bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell volume, K transport, and cell density in human erythrocytes
Authors: Brugnara C. , Tosteson D. C.
Published: December 2017

We report here studies on the regulation of cell volume and K transport in human erythrocytes separated according to density. When cell volume was increased (isosmotic swelling, nystatin technique), erythrocytes of the least dense but not of the densest fraction shrunk back toward their original volume. This process was due to a ouabain (0.1 mM) and bumetanide (0.01 mM) (OB)-resistant K loss. OB-resistant K+ efflux from the least dense fraction was stimulated by hypotonic swelling and had a bell-shaped dependence on pH (pH optimum 6.75–7.0). These pH and volume effects were not evident in the densest fraction. The swelling-induced K+ efflux from the least dense fraction was inhibited when chloride was substituted by nitrate, thiocyanate, and acetate, whereas it was stimulated by bromide. Increasing cell Mg2+ content also markedly inhibited K+ efflux from isosmotically swollen cells. N-ethylmaleimide (NEM, 1 mM) greatly increased OB-resistant K+ efflux from the least dense fraction but not from the densest fraction. These data reveal the presence, in the lease dense fraction of normal human erythrocytes, of a pathway for K+ transport that is dependent on volume, pH, and chloride, is inhibited by internal Mg2+, and possibly plays a role in determining the erythrocyte water and cation content.
Peripheral nerve as an osmometer: role of endoneurial capillaries in frog sciatic nerve
Authors: Odman S. , Levitan H. , Robinson P. J. , Michel M. E. , Ask P. , Rapoport S. I.
Published: December 2017

The sciatic nerve of the frog was perfused in vivo with isotonic Ringer solution followed by Ringer made hypertonic by addition of sucrose or of NaCl. Nerve diameter and endoneurial hydrostatic pressure fell during hypertonic Ringer perfusion. Using a model that describes the elastic and osmotic properties of the nerve, sigma sLp, the product of the osmotic reflection coefficient at endoneurial capillaries for s equals sucrose or NaCl (which approximates 1), and of capillary hydraulic conductivity, was found to equal 73 X 10(-13) cm3 X s-1 X dyn-1. The nerve is elastic. It has a compliance K of 3.7 X 10(-5) cm2 X mmHg-1, corresponding to a modulus of elasticity E of the perineurium equal to 1.2 X 10(6) dyn X cm-2. The results indicate that the nerve behaves as an osmometer during vascular perfusion, due to the low permeability of endoneurial capillaries to small solutes such as NaCl and sucrose. A low capillary hydraulic conductivity limits bulk water flow between blood and nerve, and a low compliance limits nerve swelling and edema.
Mitochondrial transmembrane ion distribution during anoxia
Authors: Aw T. Y. , Andersson B. S. , Jones D. P.
Published: December 2017

The distribution of pyruvate, phosphate, malate, citrate, K+, aspartate, glutamate, ADP, and ATP between the mitochondrial and cytosolic compartments was studied in isolated rat hepatocytes exposed to 30 min anoxia. The results show that pyruvate and citrate gradients are comparable to aerobic values, indicating that the pH gradient across the membrane under anaerobic conditions is comparable to that under normal aerobic conditions. In contrast, the distribution of phosphate, malate, ATP, ADP, aspartate, and glutamate suggests that transport of these species may be inhibited during anoxia. The results are discussed in terms of potential regulation of mitochondrial function to provide a quiescent anoxic state that is capable of recovering normal function on reoxygenation.
Vasoactive intestinal peptide stimulates alkali excretion in turtle urinary bladder
Authors: Durham J. H. , Matons C. , Brodsky W. A.
Published: December 2017

The turtle urinary bladder possesses an active transport mechanism for the electrogenic secretion of alkali. This process is independent of exogenous Cl and Na, induced by cyclic AMP (cAMP), and potentiated in bladders from NaHCO3-loaded (alkalotic) turtles. In the present study, it is shown that the serosal addition of vasoactive intestinal peptide (VIP) induces rapidly developing parallel increases in alkali secretion and in the short-circuiting current carried by this secretion. The VIP-induced increment in alkali secretion is greater in the presence than in the absence of an exogenously added phosphodiesterase inhibitor. Additions of a cAMP analog subsequent to the VIP-induced alkali secretion fail to induce any further increase in alkalinization. These results provide evidence for the action of VIP as a hormonal up regulator of alkali excretion in the turtle urinary bladder.
Effect of iron chelators on placental uptake and transfer of iron in rat
Authors: Wong C. T. , McArdle H. J. , Morgan E. H.
Published: December 2017

The uptake of radiolabelled transferrin and iron by the rat placenta has been studied using two approaches. The first involved injection of a ferrous or ferric iron chelator followed by injection of label. Neither chelator decreased the amount of labelled transferrin in the placenta after 2-h incubation and only bipyridine, a ferrous iron chelator, inhibited iron transport to the fetus. Deferoxamine (DFO), a ferric iron chelator, had no effect on iron transport to the fetus but reduced iron uptake by the liver. Both bipyridine and DFO increased iron excretion into the gut and by the urinary tract to the same degree into the gut, but there was a 10-fold greater urinary excretion with bipyridine than with DFO. Injection of iron attached to the chelators showed that neither bipyridine nor DFO could donate iron to the fetus as efficiently as transferrin. The mechanism involved was further investigated by studying the effect of the chelators on uptake of transferrin-bound iron by placental cells in culture. DFO inhibited iron accumulation more effectively than bipyridine in the cultured cells. The effect was not due to a decrease in the cycling time of the receptor. The results can be explained if the iron is released from the transferrin in intracellular vesicles in the ferrous form, where it may be chelated by bipyridine and prevented from passing to the fetus or converted to the ferric form once it is inside the cell matrix.
Characterization of Na+-H+ antiport in type II alveolar epithelial cells
Authors: Nord E. P. , Brown S. E. , Crandall E. D.
Published: December 2017

The presence of a Na+-H+ exchange pathway in the plasma membrane of type II alveolar epithelial cells was explored using the pH-sensitive fluorescent probe 2,7-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) to monitor changes in cytosolic pH. Freshly prepared pneumocytes suspended in medium at pH 7.4 had an intracellular pH of 7.07 +/- 0.07. Acid-loaded cells equilibrated in sodium-free buffer showed rapid cytoplasmic alkalinization when exposed to sodium. This response to sodium was inhibited greater than 90% by 10(-4) M amiloride. The presence of the K+ ionophore, valinomycin, had no effect on the rate of Na+-dependent alkalinization, indicating the electroneutrality of the system. Li+ partially supported the alkalinization process, but other monovalent cations, notably K+, Rb+, and Cs+, were without effect. Kinetic analysis for Na+ at the external binding site yielded KNat (dissociation constant) = 62 +/- 3 mM. Hill equation analysis of the data derived a Hill coefficient (n) = 1.2 +/- 0.1 for Na+, consistent with a 1:1 stoichiometry for Na+ and H+ for the transporter. The Ki for amiloride inhibition of proton efflux at the external locus was 0.45 microM. These findings define the transport pathway as Na+-H+ antiport, with kinetic parameters somewhat similar to those described for other cell types. Antiport activity was detected at intracellular pH (pHi) values of 6.8 or below, with no activity observed at pHi 7.0-7.2. It is suggested that Na+-H+ exchange is a major mechanism whereby pneumocytes recover from an acid load and that this transport pathway may play an important role in vectorial reabsorption of Na+ from the alveolar air spaces.
Role of sodium-calcium exchange in regulation of intracellular calcium in nerve terminals
Authors: Sanchez-Armass S. , Blaustein M. P.
Published: December 2017

Ca efflux from rat brain presynaptic nerve terminals (synaptosomes) was examined after loading the terminals with 45Ca during a brief depolarization, usually in media containing 20 microM Ca labeled with 45Ca, to assure a small (physiological) load. Efflux of 45Ca was very slow in the absence of external Na and Ca (approximately 0.5% of the load/s) and was greatly accelerated by Na and/or Ca (presumably Na+-Ca2+ and Ca2+-Ca2+ exchange, respectively). The dependence of 45Ca efflux on external Na was sigmoid, with a Hill coefficient of approximately 2.5; this implies that more than two external Na ions are required to activate the efflux of one Ca ion. The external Na (Nao)-dependent Ca efflux was inhibited by 1 mM external La, by low temperature (Q10 congruent to 2.3), and by raising external K (to depolarize the synaptosomes). With small Ca loads, the mitochondrial uncoupler, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), had negligible effect on either Ca uptake or efflux; with large loads (greater than or equal to 5 nmol/mg protein), however, FCCP reduced the depolarization-stimulated Ca uptake and increased the Nao-dependent Ca efflux. These effects may be attributed to reduction of mitochondrial Ca sequestration. Mitochondria do not appear to sequester much Ca when the loads are smaller (and more physiological). Estimations of Ca efflux indicate that approximately 20% of a small 45Ca load (approximately 0.75 nmol Ca/mg protein) may be extruded via Na+-Ca2+ exchange within 1 s; this corresponds to a net Ca efflux of approximately 110 pmol Ca X mg protein-1 X s-1.(ABSTRACT TRUNCATED AT 250 WORDS)
Beta-adrenergic stimulation of ion transport in primary cultures of avian salt glands
Authors: Lowy R. J. , Ernst S. A.
Published: December 2017

Adrenergic stimulation of transmural ion transport was identified and characterized in primary cultures of avian salt gland. Adrenergic activation was mediated by beta-receptors since stimulation of the short-circuit current (Isc) was blocked by propranolol but not phentolamine. The Isc's elicited by isoproterenol, epinephrine, and norepinephrine were dose dependent, with respective EC50 values of 1.5 X 10(-8) M, 5.0 X 10(-6) M, and 1.1 X 10(-5) M. The apparent Ki for propranolol inhibition after isoproterenol stimulation was 7.5 X 10(-10) M. 8-Br cyclic AMP (8-Br cAMP) and forskolin-elicited Isc's that were insensitive to propranolol, were potentiated by theophylline, and inhibited by furosemide or ouabain. Isoproterenol also induced an increase in ouabain-sensitive respiration in acutely dispersed cells from salt-stressed juvenile or unstressed adult animals, but not in fully salt-stressed adults. The data indicate that, in addition to the well-established cholinergic receptors, beta-adrenergic receptors can control ion transport in these glands. Furthermore, the results suggest for the first time that an intracellular effector pathway involving cAMP is present.
Disorganization by calcium antagonists of actin microfilament in aortic smooth muscle cells
Authors: Sasaki Y. , Sasaki Y. , Kanno K. , Hidaka H.
Published: December 2017

To assess the physiological role of intracellular Ca2+ in the organization of actin microfilaments in smooth muscle cells, we employed several types of Ca2+ antagonists. The rabbit aortic smooth muscle cells treated with the putative intracellular Ca2+ antagonist 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB 8) at 5-100 microM showed a loss or a decrease in size and length of the actin-containing microfilament structure in a dose-dependent manner. Similar disorganization of actin structure was observed in the smooth muscle cells treated with 1-(5-isoquinolinesulfonyl)-homopiperazine (HA 1077) at 5-100 microM, which is a new type of Ca2+ antagonist different from Ca2+ entry blocker. In contrast, 100 microM verapamil and diltiazem induced no reorganization of the actin microfilament structure. Antimycin A decreased the ATP levels in smooth muscle cells and disorganized the actin-containing structure. Unlike antimycin A, TMB 8 and HA 1077 did not lower the ATP level below the threshold needed to maintain the actin filament structure. Both TMB 8 and HA 1077 directly interacted with neither the actin monomer nor F-actin in a viscometrical assay system. Thus these reagents may induce the disorganization of actin microfilament structure in smooth muscle cells through the indirect reaction(s) with the actin, suggesting that an appropriate level of ATP and Ca2+ and/or its involving reactions may be essential for maintenance of the actin structure.
Band 3 tyrosine kinase in avian erythrocyte plasma membrane is immunologically related to pp60c-src
Authors: Hillsgrove D. , Shores C. G. , Parker J. C. , Maness P. F.
Published: December 2017

We have identified in the plasma membrane of the chicken erythrocyte a 60-kDa tyrosine-specific protein kinase immunologically related to the transforming protein pp60v-src of Rous sarcoma virus. The erythrocyte protein kinase phosphorylated heavy chains of tumor-bearing rabbit (TBR) antibodies reactive with pp60c-src at tyrosine in immune complex protein kinase assays. The kinase was identified as a 60-kDa protein by [35S]methionine labeling of erythrocytes and by autophosphorylation in immune complexes. The kinase migrated on two-dimensional gel electrophoresis with an apparent pI and molecular mass similar to pp60c-src. A plasma membrane-enriched fraction isolated from chicken red cells contained the majority of the kinase activity. The kinase was solubilized from the plasma membrane by the detergents 0.5% (wt/vol) Na-deoxycholate and 1% (vol/vol) Nonidet P-40. One molar NaCl was much less effective, indicating a strong association of the kinase with the plasma membrane. Incubation of the plasma membrane fraction with [32P]ATP resulted in tyrosine phosphorylation of the anion transport protein band 3. Band 3 phosphorylation was blocked by TBR antibodies, indicating that the kinase recognized by pp60c-src antibodies was responsible for band 3 phosphorylation. These results demonstrate that the avian erythrocyte plasma membrane contains a tightly bound tyrosine-specific protein kinase identical or closely related to pp60c-src and that this kinase is responsible for band 3 phosphorylation in vitro.
Free calcium in isolated chick embryo heart cells measured using quin2 and fura-2
Authors: Jacob R. , Murphy E. , Lieberman M.
Published: December 2017

Cytosolic free calcium (Cai) was measured using quin2 and fura-2 in isolated chick embryo heart cells. Account was taken of extracellular quin2 and fura-2 (which could not be entirely washed away) by adding Mn. Shortly after loading with quin2, Cai was 49 nM (n = 7) but then rose continuously at a rate varying between 13 and 88%/h. By varying the time between cell isolation and quin2 loading, it was ascertained that the loading was causing the rise in Cai. In one set of experiments, Cai was stable in time and the apparent Cai increased steadily from 55 to 179 nM as dye loading (quin2 or fura-2) was decreased from 1 mM to 5 microM. We conclude that although quin2 and fura-2 are useful for comparing Cai levels and determining whether Cai changes as a result of certain maneuvers, they do not provide an absolute measure of Cai in isolated embryonic heart cells.
Cl(-)-selective microelectrodes: sensitivity to anionic Cl- transport inhibitors
Authors: Chao A. C. , Armstrong W. M.
Published: December 2017

Cl(-)-selective microelectrodes, containing Corning code 477315 or 477913 liquid ion exchangers, are often used to measure extra- and intracellular Cl- activities in the presence of Cl- transport inhibitors such as furosemide, bumetanide, and the stilbene sulfonic acid derivative 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS). Because these inhibitors are anions in the physiological pH range and have relatively high lipid solubilities, they would be expected to interfere with the response to Cl- of these microelectrodes. Preliminary reports have confirmed this expectation. We examined the effect of furosemide, bumetanide, and SITS on the Cl(-)-selective barrels of double-barreled microelectrodes containing Corning code 477315 liquid anion exchanger and suitable for impaling small cells (e.g., epithelial cells). The results showed that at pH 8.2 in pure solutions of furosemide and bumetanide, these microelectrodes gave linear responses to the logarithm of furosemide or bumetanide concentrations ranging from 1 X 10(-2) to 1 X 10(-4) M. In the physiological pH range both these inhibitors (in concentrations of 0.1 mM) interfered significantly with the response of the microelectrodes to Cl- (in concentrations ranging from 100 to 1 mM). Calculated electrode sensitivities, relative to Cl-, were approximately 150 for both these compounds. Microelectrodes of this type appeared to be approximately 1,000 times as sensitive toward SITS as they were toward Cl-.
Dependence of tonic tension on extracellular calcium in rat extraocular muscle
Authors: Chiarandini D. J. , Jacoby J.
Published: December 2017

Ca2+-free saline containing 3.0 mM Mg2+ virtually abolishes the tonic tension evoked by depolarization with a high K+ concentration of the tonic multiply innervated fibers of rat extraocular muscles. The tonic tension abolished by Ca2+ withdrawal is restored when Ca2+ is substituted by Sr2+ but not by Ni2+. The increase of Mg2+ reduces the tonic tension and displaces the tension-log K+ relationship to the right. Cd2+ significantly reduces the tension amplitude but does not shift the tension-log K+ relationship. The organic blocker of Ca2+ channels, nifedipine (1-10 microM), has no effect on the tonic tension. In contrast, diltiazem (20 microM) reduces the amplitude of the responses without changing the tension-log K+ relationship. Both foreign anions NO3- and SCN- potentiate tonic tension without changing the tension-log K+ relationship. SCN- increases the resting tension of the muscle; this effect depends on Ca2+. In conclusion, the disappearance of tonic tension after Ca2+ withdrawal is not due to depolarization of the fibers or inactivation of the contractile responses. It is suggested that entry of extracellular Ca2+, via a voltage-dependent Ca2+ conductance, or specific interactions of Ca2+ with membrane sites involved in the regulation of excitation-contraction coupling play a role in evoking tension in tonic fibers.
Regulation of ribosome synthesis during compensatory renal hypertrophy in mice
Authors: Ouellette A. J. , Moonka R. , Zelenetz A. D. , Malt R. A.
Published: December 2017

Ribosomal synthesis was studied at the transcriptional and translational levels to investigate the mechanisms of ribosome accretion during compensatory renal hypertrophy. As measured by in vitro transcriptional runoff comparisons 6-48 h after surgery, nuclei from the kidney remaining after contralateral nephrectomy show an increase of up to 150% in the rate of synthesis of ribosomal precursor RNA. The rate of rDNA transcription is 40-50% greater than control values as early as 6 h after nephrectomy; by 48 h, the rate returns to normal. In contrast to the stimulated transcription of rDNA and accretion of rRNA, the steady-state levels and the cytoplasmic distribution of ribosomal protein mRNAs S16 and L10 remain unchanged during induced renal growth. Thus coordinate production of adequate protein for increased assembly of ribosomes during induced renal growth appears to be accomplished by increasingly efficient translation of existing ribosomal protein mRNAs or by post-translational stabilization of ribosomal proteins. The rate of rDNA transcription may be regulated by accelerating the transcription of already functioning genes or, more likely, by recruiting transcription units that are transcriptionally inactive in the normal kidney.
Cl- secretagogues increase basolateral K+ conductance of frog corneal epithelium
Authors: Wolosin J. M. , Candia O. A.
Published: December 2017

The stromal-to-tear transport of Cl- by the corneal epithelium of the frog is increased by pharmacological effectors (secretagogues) that are known to raise the intracellular levels of cyclic AMP or Ca2+. It has been shown in the past that the Cl- secretagogues increase the apical membrane permeability to Cl- and thus facilitate the cell-to-tear flux of the anion. In this report, we combine transepithelial and microelectrode studies to show that three of these secretagogues, epinephrine, the Ca2+ ionophore A23187, and forskolin, also increase the K+ conductance of the basolateral membrane by two- to threefold. The increase in the K+ conductance is not dependent on membrane potential, since this increase occurred equally when the basolateral membrane potential either exceeded 60 mV, as measured with microelectrodes, or was depolarized by voltage clamping after apical permeabilization with amphotericin B. It is proposed that both Cl- and K+ conductances are under the control of intracellular mediators that act independently on each pathway. The increase in basolateral K+ conductance favors the Cl- secretory process.
Cardiac myocyte guanosine transport and metabolism
Authors: Geisbuhler T. P. , Johnson D. A. , Rovetto M. J.
Published: December 2017

Guanosine transport and metabolism were examined in adult rat cardiac myocytes. Myocytes transported guanosine via saturable [Km = 18 microM, maximum velocity (Vmax) = 3.61 pmol.mg-1.s-1] and nonsaturable (rate constant = 1.47 X 10(-2] processes. The saturable process was inhibited by nitrobenzyl-thioinosine, inosine [inhibition constant (Ki) = 180 microM], and adenosine (Ki = 112 microM). Extracellular guanosine taken up by myocytes was slowly phosphorylated to guanine nucleotides. The majority of guanosine (98%) existed as free intracellular guanosine after 60 s. Countertransport of nucleosides could not be demonstrated in these cells at physiological concentrations in the presence of up to a 10-fold gradient of nucleoside. These studies indicate that adult rat cardiac myocytes can be used to assess myocardial guanosine transport separate from its metabolism. Comparable inhibition of guanosine and adenosine transport by each other and by inosine support the hypothesis that guanosine and adenosine are transported by a common carrier.
Effect of plasma membrane fluidity on serotonin transport by endothelial cells
Authors: Block E. R. , Edwards D.
Published: December 2017

To evaluate the effect of plasma membrane fluidity of lung endothelial cells on serotonin transport, porcine pulmonary artery endothelial cells were incubated for 3 h with either 0.1 mM cholesterol hemisuccinate, 0.1 mM cis-vaccenic acid, or vehicle (control), after which plasma membrane fluidity and serotonin transport were measured. Fluorescence spectroscopy was used to measure fluidity in the plasma membrane. Serotonin uptake was calculated from the disappearance of [14C]-serotonin from the culture medium. Cholesterol decreased fluidity in the subpolar head group and central and midacyl side-chain regions of the plasma membrane and decreased serotonin transport, whereas cis-vaccenic acid increased fluidity in the central and midacyl side-chain regions of the plasma membrane and also increased serotonin transport. Cis-vaccenic acid had no effect on fluidity in the subpolar head group region of the plasma membrane. These results provide evidence that the physical state of the central and midacyl chains within the pulmonary artery endothelial cell plasma membrane lipid bilayer modulates transmembrane transport of serotonin by these cells.
Phenamil, an amiloride analogue, inhibits differentiation of Friend murine erythroleukemic cells
Authors: Lannigan D. A. , Bennington J. B. , Cragoe E. J. , Knauf P. A.
Published: December 2017

Amiloride has been reported to inhibit Friend murine erythroleukemic (MEL) cell commitment to differentiate by inhibiting the MEL cell plasma membrane Na+-Ca2+ antiporter (R. L. Smith, I. G. Macara, R. Levenson, D. Housman, and L. Cantley. J. Biol. Chem. 257: 773-780, 1982). We therefore screened a series of amiloride analogues to determine whether a more potent and specific inhibitor of MEL cell differentiation could be found. In our experiments, as in those of Lubin (J. Cell. Physiol. 124: 539-544, 1985), amiloride itself did not inhibit MEL cell differentiation. However, we did find that the amiloride analogue phenamil reversibly inhibits dimethyl sulfoxide (DMSO)-induced MEL cell commitment to differentiate with a K1/2 of 2.5-5.0 microM (in plasma clot assay). At an extracellular concentration of 15 microM, phenamil inhibits commitment to differentiate by approximately 90% in the plasma clot assay while having a minimal effect on growth. Phenamil is not metabolized but is rapidly taken up by MEL cells. Phenamil was most effective as an inhibitor when present during the first 12 h of DMSO treatment, indicating that phenamil affects the early commitment process rather than later steps involved in hemoglobin synthesis. Phenamil does not, however, inhibit the early differentiation-induced decrease in [Na+]i and the concomitant drop in the Na+-K+ pump rate. A specific binding site for phenamil is suggested because some analogues in which the phenamil structure is slightly modified are unable to inhibit differentiation.
Calcium diffusion through perineurium of frog sciatic nerve
Authors: Wadhwani K. C. , Levitan H. , Rapoport S. I.
Published: December 2017

Calcium and sucrose permeabilities (PCa or Psucrose) were calculated from the fluxes of 45Ca and [3H]sucrose across perfused everted and normal configurations of the perineurial cylinder isolated from the frog sciatic nerve and from fluxes into an intact nerve segment bathed in Ringer. Mean PCa for influx across the isolated perineurium equaled 10.2 +/- 0.6 X 10(-7) cm/s (n = 16) compared with Psucrose = 7.4 +/- 0.4 X 10(-7) cm/s. For efflux, PCa = 27.5 +/- 5.0 X 10(-7) cm/s and Psucrose = 23.2 +/- 4.7 X 10(-7) cm/s. The mean ratio of PCa for efflux to PCa for influx (2.7 +/- 0.5) was not significantly different from the flux ratio for sucrose (3.1 +/- 0.7). No effect on PCa or Psucrose was observed when the calcium concentration in the bath was varied from 0.5 to 20 mM, when Na-free Ringer was perfused, or when ouabain, La3+, or 2,4-dinitrophenol was applied. Asymmetrical fluxes across the perineurial cylinder were due presumably to bulk flow and resultant solvent drag out of the lumen caused by perfusion pressure. Calcium accumulated in the perineurial tissue in a saturable manner with a Km of 80 microM and a Bmax of 0.22 mumol/g wet wt. The half time for calcium exchange from the external medium to the nerve was calculated as 3 h. This long half time and the calcium-sequestering ability of the perineurium suggest that the perineurium can stabilize endoneurial calcium during transient changes in the calcium concentration of plasma.
A simple method for continuous measurement of the volume of cells in suspension
Authors: Blumenfeld J. D. , Gullans S. R. , Hebert S. C.
Published: December 2017

A simple electrical method for continuous monitoring of the volume of cells in suspension is described. The procedure consists of placing cells in a medium containing an impermeant quaternary ammonium ion, such as tetramethylammonium (TMA), which behaves as an extracellular volume marker, and monitoring the activity of this ion with a quaternary ammonium ion-specific minielectrode. Changes in the activity of the cell impermeant TMA ion in the extracellular medium quantitatively reflect movements of water into, or out of, cells and, therefore, provide a direct measure of alterations in cell volume. A direct linear relation between the fractional decrease in rat red blood cell volume and the fractional increase in medium osmolality was observed using this method. The osmotically active water volume of these red blood cells, calculated from the slope of this relationship, was 73.6 +/- 0.7% of total cell volume, a value similar to that defined by other techniques. This method should be easily adaptable for use with virtually any type of separated cell, including epithelial cells.
Protein kinase activator 1-oleoyl-2-acetyl-sn-glycerol inhibits two types of calcium currents in GH3 cells
Authors: Marchetti C. , Brown A. M.
Published: December 2017

Two types of Ca2+ currents, high-threshold, long-lasting, or L currents and low-threshold, transient, or T currents, are present in many excitable cells. L-type Ca2+ current is modulated by, among others, beta- and alpha-adrenoreceptors and intracellular Ca2+, but modulation of T-type Ca2+ current is less well established. 1-Oleoyl-2-acetyl-sn-glycerol (OAG), a synthetic activator of protein kinase C (PKC), modulates whole cell Ca2+ currents in a variety of excitable cells. Whether activators of PKC affect preferentially L and T types of Ca2+ currents is unknown. We tested OAGs effects on whole cell Ca2+ currents in the clonal GH3 line of anterior pituitary cells. The currents were measured using the whole cell patch-clamp method. Four to 60 microM OAG reversibly reduced Ca2+ currents produced by test potentials to 10 mV, and the inhibition was half maximal at approximately 25 microM. Such concentrations depress Ca2+ currents in chick embryo dorsal root ganglion (DRG) cells and clonal AtT-20 pituitary cells. To test whether OAG acted preferentially on L or T current, we separated the two using depolarizing prepulses to inactivate T current. OAG (40 microM) attenuated T currents by 60% and L currents by 50%. The current waveforms were not changed and were simply scaled, and the effects on both occurred approximately 15 s after OAG was applied. In chick embryo DRGs OAG inhibited the T current by 30% and the L current by 50%. We conclude that PKC modulates Ca2+ currents by acting on both L and T Ca2+ channels.
cAMP stimulates the Na+-K+ pump in frog retinal pigment epithelium
Authors: Hughes B. A. , Miller S. S. , Joseph D. P. , Edelman J. L.
Published: December 2017

Adenosine 3', 5'-cyclic monophosphate (cAMP) induced increases in active Na+ secretion and K+ absorption that were blocked by apical ouabain (10(-4) M), suggesting stimulation of the Na+-K+ pump. cAMP also produced rapid membrane voltage and resistance changes that could be divided chronologically into three phases. In phase 1, the basolateral membrane depolarized at a faster rate than the apical membrane, probably as a result of an increase in basolateral membrane conductance. In phase 2, the apical membrane repolarized toward control faster than the basal membrane, whereas in phase 3 the basolateral membrane repolarized faster than the apical membrane. Apical ouabain completely inhibited the cAMP-induced repolarization of the apical membrane during phase 2. Thus the stimulation of the Na+-K+ pump occurs within minutes of cAMP elevation. Na+ removal from the basal side did not block the cAMP-induced voltage changes, indicating that the initial conductance increase is not due to Na+. In contrast, Na+ removal from the apical bath inhibited all phases of the cAMP response. This suggests that apical membrane Na+-dependent transport mechanisms mediate the stimulation of the Na+-K+ pump. cAMP also caused a significant drop in intracellular K+ activity (approximately 5 mM) that preceded phase 2. This drop could stimulate the Na+-K+ pump, as suggested by previous experiments.
Smooth muscle intracellular pH: measurement, regulation, and function
Authors: Wray S.
Published: December 2017

Smooth muscle performs many functions that are essential for the normal working of the human body. Changes in pH are thought to affect many aspects of smooth muscle. Despite this, until recently little was known about either intracellular pH (pHi) values or pHi regulation in smooth muscle. Recent work measuring pHi with either microelectrodes or nuclear magnetic resonance spectroscopy is now providing some of this much needed information for smooth muscles. From these studies, it can be concluded tentatively that pHi is the same in different smooth muscles, approximately 7.06 (37 degrees C). This value is very close to those obtained in cardiac and skeletal muscle. It is clear that H+ is not in equilibrium across the smooth muscle membrane; i.e., pHi is regulated. Preliminary results in smooth muscle suggest that certain aspects of this regulation are different from that described for other muscle types. Changes in pHi have been found to produce marked effects on contraction in smooth muscle. Of particular interest is the fact that, unlike striated muscles, some smooth muscles can product more force during an intracellular acidification.
Thyroid hormone coordinately regulates Na+-K+-ATPase alpha- and beta-subunit mRNA levels in kidney
Authors: McDonough A. A. , Brown T. A. , Horowitz B. , Chiu R. , Schlotterbeck J. , Bowen J. , Schmitt C. A.
Published: December 2017

Synthesis of the sodium pump, Na+-K+-ATPase, is regulated by thyroid hormone in responsive tissues. The purpose of this study was to determine if triiodothyronine (T3) regulates the concentration of the mRNAs coding for the two enzyme subunits, alpha and beta, and the time course of the response. A single dose of T3 (250 micrograms/100 g body wt) was administered to hypothyroid rats that were killed at various times after injection. In the kidney cortexes of the T3-injected animals, as well as hypothyroid and euthyroid rats, alpha- and beta-mRNA concentrations were measured by dot blot using cDNAs corresponding to the two mRNAs; alpha-subunit abundance was measured by Western blot using antibodies to the enzyme, and Na+-K+-ATPase activity was measured enzymatically. alpha- and beta-mRNAs increased coordinately, after a 6-h time lag to 1.6-fold over hypothyroid levels by 12 h after T3. alpha-Subunit abundance increased significantly by 48 h and to 1.4-fold over hypothyroid by 72 h after T3. Na+-K+-ATPase activity increased with the same time course as the increase in alpha-subunit abundance to 1.3-fold over hypothyroid by 72 h after T3. We conclude that T3 regulates Na+-K+-ATPase synthesis and activity by coordinately increasing the mRNAs of both the alpha- and beta-subunits of the enzyme.
High-pressure pulsation of central and microvessel pulmonary artery endothelial cells
Authors: Rabinovitch M. , Bothwell T. , Mullen M. , Hayakawa B. N.
Published: December 2017

We developed an in vitro method of pulsating central and microvessel pulmonary artery endothelial cells that would allow us to study the effects of increased distending pressures over a prolonged period of time. Preservation of the contact-inhibited monolayer was assessed on phase contrast microscopy and, in addition, scanning and transmission electron microscopy (SEM, TEM) were used to determine whether there were alterations in the surface characteristics or intracytoplasmic organelles that suggested cellular damage. The cells used were obtained from Rambouillet lambs, age 3-5 days, anesthetized with halothane and ventilated. The endothelium was harvested from the central pulmonary artery (CPA) by scraping the luminal surface and from the microvessels (MPA) by infusing microcarrier beads 40-140 microns external diameter. After the second passage in culture, the cells were seeded onto the translucent, flexible polyvinylchloride membrane of a transducer dome and grown to confluence. The cell dome was then connected to a blank dome with an attached quartz transducer, to a reservoir, and to stainless steel bellows tubing, all filled with culture medium and affixed to a pulsation generator. By varying the height of the reservoir, the amplitude of excursion of the bellows tubing, and the rate, the cells could be pulsated at a given distending pressure and frequency. Confluent CPA endothelial cells from three lambs and MPA cells from two others were studied after pulsation at both 100/60 and 20/10 mmHg, 60 times/min for 48 h and after nonpulsation. On phase contrast light microscopy and on SEM, the cells remained confluent.(ABSTRACT TRUNCATED AT 250 WORDS)
Cl(-)-HCO3- exchange is present with Na+-K+-Cl- cotransport in rabbit parotid acinar basolateral membranes
Authors: Turner R. J. , George J. N.
Published: December 2017

The presence of a sodium-independent electroneutral Cl(-)-anion exchanger in a basolateral membrane vesicle preparation from the rabbit parotid is demonstrated. This exchanger is shared by HCO3-, NO3-, Br-, F-, and formate, but not by thiocyanate, acetate, methylsulfate, gluconate, or hydroxyl ions. In order of relative potency, the exchanger is inhibited by SITS greater than or equal to phloretin greater than furosemide greater than bumetanide greater than or equal to phlorizin. A Na+-K+-dependent component of chloride flux, presumably due to the Na+-K+-Cl- cotransporter already characterized in this preparation, was also observed. 36Cl uptake into vesicles loaded with KCl exhibited an "overshoot" of intravesicular [36Cl] due to 36Cl-Cl exchange. However, when vesicles were loaded with both KCl and NaCl the height of the overshoot was considerably decreased indicating a Na+-K+-dependent dissipation of the intravesicular to extravesicular chloride gradient. This experiment provides strong evidence that the Na+-K+-Cl- cotransporter and the Cl(-)-HCO3- exchanger are present in the same membrane vesicles. These results indicate that Cl(-)-HCO3- exchange is present in the basolateral membrane of parotid acinar cells and thus that this transporter may play a significant role in salivary secretion.
Na+-Ca2+ exchange in sarcolemmal vesicles from bovine superior mesenteric artery
Authors: Kahn A. M. , Allen J. C. , Shelat H.
Published: December 2017

These studies were designed to determine whether a Na+-Ca2+ exchanger is present in sarcolemmal vesicles from bovine superior mesenteric artery and, if so, to determine whether this transport system is qualitatively similar to that found in other excitable tissues. Vesicles, preferentially enriched in sarcolemma, were prepared by a Mg2+ aggregation and differential centrifugation technique. An inwardly directed Ca2+ gradient stimulated 22Na+ efflux and an outwardly directed Ca2+ gradient stimulated 22Na+ uptake. Similarly, an inwardly directed Na+ gradient stimulated 45Ca2+ efflux, and an outwardly directed Na+ gradient stimulated 45Ca2+ uptake. Ca2+ gradient-stimulated Na+ transport and Na+ gradient-stimulated Ca2+ transport were not due to voltage coupling between the two ions. Hence, a Na+-Ca2+ exchanger is present in these vesicles. The Na+ gradient-dependent component of Ca2+ uptake (Na+-Ca2+ exchange) was stimulated by rendering the vesicles electropositive inside, and Na+-Ca2+ exchange activity was inhibited by amiloride and quinidine in a dose-dependent fashion. These data demonstrate similarities between this mesenteric arterial smooth muscle Na+-Ca2+ exchanger and that found in other excitable tissues. In the absence of added Ca2+, amiloride-sensitive 22Na+ uptake in the vesicles was stimulated by an outwardly directed proton gradient, and an inwardly directed Na+ gradient stimulated proton efflux. Thus these vesicles also contain a Na+-H+ exchanger, which has been found in the sarcolemma of other vascular smooth muscle cells. When Na+ uptake was stimulated via Na+-H+ exchange, the subsequent uptake of Ca2+ via Na+-Ca2+ exchange was tripled. In conclusion, these studies unequivocally demonstrate that sarcolemmal-enriched vesicles from bovine superior mesenteric artery contain a Na+-Ca2+ exchanger.(ABSTRACT TRUNCATED AT 250 WORDS)
Lectin-gold labeling of glycoconjugates in normal and Brattleboro rat papilla: effect of vasopressin
Authors: Weyer P. , Brown D. , Orci L.
Published: December 2017

Some reports suggest that the plasma membrane glycocalyx of collecting duct epithelial cells, as well as interstitial glycoconjugates, may be involved in vasopressin action and urinary concentration. In view of this, we have used the lectin-gold technique to map and quantify Helix pomatia lectin (HPL)-binding sites in the inner medulla of kidneys from normal Long-Evans rats, vasopressin-deficient Brattleboro rats, and Brattleboro rats treated for up to 5 wk with exogenous vasopressin. The results show that the labeling of epithelial cell plasma membranes from collecting ducts and thin limbs of Henle is not different between normal and Brattleboro rats, and the labeling is not modified by chronic vasopressin treatment. In contrast, the heavy interstitial labeling seen in normal rats is virtually absent from Brattleboro rats, but it is progressively restored by chronic vasopressin treatment of Brattleboro rats. These results show that vasopressin does not modify HPL-binding glycoconjugates on epithelial cell plasma membranes, but that vasopressin treatment has a major effect on HPL-binding glycoconjugates in the medullary interstitium.
Osmotic stability of red cells in renal circulation requires rapid urea transport
Authors: Macey R. I. , Yousef L. W.
Published: December 2017

Urea transport by the human erythrocyte occurs via an asymmetric-facilitated diffusion system with high Michaelis constants and high maximal velocities; the equivalent permeability in the limit of zero urea concentration is approximately 10(-3) cm/s (J. Gen. Physiol. 81: 221-237, 239-253, 1983). A physiological role for this system is revealed by numerical integration of the appropriate equations that show that rapid urea transport is essential for red cell stability in passing through the renal medulla. The calculation compares two cells. Cell A transports urea with permeability characteristics of normal red cells; cell B has urea permeability similar to lipid bilayers. On entering the hypertonic medulla, both cells shrink, but only B swells on leaving the medulla. The osmotic stress for cell B is greater than for A. Cell B is close to hypertonic hemolysis in the medulla and to hypotonic hemolysis in the cortex. Cell B remains swollen for some time after its exit; the resulting decreased deformability presents a hazard if B reenters the microcirculation. Furthermore, cell B removes a significant fraction of the filtered load of urea and compromises the osmotic gradients in the medulla.
Electrical activity in chromaffin cells of intact mouse adrenal gland
Authors: Nassar-Gentina V. , Pollard H. B. , Rojas E.
Published: December 2017

Membrane potentials of medullary chromaffin cells of the adrenal gland of the mouse were measured in situ. Resting potential (-54.3 +/- 8.8 mV) depended on extracellular [K+] as predicted by the constant-field equation with a permeability ratio, PNa/PK, of 0.09. Current-voltage (I-V) relationships showed that the current is rectified across the chromaffin cell membrane. A rectification ratio of 0.4 was calculated from the slopes of the I-V curves for positive (41 +/- 26 M omega) and negative (103 +/- M omega) currents. Because input resistance for a resting chromaffin cell in isolation is approximately 5 G omega, the chromaffin cells in situ behave as if they were electrically coupled. Most cells responded to depolarizing current pulses with repetitive action potentials, but only 50% of them showed spontaneous electrical activity. Spontaneous activity was often seen in the presence of tetrodotoxin (3 microM). Although the application of the K+-channel blockers tetraethylammonium and Ba2+ greatly increased the amplitude of the action potentials, only Ba2+ induced continuous electrical activity. Application of acetylcholine (ACh) always depolarized the cell membrane. This effect was blocked by atropine but not by D-tubocurarine, suggesting that ACh stimulation of chromaffin cells in the mouse involves activation of muscarinic receptors.
Optical absorbance changes induced by CCK-8 under limited O2 supply in isolated perfused rat pancreas
Authors: Matsumoto T. , Kanno T.
Published: December 2017

Optical absorbance spectra were recorded simultaneously and continuously in the isolated rat pancreas arterially perfused at various flow rates. This was done to explain how optical absorbance changes corresponding to parallel reduction of cytochromes aa3, b, and cc1 were observed in perfused pancreas stimulated with high concentration of an exocrine secretagogue such as cholecystokinin-(26–33) (CCK-8). With perfusion flow rate between 1.5 and 3.0 ml/min, there were no optical absorbance changes corresponding to cytochrome reduction, but these optical absorbance changes occurred when the perfusion flow rate was decreased to 1.0 ml/min. These optical absorbance changes were not observed during exocrine secretion stimulated by CCK-8 (30 pM, 100 pM, and 1 nM) at the perfusion flow rate of 3.0 ml/min. Transient but slight changes in optical absorbances, which correspond to reduction of cytochromes, were observed in the glands perfused at the flow rate of 2.0 ml/min when secretion was stimulated by 1 nM CCK-8. When the perfusion flow rate was decreased to 1.0-1.5 ml/min, these optical absorbance changes corresponding to reduction of cytochromes occurred in glands stimulated by CCK-8 (30 pM, 100 pM, and 1 nM). Optical absorbance changes corresponding to reduction of mitochondrial cytochromes during secretion stimulated with CCK-8 may indicate local hypoxia in the perfused organ.
Muscle force and moment arm contributions to torque production in frog hindlimb
Authors: Lieber R. L. , Boakes J. L.
Published: December 2017

The relative contribution of maximum muscle tetanic tension (Po) and muscle moment arm to maximum knee flexion torque was investigated in the frog hindlimb. Isometric torque was measured in frog semitendinosus muscle-bone complexes throughout the range of 0-160 degrees of flexion. Optimal joint angle (the angle at which isometric torque was maximum) was observed at 140 degrees of flexion. After torque measurements, the muscle was excised and the muscle length-tension relationship measured for determination of Po and optimal muscle length. In addition, the kinematics of the knee joint and therefore, the muscle moment arm was measured as a function of joint angle using principles of rigid body kinematics. Stepwise linear regression indicated that maximum torque was most highly correlated with Po (r = +0.77, P less than 0.01) and accounted for approximately 75% of the measured torque. In addition, there was no significant correlation between maximum torque and maximum muscle moment arm (r = +0.11, P greater than 0.7) suggesting that muscle force, not musculoskeletal anatomy, represents the major determinant of maximum torque production in the frog hindlimb.
Liver and heart mitochondria in rats submitted to chronic hypobaric hypoxia
Authors: Costa L. E. , Boveris A. , Koch O. R. , Taquini A. C.
Published: December 2017

Mitochondrial mass was determined in the heart and liver of rats submitted to 4,400 m (simulated altitude) for 9 mo and their controls at sea level. This was done 1) by evaluation of isolated mitochondrial protein per gram of tissue, 2) by evaluation of the ratio between cytochrome oxidase activity in tissue homogenate and in isolated mitochondria, and 3) by evaluation of mitochondrial numerical and volume density in fixed tissues analyzed by electron microscopy. An increase in mitochondrial mass and a more homogeneous distribution of mitochondria were found in liver. In cardiac tissue an increase in numerical density of mitochondria accompanied by a slight decrease in their mean volume was observed. Maximal physiological rate of mitochondrial respiration (state 3, active respiration), resting respiration, ADP/O, and acceptor control ratio were determined in the isolated mitochondria. No differences were found in the intrinsic properties of mitochondria. The results suggest that chronic mild hypoxia promotes tissue adaptation by increasing the mitochondrial mass or number in liver and heart, respectively, and improves intracellular O2 diffusion by adopting a more homogeneous intracellular distribution of mitochondria in the liver.