bluuebunny/crossref_metadata_2025 huggingface dataset:
Metabolic control in a state of decreased activation: modulation of red cell metabolism
Authors: Jevning R. , Wilson A. F. , Pirkle H. , O'Halloran J. P. , Walsh R. N.
Published: December 2017

Very little is known in depth of the biochemical and physiological changes induced at the cellular level by human behavioral states. For study of the physiology of behavior at this level, the erythrocyte may be useful, because it is readily available and its metabolism and metabolic control are comparatively well understood. In this report we describe a marked decline of red cell glycolytic rate induced by the transcendental meditation technique (TM). This decline was significantly correlated with decreased plasma lactate concentration and with relaxation as indicated by electrodermal response. The occurrence of sleep was not correlated with the metabolic changes. The observed lack of variation of blood pH, blood gases, glucose, and hematocrit in this behavior implies that the decrease of erythrocyte metabolism is not an epiphenomenon of respiratory change or substrate availability. Based upon further measurements indicating persisting alteration of the red blood cell, we suggest the possibility of attachment of a humoral agent(s) to the cell in the mechanism of this effect. This behavioral effect is unique, and the effector(s) responsible may increase our understanding of metabolic control of the erythrocyte and of TM.
The application of nuclear magnetic resonance to the study of cellular physiology
Authors: Balaban R. S.
Published: December 2017

Nuclear magnetic resonance (NMR) is a form of absorption spectroscopy that can noninvasively monitor the intracellular concentration and several kinetic properties of numerous organic and inorganic compounds. Utilizing these characteristics, investigators have demonstrated that NMR is a useful tool in the study of cellular physiology. In this review, the techniques for using NMR to study isolated cells are outlined with suggestions for the determination of cellular viability within the NMR spectrometer. Whenever feasible, cell preparations that are continuously perfused are preferred, because they can be constantly fed and controlled. Results of various NMR experiments on isolated cells using several nuclides are reviewed to highlight the type of information NMR can provide about cellular physiology. Several important differences between NMR and chemical extraction data are noted. The reason for these differences is probably related to the chemical extraction techniques determining the total amount of a compound within the cell in comparison to NMR, which is somewhat more specific, theoretically, detecting only the free species within the cytosol.
Effects of temperature on protein turnover in isolated rat skeletal muscle
Authors: Baracos V. E. , Wilson E. J. , Goldberg A. L.
Published: December 2017

To understand the net loss of muscle protein during fever and the possible changes in body protein balance with hyperthermia, we investigated the influence of temperature on protein synthesis and degradation in rat skeletal muscles. In the incubated soleus, extensor digitorum longus, or diaphragm, net protein degradation increased by about 11%/degrees C between 33 and 42 degrees C. This loss of muscle protein resulted from an increase in protein degradation (172% between 33 and 42 degrees C). By contrast, protein synthesis increased by only 25% between 33 and 39 degrees C and fell markedly by 42 degrees C. Unlike muscle, in isolated rat hepatocytes, protein breakdown did not increase significantly between 36 and 39 degrees C. The stimulation of protein degradation between 36 and 39 degrees C was not reduced by leupeptin or Ep-475, which inhibit lysosomal thiol proteases and reduce net protein degradation in the incubated muscles. Prostaglandin E2 (PGE2) has been implicated in the accelerated muscle proteolysis during fever. However, PGE2 release by muscles was unchanged between 33 and 42 degrees C, and inhibition of PGE2 synthesis by indomethacin did not reduce the stimulation of proteolysis at 40 degrees C. This catabolic effect of increased temperature may contribute to the negative nitrogen balance during fever.
Enhancement by GTP of cAMP binding to hepatic nuclei and cytosol
Authors: Rosenberg E. M. , Goodman A. D. , Lipinski T. L.
Published: December 2017

In the present study we have demonstrated specific binding of 3H-labeled adenosine 3',5'-cyclic monophosphate (cAMP) to a nuclear extract from rat liver. GTP, GDP, and low concentrations of ATP and ADP increased nuclear binding of [3H]cAMP, and AMP inhibited [3H]cAMP binding. Photoaffinity labeling studies employing [32P]cAMP revealed four nuclear binding proteins [relative molecular weight (Mr) 36,000, 49,000, 54,000 and 57,000]. Unlabeled cAMP decreased [32P]cAMP binding to all four proteins, whereas GTP increased binding to the 57,000 protein. We also observed specific binding of [3H]cAMP in the liver cytosol, which was stimulated by GTP but not by ADP or ATP. Photoaffinity labeling studies of the cytosol in the absence of unlabeled nucleotides revealed three cAMP-binding proteins (Mr 36,000, 49,000, and 54,000). Unlabeled cAMP inhibited binding of [32P]cAMP to all three proteins, whereas in the presence of GTP there was binding of [32P]cAMP to a Mr 57,000 protein. Using DEAE-cellulose, we isolated from the nuclear extract and cytosol a cAMP-binding protein that responded to GTP with an increase in cAMP binding but was unaffected by GDP, ATP, ADP, and AMP. Guanosine imidodiphosphate did not affect cAMP binding, suggesting that the stimulatory effect of GTP may be mediated by phosphorylation. We speculate that alterations in intracellular GTP in vivo may modulate the binding of cAMP to a protein in the nucleus and cytosol.
Phosphoinositide breakdown in blowfly salivary glands
Authors: Litosch I. , Lee H. S. , Fain J. N.
Published: December 2017

In blowfly salivary glands, 5-hydroxytryptamine stimulated a rapid and sustained loss of [3H]inositol, [32P]phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate. There was a corresponding increase in labeled inositol phosphates. In the absence of Ca2+, 5-hydroxytryptamine stimulated a rapid but transient loss of labeled phosphatidylinositol 4,5-bisphosphate. By 5 min, the amount of labeled phosphatidylinositol 4,5-bisphosphate recovered to control values. The divalent ionophore A23187 stimulated loss of labeled phosphatidylinositol 4,5-bisphosphate and increased the amount of labeled phosphatidylinositol. In homogenates, Ca2+ stimulated phosphatidylinositol 4,5-bisphosphate breakdown but not phosphatidylinositol breakdown. These results suggest that hormone-stimulated breakdown of labeled phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate occurs through a phospholipase C and is relatively independent of extracellular Ca2+. There is also a Ca2+-activated conversion of phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol.
Decreased bone formation and mineralization in virus-induced diabetes mellitus
Authors: Yoon J. W. , Reddi A. H.
Published: December 2017

Severe hyperglycemic and hypoinsulinemic mice were selected at 30 and 180 days after infection with the D variant of encephalomyocarditis (EMC) virus. In these animals, although the concentration of calcium and phosphate in serum was not changed, the alkaline phosphatase activity and the rate of mineralization in the proximal tibial epimetaphysis were markedly decreased in the diabetic mice for 30 and 180 days compared with those of the age-matched uninfected animals. Viral specific antigens were not found in these tissues at either earlier or later infection with the virus. It is concluded that endochondral bone formation and mineralization are greatly impaired in EMC virus-induced diabetic mice, and the decreased bone formation and mineralization are not due to the virus-induced tissue damage, but to the persistent metabolic alterations.
Effect of temperature on intracellular pH in crayfish neurons and muscle fibers
Authors: Rodeau J. L.
Published: December 2017

Intracellular pH microelectrodes were used to determine the effects of temperature (13-26 degrees C) on the in vitro regulation of intracellular acid-base status of neurons and muscle fibers of the crayfish Astacus leptodactylus. The values of the temperature coefficients delta pH/delta T (pH unit/degrees C) were -0.019 and -0.026 for muscles and neurons, respectively, values which are close to the temperature coefficient (-0.019) of the pK' of protein imidazole buffer groups. When temperature varies, the dissociation ratio of imidazole groups is thus maintained by the cellular regulation of cytoplasmic pH. According to the alphastat regulation hypothesis, this constancy would minimize the temperature effects on enzymic systems.
Nitrogen narcosis and pressure reversal of anesthetic effects in node of Ranvier
Authors: Kendig J. J.
Published: December 2017

To compare sodium channel block by hyperbaric nitrogen with that induced by other anesthetics and to examine the basis for pressure antagonism to anesthetic condition block, voltage clamped nodes of Ranvier were exposed to nitrogen at pressures at 1-14 atm alone and in combination with helium to a total pressure of up to 100 atm. At 7 and 14 atm nitrogen, sodium currents were reversibly depressed without accompanying changes in the current-voltage relation. The curve relating steady-state inactivation (h infinity) to voltage was shifted in the hyperpolarizing direction, as is the case with other general anesthetic agents. The time constant of inactivation (tau h) was slightly decreased at depolarized potentials. The preceding companion paper demonstrated an opposite effect of hyperbaric helium on the properties of sodium inactivation. Addition of helium pressure in the presence of nitrogen at 14 atm did not increase peak sodium current with inactivation maximally removed, but it did shift the h infinity curve back toward control levels, thus increasing sodium current at points on the slope of the curve. It is proposed that these opposing shifts in steady-state inactivation levels are the basis for pressure antagonism to anesthetic conduction block. In the case of inert gases and volatile anesthetic agents, the antagonism may be direct but has not been shown to be so. In the case of the local anesthetic benzocaine, differences in the voltage dependence of anesthetic and pressure-induced changes in tau h indicate the antagonism is indirect.
Release of cAMP from a renal epithelial cell line
Authors: Strewler G. J.
Published: December 2017

LLC-PK1 is an established porcine renal cell line with epithelial characteristics. Upon hormonal stimulation by vasopressin, LLC-PK1 cells release adenosine 3',5'-cyclic monophosphate (cAMP) into the medium. Release of cAMP is inhibited by the organic anion transport inhibitor probenecid and by cold phosphodiesterase inhibitors and iodoacetate but not by prostaglandins A1 or E1. The kinetics of release are first order, and cAMP analogues do not induce the release of cAMP. When grown on cellulose filters, monolayers of LLC-PK1 have morphological characteristics of transporting epithelia (apical microvilli and intercellular tight junctions) and maintain a transepithelial potential difference. Stimulation of such monolayers by vasopressin elicits probenecid-sensitive release of cAMP into the medium bathing the apical surface. Smaller quantities of cAMP are released from the basolateral surface, but release in this direction is not inhibited by probenecid. In contrast, release of cAMP from the nonepithelial cell line BHK is symmetrical and is symmetrically inhibited by probenecid. Probenecid-sensitive release of cAMP from LLC-PK1 is thus a function of the apical (brush-border) membrane.
Kinetics of CO2 exchange in human erythrocytes analyzed by 13C-NMR
Authors: Shporer M. , Forster R. E. , Civan M. M.
Published: December 2017

The 13C-nuclear magnetic resonance signals of CO2 and bicarbonate have been studied in suspensions of mature human erythrocytes, in lysed suspensions, and in supernatant solutions. This technique requires no chemical perturbation of the samples analyzed. When care is taken to avoid technical artifacts, lysis produces only a slight broadening of the 13CO2 spectral line. The results are consistent with the concept that CO2 exchange is primarily determined by the kinetics of intracellular carbonic anhydrase and not by the permeability of the erythrocyte membrane.
Mechanism of action of barium ion on rat aortic smooth muscle
Authors: Hansen T. R. , Dineen D. X. , Petrak R.
Published: December 2017

The mechanism of action of barium ion on the aortic smooth muscle of the normal rat was investigated using in vitro calcium-depleted aortic strips. Aortic strips were depleted of calcium by repeated exposure to norepinephrine in a calcium-free bathing solution. Although calcium depletion abrogated the response of strips to catecholamines and depolarizing agents, the response to barium chloride remained quantitatively intact. The calcium influx blocker D 600 prevented the contractile response to barium but not to catecholamines, whereas phentolamine prevented the response to catecholamines but not barium. The strip response to barium was depressed by a twofold increase in extracellular magnesium concentration whether the strip was intact or calcium depleted. Although increased concentrations of calcium in the extracellular medium inhibited the contractile response to potassium ion, increases in barium merely potentiated the potassium contracture. These findings indicate that barium produces its contractile effect on vascular smooth muscle by a direct intracellular interaction with the contractile or regulatory proteins. Barium enters these cells via calcium influx channels and is probably not sequestered in a physiologically releasable pool. Unlike calcium, barium does not stabilize the smooth muscle sarcolemma when present in high concentration.
Potassium transport by turtle colon: active secretion and active absorption
Authors: Halm D. R. , Dawson D. C.
Published: December 2017

Measurement of transepithelial potassium fluxes in the absence of transmural electrochemical potential gradients showed that the isolated turtle colon can actively absorb and actively secrete K+. Under short-circuit conditions the active secretory flow was inhibited by mucosal amiloride, whereas the absorptive flow was unaffected by the diuretic. The effects of ouabain and barium on secretory flow were consistent with a simple model involving basolateral uptake by an Na+-K+-ATPase and conductive exist across the apical and basolateral membranes. The active absorptive flux was blocked by mucosal ouabain and by serosal barium. The opposing active flows clearly represented cellular K+ transport, whereas paracellular K+ flows behaved as expected for a free-solution shunt.
Electronic device for microelectrode recordings in epithelial cells
Authors: Garcia-Diaz J. F. , Stump S. , Armstrong W. M.
Published: December 2017

A device is described that permits continuous measurement of electrophysiological parameters in epithelial tissues in the open-circuit mode. Transepithelial potential (VT) and microelectrode (either conventional or ion-selective) potential (VM) are directly measured. Application of transepithelial current pulses allows continuous monitoring of transepithelial resistance (RT) and the ratio between the changes in VM and VT induced by these pulses. Measurement of this ratio, which under some circumstances reflects the apical fractional resistance of the cellular pathway, is important in assessing membrane damage during microelectrode impalement and/or as an index that the microelectrode tip is inside a cell. This is particularly useful when the change in VM during impalement is small. Application of 0.5-nA current pulses through open-tip microelectrodes allows continuous recording of the microelectrode resistance (RM). In epithelia where the individual cells are electrically coupled this permits acceptable impalements (RM remains nearly constant) to be distinguished from those affected by tip potential artifacts due to plugging of the microelectrode tip (RM increases after penetration of the cell membrane). The device provides compensation for the IR voltage drop in the solution between the potential measuring salt bridges and the epithelial surfaces. The microelectrode electrometer has an input impedance greater than 10(15) and is provided with stray capacitance neutralization.
Insulin-specific receptor-mediated slowing of beat rate in embryonic heart cells
Authors: DeHaan R. L. , Goodrum G. , Strumlauf E. , Elsas L. J.
Published: December 2017

Spheroidal aggregates of embryonic heart cells showed their spontaneous beat rate when exposed to insulin. The concentration that produced a half-maximal response (1.7 nM) corresponded to the dissociation constant of binding to a specific high-affinity insulin receptor. The pace-maker phase of action potentials recorded during insulin perfusion was preceded by a prolonged or flattened after hyperpolarization, and its slope was less steep than controls. The action potential duration was also prolonged. These results indicate that physiological concentrations of insulin can regulate the embryonic heart rate.
Growth of kidney epithelial cells in culture: evidence for autocrine control
Authors: Mordan L. J. , Toback F. G.
Published: December 2017

The factors that stimulate kidney growth in K+-deficient animals are unknown. Cultures of renal epithelial cells (BSC-1 line) were used to study this phenomenon because their growth is accelerated in medium containing a reduced K+ concentration. We tested the hypothesis that growth induced by low-K+ medium is mediated by factors produced by the cells; i.e., is subject to autocrine control. Low-K+ (3.2 mM) or control (5.4 mM) medium was conditioned by placing it on confluent cultures of BSC-1 cells for 1 h and was then collected. The K+ concentration of the low-K+ conditioned medium was then adjusted to the control value by addition of KCl. This conditioned medium stimulated growth of fresh cultures of cells to the same extent as did unconditioned low-K+ medium. The appearance of growth-promoting activity was maximal at a K+ concentration of 3.2 mM during conditioning of the medium. Low-K+ conditioned medium, corrected to a K+ concentration of 5.4 mM, required 6 h to commit cells to enhanced proliferation. Growth-stimulating activity in low-K+ conditioned medium was antagonized by a purified growth inhibitor produced by the cells. These observations are consistent with the hypothesis that autocrine products with opposite effects on growth can regulate proliferation of renal epithelial cells.
Electrical properties of dog tracheal epithelial cells grown in monolayer culture
Authors: Coleman D. L. , Tuet I. K. , Widdicombe J. H.
Published: December 2017

Epithelial cells from dog trachea, when grown in tissue culture, formed confluent monolayers 5-6 days after plating. After 8-10 days, domes [mean diam 356 +/- (SE) 53 micron] appeared in monolayers grown in collagen-coated flasks. When grown on polycarbonate filters coated with collagen, a detectable resistance (greater than 5 omega X cm2) and transepithelial potential difference (PD) (greater than 0.1 mV) developed 6 days after plating and increased to approximately 15 omega X cm2 and 15 mV at 10 days. Serosal ouabain (10(-4) M) abolished PD and short-circuit current (Isc). Luminal ouabain had no effect. Luminal amiloride (10(-4) M) and serosal bumetanide (10(-4) M) each decreased PD and Isc. However, a combination of both of these drugs did not abolish Isc. Isoproterenol (10(-5) M), dibutyryl adenosine 3',5'-cyclic monophosphate (10(-3) M), vasoactive intestinal peptide (10(-7) M), prostaglandin (PG) E2 (10(-5) M), PGF2 alpha (10(-5) M), and bradykinin (10(-5) M) each increased PD and Isc. Thus these monolayer cultures maintain electrical properties resembling those of the original tissue. This preparation may prove useful for the study of water and ion transport by airway epithelia.
Analysis of Cl- -HCO3(-) exchange during recovery from intracellular acidosis in cardiac Purkinje strands
Authors: Vanheel B. , de Hemptinne A. , Leusen I.
Published: December 2017

The possible role of a Cl- -HCO3(-) exchange mechanism in the recovery from intracellular acidosis of isolated cardiac Purkinje strands was investigated. Intracellular pH (pHi) was measured using double-barreled pH-sensitive microelectrodes. Acidifications were produced by withdrawing 20 meq NH+4 from the superfusate. Experiments were performed in normal CO2-HCO3(-)-buffered, in HCO3(-)free, and in Cl-free solutions and also in the presence of 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), a blocker of Cl--HCO3(-) exchange. In the absence of external HCO3(-), the apparent rate of acid extrusion following induced acidification was only slightly decreased, but the observed effect does not necessarily imply the intervention of a Cl--HCO3(-) exchange mechanism. SITS had little effect on the response to acidification. In zero-Cl- solutions, recovery of pHi from acidosis was not impaired. These observations suggest that in Purkinje fibers, [Cl-]i-[HCO3(-)]o exchange plays no significant role in recovery from intracellular acidification. Moreover, additional evidence is presented in favor of a passive HCO3(-) efflux at steady-state pHi in the normal superfusate. The apparent membrane permeability to HCO-3 was estimated to be 3.2 X 10(-8) cm X s-1.
Na+ and K+ cell concentrations in collagenase-treated rat kidney tubules incubated at various temperatures
Authors: Sudo J. , Morel F.
Published: December 2017

Na+ and K+ cell contents were measured in single pieces of tubule (0.4–0.8 mm/sample) micro-dissected from the outer medulla of collagenase-treated rat kidney tissue. Extracellular cations were washed out by rinsing the tubules in ice-cold choline-chloride solution. Na+ and K+ cell contents were measured by emission flame microphotometry after appropriate treatment of the samples. Tubular volumes were calculated from photographic pictures taken before (at 4 degrees C) and after incubation of the samples. Medullary collecting tubules (MCT) and medullary thick ascending limbs of Henle (MAL) were used in this study. When kept at 4 degrees C for 2 h or more, Na+ and K+ concentrations (meq/l cell volume) were 86.3 and 30.6, respectively, in MCT and 16.2 and 94.3, respectively, in MAL. After about 5 min of incubation at 30 degrees C, MCT samples inverted their cation contents up to new steady-state concentrations (Na+ 17.4 and K+ 97.5). During incubation, the volume of MCT samples decreased slowly and in an exponential way, the rate of which was highly temperature dependent. Na+ and K+ cell concentrations in such incubated MCT samples, however, remained fairly constant between 20 and 37 degrees C. In contrast, when MAL samples were incubated at 30 degrees C, Na+ and K+ concentrations (15.9 and 90.4, respectively) remained equal to those measured at 4 degrees C and no change in volume was observed in MAL samples.
Hormonal regulation of protein phosphorylation in isolated rat heart cells
Authors: Blackshear P. J. , Nemenoff R. A. , Bonventre J. V. , Cheung J. Y. , Avruch J.
Published: December 2017

We used a recently developed preparation of calcium-tolerant isolated rat cardiac ventricular cells to investigate certain aspects of hormone-mediated protein phosphorylation in heart tissue. Isoproterenol or dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) promoted the phosphorylation of at least 13 proteins and promoted the dephosphorylation of a single protein of relative molecular weight (Mr) 21,000, whose phosphorylation appeared to be stimulated by insulin. The isoproterenol-induced protein phosphorylations reached maximum levels for most proteins within 5 min at slightly different rates. However, when excess propranolol was added to the cells after exposure to isoproterenol, there appeared to be two major patterns of dephosphorylation: proteins that remained fully phosphorylated after propranolol addition, exemplified by proteins tentatively identified as troponin I and C-protein, and proteins that were rapidly dephosphorylated after propranolol, exemplified by phospholamban, the modulator of the sarcoplasmic reticulum calcium-dependent ATPase. The Mr 21,000 protein was rapidly dephosphorylated in response to isoproterenol and was rephosphorylated after addition of propranolol. This protein remains unidentified; it is not the Mr 19,000 myosin light chain whose phosphorylation state was unaffected by isoproterenol. This preparation of isolated heart cells provides a convenient way to investigate the biochemical effects resulting from exposure of the heart to hormones and can separate direct hormonal effects from those resulting from changes in contractility or heart rate.
Electrical and anatomic characteristics of cells of ferret paratracheal ganglion
Authors: Cameron A. R. , Coburn R. F.
Published: December 2017

A preparation has been developed for study of the electrical properties of the neurons of the ferret paratracheal ganglia. Two cell types were identified. AH cells were characterized by the presence of a single action potential in response to cathodal current pulses, followed by a profound after hyperpolarization that lasted for several hundred milliseconds. Electrical stimulation of branches of the laryngeal nerves produced a fast excitatory postsynaptic potential (EPSP) that was often followed by an apparent inhibitory postsynaptic potential (IPSP). A similar response was seen with electrical stimulation of interganglionic nerve trunks. Hexamethonium reversibly inhibited fast EPSPs in these cells. Type B cells could not be excited by intracellular current injections. At least some of these cells appear to be neurons as electrical stimulation of nerve trunks resulted in a slow EPSP and, rarely, a fast IPSP. Ganglia have between 10 and 20 cell bodies, and their diameters are between 15 and 40 microns. Two types of nerve endings were seen: 1) those with mainly round agranular vesicles 50–60 nm in diameter, and 2) those with large dense-cored vesicles of approximately 100 nm with an electron-lucent halo around the core.
Catecholamine-angiotensin II receptor interaction in primary cultures of rat brain
Authors: Sumners C. , Raizada M. K.
Published: December 2017

Neuron-enriched primary cultures from 1-day-old rat brains have been used to study the influence of catecholamines on angiotension II receptors. Treatment of cultures with alpha-methyl-p-tyrosine caused a time- and concentration-dependent increase in the specific binding of 125I-angiotensin II and a decrease in neuronal norepinephrine and dopamine contents. A maximum increase of 75% in the binding and a decrease of 67% in catecholamine content was observed with 400 microM alpha-methyl-p-tyrosine. Removal of alpha-methyl-p-tyrosine from cultures resulted in the recovery of catecholamine levels and decrease in 125I-angiotensin II binding to control levels. In contrast, treatment of cultures with pargyline caused decreases in the binding of 125I-angiotensin II and increases in catecholamine levels in neuronal cultures. Scatchard analysis demonstrated that an increase in 125I-angiotensin II binding by alpha-methyl-p-tyrosine was due to an increase in the affinity of receptors for angiotensin II without changes in the number of angiotensin II binding sites. The results obtained here indicate that angiotensin II binding to neuron-enriched cultures is reciprocally related to the levels of neuronal catecholamines.
Stimulation of oxyntic cell triggers K+ and Cl- conductances in apical H+-K+-ATPase membrane
Authors: Wolosin J. M. , Forte J. G.
Published: December 2017

Vesicles isolated from the apical membrane of stimulated oxyntic cells [stimulation-associated (SA) vesicles] are highly permeable to KCl. The KCl flux is coupled to an electroneutral ATP-driven H+-K+ exchange (the H+-K+-ATPase) to produce net intravesicular HCl accumulation. In the past, we observed that rates of KCl transport were not accelerated by valinomycin and that dissipation of preformed H+ gradients in the presence of a protonophore (carbonyl cyanide, m-chlorophenylhydrazone, 10 microM) required the simultaneous presence of valinomycin. Consequently the fast KCl transport was attributed to an electroneutral cotransport system. Now we have been able to elicit fast H+ gradient dissipation in the absence of valinomycin by using the protonophore tetrachlorosalicylanilide. Experiments carried out in the absence of Cl- demonstrated the existence of a specific high-conductance pathway for K+. Experiments in K+-free medium demonstrated the existence of a high Cl- conductance. Parallel experiments in the equivalent H+-K+-ATPase-rich vesicles from nonsecreting oxyntic cells showed very little K+ and Cl- conductivity, suggesting that the appearance of large ionic conductance in the membrane is associated with the stimulation of the cell.
[3H]ouabain binding to cultured rat vascular smooth muscle cells
Authors: Khalil F. , Hopp L. , Searle B. M. , Tokushige A. , Tamura H. , Kino M. , Aviv A.
Published: December 2017

The number of Na+ pump units (Bmax) and the equilibrium dissociation constant (Kd) for ouabain as well as parameters of K+ binding to the Na+ pump were examined in in vitro-grown vascular smooth muscle cells ( VSMC ) derived from Sprague-Dawley rats. The technique to measure these variables utilizes analyses of [3H]ouabain displacement from its VSMC receptors by nonlabeled ouabain and K+. The mean values for Bmax and Kd in the cultured VSMCs were 1.95 X 10(5) receptor sites per single VSMC and 2.68 X 10(-6) M, respectively. The equilibrium dissociation constant for K+ (Ki) was 0.92 mM. K+ binding to the cultured VSMCs demonstrated positive cooperativity with a Hill coefficient (n) of 1.78.
Activity of circular muscle of rat uterus at different times in pregnancy
Authors: Bengtsson B. , Chow E. M. H. , Marshall J. M.
Published: December 2017

Page C216: B. Bengtsson, E. M. H. Chow, and J. M. Marshall. “Activity of circular muscle of rat uterus at different times in pregnancy.” Page C216: the citation of the journal should read: Am. J. Physiol. 246 (Cell Physiol. 15): C216– C223, 1984.
Regulation of glycolysis in rat aorta
Authors: Kutchai H. , Geddis L. M.
Published: December 2017

Certain factors that might contribute to the regulation of the rate of glycolysis by rat aorta were investigated. Rat aortic rings were incubated with [14C]glucose, and the release of [14C]lactate was determined. There was good agreement between the lactate production estimated by enzymatic assay and by [14C]lactate release, suggesting that almost all the lactate produced under our experimental conditions was derived from exogenous glucose. When the glucose concentration in the medium was 10 mM or higher, the rate of glucose transport did not limit the rate of lactate production. In most cases studies were done both aerobically and anaerobically. In Hanks' Balanced Salt Solution the aerobic rate of lactate production was 18% of the anaerobic rate. We tested the effects on glycolysis of agents that alter ATP generation by mitochondria or ATP splitting by Na+-K+-ATPase or the mitochondrial ATPase. Under aerobic conditions, ouabain (5 mM) caused a 54% decrease in lactate production, and gramicidin (5 micrograms/ml) caused a 45% increase. Under anaerobic conditions, neither ouabain nor gramicidin affected lactate production. Aerobically dinitrophenol (25 microM) and carboxyatractyloside (0.5 mM) caused substantial increases in lactate production, 72 and 98% respectively. Under anaerobic conditions the effects of dinitrophenol and carboxyatractyloside were much smaller, with dinitrophenol causing a 15% increase and carboxyatractyloside a 12% decrease in lactate production. Increasing the concentration of phosphate in the incubation medium caused marked increases in lactate production. Both aerobically and anaerobically, shifting from 1.3 to 50 mM phosphate in the incubation medium caused a 3.5-fold increase in lactate production.(ABSTRACT TRUNCATED AT 250 WORDS)
Angiotensin II in neuronal cultures from brains of normotensive and hypertensive rats
Authors: Raizada M. K. , Stenstrom B. , Phillips M. I. , Sumners C.
Published: December 2017

Primary neuronal cultures from 1-day-old rat brains, which contain angiotensin II (ANG II) immunoreactivity within the neurons and are capable of de novo synthesis of this immunoreactivity, have been used in this study to determine the nature of this immunoreactivity by high-performance liquid chromatography (HPLC). Neuronal cultures from the brains of normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SH) rats were found to contain ANG II immunoreactivity, which co-migrated with authentic ANG II on HPLC. The angiotensin detected in brain cultures was not derived from the growth medium, and its level was significantly decreased by incubating the cultures with captopril. A 71% decrease in the levels of ANG II was observed in neuronal cultures of SH rat brains compared with those from WKY controls. These observations show that differences in brain angiotensin between SH and WKY are present before differences in blood pressure are manifested.
Stimulation of Na+-K+ pump in sheep red blood cells by heteroimmune anti-sheep red cell antibodies
Authors: Dunham P. B. , Farquharson B. E. , Bratcher R. L.
Published: December 2017

In the HK-LK polymorphism of sheep red blood cells, alloimmune antiserum against the L antigen on LK cells is known to stimulate the Na+-K+ pump in low K+ (LK) cells, but alloimmune antiserum against the M antigen of high K+ (HK) cells does not. We have shown for the first time that heteroimmune antibodies against sheep red blood cells raised in mice can stimulate the pump. Heteroimmune antibodies against both LK(L) cells and HK(M) cells stimulated active K+ transport in LK cells. Furthermore heteroimmune antibodies against LK(L) cells also stimulated the pump in HK cells. As expected, alloimmune and heteroimmune antibodies acted at different sites in stimulating transport in LK cells.
Electrical potentials and cell-to-cell dye movement in mouse mammary gland during lactation
Authors: Berga S. E.
Published: December 2017

Stable potentials were recorded with microelectrodes in an in vivo preparation of the mammary gland from the anesthetized lactating mouse. Location of the microelectrode tip was determined by ionophoretic injection of the fluorescent dye Lucifer yellow CH. Fifteen dye injections were localized to mammary alveolar cells; the average recorded potential for these penetrations was -49 +/- 2 mV. Cell-to-cell dye transfer between alveolar cells was observed with all intracellular Lucifer yellow injections. Ten dye injections were localized to the alveolar lumina with an average recorded potential of -35 +/- 2 mV. With these penetrations Lucifer yellow spread rapidly to many alveolar lumina. These findings indicate that stable potentials can be obtained from both cells and lumina in the in vivo mammary gland, demonstrating the feasibility of electrophysiological studies of the mammary epithelium. The presence of a large transepithelial potential provides evidence for physiologically tight junctions between mammary alveolar cells. In addition, the distribution of Lucifer yellow shows that mammary alveolar cells are coupled and suggests that milk flows freely between alveolar lumina.
Effect of mitochondrial clustering on O2 supply in hepatocytes
Authors: Jones D. P.
Published: December 2017

Isolated rat hepatocytes were treated with digitonin to selectively disrupt the plasma membrane and allow study of the O2 dependence of mitochondria within the cell cytoskeleton, but without cytosol. Half-maximal oxidation of cytochrome c occurred at 2.0 microM O2 in treated cells incubated under State 3 conditions, whereas in intact cells it was 6.0 microM and in isolated mitochondria (State 3) 0.69 microM. the intermediate value for treated cells indicates that both the geometry of mitochondrial packing and the intracellular diffusion coefficient are important in determining intracellular mitochondrial O2 dependence. Analysis of intracellular diffusion, assuming that the mitochondrial clustering increases the effective mitochondrial radius, indicates that an intracellular diffusion coefficient of between 10(-6) and 4 X 10(-6) cm2 . s-1 and an effective mitochondrial radius of approximately 2 micron would account for the observed intracellular O2 dependence of mitochondrial function.
Aggregate-carrying membranes during ADH stimulation and washout in toad bladder
Authors: Muller J. , Kachadorian W. A.
Published: December 2017

Aggregates of intramembrane particles are found in cytoplasmic structures that we now term “aggrephores.” Antidiuretic hormone (ADH) causes aggrephores to fuse with the luminal membrane. Aggregates subsequently become dispersed in the membrane and behave as sites for water flow. In the present studies we examined further the behavior of aggrephores. 1) Bladders stimulated for 10, 30, or 60 min in the absence of an osmotic gradient had two to three times more fusions as those stimulated in the presence of a gradient, indicating that aggrephore fusion frequency is influenced by water flow. 2) ADH stimulation did not change the projected luminal surface area of granular cells. Thus fused aggrephores remain fixed while aggregates move from the aggrephore to flat areas of luminal membrane. 3) Horseradish peroxidase, present in the mucosal baths of bladders stimulated with and then washed of ADH, was found in the lumen of cytoplasmic aggrephores; aggrephores therefore detach from the luminal membrane during washout. The same bladders had about twice as many multivesicular body-type lysosomes as unstimulated bladders, suggesting that, after detachment, aggrephores may join or become lysosomes. 4) Colchicine did not affect the rate of disappearance of fusion sites during washout, whereas cytochalasin B slowed it, suggesting that aggrephore detachment depends of microfilaments, but not microtubules.
Differential effects of cysteine on protein and coenzyme A synthesis in rat heart
Authors: Chua B. H. , Giger K. E. , Kleinhans B. J. , Robishaw J. D. , Morgan H. E.
Published: December 2017

The effect of cysteine availability on protein and coenzyme A (CoA) synthesis in perfused rat heart was incompletely evaluated in earlier experiments because rapid conversion of cysteine to cystine occurred when the perfusion buffer was oxygenated. This conversion was minimized by addition of an excess of reducing agents such as dithiothreitol or mercaptodextran or by provision of bathocuproine disulfonate, a copper chelator. Dithiothreitol was not a suitable protective agent because it reduced ATP and creatine phosphate contents. Perfusion of hearts with [35S]cystine or [35S]cysteine in the presence of mercaptodextran resulted in a 22-fold or 5-fold increase, respectively, in incorporation of [35S] into protein and a 5-fold or 8-fold increase, respectively, in incorporation into CoA compared with hearts supplied [35S]cystine or [35S]cysteine without the reducing agent. When compared with hearts perfused at an aortic pressure of 90 mmHg with bicarbonate buffer that contained 15 mM glucose, 25 mU insulin/ml, 0.4 mM [14C]phenylalanine, no cysteine and plasma levels of other amino acids, provision of 0.09 or 0.2 mM cysteine alone or in the presence of mercaptodextran, or bathocuproine disulfonate enhanced rates of protein synthesis 16-35%. When 0.2 mM cysteine was added to bicarbonate buffer containing 7 microM pantothenic acid, supplementation with mercaptodextran or bathocuproine disulfonate was required to raise CoA content. These results indicated that an exogenous supply of cysteine was needed to maintain maximal rates of protein and CoA synthesis in the perfused rat heart. Protective compounds were required to obtain the cysteine effect on CoA but not on protein synthesis.
Freezing of living cells: mechanisms and implications
Authors: Mazur P.
Published: December 2017

Cells can endure storage at low temperatures such as--196 degrees C for centuries. The challenge is to determine how they can survive both the cooling to such temperatures and the subsequent return to physiological conditions. A major factor is whether they freeze intracellularly. They do so if cooling is too rapid, because with rapid cooling insufficient cell water is removed osmotically to eliminate supercooling. Equations have been developed that describe the kinetics of this water loss and permit one to predict the likelihood of intracellular freezing as a function of cooling rate. Such predictions agree well with observations. Although the avoidance of intracellular freezing is usually necessary for survival, it is not sufficient. Slow freezing itself can be injurious. As ice forms outside the cell, the residual unfrozen medium forms channels of decreasing size and increasing solute concentration. The cells lie in the channels and shrink in osmotic response to the rising solute concentration. Prior theories have ascribed slow freezing injury to the concentration of solutes or the cell shrinkage. Recent experiments, however, indicate that the damage is due more to the decrease in the size of the unfrozen channels. This new view of the mechanism of slow freezing injury ought to facilitate the development of procedures for the preservation of complex assemblages of cells of biological, medical, and agricultural significance.
Effects of cadmium and zinc on calcium uptake in human red blood cells
Authors: Plishker G. A.
Published: December 2017

Cadmium and zinc increased the accumulation of calcium in human red blood cells by increasing passive influx without enhancing the permeability to other ions. The effect of cadmium and zinc appeared specific to these metals, because barium, magnesium, cobalt, strontium, manganese, and nickel had no effect. Changes in calcium uptake by extracellular sodium, potassium, and pH were not altered by zinc and cadmium. Inhibition of calcium uptake by quinine, oligomycin, and iodoacetate was not affected by cadmium or zinc. These results suggest that cadmium and zinc increase calcium movement through normal influx pathways. Cadmium and zinc acted synergistically apparently by different mechanisms. Zinc and cadmium differentially affected calcium uptake in different extracellular calcium concentrations. The cadmium effect was increased by low concentrations of 2-mercaptoethanol and above pH 8.0, while the zinc effect was less sensitive to these factors. These findings suggest that the cadmium effect involves a disulfide bond between cysteinyl residues and the zinc effect involves a different site.
Inhibition of endogenous pyrogen-induced fever by a muramyl dipeptide derivative
Authors: Parant M. , Riveau G. , Parant F. , Chedid L.
Published: December 2017

N-acetylmuramyl-L-alanyl-D-isoglutamine, or muramyl dipeptide (MDP), is a synthetic immunoadjuvant analogue of a bacterial peptidoglycan subunit that has a definite pyrogenic effect in the rabbit. Some adjuvant-active derivatives such as murabutide [MDP(Gln)-OnBu] or murametide [MDP(Gln)-OMe] are not pyrogenic. Murabutide did not stimulate human or rabbit cells to release endogenous pyrogen (EP), but murametide induced EP production at the same dosage levels as MDP. Moreover, plasma from rabbits treated with murametide transferred into untreated recipients elicited a febrile response typical of EP fever and comparable with that induced by plasma from MDP-treated animals. Murametide not only inhibited the central effect of EP that is generated but also the effect of an extra dose of EP administered later by the intravenous route. Moreover, pretreatment of rabbits with murametide decreased fever responses induced by certain high-molecular-weight exogenous pyrogens as mediated through the production of EP.
Ion transport in dog tracheal mucosa: interaction between calcium and prostaglandins
Authors: Al-Bazzaz F. J. , Yadava V. P.
Published: December 2017

Prostaglandins E1 and F2 alpha (PGE1 and PGF2 alpha) stimulate short-circuit current (SCC), tissue conductance (G), and net Cl secretion by canine tracheal mucosa. To determine if these actions of prostaglandins require extracellular Ca we tested the effects of PGE1 and PGF2 alpha on tracheal mucosa mounted in Ussing chambers when one side of the preparation was exposed to 0.2 mM ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid in Krebs-Henseleit solution without Ca. Lack of Ca from the mucosal bath was associated with increased G, lowered potential difference (PD), and severalfold increase of 22Na and 36Cl fluxes in both directions. Addition of 1 microM of PGE1 or PGF2 alpha to the Ca-free mucosal bath raised PD by 6 mV and SCC by 15 microA X cm-2 but decreased G by 1.4 mS X cm-2. Unidirectional 22Na and 36Cl fluxes decreased by 32-49% (n = 7, P less than 0.05). These findings suggest that the increase in G, most likely of the paracellular pathway and brought about by the lack of mucosal Ca, was partially reversible by PGE1 and PGF2 alpha. In contrast, when tissues were exposed to Ca-free solution on their submucosal side, PGE1 or PGF2 alpha were not able to reverse the progressive decline in PD and tissue resistance (n = 8). [14C]mannitol flux increased when Ca was absent from the mucosal bath; then addition of 1 microM PGF2 alpha caused a 37% decline in flux (n = 5). In contrast, the increase in the flux of [14C]mannitol found when the submucosal bath lacked calcium was not reversed by 1 microM PGE1.(ABSTRACT TRUNCATED AT 250 WORDS)
Estimation of cellular pH gradients with 31P-NMR in intact rabbit renal tubular cells
Authors: Adler S. , Shoubridge E. , Radda G. K.
Published: December 2017

To determine intracellular pH gradients rabbit renal cortical tubular cells were prepared by collagenase separation, suspended in a Krebs-Ringer buffer solution, and gassed with 95% O2-5% CO2 in a special nuclear magnetic resonance (NMR) probe. Renal tubular cellular pH was determined simultaneously from the distribution of 14C-dimethadione (DMO) (pHDMO) or the chemical shift of inorganic phosphate (pHNMR). Experiments were performed at different external pH values (pHe) ranging between 6.52 and 7.20. pHNMR, a measure of cytoplasmic pH, changed by an amount equal to the change in pHe. pHDMO, however, a measure of cytoplasmic plus mitochondrial pH, changed less than pHe as the latter increased. pHDMO, higher than pHNMR at low pHe, became equal to pHNMR at higher pHe values. By use of assumed mitochondrial volumes of 30-40% mitochondrial pH was calculated from pHDMO and pHNMR. Mitochondrial pH remained relatively constant over the entire pHe range studied. Since cytoplasmic pH fell as pHe was lowered, the transmitochondrial pH gradient increased at low pHE values. These findings suggest that the transmitochondrial pH gradient may be important in regulating metabolism.
Regulation of protein turnover by glucose, insulin, and amino acids in adipose tissue
Authors: Tischler M. E. , Ost A. H. , Spina B. , Cook P. H. , Coffman J.
Published: December 2017

Protein synthesis and degradation were measured simultaneously in epididymal fat pads of rats by use of the incorporation of [14C]phenylalanine into protein and the sum of net protein breakdown and protein synthesis, respectively. Neither glucose nor insulin altered protein synthesis, but together they promoted this process; pyruvate could be substituted for glucose. Separately, glucose or insulin diminished proteolysis, and these effects were additive. In the presence of glucose and insulin, leucine, alanine, glutamine, glutamate, and aspartate lowered protein degradation to varying degrees but did not alter protein synthesis. Glutamate, but not leucine or alanine, was inhibitory without glucose and insulin present. When aminooxyacetic acid was provided to decrease the rate of transamination of amino acids, the inhibitory effects of leucine, alanine, and aspartate, but not of glutamate, appeared to be diminished. alpha-Ketoglutarate, but neither alpha-ketoisocaproate nor pyruvate, could diminish proteolysis. Inhibition of proteolysis was associated with a higher tissue content of glutamate and a greater production of glutamate and glutamine. These results suggest that glutamate itself may inhibit proteolysis in adipose tissue and mediate, at least in part, the effects of other amino acids.