adenylate cyclase, caused a marked inhibition of endocytic uptake of the fluid-phase marker horseradish peroxidase (HRP) and the adsorptive marker wheat germ agglutinin conjugated to HRP. Similar inhibition was obtained with vasoactive intestinal peptide, a secretagogue whose receptor is coupled to adenylate cyclase, and 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, a membrane-permeable cAMP analogue. 1,9-Dideoxy-forskolin, a forskolin analogue that fails to activate adenylate cyclase, was without effect on endocytosis. Our data show that the net rate of endocytosis, as measured by fluid-phase uptake, is decreased by a cAMP-mediated mechanism. Because the number of Cl- channels or associated regulatory proteins in the plasma membrane reflects a balance between their exocytic insertion and endocytic retrieval, we propose that the cAMP-mediated decrease in endocytosis could contribute to the concomitant increase in plasma membrane Cl- permeability.
Recovery from exercise-induced desaturation in the quadriceps muscles of elite competitive rowers
Authors: Chance B. , Dait M. T. , Zhang C. , Hamaoka T. , Hagerman F.
Published: December 2017

A simple muscle tissue spectrophotometer is adapted to measure the recovery time (TR) for hemoglobin/myoglobin (Hb/Mb) desaturation in the capillary bed of exercising muscle, termed a deoxygenation meter. The use of the instrument for measuring the extent of deoxygenation is presented, but the use of TR avoids difficulties of quantifying Hb/Mb saturation changes. The TR reflects the balance of oxygen delivery and oxygen demand in the localized muscles of the quadriceps following work near maximum voluntary contraction (MVC) in elite male and female rowers (a total of 22) on two occasions, 1 yr apart. TR ranged from 10 to 80 s and was interpreted as a measure of the time for repayment of oxygen and energy deficits accumulated during intense exercise by tissue respiration under ADP control. The Hb/Mb resaturation times provide a noninvasive localized indication of the degree of O2 delivery stress as evoked by rowing ergometry and may provide directions for localized muscle power output improvement for particular individuals in rowing competitions.
Kidney aldose reductase gene transcription is osmotically regulated
Authors: Smardo F. L. , Burg M. B. , Garcia-Perez A.
Published: December 2017

Cells generally adapt to long-term hypertonic stress by accumulating organic osmolytes. PAP-HT25 renal medullary cells in hypertonic medium accumulate sorbitol through a reaction catalyzed by aldose reductase and betaine through osmotically regulated transport. Hypertonicity increases aldose reductase protein synthesis rate by elevating its mRNA abundance. To test whether the rise in aldose reductase mRNA is due to enhanced transcription, PAP-HT25 cells adapted to isotonic medium were switched to hypertonic medium, and transcription rate was measured by nuclear run-on. Aldose reductase transcription rate peaked at 17-fold the isotonic level after 12 h of hypertonicity. Then, transcription fell as sorbitol and betaine accumulated. Transcription stabilized at fivefold the isotonic level within days. Aldose reductase mRNA stability was not significantly different between the hypertonic and isotonic steady states. Thus aldose reductase mRNA is osmotically regulated through changes in its transcription. The osmotically induced rise in aldose reductase transcription is blunted by the accumulation of intracellular betaine and is exaggerated and prolonged by preventing the accumulation of both sorbitol (by aldose reductase inhibition) and betaine (by removal from the medium). This study presents the first description of osmoregulated gene transcription in animal cells.
Transport and binding in calcium uptake by microvillous membrane of human placenta
Authors: Kamath S. G. , Kelley L. K. , Friedman A. F. , Smith C. H.
Published: December 2017

Calcium entry across the microvillous membrane of the human placental syncytiotrophoblast is the first step in the transfer of this important nutrient to the fetus. Calcium uptake by isolated microvillous membranes was time dependent. Equilibrium uptake was very much greater than could be explained by equilibration of the vesicle space with medium, indicating that calcium is bound to internal sites. Addition of the ionophore A23187 greatly increased the rates of influx and efflux, indicating that transport across the plasma membrane is rate limiting in entry. Concentration dependence data for calcium transport at 4 s fit well to a Michaelis-Menten equation having two saturable sites and diffusion. Calcium entry by both transporters was unaffected by calcium channel blockers but was strongly inhibited by the group II metals. The distinct inhibition constant values for strontium inhibition provided additional evidence for two transporters. Calcium binding fit well to a single-site model saturable in the micromolar range. In vivo, the saturable transport processes may mediate calcium entry into the syncytiotrophoblast and binding may regulate concentration within the placental microvilli.
Stress-induced alterations in autophagic pathway: relationship to ubiquitin system
Authors: Schwartz A. L. , Brandt R. A. , Geuze H. , Ciechanover A.
Published: December 2017

The autophagic response of the cell to nutrient deprivation or heat stress is characterized by an increase in the rate of cellular protein degradation. Using temperature-sensitive mutant cell lines that harbor a mutation in the ubiquitin pathway, we have recently shown that this response is dependent on a functional ubiquitin-activating enzyme E1. The ubiquitin pathway is involved in a multitude of cellular events including protein degradation, the best understood of these. Herein the activation of the ubiquitin molecule via E1 is followed by its covalent conjugation to acceptor proteins followed by proteolysis. It is therefore important to study the linkage between the autophagic response and E1. Using these same cell lines, CHO E36 and CHO ts20, we demonstrate that after heat stress or nutrient deprivation there is a rapid and reversible decrease in the buoyant density of subcellular vesicles containing lysosomal hydrolases, a characteristic found to accompany autophagy. This stress-induced change is found in all cell lines examined, independent of the activity of the E1. The light-density vesicles, which comigrate with endosomes on colloidal silica gradients, are not accessible to the endocytic marker transferrin-horseradish peroxidase (HRP) after cellular uptake and subsequent HRP-mediated density shift analysis. Furthermore, morphology of the isolated fractions from control and stress-induced cells was similar. These results thus demonstrate the changes in hydrolase-containing intracellular vesicles that accompany nutritional deprivation or heat stress and support the notion that the linkage of the autophagic response to the ubiquitin system is at a step in autophagy which does not affect the formation of autophagic vesicles.
Contraction of glycerinated rabbit slow-twitch muscle fibers as a function of MgATP concentration
Authors: Pate E. , Lin M. , Franks-Skiba K. , Cooke R.
Published: December 2017

We have measured the isometric tension and force-velocity relationships of glycerinated rabbit slow-twitch semimembranosus muscle as a function of MgATP concentration ([MgATP]) and have compared the results with those obtained previously from fast-twitch psoas muscle. We find that isometric tension decreases as [MgATP] increases. The magnitude of the decrease is not as great as observed in psoas. Maximum shortening velocity (Vmax) exhibits classical Michaelian saturation behavior with respect to [MgATP] with a Michaelis constant (Km) for half-maximal velocity of 18 microM and a value at saturating [MgATP] of 0.6 muscle lengths/s. Similar values were observed in fibers from soleus, another slow-twitch muscle. The corresponding values in rabbit psoas muscle are 150 microM and 1.6 lengths/s. Compared with psoas, in semimembranosus muscle Km decreases by a factor of approximately 10, whereas Vmax decreases by about a factor of 3. Thus, although in a nonphysiological regime, at low [MgATP], a "fast" muscle actually has a lower shortening velocity than a "slow" muscle.
Evidence of two distinct epithelial cell types in primary cultures from human sweat gland secretory coil
Authors: Reddy M. M. , Bell C. L. , Quinton P. M.
Published: December 2017

The human sweat gland secretory coil (SC) is comprised of myoepithelial (ME) and two types of secretory epithelial cells. The secretory cells include beta-adrenergic-sensitive (beta-S) cells [responsive to the beta-adrenergic agonist isoproterenol (IPR)] and beta-adrenergic insensitive (beta-I) cells. We have grown segments of SC in primary culture and found that under the conditions described here, only epithelial cells form outgrowths as indicated by morphological and physiological properties. As in the native SC epithelium, the secretory cells in primary culture were comprised of polygonal epithelial cells with a characteristic hyperpolarization of cell potentials (Vm) to cholinergic stimulation by mecholyl (magnitude of change of Vm = delta Vm = 21.5 +/- 1.3 mV, mean +/- SE, n = number of cells = 44). We have found both beta-S and beta-I cells as determined by unstimulated membrane potentials, sensitivity to IPR, and K+ conductance (GK+). The frequency distribution of unstimulated cells indicated two distinct populations of cells, one with high membrane potentials (Vm = -63 +/- 2.6 mV), which correlated with beta-S cells, and a second with low membrane potentials (Vm = -22 +/- 1.5 mV), which correlated with the beta-I cells. IPR depolarized the Vm of beta-S cells (delta Vm = 11.0 +/- 0.8 mV, n = 25) without affecting the Vm of beta-I cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Lymphocyte adhesion can be regulated by cytoskeleton-associated, PMA-induced capping of surface receptors
Authors: Haverstick D. M. , Sakai H. , Gray L. S.
Published: December 2017

Intercellular adhesion in lymphocytes is mediated in part by the interaction of the integrin lymphocyte function-associated antigen-1 (LFA-1) with intercellular adhesion molecule-1 (ICAM-1). The B lymphoblastoid line JY expresses both LFA-1 and ICAM-1, and intercellular adhesion is enhanced by treatment with the phorbol ester phorbol 12-myristate 13-acetate (PMA), which also induced capping of LFA-1, ICAM-1, and human leukocyte antigen. Capping of LFA-1 is likely to result from protein kinase C (PKC) activation because receptor-mediated stimulation of PKC also led to capping. Additionally, adhesion mediated by PMA or lipopolysaccharide was blocked by either of two PKC inhibitors, calphostin C and staurosporine. PMA induced the apparent condensation of cytoskeletal elements that colocalized with the membrane protein cap. Cytoskeletal condensation and capping occurred in the absence of intercellular adhesion. Alteration in the distribution of cytoskeletal components and membrane redistribution of LFA-1 were inhibited by cytochalasin D, which also abolished intercellular adhesion. Taken together, these data suggest that intercellular adhesion is the result of PKC-mediated membrane redistribution of LFA-1 and ICAM-1, which is in turn associated with modification of the actin-based cytoskeleton.
Growth factors downregulate vascular smooth muscle thromboxane receptors independent of cell growth
Authors: Dorn G. W. , Becker M. W.
Published: December 2017

Growth factors, in addition to being mitogenic, may modulate vascular smooth muscle differentiation. We tested whether serum or defined growth factors could regulate thromboxane A2 (TxA2) receptors in cultured rabbit aorta smooth muscle cells. Fetal bovine serum (10%) stimulated cell proliferation and DNA synthesis in subconfluent cell cultures. Binding of the thromboxane A2 agonist [1S-(1 alpha 2 beta(5Z),3 alpha(1E,3S),4 alpha)]-7-[3-(3-hydroxy-4-p- iodophenoxy-1-butenyl)-7-oxabicyclo[2.2.1]heptan-2-yl]-5-hep tenoic acid showed a 41% decrease in TxA2 receptors in cells treated with 10% serum compared with serum-deprived (0.1%) controls. Receptor downregulation by serum was gradually reversible upon serum withdrawal. Compared with serum-deprived cells, those exposed to 10% serum also had diminished TxA2-stimulated phosphatidylinositol hydrolysis. Regulatory actions of serum on TxA2 receptors were distinguished from mitogenic effects with heparin, which prevented cell growth but did not inhibit serum-induced downregulation of TxA2 receptors. Furthermore, low concentrations of platelet-derived growth factor and basic fibroblast growth factor decreased TxA2 receptors without stimulating cell proliferation or DNA synthesis. These observations describe a previously unrecognized regulatory action of growth factors on a vascular smooth muscle vasoconstrictor receptor, an action which is independent of effects on cell proliferation or DNA synthesis.
ATP activates a cation-permeable pathway in rat parotid acinar cells
Authors: Soltoff S. P. , McMillian M. K. , Talamo B. R.
Published: December 2017

Effects of several purinergic receptor agonists were examined on rat parotid acinar cells. Extracellular ATP stimulated 45Ca2+ uptake into isolated rat parotid acinar cells in a concentration-dependent fashion (EC50 approximately 125 microM ATP) at a maximum rate of approximately 6 nmol.mg protein-1.min-1. In the absence of extracellular Na+, ATP increased the uptake rate by greater than 100%. Increasing concentrations of extracellular Na+ reduced the ATP-stimulated rate of 45Ca2+ entry in a graded fashion (IC50 16.6 mM), suggesting that Ca2+ and Na+ compete for entry. Uptake rate was not reduced when intracellular Ca2+ was buffered with 1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid, indicating that the effects of ATP were not initiated by an elevation in intracellular free Ca2+ concentration. 3-O-(4'-benzoyl)benzoyl-ATP was much more potent (EC50 approximately 4 microM) and stimulated Ca2+ influx at a greater rate (approximately 12 nmol.mg protein-1.min-1) than ATP. Other nucleotide analogues, including adenosine 5'-O-(3-thiotriphosphate), 2-methylthio-ATP, and 5'-adenylylimidodiphosphate, were much less effective than ATP. ATP produced a biphasic effect on membrane potential: an initial hyperpolarization was followed by a rapid depolarization. The depolarization was greatly reduced in the absence of extracellular Na+, but not in the absence of extracellular Ca2+, indicating that the majority of the depolarizing current was due to Na+ entry. Effects of ATP on the membrane potential were distinguishable from those of the Ca2+ ionophore ionomycin and the muscarinic agonist carbachol. Depolarization of the cells by gramicidin or K+ did not produce an increase in 45Ca2+ uptake.(ABSTRACT TRUNCATED AT 250 WORDS)
Effect of trypsin on a Ca(2+)-activated K+ channel reconstituted into planar phospholipid bilayers
Authors: Salomao L. , Wark G. , Dubinsky W. P. , Schultz S. G.
Published: December 2017

Exposure of the cytoplasmic side of calcium-activated, high (maxi)-conductance potassium [BK(Ca)] channels in basolateral membrane vesicles from rabbit colonocytes incorporated into planar phospholipid bilayers to trypsin rapidly reduces, but does not abolish, the sensitivity of this channel to activation by calcium without affecting its conductance or high selectivity for K+ over Cl-. The results of these studies also indicate that this BK(Ca) channel does not have intrinsic voltage-gating properties but that its voltage sensitivity is related to its ability to interact with calcium. This conclusion is consistent with the model proposed by Moczydlowski and Lattore (J. Gen. Physiol. 82: 511-542, 1983) for the role of membrane voltage in modulating the interaction between calcium and the BK(Ca) channel in rat skeletal muscle.