bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular physiology of olfaction
Authors: Anholt R. R.
Published: December 2017

Olfactory reception is mediated by olfactory receptor cells located in the olfactory epithelium. These cells are bipolar neurons that extend a dendrite toward the nasal lumen and an axon toward the olfactory bulb in the brain. The dendrite possesses a group of apical cilia embedded in mucus. Odorant recognition and signal transduction are initiated at the membranes of these chemosensory cilia and culminate in excitation of the olfactory receptor cell. Differential activation by odorants of distinct groups of olfactory receptor cells generates patterns of neuronal activity that encode odor quality and concentration. The identities of primary odorant recognition sites at the ciliary membrane remain to be established. However, a significant body of information has become available with respect to olfactory transduction mechanisms. It is now becoming clear that olfactory transduction involves the interplay of several second messenger systems to control the responses of these exquisitely sensitive chemosensory neurons.
Linear dependence of muscle phosphocreatine kinetics on total creatine content
Authors: Meyer R. A.
Published: December 2017

Phosphorus nuclear magnetic resonance (NMR) spectra and twitch tension were recorded during stimulation of gastrocnemius muscles of pentobarbital sodium-anesthetized rats which had been fed the creatine analogue beta-guanidinopropionic acid (beta-GPA, 2% diet) for periods from 0 (control) to 8 wk. Total creatine content of unstimulated muscles decreased by 42, 67, 82, and 88% compared with controls after 2-, 4-, 6-, and 8-wk feeding, respectively. The staircase effect observed in control muscles during 8 min of twitch stimulation at 0.25, 0.5, and 0.75 Hz was reduced after 2- to 8-wk beta-GPA feeding. However, after 6- to 8-wk feeding, the twitch force at the end of 8 min of stimulation was not different from controls. The time constant for phosphocreatine (PCr) changes at the onset of and during recovery after stimulation was proportional to total creatine content. The relationship between PCr content and twitch rate times force at the end of stimulation was linear, with slope proportional to total creatine content. PCr content in beta-GPA-fed animals was transiently greater during recovery than before stimulation, suggesting a regulatory effect of the inorganic phosphate released by hydrolysis of phosphorylated beta-GPA. The results are consistent with linear models of respiratory control in which the creatine kinase reaction acts as a simple buffer of adenine nucleotide levels.
Cytotoxic properties of salivary oxidants
Authors: Grisham M. B. , Ryan E. M.
Published: December 2017

Salivary peroxidase and to a lesser extent myeloperoxidase are present in significant concentrations in saliva and catalyze the oxidation of thiocyanate anion (SCN-) by H2O2 to yield the potent oxidants hypothiocyanous acid (HOSCN) and its conjugate base hypothiocyanite anion (OSCN-). The objective of this study was to characterize the cytotoxic potential of peroxidase-generated HOSCN/OSCN- toward human erythrocytes. We found that HOSCN/OSCN- (0.25 mM) generated by the peroxidase-H2O2-SCN- system caused significant hemolysis at pH 6.0 but not at pH 6.5, 7.0, or 7.4. Erythrocyte hemoglobin (OxyHb) was oxidized to methemoglobin (MetHb) at all pH values tested; however, the rate of MetHb formation was dramatically increased at low pH and was not affected by inosine hexaphosphate, suggesting that hemoglobin was oxidized primarily by HOSCN. Concurrent with oxidation of hemoglobin (Hb), there was a pH-dependent consumption of HOSCN/OSCN- with more of the oxidant consumed at pH 6.0 compared with pH 6.5, 7.0, or 7.4. The enhanced oxidation of Hb at acidic pH was not due simply to increased membrane permeability by the uncharged species (HOSCN), since both erythrocyte lysate Hb and purified Hb were oxidized to the same extent at low pH as were intact erythrocytes. It is concluded that both OSCN- and HOSCN enter human erythrocytes where the protonated oxidant (HOSCN) mediates hemolysis and oxidizes OxyHb to MetHb, whereas both HOSCN and OSCN- oxidize glutathione (GSH). These data suggest that the extracellular pH may play an important role in modulating the cytotoxic properties of salivary oxidants.
Effects of NaCl, glucose, and aldose reductase inhibitors on cloning efficiency of renal medullary cells
Authors: Yancey P. H. , Burg M. B. , Bagnasco S. M.
Published: December 2017

To analyze the effects of sorbitol accumulation on the survival and growth of epithelial cells from rabbit renal inner medulla, cloning efficiency (an index of cell viability) was measured at normal and high glucose and NaCl concentrations and when sorbitol accumulation was prevented by Tolrestat and Sorbinil, which inhibit aldose reductase. With PAP-HT25 cells grown to near confluence, high NaCl increases aldose reductase activity, causing enough rise in cell sorbitol concentration to balance most of the increased osmolality of the high extracellular NaCl. Inhibition of aldose reductase prevents both the increased enzyme activity and sorbitol accumulation in a dose-related manner. Paralleling this, colony-forming efficiency is not affected by the inhibitors at a normal NaCl concentration but is greatly reduced when extracellular NaCl is high. On the other hand, high glucose levels, as occur in diabetes, increase sorbitol content well above the concentration required for osmotic balance and inhibit colony-forming efficiency. Under those conditions, aldose reductase inhibitors lower cell sorbitol and reverse (at 300-350 mosmol/kgH2O) or reduce (at 500-550 mosmol/kgH2O) the decrease in colony-forming efficiency caused by high glucose. Thus sorbitol accumulation is necessary for osmoregulation when induced by high osmolality but is harmful when induced by high glucose.
Intracellular pH measurement using single excitation-dual emission fluorescence ratios
Authors: Bassnett S. , Reinisch L. , Beebe D. C.
Published: December 2017

In the present paper, laser spectroscopy was used to evaluate the utility of a new fluorochrome, carboxyseminaphthorhodafluor-1 (Snarf-1), for single excitation-dual emission ratio measurement of intracellular pH (pHi). The emission spectrum of Snarf-1 showed clear pH-dependent shifts, and emission ratios calculated from the 640 and 587 nm maxima were a sensitive indicator of pH. When irradiated in Cunningham chambers, solutions of Snarf-1 were rapidly bleached, and at pH 7.3 or higher, this bleaching led to a decrease in the 640/587 nm emission ratio. These ratio changes were also observed in intracellular measurements on lens embryonic epithelial cells under conditions in which the entrapped dye was rapidly bleached. As the laser dosage was reduced (by increasing the step size between sample points), bleaching could be reduced to very low levels, and under these conditions, the ratio remained constant. Snarf-1 loaded into lens epithelial explants was calibrated intracellularly using nigericin. Intracellular calibration curves were shifted to more alkaline values than in vitro curves. Intracellular calibration allowed estimates of pHi that were in reasonable agreement with previously published values for lens tissue. Potential artifacts arising from differential photobleaching and intracellular-in vitro calibration are discussed.
Multiple calcium mobilization pathways in single avian salt gland cells
Authors: Stuenkel E. L. , Ernst S. A.
Published: December 2017

Agonist-induced changes in intracellular Ca2+ concentration ([Ca2+]i) in individual secretory cells from the avian salt gland were detailed using dual-wavelength microspectrofluorimetry of the Ca2(+)-sensitive fluorescent probe fura-2. Resting [Ca2+]i averaged 42 +/- 5 nM. Stimulation with the cholinergic agonist carbachol (1 microM) resulted in a rapid increase in [Ca2+]i to 308 +/- 26 nM, which was sustained at a nearly constant elevated level (328 +/- 31 nM) throughout agonist application. In the absence of extracellular Ca2+ or in the presence of an inorganic blocker of Ca2+ entry (Ni2+, 1 mM), only a transient increase in [Ca2+]i occurred on agonist stimulation, whereas subsequent readmission of Ca2+ or washout of Ni2+ reinitiated a sustained increase in [Ca2+]i. The initial transient response results from Ca2+ release from intracellular stores, whereas the sustained phase represents entry of extracellular Ca2+ into the cytoplasm. Repetitive stimulations in Ca2(+)-free medium alternating with Ca2(+)-containing medium were performed to examine the mechanisms involved in refilling of the agonist-sensitive intracellular pool. After depletion of the intracellular pool by stimulation in Ca2(+)-free medium, removal of the agonist and readmission of Ca2+ resulted in a rapid transient increase in [Ca2+]i that could be blocked by Ni2+, La3+, or elevated K+. Subsequent removal of extracellular Ca2+ and restimulation nonetheless showed that complete refilling of the intracellular pool had occurred in each case. These results suggest that two separate Ca2(+)-entry mechanisms, one sensitive to Ni2+, La3+, and elevated K+ and responsible for the agonist-induced increase in [Ca2+]i and one insensitive to the blockers and involved in refilling of the intracellular pool, may exist in salt gland cells. Spontaneous oscillations of [Ca2+]i that are independent of extracellular Ca2+ have also been observed in 10% of the cells. The abolition of the oscillations by depletion of the agonist-sensitive pool suggests this pool as the Ca2+ source for the oscillations.
Smooth muscle energetics and theories of cross-bridge regulation
Authors: Paul R. J.
Published: December 2017

The energetics of smooth muscle is characterized by low tension cost (rate of ATP utilization per isometric force/cross-section area), ranging from 100- to 500-fold less than skeletal muscle. The efficiency (ATP usage per work) of smooth muscle, although less well documented, is also somewhat (4-fold) less than skeletal muscle. Another well-known characteristic of smooth muscle is the linear relation between the steady-state of ATP utilization (JATP) and isometric force. Recently, Murphy and colleagues [C.-M. Hai and R. A. Murphy. Am. J. Physiol. 254 (Cell Physiol. 23) C99-C106, 1988] have put forth a kinetic model of cross-bridge regulation that predicts the time course of stress and myosin light chain phosphorylation (MLC-Pi). The energetics consequences of this model, in brief, are that the low tension cost is partly attributed to a slow detachment rate of the myosin cross bridge when dephosphorylated when attached to actin ("latch state"), whereas the lower efficiency is ascribed to a high rate of myosin phosphorylation-dephosphorylation inherent to a fit of data to this kinetic scheme. This latter corollary is somewhat controversial in light of current interpretations of smooth muscle energetics data. Using SCoP software (National Biomedical Simulation Resource, Duke University), we tested this model in terms of fitting existing data with respect to 1) is a high myosin-dephosphorylation adenosine triphosphatase (ATPase) necessary to fit the available data on the time course of stress and MLC-Pi?; and 2) can this model predict the observed linear relation between the steady-state rate of ATP hydrolysis (JATP) and isometric force?(ABSTRACT TRUNCATED AT 250 WORDS)
Growth factor activity of endothelin on vascular smooth muscle
Authors: Bobik A. , Grooms A. , Millar J. A. , Mitchell A. , Grinpukel S.
Published: December 2017

Endothelin is a novel peptide secreted by endothelial cells, the vasoconstrictor effects of which appear dependent on the activation of phospholipase C. We examined in tissue culture its potential as a growth factor for vascular smooth muscle. In quiescent cultures of rat aortic smooth muscle cells, endothelin rapidly elevated levels of c-fos and c-myc mRNA. Peak effects on c-fos mRNA occurred between 15 and 30 min and were completely gone after 2 h. The elevation in c-fos mRNA was, in part, dependent on protein kinase C, since phorbol myristate acetate (PMA) also elevated c-fos mRNA and further increased c-fos mRNA expression by endothelin, but the effects were not additive. Furthermore, the endothelin-induced elevation in c-fos mRNA was attenuated but not abolished in protein kinase C-depleted cells. Maximum levels of c-myc mRNA occurred between 15 and 30 min after exposing the cells to endothelin and persisted for at least 6 h. The effects of simultaneous addition of endothelin and PMA on c-myc mRNA levels were essentially similar to those observed with c-fos mRNA. [3H]thymidine incorporation into DNA occurred 8 h after exposing the cells to endothelin. The mitogenic effect of endothelin was smaller than that observed with either fetal calf serum or epidermal growth factor and was dependent on both pertussis toxin-insensitive and -sensitive pathways. Sensitivity to the latter pathway did not appear dependent on attenuation of phospholipase C activity, since neither peak intracellular calcium concentrations nor c-fos mRNA levels were reduced in pertussis toxin-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Expression of Na(+)-H+ exchange and ATP-dependent proton extrusion in growing rat IMCD cells
Authors: Stanton R. C. , Boxer D. C. , Seifter J. L.
Published: December 2017

As the last step of urinary acidification, the inner medullary collecting duct (IMCD) is thought to secrete protons into the tubular lumens by means of a H(+)-translocating adenosinetriphosphatase (H(+)-ATPase). However, recent studies have also shown the existence of Na(+)-H+ exchange activity in IMCD cells. Although the physiological function of the antiporter in IMCD cells is unknown, activation of Na(+)-H+ exchange in other cell-culture systems has been suggested to be closely associated with the process of cell growth. Thus presence of Na(+)-H+ exchange may relate to the growth phase of these cells. To examine intracellular pH (pHi) regulation in growing IMCD cells, we studied proton transport by Na(+)-dependent and Na(+)-independent mechanisms by microfluorimetry using the pHi-sensitive dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester (BCECF/AM). Actively growing cells, defined by [3H]thymidine incorporations, demonstrated an amiloride-sensitive Na(+)-dependent pHi recovery after an acid load. No pHi recovery was evident in the absence of Na+, indicating the importance of Na(+)-H+ exchange for pHi recovery. However, when evaluated in quiescent cells, Na(+)-dependent pHi recovery appeared to be diminished. Instead, a Na(+)-independent pHi recovery which was inhibitable by ATP depletion and by 1 mM N-ethylmaleimide was present, suggesting function of a H(+)-ATPase. These findings indicate that Na(+)-dependent proton extrusion activity (Na(+)-H+ exchange) but not Na(+)-independent proton extrusion activity is expressed during the rapid growth phase of IMCD cells, whereas the more quiescent cells express Na(+)-independent ATP-dependent proton extrusion activity and a possibly less active Na(+)-H+ exchanger.
Increased de novo purine synthesis by insulin through selective enzyme induction in primary cultured rat hepatocytes
Authors: Tsuchiya M. , Yoshikawa H. , Itakura M. , Yamashita K.
Published: December 2017

The proliferative effect of insulin on de novo purine synthesis and on the expression of various enzymes of purine metabolism were studied in primary cultured rat hepatocytes. Insulin greater than 1.5 x 10(-8) M increased DNA and de novo purine synthesis to 260-390 and 270-420%, respectively, 24 and 8 h after the administration. Insulin at 1.5 x 10(-7) M increased the specific activity of amidophosphoribosyltransferase (ATase) to 154-180%, hypoxanthine-guanine phosphoribosyltransferase to 129%, and adenine phosphoribosyltransferase (APRT) to 205%, in contrast to unchanged xanthine dehydrogenase at 80%. Enzyme induction was supported by the results of kinetic analysis and the inhibition of the insulin-induced increase in enzyme activities by protein synthesis inhibitors. Insulin increased ATP to 127% and decreased AMP, ADP, 5'-guanylic acid (GMP), and guanosine 5'-diphosphate (GDP), respectively, to 73, 69, 73, and 69%. Insulin increased adenylate energy charge from 0.83 to 0.90 without changing total feedback inhibitory potential on ATase. No obvious increase of 5-phosphoribosyl-1-pyrophosphate supply was suggested, although its apparent availability for purine ribonucleotide synthesis was increased to 208-245%, reflecting mainly induced APRT activity to 205%. It is concluded that hepatocyte proliferation by insulin, as evidenced by purine metabolism, is mediated by the selective gene activation of anabolic enzymes and increased ATP as the basis to activate multiple metabolic pathways without remarkable changes of substrate availability or feedback inhibition.
Voltage-activated cation permeability in high-potassium but not low-potassium red blood cells
Authors: Halperin J. A. , Brugnara C. , Van Ha T. , Tosteson D. C.
Published: December 2017

We have recently reported that voltage-activated fluxes of Na, K, and Ca occur in human red blood cells [J.A. Halperin, C. Brugnara, M. Tosteson, T. Van Ha, and D. C. Tosteson. Am. J. Physiol. 257 (Cell Physiol. 26): C986-C996, 1989]. The cation permeability increases progressively as the membrane potential becomes more inside positive above +20 mV. In this paper we show that this effect also occurs in high-potassium (HK), but not in low-potassium (LK), sheep and dog red blood cells. This result suggests that the voltage-activated cation transport pathway is not the result of nonspecific dielectric breakdown of the lipid bilayer but, rather, relates to some membrane component, presumably a protein, that is expressed in HK human and sheep but not in LK sheep and dog red blood cells.
Na(+)-H+ and Na(+)-dependent Cl(-)-HCO3- exchange control pHi in vascular smooth muscle
Authors: Kahn A. M. , Cragoe E. J. , Allen J. C. , Halligan R. D. , Shelat H.
Published: December 2017

The mechanisms that control intracellular pH (pHi) in vascular smooth muscle are not fully understood. These studies were performed to determine the identity and relative importance of the sarcolemmal transport systems that mediate net acid efflux in primary cultured vascular smooth muscle cells from canine femoral artery. In HEPES- or HCO3(-)-buffered physiological salt solution (HEPES-PSS, HCO3(-)-PSS), recovery from an acute acid load was totally dependent on external Na+. 5-[N-ethyl-N-isopropyl]amiloride (EIPA, 50 microM) inhibited pHi recovery 100 and 68% in HEPES-PSS and HCO3(-)-PSS, respectively. EIPA-insensitive pHi recovery in HCO3(-)-PSS was inhibited 48% by 4,4'-diisothyocyanostilbene-2,2'-disulfonic acid (DIDS). An outwardly directed H+ gradient stimulated amiloride-sensitive 22Na+ uptake, and an inwardly directed HCO3- gradient stimulated amiloride-insensitive 22Na+ uptake. The latter was inhibited by DIDS or prior depletion of cell Cl-. In HEPES-PSS, resting pHi was 7.17 +/- 0.03, was not affected by DIDS, but was lowered by EIPA or by removing extracellular Na+. In HCO3(-)-PSS, resting pHi was 7.25 +/- 0.02 (P less than 0.05) and was not affected by EIPA. Removing extracellular Na+ in the presence of EIPA decreased pHi in HCO3(-)-PSS but not in HEPES-PSS. DIDS lowered resting pHi in HCO3(-)-PSS, after which EIPA further lowered pHi. We conclude that acid efflux from these cells is mediated by a Na(+)-H+ exchanger and a Na(+)-dependent Cl(-)-HCO3- exchanger. In HEPES-PSS, acid efflux via the Na(+)-H+ exchanger maintains resting pHi. In HCO3(-)-PSS, additional acid efflux via the Na(+)-dependent Cl(-)-HCO3- exchanger results in a higher pHi. Although the Na(+)-H+ exchanger is primarily responsible for acid efflux after an acute acid load, the Na(+)-dependent Cl(-)-HCO3- exchanger is responsible for acid efflux under physiological conditions.
Muscle fiber formation and fiber hypertrophy during the onset of stretch-overload
Authors: Alway S. E. , Gonyea W. J. , Davis M. E.
Published: December 2017

The contributions of fiber hypertrophy and new fiber formation to the onset of stretch-induced muscle enlargement were evaluated in the anterior latissimus dorsi (ALD) of adult Japanese quails, because it was not known whether the mechanisms which initiate new fiber formation were dependent on first achieving significant fiber hypertrophy. A weight corresponding to 10% of the bird's body mass was attached to one wing, and eight birds were killed after each day during the first week of stretch. Muscle mass was significantly increased after 48 h of stretch; however, the elevation in nonmuscle tissue accounted for this increase. Muscle mass corrected for non-muscle tissue was significantly greater than the intra-animal control by the fourth day of stretch. Mean fiber cross-sectional area did not change during days 0-6, but cross-sectional area was 30.0 +/- 17.2% greater than the intra-animal control areas at day 7. Fiber number determined after nitric acid digestion of connective tissue was 27.1 +/- 5.8% greater than the intra-animal control at days 5-7 of stretch, but the number of fibers in the control muscles at days 5 and 6 were lower than at day 0. Thus new fiber formation was not preceded by significant fiber hypertrophy. These results fail to support a mechanism for new fiber formation which involves fiber splitting from hypertrophied myofibers during the first week of stretch.
Energy dependence of sodium-calcium exchange in vascular smooth muscle cells
Authors: Smith J. B. , Smith L.
Published: December 2017

Three different types of mitochondrial poisons (oligomycin, antimycin A, and dinitrophenol) strongly inhibited Na(+)-Ca2+ exchange in aortic myocytes. Exchange activity was assayed as 45Ca2+ uptake that depended on inverting the Na+ gradient and was inhibited by 25 microM dimethylbenzamil. Glucose markedly decreased the inhibition of exchange activity by these three poisons. Glucose also prevented rotenone from inhibiting exchange and depleting cellular ATP. In the absence of glucose, rotenone decreased ATP and exchange activity with half-times of 0.8 and 0.9 min, respectively. Almost eliminating cellular ATP with rotenone maximally inhibited exchange by 80%. Repletion of ATP with glucose substantially restored Na(+)-Ca2+ exchange activity. Ca2+ uptake by organelles, subsequent to entry via exchange for Na+, does not appear to contribute significantly to exchange activity as assayed in intact myocytes. The specific activity of Na(+)-Ca2+ exchange was approximately 30 nmol.min-1.mg protein-1. These findings suggest that ATP modulates exchange activity and that there are approximately 150,000 Na(+)-Ca2+ exchangers per cell, assuming that the turnover number is 1,000 s-1.
Internalization of fluorescent vasotocin-receptor agonist and antagonist in the toad bladder
Authors: Eggena P. , Lu M. , Buku A.
Published: December 2017

This study compares hydrosmotic action, receptor binding, and fluorescent uptake of an agonist, d9phe(flu)AVT, and an antagonist, d4lys(flu)AVT, in the toad bladder. D9phe(flu)AVT increased osmotic water flow across the bladder with a 50% effective dose of 2 nM, whereas d4lys(flu)AVT inhibited water flow with a 50% effective dose of 0.1 microM. D9phe(flu)aVT displaced 10 nM [3H]arginine vasopressin (AVP) from plasma membranes by 50% (IC50) with 10 nM, whereas d4lys(flu)AVT had an IC50 of 3 microM. The fluorescent agonist induced a persistent increase in membrane permeability to water after removal from the serosal bathing solution. This residual response was diminished by preincubation with an agonist (AVP), but not with an antagonist [d4lys(N3)AVT]. The agonist, d9phe(flu)AVT, was internalized into toad bladder epithelial cells, as seen by epifluorescence microscopy, and this uptake was blocked by d4lys(N3)AVT. The antagonist, by contrast, was not internalized but remained at the cell surface. After stimulation with forskolin, however, the fluorescent antagonist was also internalized. These experiments suggest that agonists, but not antagonists, form functional complexes with receptors that, via formation of cAMP, trigger not only an increase in membrane permeability to water but also facilitate the clearance of hormone from the cell surface by endocytic uptake.
Gadolinium blocks low- and high-threshold calcium currents in pituitary cells
Authors: Biagi B. A. , Enyeart J. J.
Published: December 2017

The inhibition of L- and T-type Ca2+ currents by Gd3+ was studied in the rat pituitary GH4C1 cell line. In whole cell patch recordings, Gd3+ at concentrations of 50 nM to 5 microM blocked Ca2+ current through L-type channels. Block was promoted by prolonged channel activation. With 4.5-s test pulses to + 10 mV, Gd3+ at concentrations as low as 200 nM produced near-complete block of L current. At higher Gd3+ concentrations (5 microM), complete block occurred with short test pulses and appeared to be independent of channel activation. Gd3+ also blocked current through low-threshold T channels in GH4C1 cells. Two other trivalent elements, La3+ and Y3+, blocked L-type Ca2+ channels in GH4C1 cells with potency similar to Gd3+. These results indicate that these trivalent cations are effective nonselective inhibitors of both low- and high-threshold Ca2+ channels in endocrine cells. In this regard, they are among the most potent inorganic Ca2+ antagonists yet discovered.
Cholinergic modulation of apical Na+ channels in turtle colon: analysis of CDPC-induced fluctuations
Authors: Wilkinson D. J. , Dawson D. C.
Published: December 2017

Current fluctuation analysis was used to investigate the properties of apical Na+ channels during muscarinic inhibition of active Na+ absorption. A reversible Na+ channel blocker, 6-chloro-3,5-diaminopyrazine-2-carboxamide (CDPC), was used to induce fluctuations in the short-circuit current (I(sc)). Power density spectra of the CDPC-induced fluctuations exhibited a clearly discernible Lorentzian component, characterized by a corner frequency that was linearly related to CDPC concentration between 20 and 100 microM. The on (k'on) and off (k(off)) rate coefficients for the CDPC blocking reaction were k'on = 11.1 +/- 0.8 rad.s-1.microM-1 and k(off) = 744 +/- 53 rad/s, and the microscopic inhibition constant was 67 microM (n = 11). CDPC blocking kinetics were not significantly different after inhibition of Isc by 5 microM serosal carbachol. Single-channel Na+ current (iNa) and the density of open and blocked Na+ channels (N(ob)) were estimated from the fluctuations induced by 40 microM CDPC. Under control conditions, iNa was 0.43 +/- 0.05 pA and N(ob) was 251 +/- 42 X 10(6)/cm2 (n = 10). After exposure to serosal carbachol (2-10 microM) for 60 min, Na+ current and N(ob) were reduced by approximately 50%, but iNa was not changed significantly. These results indicate that muscarinic inhibition of electrogenic Na+ absorption was associated with a reduction in the number of open Na+ channels in the apical membrane. They also suggest that this downregulation of transport involved a coordinated decrease in both apical and basolateral membrane conductances.
Platelet-activating factor and related acetylated lipids as potent biologically active cellular mediators
Authors: Snyder F.
Published: December 2017

Platelet-activating factor (PAF or 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is the most potent lipid mediator yet discovered. It is known to stimulate a wide span of biological responses ranging from aggregation and degranulation of platelets and neutrophils to a variety of cellular effects involving the stimulation of chemotaxis; chemokinesis; superoxide formation; protein phosphorylation; activation of protein kinase C, arachidonic acid, and phosphoinositide metabolites; glycogenolysis; and tumor necrosis factor production. Obviously, with such a diversity of biological activities, it is not surprising that PAF has been considered to be a key component in numerous diseases related to hypersensitivity and inflammatory responses. Evidence has also been presented for the role of PAF in physiological processes, particularly those involving reproduction and fetal development. Furthermore, because of its potent hypotensive action, PAF has been implicated as a contributing factor in blood pressure regulation. PAF is produced by two independent enzymatic pathways. The remodeling route involves the structural modification of a membrane lipid (1-alkyl-2-acyl-sn-glycero-3-phosphocholine) by replacement of the acyl moiety with an acetate group. An alternate route is the de novo synthesis of PAF from an O-alkyl analogue of a lysophosphatidic acid that requires a reaction sequence of acetylation, dephosphorylation, and phosphocholine addition steps. Hypersensitivity and other pathophysiological reactions are thought to be caused by activation of the remodeling pathway, whereas the de novo route is believed to be the source of endogenous levels of PAF required for physiological functions. Inactivation of PAF occurs when the acetate group is hydrolyzed by an acetylhydrolase that is present in both extra- and intracellular compartments, although the catalytic activity of the two forms of acetylhydrolase are identical, some of their properties differ. The control of PAF metabolism is very complex, but acetylhydrolase, Ca2+, phosphorylation/dephosphorylation of enzymes, and fatty acids (especially polyunsaturates) appear to be important regulatory factors. Specific PAF receptors have clearly been demonstrated on several different types of cells, and although the mechanism of PAF actions is poorly understood, it appears that the PAF/receptor-induced responses are closely associated with the signal transduction process; both G proteins and adenyl cyclase appear to be involved. Because significant quantities of PAF are often retained within certain cells, the possibility of PAF serving as an intracellular mediator has also been proposed.
Heat induces intracellular acidification in human A-431 cells: role of Na(+)-H+ exchange and metabolism
Authors: Kiang J. G. , McKinney L. C. , Gallin E. K.
Published: December 2017

The resting intracellular pH (pHi) of A-431 cells at 37 degrees C in Na+ Hanks' solution is 7.23 +/- 0.02. In the presence of amiloride (100 microM) pHi decreases to 7.08 +/- 0.03. Hyperthermia induces a temperature- and time-dependent intracellular acidification of 0.2 pH units in either bicarbonate-free or bicarbonate-buffered solutions. After heat treatment (45 degrees C, 10 min) pHi returns to normal 1 h after incubation at 37 degrees C. The activity of the Na(+)-H+ exchanger was examined in heated and unheated cells in the absence of bicarbonate. Unheated cells recover from an acid load in a [Na+]o-dependent and amiloride-sensitive manner. The apparent Michaelis constant for extracellular Na+ is 38 +/- 9 mM, and the apparent mean affinity constant for amiloride is 11 +/- 3 microM. In heated cells the apparent affinity of the Na(+)-H+ exchanger for extracellular Na+ is not changed, but the maximal recovery rate is approximately 40% slower than that of unheated cells. The rate of recovery from acid loading returns to normal 2 h after heat treatment. [Na+]i and intrinsic buffering power in heated cells are the same as those in unheated cells. Decreases in both intracellular ATP and lactic acid are observed in heated cells. 2-Deoxy-D-glucose and sodium azide induce an intracellular acidification but prevent most of the acidification induced by heat. Heat treatment causes no further acidification in cells that are acidified by both amiloride and 2-deoxy-D-glucose together. These data are the first to suggest that thermally induced intracellular acidification is due to both an inhibition of Na(+)-H+ exchange and an inhibition of metabolic pathways.
Tricarboxylic acid cycle intermediates in human muscle during prolonged exercise
Authors: Sahlin K. , Katz A. , Broberg S.
Published: December 2017

Seven subjects cycled to fatigue [75 +/- 5 (SE) min] at a work load corresponding to approximately 75% of their maximal oxygen uptake. Biopsies were taken from the quadriceps femoris muscle at rest and during exercise. Muscle glycogen decreased from a preexercise level of 445 +/- 33 mmol glucosyl units/kg dry wt to 50 +/- 14 at fatigue. The sum of the measured tricarboxylic acid cycle intermediates (TCAI = malate + citrate + fumarate + oxaloacetate) was 0.49 +/- 0.05 mmol/kg dry wt at rest, increased to 4.41 +/- 0.23 after 5 min of exercise, and then decreased continuously to 3.33 +/- 0.29 and to 2.83 +/- 0.27 mmol/kg dry wt after 40 min of exercise and at fatigue (P less than 0.05 vs. 5 min), respectively. The point of fatigue was characterized by an enhanced deamination of AMP (judged by increase in IMP) and reduced contents (vs. 5 min of exercise) of lactate, pyruvate, and alanine. In contrast, acetylcarnitine (reflects the availability of acetylunits) increased threefold at the onset of exercise and was maintained approximately at this level until fatigue. It is concluded that prolonged exercise to fatigue at moderate work loads results in glycogen depletion, energy deficiency (increased AMP deamination), reduced levels of three-carbon compounds and TCAI (compared with the initial phase of exercise) but in maintained levels of acetylunits. The present data indicate that carbohydrate depletion may impair aerobic energy production by reducing the level of TCAI.
Neuromuscular adaptations to cross-reinnervation in 12- and 29-mo-old Fischer 344 rats
Authors: Clark K. I. , White T. P.
Published: December 2017

The aim was to test hypotheses regarding the adaptive response of the extensor digitorum longus (EDL) muscle of 12- and 29-mo rats following denervation and cross-reinnervation by the soleus nerve. The mass of cross-reinnervated EDL muscle was 87 and 86% of self-reinnervated control values in 12-mo (99 +/- 3 mg) and 29-mo (74 +/- 3 mg) rats, respectively. Cross-reinnervated EDL fiber area was 56 and 67% of self-reinnervated values in 12-mo (1,733 +/- 253 microns 2) and 29-mo (1,264 +/- 71 microns 2) rats, respectively. Cross-reinnervation increased the density of neural contact 26% in 12-mo rats and decreased density by 50% in 29-mo animals. In 12-mo rats 17% of motor end plates (MEP) were void of terminal nerves following cross-reinnervation compared with 48% in 29-mo rats. In cross-reinnervated muscles, slow myosin heavy chain (MHC) was 65 +/- 9 and 25 +/- 3% of total MHC in 12- and 29-mo rats, respectively. The percentage of type I fibers derived histochemically was 65 +/- 8% in 12-mo rats and 18 +/- 1% in 29-mo rats. In conclusion, there is an age-associated decrease in the ability of neurons to reinnervate the MEP area after nerve section. The conversion of fiber type in innervated fibers in response to cross-reinnervation may not differ due to age.
Immunolocalization of gluco- and mineralocorticoid receptors in rabbit kidney
Authors: Farman N. , Oblin M. E. , Lombes M. , Delahaye F. , Westphal H. M. , Bonvalet J. P. , Gasc J. M.
Published: December 2017

The localization of glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) was determined in the rabbit kidney by immunohistochemistry with the use of a monoclonal, anti-GR antibody and a monoclonal, anti-idiotypic, anti-MR antibody. Immunostaining was performed on serial histological sections from normal and adrenalectomized rabbits. The specificity of immunostaining was assessed for MR by in situ competition studies with steroids and for GR by presaturation of the antibody with GR preparation. Immunostaining by both the anti-MR and the anti-GR antibodies was present in all parts of the distal nephron (beyond proximal tubule) and absent in the glomerulus and proximal tubule. The absence of staining by the anti-GR antibody in the proximal tubule suggests that the effects of glucocorticoids in this structure involve either a GR different from that of distal structures or a non-receptor mediated mechanism of action. MR immunostaining predominates in the distal and all along the collecting tubule in its cortical, medullary, and papillary portions. GR immunostaining was most abundant in the medullary ascending limb and distal tubule. Immunostaining by both antibodies was present in papillary interstitial cells and cells of the epithelium lining the papilla. Fifteen to twenty percent of the cells of the cortical collecting tubule, presumably intercalated cells, were devoid of MR and GR immunostaining. Immunostaining was present in both nuclear and cytoplasmic cell compartments. No clear difference was observed between normal and adrenalectomized rabbits. This study is the first report on renal immunolocalization of GR compared with MR. In addition, we show evidence for new targets for corticosteroid hormones such as papillary interstitial cells and papillary epithelium.
Evaluation of the electroinjection method for introducing proteins into living cells
Authors: Wilson A. K. , Horwitz J. , De Lanerolle P.
Published: December 2017

The introduction of impermeant probes such as antibodies and other proteins into living cells without compromising physiological function is an important approach for studying cellular regulatory mechanisms. Many techniques including direct microinjection, liposome-mediated delivery, fusion of red cell ghosts, and osmotic lysis of pinocytic vesicles have been used to introduce proteins into intact cells. We have used a modification of the voltage-discharge technique to introduce antibodies and other proteins into living physiologically responsive pheochromocytoma and other cultured cells. In this technique, called electroinjection, a single discharge of relatively low field strength is used to transiently permeabilize the plasma membrane. Our experiments demonstrate that electroinjection permits the introduction of large amounts (microM) of probe into 2-5 x 10(6) cells simultaneously without compromising cell viability or physiological responsiveness when performed under carefully defined conditions. They also demonstrate that electroinjection results in a single population of loaded cells and that protein incorporation is a function of field strength, capacitance, molecular weight of the protein, and the concentration of the protein in the electroinjection buffer. Interestingly, a significant fraction of the protein electroinjected into cells is trapped in the plasma membrane when cells are shocked at high capacitance. These results demonstrate that electroinjection appears to be an efficient method for loading exogenous proteins into cells while maintaining the integrity of the physiological properties of the cell.
Voltage gating of the mitochondrial outer membrane channel VDAC is regulated by a very conserved protein
Authors: Liu M. Y. , Colombini M.
Published: December 2017

Soluble protein preparations obtained from the mitochondrial fractions of three very different organisms, Neurospora crassa, rat, and potato, were discovered to greatly enhance the voltage sensitivity of the mitochondrial outer membrane channel, VDAC. The active ingredient, referred to as the VDAC modulator, increased the rate of voltage-dependent channel closure by approximately 10-fold. The modulator from one species increased the closing rate of VDAC channels from all three species. The activity is pronase sensitive and not mimicked by another negatively charged protein, BSA. The highly conserved property of this modulator suggests an important physiological role in regulating mitochondrial function.
Distinctive gap junction channel types connect WB cells, a clonal cell line derived from rat liver
Authors: Spray D. C. , Chanson M. , Moreno A. P. , Dermietzel R. , Meda P.
Published: December 2017

Gap junctions, dye coupling, and junctional conductance were studied in a cell line (WB) that is derived from rat liver and displays a phenotype similar to “oval” cells. In freeze-fracture replicas, two distinctive particle sizes were detected in gap junctional plaques. Immunocytochemical studies indicated punctate staining at membrane appositions using antibodies to connexin 43 and to a brain gap junction-associated antigen (34 kDa). No staining was observed using antibodies prepared against rat liver gap junction proteins (connexins 32 and 26). Pairs of WB cells were electrically and dye coupled. Junctional conductance (gj) between cell pairs averaged approximately 10 nS; occasionally, gj was low enough that unitary junctional conductances (gamma j) could be detected. Using a CsCl-containing electrode solution, distinctive gamma j values were recorded: approximately 20-30 pS, approximately 80-90 pS, and the sum of the other sizes. The largest gamma j events were apparently due to random coincident openings or closures of the smaller channels. Several treatments reduced gj. Frequency distributions of gamma j were unaltered by 2 mM halothane or 3.5 heptanol, but the sizes of intermediate and largest events were reduced slightly by 100 nM phorbol ester, and the relative frequency of the largest events was increased by 10 microM glutaraldehyde. We conclude that the distinctive gamma j values represent openings and closures of two distinct types of gap junction channels rather than substates of a single channel type; these unitary conductances may correspond to the dual immunoreactivity and to the two particle sizes seen in freeze fracture.
Intermediary metabolism and its relationship with ion transport in isolated guinea pig colonic epithelial cells
Authors: Del Castillo J. R. , Ricabarra B. , Sulbaran-Carrasco M. C.
Published: December 2017

We describe a method to isolate surface cells from guinea pig distal colon that obtains a good yield and high viability, as demonstrated by a 99% exclusion of trypan blue, only a 10% liberation of lactate dehydrogenase after 30-min incubation at 37 degrees C, and the inability of succinate to stimulate oxygen consumption before plasma membrane permeabilization. Oxygen consumption (QO2) measured after the sequential addition of the following drugs showed that oligomycin inhibited QO2 by 67%, carbonyl cyanide p-trifluoromethoxyphenylhydrazone increased QO2 by approximately 70% of the basal consumption, and rotenone inhibited QO2 by 90%. Cells at 37 degrees C for 30 min maintained intracellular Na+ and K+ concentrations of 25 and 120 mM, respectively. The ATP consumed by the Na(+)-K+ pump was derived from oxidative phosphorylation (79%) and from glycolysis (21%). Initial rates for Na+ and K+ transported by the pump were 105 +/- 10 and 65 +/- 5 nmol.mg protein-1.min-1, respectively. The rates of Na+ and K+ transported per ATP consumed were estimated to be 2.5 Na+/ATP and 1.6 K+/ATP.
Atrial natriuretic factor decreases cell volume of rabbit atrial and ventricular myocytes
Authors: Clemo H. F. , Baumgarten C. M.
Published: December 2017

The effect of atrial natriuretic factor (ANF) on cell volume was studied using video microscopy of rabbit atrial and ventricular myocytes. Each cell served as its own control, and relative cell volumes were determined. ANF (1 microM) significantly decreased relative cell volume to 0.929 +/- 0.006 (n = 7) in atrial and 0.930 +/- 0.013 (n = 5) in ventricular myocytes (normalized to volume without ANF). Reduction of volume was detectable at greater than or equal to 0.01 microM ANF, and the ED50 was 0.072 +/- 0.007 microM (n = 15). The effect of ANF also was examined under hypotonic (0.55T, 168 mosmol/l) and hypertonic (1.82T, 560 mosmol/l) conditions; osmolarity was adjusted using mannitol with NaCl fixed at 65 mM. In 0.55T, 1 microM ANF decreased cell volume to 0.941 +/- 0.014 (n = 5) in atrial and 0.942 +/- 0.017 (n = 7) in ventricular cells (normalized to 0.55T without ANF). In contrast, 1 microM ANF had no effect on atrial (n = 13) or ventricular (n = 11) cell volume in 1.82T. The hypothesis that ANF decreases cell volume by inhibiting Na(+)-K(+)-2Cl- cotransport was tested by blocking the cotransporter with bumetanide (10 microM). After inhibition of Na(+)-K(+)-2Cl- cotransport, 1 microM ANF failed to reduce cell volume in either atrial (n = 6) or ventricular myocytes (n = 6). Block of ANF-induced cell shrinkage by bumetanide was not due to changes in cell volume, since similar results were obtained using atrial (n = 7) and ventricular (n = 7) cells swollen in hypotonic (0.80T, 244 mosmol/l) solution. Replacement of Na+ with N-methyl-D-glucamine or Cl- with methanesulfonate abolished the ability of both ANF and bumetanide to decrease volume of atrial and ventricular cells in 1T and 0.8T solution. These data suggest that ANF can decrease the volume of atrial and ventricular cells under isotonic and hypotonic conditions by a mechanism that may involve Na(+)-K(+)-2Cl- cotransport. An ANF-induced decrease in cell volume may act as negative feedback and inhibit the stretch-induced release of ANF from atrial and ventricular cells. Furthermore, it may contribute to cell volume maintenance in myocytes in the setting of congestive heart failure or myocardial hypoxia when ANF release is elevated.
Metabolic and contractile responses of rat fast-twitch muscle to 10-Hz stimulation
Authors: Hood D. A. , Parent G.
Published: December 2017

Contractile and metabolic responses of rat fast-twitch gastrocnemius-plantaris muscles were studied. Acute in situ 10-Hz stimulation (STIM) for two 60-min periods, separated by 60 min of recovery (REC), was used. Muscles were removed at 1, 3, 15, 60, 75, 120, 123, or 180 min for metabolite measurements. Twitch and tetanic tensions were reduced to 36 and 28% of initial during the first 60 min of STIM. During REC, these tensions returned only to 56-58% of initial by 120 min. These contractile responses did not parallel changes in metabolites in mixed muscle. pH was reduced from 7.0 to 6.4 by 1 min, but by 15 min of STIM had returned to resting levels. Free ADP and AMP increased 3- and 15-fold during STIM, then decreased to resting levels by 3 min of REC. The most sensitive indicator of metabolic stress during STIM and REC was the phosphorylation potential, which varied up to 40-fold. After initial phases of depletion, ATP and phosphocreatine levels were partially restored despite ongoing STIM. Approximately 75% of the change in ATP level could be accounted for by IMP. In red gastrocnemius [fast-twitch red (FTR)] muscle, IMP was increased by 3 min of STIM but returned to control values by 60 min. Thus reamination of IMP occurred during contractions of FTR muscle. Metabolic and contractile responses during the second STIM period (120-180 min) were similar to the first. This cycle of metabolic and contractile responses occurs in fast-twitch muscle which, with chronically repeated STIM and REC periods, undergoes large phenotypic changes as a result of use.
Mitochondrial adaptations in denervated muscle: relationship to muscle performance
Authors: Wicks K. L. , Hood D. A.
Published: December 2017

We have studied mitochondrial adaptations in muscle subject to chronic denervation, and their relationship to muscle performance, using a model of unilateral sciatic nerve denervation in rats over periods of 2, 5, 8, 14, 21, 28, 35, and 42 days (n = 5-9 rats/day). Time to peak tension (TPT), one-half relaxation time (1/2RT), and endurance performance were evaluated during in situ stimulation of denervated and contralateral gastrocnemius-plantaris muscles. Denervation led to a 70% decline in muscle mass after 42 days. TPT and 1/2RT increased 17 and 30%, respectively, indicating a transformation toward slower muscle. The activities of the enzymes cytochrome-c oxidase (CYTOX), succinate dehydrogenase, and citrate synthase were decreased by 8-14 days, and by 42 days these were 34-58% of control. The mitochondrial phospholipid cardiolipin was reduced earlier, by 5 days, and gradually decreased to 37% of control. Thus phospholipid removal appears to precede the loss of enzyme activity during decreases in mitochondrial content. Endurance performance was reduced in parallel with decreases in enzyme activity and cardiolipin. Cytochrome c mRNA levels decreased to 52% of control by 5 days. Denervation resulted in coordinated changes in mRNA levels encoding the nuclear-derived CYTOX subunit VIc and the mitochondrially derived CYTOX subunit III. However, changes in CYTOX activity did not always parallel alterations in subunit mRNA levels. Thus transcriptional and translational mechanisms operate in regulating mitochondrial gene expression during denervation.