bluuebunny/crossref_metadata_2025 huggingface dataset:
Neutral amino acid influx in developing rabbit blastocysts
Authors: Bell J. E. , Begg K. E. , Sin Y. , Biggers J. D. , Benos D. J.
Published: December 2017

The influx of the neutral amino acids glycine, aminoisobutyric acid (AIB), and leucine into rabbit blastocysts was measured. In day 6 postcoitus (pc) embryos, glycine influx was Na+ independent, whereas AIB and leucine influx involved both Na+-dependent and independent components. From days 5 to 7 pc, the leucine and AIB influx remained constant, although the Na+-dependent fraction decreased and the Na+-independent fraction increased with age. None of the Na+-independent influx was inhibited by methylaminoisobutyric acid (MeAIB), an amino acid analogue specific for the system A of neutral amino acid uptake. In addition, MeAIB influx was Na+ independent, implying that system A is not involved in leucine or AIB uptake. All Na+-dependent influx is thus considered to occur via system ASC. System L contributed only to the influx of leucine at days 6 and 7 pc, as measured by inhibition of Na+-independent influx by 2-amino-bicyclo-(2,2,1)-heptane-2-carboxylic acid.
Two types of collecting duct mitochondria-rich (intercalated) cells: lectin and band 3 cytochemistry
Authors: Schuster V. L. , Bonsib S. M. , Jennings M. L.
Published: December 2017

Anion exchange plays an important role in renal ion transport and acidification. To further understand the molecular nature of renal epithelial anion exchange, we used a monoclonal antibody to the membrane domain (52 kDa) of human erythrocyte band 3 protein to immunocytochemically search for this polypeptide in the rabbit kidney. In cryostat sections, a subpopulation of cells in the cortical and outer medullary collecting tubules showed immunoreactivity; labeling was restricted to the basolateral membrane. Proximal tubules and thick and thin limbs of Henle showed no immunoreactivity. Approximately 11% of cells in the cortical, but 43% of cells in the medullary, collecting tubule were positive for band 3. To determine the type of cells that were band 3 positive, mitochondria-rich (intercalated) cells were identified by their positive histochemical staining for succinic dehydrogenase activity and by their ability to bind peanut lectin at the apical membrane. In the cortical collecting tubule, the majority of mitochondria-rich cells bound peanut lectin but were band 3 negative; the remainder were band 3 positive but lectin negative. This distribution was reversed in the inner stripe of the outer medulla: all mitochondria-rich cells were band 3 positive and lectin negative. Thus mitochondria-rich cells are of at least two types, each of which has a distinct axial distribution pattern. Given available information about in vitro HCO3 transport properties of rabbit collecting tubules, it is likely that the lectin-positive, band 3-negative mitochondria-rich cells secrete HCO3, whereas the lectin-negative, band 3-positive cells reabsorb HCO3 (secrete H).
Activation of Cl-dependent K transport in Ehrlich ascites tumor cells
Authors: Kramhoft B. , Lambert I. H. , Hoffmann E. K. , Jorgensen F.
Published: December 2017

N-ethylmaleimide (NEM) treatment of steady-state Ehrlich cells induces a substantial net loss of cellular KCl and cell shrinkage. The majority of the initial K loss is Cl dependent. From estimates of membrane potential it is concluded that the NEM-induced KCl loss is electroneutral. The effect of NEM on H extrusion by cells in 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-containing medium showed that only an insignificant part of the K loss could be attributed to an activation of a K-H exchange system. Consequently, NEM appears to activate a K-Cl cotransport, which causes cell shrinkage. The anion preference of the K loss is Cl greater than Br much greater than SCN = NO3. NEM also seems to inhibit a Cl-dependent Na uptake previously described in shrunken cells. Addition of NEM to cells undergoing regulatory volume decrease after swelling in hyposmotic media results in a Cl-dependent acceleration of cell shrinkage, suggesting that a Cl-dependent component of K efflux is induced by NEM also in swollen cells. A Cl-dependent K efflux is also activated in Ca-depleted cells or at reduced extracellular pH after cell swelling. Under isotonic conditions activation of Cl-dependent K flux after Ca depletion or pH reduction could not be demonstrated. The combined results show that Ehrlich cells possess a latent K-Cl cotransport that becomes active after changes in the state of SH groups, regardless of the initial cell volume. A similar K-Cl cotransport is activated in hypotonically swollen cells after Ca depletion or after reduction of the extracellular pH.
Evaluation of granule exocytosis in toad urinary bladder
Authors: Wade J. B. , McCusker C. , Coleman R. A.
Published: December 2017

Addition of phorbol myristate acetate (PMA) to the mucosal bathing solution induces exocytosis of the granules found in the apical cytoplasm of toad urinary bladder epithelial cells. Because mucosal application of PMA also increases epithelial water permeability in the complete absence of vasopressin, these observations have suggested that granules might have a role in mediating the permeability response to vasopressin. From electron microscopic immunolocalization studies we have identified a 70-kDa protein as a component of the apical surface glycocalyx that is stored in the granules. Using antibodies to this protein in a competitive immunoassay, we found that addition of PMA to the mucosal side results in more than a fourfold increase in antigen release into the mucosal bath. The amount of antigen detectable on the mucosal surface of the cells was doubled by mucosal PMA exposure. Serosal application of PMA or addition of the inactive analogue phorbol didecanoate to either surface failed to produce significant antigen release. These results are consistent with electron microscopic studies showing exocytosis of granules only with mucosal exposure to PMA. However, immunoassay of granule antigen after vasopressin stimulation of water permeability failed to show a detectable change in granule exocytosis. We conclude that granule exocytosis does not play an important role in mediating vasopressin-induced changes in permeability.
Segregation of gastric Na and Cl transport: a vibrating probe and microelectrode study
Authors: Demarest J. R. , Scheffey C. , Machen T. E.
Published: December 2017

The short-circuit current (Isc) of resting Necturus gastric mucosa (approximately 20 microA/cm2) can be attributed to the algebraic sum of the net Cl- secretion and amiloride-inhibitable net Na+ absorption. We have attempted to identify the cell types [surface epithelial cells (SCs) or oxyntic cells (OCs)] responsible for the transport of these ions in Necturus gastric mucosa using microelectrodes (ME) and a vibrating probe (VP). Mucosae were mounted horizontally in an open-topped Plexiglas chamber either serosal side up for basolateral ME impalements of OCs or mucosal side up for apical impalements of SCs and VP measurements. Cell impalements were made under open-circuit conditions, and VP measurements were performed under short-circuit conditions. Impalements of OCs indicate that neither the ratio of their apical to basolateral cell membrane resistances (Ra/Rb = 1.3 +/- 0.2) nor their cell membrane potentials were affected by 10(-6) M mucosal amiloride. In contrast, impalements of SCs indicate that amiloride increased their Ra/Rb from 3.5 +/- 0.2 to 15.6 +/- 1.8 and hyperpolarized both cell membrane potentials by greater than 20 mV. VP measurements showed that the amiloride-induced change in the current from SCs (5.6 microA/cm2) accounted for the amiloride-induced change in the Isc (5.5 microA/cm2). A non-zero current (4.4 +/- 1.0 microA/cm2) measured over SCs in the presence of amiloride was due to contamination from current arising from the gastric crypts that contain the OCs.(ABSTRACT TRUNCATED AT 250 WORDS)
Embryonic chick allantois: functional isolation and development of sodium transport
Authors: Graves J. S. , Dunn B. E. , Brown S. C.
Published: December 2017

By removing the shell membranes from the chorioallantoic membrane, the chorion is damaged, as visualized by electron microscopy, and rendered permeable, as evidenced by penetration of horseradish peroxidase and increased inhibition of the allantoic Na+-K+ pump by ouabain applied on the chorionic side. The short-circuit current (SCC) of this functionally isolated allantoic epithelium is augmented by nystatin, a channel-forming ionophore, when applied to the mucosal surface. Electrical parameters were determined for three age groups between 12 and 19 days of incubation. The SCC approximately doubled from the youngest (12-13 days) to the oldest (18-19 days) groups, whereas the transepithelial resistance (Re) of 700-900 omega X cm2 remained the same. Amiloride, an inhibitor of apical Na+ uptake, inhibited 98-100% of the SCC at 10(-4) M in both 15-16 and 18-19 day epithelia. In the 12- to 13-day preparation 20-25% of the SCC was insensitive to 10(-3) M amiloride. The Ki's for amiloride were similar in all preparations, at about 5 X 10(-7) M. Determination of the Hill coefficients for inhibition revealed a lower value (0.75 +/- 0.03) for the 12-13 day preparation compared with the two older preparations with coefficients not significantly different from unity. Replacing Na+ in the bathing solutions abolished the SCC of 18-19 day epithelia, whereas about 15% of the SCC remained at 12-13 days. Thus, during development, the SCC of the allantoic epithelium increases in magnitude and becomes increasingly (to 100%) amiloride-sensitive and Na+-dependent.
Jejunal epithelial glucose metabolism: effects of Na+ replacement
Authors: Mallet R. T. , Jackson M. J. , Kelleher J. K.
Published: December 2017

The objective of this study was to characterize the effects of replacement of extracellular Na+ with a nontransportable cation, N-methyl-D-glucamine (NMDG+) on jejunal epithelial glucose metabolism. Jejunal epithelium isolated from male Sprague-Dawley rats was incubated in media containing 5 mM glucose, 0.5 mM glutamine, 0.5 mM beta-hydroxybutyrate, and 0.3 mM acetoacetate as the principal carbon sources. O2 consumption and total glucose utilization were reduced 30 and 50%, respectively, when Na+ was replaced with NMDG+. In both media, approximately 75% of utilized glucose carbon was converted to lactate. The rate of glucose metabolism via the hexose monophosphate shunt, as evaluated using specific 14CO2 yields from [1-14C]glucose and [6-14C]glucose, was not appreciably altered by Na+ replacement. Tricarboxylic acid (TCA) cycle flux was evaluated using 14CO2 production from [14C]glucose and [14C]pyruvate radioisotopes. Approximately 50% of TCA cycle flux was shunted into products other than CO2 in both media. The majority of the acetyl-CoA oxidized in the TCA cycle was derived from cytosolic pyruvate. It is concluded that removal of Na+ from the bathing medium substantially reduced glucose utilization via the Embden-Meyerhof pathway and TCA cycle in the jejunal epithelium.
Effects of phenothiazines on binding and processing of epidermal growth factor in 3T3 cells
Authors: Selinfreund R. , Lin P. H. , Cooper J. L. , Wharton W.
Published: December 2017

Chlorpromazine (CPZ) or the functionally related N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide caused a rapid decrease in binding of 125I-epidermal growth factor (EGF) that was due to a specific decrease in receptor affinity. The decrease in ligand binding was observed when cells were exposed to CPZ at either 4 degrees C or 37 degrees C but a rapid reversal of CPZs effects was observed only during a 37 degrees C incubation. In contrast to the decrease in 125I-EGF binding seen after short (30 min) accumulations at 37 degrees C, the presence of CPZ caused a large increase in the amount of cell-associated radioactivity after longer periods (over 1 h) of accumulation. Although the CPZ-induced effect was similar in extent to that observed after the addition of methylamine, the increased accumulation after CPZ was probably not due to a nonspecific ionic neutralization of the lysosomes. CPZ did not lower EGF binding in cultures chronically treated with a phorbol ester to reduce protein kinase C levels, although the CPZ-induced increases in accumulation were still observed in cells with reduced protein kinase C activity.
ATP-dependent calcium transport across basal plasma membranes of human placental trophoblast
Authors: Fisher G. J. , Kelley L. K. , Smith C. H.
Published: December 2017

As a first step in understanding the cellular basis of maternal-fetal calcium transfer, we examined the characteristics of calcium uptake by a highly purified preparation of the syncytiotrophoblast basal (fetal facing) plasma membrane. In the presence of nanomolar concentrations of free calcium, basal membranes demonstrated substantial ATP-dependent calcium uptake [K0.5 = 119 nM, maximum velocity (Vmax) = 2 nM X min-1 X mg-1]. This uptake required magnesium, was not significantly affected by Na+ or K+ (50 mM), or sodium azide (10 mM). Intravesicular calcium was rapidly and completely released by the calcium ionophore A23187. Calcium transport was significantly stimulated by the calcium-dependent regulatory protein calmodulin. Placental membrane fractions enriched in endoplasmic reticulum (ER) and mitochondria also demonstrated ATP-dependent calcium uptake. In contrast to basal membrane, mitochondrial calcium uptake was completely inhibited by azide. The rate of calcium uptake by the ER was only 20% of that of basal membranes. We conclude that the placental basal plasma membrane possesses a high-affinity calcium transport system similar to that found in plasma membranes of a variety of cell types. This transporter is situated to permit it to function in vivo in maternal-fetal calcium transfer.
Energy expenditure of longitudinal smooth muscle of rabbit urinary bladder
Authors: Wendt I. R. , Gibbs C. L.
Published: December 2017

Heat production, oxygen consumption, and lactate production of longitudinal smooth muscle from rabbit urinary bladder has been measured at 27 degrees C. In isometric contractions (initiated by 1-Hz electrical stimulation) ranging in duration from 2 to 300 s, total energy expenditure correlated linearly with the force-time integral. For any given force-time integral oxygen consumption could account for only approximately 60% of the total energy measured as heat production. A substantial contribution of aerobic lactate production to the total energy flux was observed. This lactate production was also linearly correlated with force-time integral and was of sufficient magnitude to account for the discrepancy between total energy expenditure determined as heat production and oxygen consumption. The suprabasal rate of energy expenditure during the maintenance of force was approximately 2.6 mW/g and remained constant throughout contractions of up to 5-min duration, suggesting that in this muscle there is no change in the energetic cost of force maintenance with increasing duration of contraction. The rate of energy expenditure during the initial period of force development was, however, about twofold greater than that during subsequent force maintenance, indicating that there is an extra energy cost associated with the activation of contraction and development of force above that required for force maintenance.
Faster ribosome synthesis induced by elevated aortic pressure in rat heart
Authors: Chua B. H. , Russo L. A. , Gordon E. E. , Kleinhans B. J. , Morgan H. E.
Published: December 2017

An increase in aortic pressure from 60 to 120 mmHg accelerated ribosomal protein synthesis in rat hearts during 1 or 2 h of labeling with 0.4 mM [3H]phenylalanine. When hearts were perfused with buffer that contained 20 mM glucose and normal plasma concentrations of 19 other amino acids without added insulin, ribosomal protein synthesis relative to the rate of total protein synthesis increased from approximately 0.22 to 0.36 and 0.30 as aortic pressure was raised from 60 to 120 mmHg during 1 or 2 h of labeling, respectively. With the addition of insulin, the relative rate of ribosomal protein synthesis averaged 0.33 at an aortic pressure of 60 mmHg and increased to 0.42 when aortic pressure was raised to 120 mmHg. These results indicate that elevation of aortic pressure has a preferential effect on synthesis of new ribosomes. This response appears to be an early and physiologically significant event in cardiac hypertrophy.
Compartmentation of carbohydrate metabolism in vascular smooth muscle
Authors: Lynch R. M. , Paul R. J.
Published: December 2017

In vascular smooth muscle, oxidative phosphorylation and glycolysis are independently regulated. Previous studies indicated that the independent regulation of these pathways was related to a compartmentation of carbohydrate metabolism. To further study carbohydrate metabolism, glucose transport and the incorporation of radiolabel from glucose into glycogen and lactate were measured after the oxidative and glycolytic pathways were independently altered. Ouabain stimulated mechanical activity, oxygen consumption, and glycogenolysis, whereas lactate production was decreased. Although glycogenolysis was substantial, glucose was the only substrate for lactate, indicating that intermediates derived from glycogen do not mix with those from glucose uptake. Thus glycogenolysis and glycolysis are carried out by independent enzymatic pathways. Insulin-stimulated lactate production and glucose transport without affecting the other parameters. Again, lactate was produced only from glucose. Phenytoin decreased isometric tension and oxygen consumption, whereas stimulating lactate production and glycogenolysis. Glycogen was the primary substrate for the lactate produced. Our findings indicate that the compartmentation of substrate utilization is ascribable to the coordination of glycogenolysis with increases in oxygen consumption and the coupling of glycolysis to the Na-K-adenosine triphosphatase. The coupling of independent energy providing pathways to specific endergonic processes indicates a mechanism by which cellular energetic efficiency may be optimized.
Effect of N6-(L-2-phenylisopropyl)adenosine and insulin on cAMP metabolism in 3T3-L1 adipocytes
Authors: Elks M. L. , Jackson M. , Manganiello V. C. , Vaughan M.
Published: December 2017

The antilipolytic effect of N6-(L-2-phenylisopropyl)-adenosine (PIA), an adenosine analogue thought to act via cell surface receptors, was investigated in 3T3-L1 adipocytes. PIA (1 microM) was as effective as 1 nM insulin in reducing lipolysis stimulated by 1 nM isoproterenol and more effective than insulin at higher isoproterenol concentrations. In intact adipocytes, PIA reduced isoproterenol-induced cyclic AMP (cAMP) accumulation and increased particulate cAMP phosphodiesterase. In particulate preparations PIA suppressed isoproterenol stimulation of adenylate cyclase. PIA was more effective than 5'-N-ethylcarboxamide adenosine (NECA) or adenosine in inhibiting adenylate cyclase and activating phosphodiesterase. In intact adipocytes, two agents with so-called “insulin-like” activities, i.e., anti-insulin receptor antibodies and wheat germ agglutinin (WGA), also increased particulate cAMP phosphodiesterase. Pertussis toxin, which inhibits stimulation of the particulate cAMP phosphodiesterase by insulin (but not by isoproterenol), also inhibited the effects of PIA, anti-insulin receptor antibodies, and WGA. In 3T3-L1 cells, PIA appears to inhibit lipolysis by inhibiting adenylate cyclase and stimulating phosphodiesterase; these effects of PIA, as well as those of anti-insulin receptor antibodies and WGA on phosphodiesterase, may be mediated via guanyl nucleotide-binding proteins.
Mitochondrial transmembrane potential and pH gradient during anoxia
Authors: Andersson B. S. , Aw T. Y. , Jones D. P.
Published: December 2017

The effect of anoxia on the mitochondrial transmembrane potential and pH gradient was studied in a preparation of isolated hepatocytes. Transmembrane potential (delta psi) was calculated from the distribution of triphenylmethylphosphonium between the mitochondrial, cytosolic, and extracellular compartments, which were separated by digitonin fractionation and centrifugation. Mitochondrial and cytosolic pH values were calculated from the distribution of the weak acid, dimethadione, which was determined similarly. After 30 min anoxia, the magnitude of mitochondrial delta psi was decreased from -163 to -133 mV and the delta pH (mitochondria vs. cytoplasm) was essentially unchanged (aerobic, 0.78 +/- 0.08; anaerobic, 0.76 +/- 0.11). Thus the protonmotive force (delta p = delta psi-Z delta pH), is largely retained even in the absence of electron flow and ATP synthesis. Inhibitors of the ATP synthase (oligomycin), mitochondrial adenine nucleotide carrier (atractyloside), and glycolytic pathway (2-deoxy-D-glucose) do not affect the ability of the cell to maintain delta psi during anoxia. Therefore, the results indicate that retention of the protonmotive force is not due to utilization of ATP produced by glycolysis and suggest that mechanisms exist to preserve ion distribution during anoxia.
Suppression of mitochondrial respiratory function after short-term anoxia
Authors: Aw T. Y. , Andersson B. S. , Jones D. P.
Published: December 2017

Exposure of rat hepatocytes to 30 min anoxia resulted in a substantial decrease in O2 consumption on reoxygenation. Measurement of the sequestered Ca2+ pool of mitochondria by selective release with the protonophore, carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and quantitation with the metallochromic indicator, arsenazo III, showed that anoxia caused a marked decrease in mitochondrial Ca2+. This loss could, in part, be due to decreased electrophoretic uptake resulting from a 20% decrease in the magnitude of the mitochondrial transmembranal potential. The decrease was associated with a decrease in ATP synthase activity as expected from the Ca2+ dependence of endogenous inhibitor binding to the ATP synthase. These results show that short-term anoxia suppresses mitochondrial function in hepatocytes and suggest that mitochondrial Ca2+ content may be important in this regulation. Regulation of the ATP synthase and other ion transport systems may provide a means to preserve ion distribution and protonmotive force and thereby prolong the period during which cells can tolerate anoxia.
Two types of cells with central innervation in pineal gland of guinea pigs
Authors: Parkington H. C. , McCance I. , Coleman H. A.
Published: December 2017

Cells within pineal glands isolated from young, male guinea pigs were impaled with intracellular microelectrodes and their responses to stimulate the nerve supply to the gland were studied. Two types of cells were identified. The response of cells of type I was a depolarization on which spikes were superimposed. Blockers of alpha-adrenoceptors abolished the spikes while beta-adrenoceptor blockers reduced the depolarization to 27%, leaving a small tetrodotoxin-sensitive depolarization. After bilateral removal of the superior cervical ganglia (SCG) the beta-mediated depolarization was not observed while the spikes and the smaller depolarization persisted. The response of cells of type II was an initial large, transient depolarization followed by a smaller depolarization. Both components were reversibly blocked by tetrodotoxin. The only agents found to have any effect on these cells were oxytocin, vasopressin, and vasotocin. These peptides caused depolarization similar in amplitude to the larger response to nerve stimulation, although more prolonged. The large depolarization was not observed following ganglionectomy, but the smaller one persisted. It is concluded that cells of type I and II both receive inputs from nerves whose cell bodies lie in the SCG. Cells of both types are also innervated through another pathway.
Insulin action on electrophysiological properties of apical and basolateral membranes of frog skin
Authors: Schoen H. F. , Erlij D.
Published: December 2017

We measured the effects of insulin on the current-voltage (I-V) relations of frog skins impaled with an intracellular microelectrode. The current across the cell membranes was assumed to be equal to the amiloride-inhibitable current. Insulin increased short-circuit current (Isc) approximately 40% from the control value. The increase in Isc was associated with a depolarization of the cell membrane. In addition there was an increase in the value of the parameters that describe the ease of movement of Na+ across the apical membrane, namely, slope conductance (ga), chord conductance (Ga), and permeability (PNa). The values of these parameters show remarkable linear correlations with membrane current both before and after stimulation. Intracellular Na+ activity (acNa) was determined from the I-V relations of the apical membrane. Insulin did not significantly modify acNa. Insulin also increased the value of the basolateral membrane conductance, however, the relationship between this parameter and current was complex. These experiments show that the stimulatory effect of insulin on Isc is associated with an increase in the conductance of both the apical and basolateral membranes.
Measurements on permeabilized skeletal muscle fibers during continuous activation
Authors: Sweeney H. L. , Corteselli S. A. , Kushmerick M. J.
Published: December 2017

A mechanical stabilization technique involving the cycling of calcium activated rabbit psoas fibers between states of isometric force development and isovelocity shortenings at near maximal velocity (Vmax) was evaluated. This protocol succeeded in stabilizing both the sarcomere striation pattern and mechanical performance (isometric force and velocity of unloaded shortening) of the fibers for up to 10 min of continuous maximal calcium activation at 12 degrees C and for greater than 15 min at half-maximal activation. Between these cycling events, a variety of mechanical measurements can be made. A description of the strategies involved in the measurements and specific illustrations are presented. A general description of a computerized fiber mechanics system, which utilizes separate microprocessors for experimental control and data collection, is given. The techniques should be of general applicability to muscle physiologists studying permeabilized preparations and offer the great advantage of providing reproducibility of measurement in a stable preparation.
Angiotensin II stimulates phospholipases C and A2 in cultured rat mesangial cells
Authors: Schlondorff D. , DeCandido S. , Satriano J. A.
Published: December 2017

Angiotensin II stimulates prostaglandin (PG) E2 formation in mesangial cells cultured from rat renal glomeruli. The interactions between angiotensin II and PGE2 are important in modulating glomerular function. We examined the mechanism for stimulation of PGE2 production in mesangial cells using the putative diacylglycerol-lipase inhibitor RHC 80267 and trifluoperazine (TFP), an agent interfering with Ca2+-CaM-mediated processes. Although RHC 80267 inhibited diacylglycerol-lipase activity in mesangial cells, it did not influence PGE2 production in response to either angiotensin II or A23187. In contrast, TFP (50 microM) inhibited basal PGE2 production and stimulation by angiotensin II and A23187. TFP also decreased 14C release in response to angiotensin from cells prelabeled with [14C]arachidonic acid, which was associated with inhibition of 14C loss from phosphatidylinositol. In cells prelabeled with 32P, orthophosphate angiotensin II caused a rapid hydrolysis of phosphatidylinositol 4,5-bisphospate. TFP enhanced 32P labeling of phosphatidylinositides, but did not prevent the loss of phosphatidylinositol 4,5-bisphosphate in response to angiotensin. This was verified in cells prelabeled with myo-[3H]inositol where angiotensin stimulated formation of [3H]inositol trisphosphate. TFP enhanced formation of [3H]inositol trisphosphate both under basal- and angiotensin II-stimulated conditions. Thus TFP did not inhibit phospholipase C activation by angiotensin. Angiotensin II caused marked increases in [32P]lysophospholipids, indicating activation of also phospholipase A2. This process was inhibited by TFP. Taken together, these results are consistent with stimulation of both phospholipase C and A2 by angiotensin, the latter step responsible for the release of arachidonic acid and PGE2 formation. The activation of phospholipase A2, but not that of phospholipase C, is inhibited by TFP, perhaps by interference with calmodulin-dependent steps.
Flow cytometric analysis of intracellular pH in 3T3 cells
Authors: Gillies R. J. , Cook J. , Fox M. H. , Giuliano K. A.
Published: December 2017

Techniques to determine intracellular pH generally report the average pH of population and do not indicate whether or not there is significant variance among cells within the population. Population variance is important to ascribe pH changes on a per cell basis. The magnitude of the pH change in individual cells is important to ascribe physiological function to changes in pH. To determine the variability of cell responses, we have used dual wavelength fluorescence emission spectroscopy of intracellular dicyanohydroquinone monitored with flow cytometry to determine the pH of normal and transformed 3T3 cells in response to serum or serum components. All cells were mechanically harvested from subconfluent cultures. Large differences in pH were observed between serum-deprived and serum-conditioned normal, but not transformed, cells. Addition of serum caused cytosolic alkalinization, with the serum-deprived cells responding more slowly. Titration of cells with submaximal doses of serum indicate that the response of pH is graded, that all cells respond in similar manner, and that the relative affinity of transformed cells for the serum components causing the pH effect is about twice that of normal cells.
La3+, Mn2+, and Ni2+ effects on Ca2+ pump and on Na+-Ca2+ exchange in bullfrog ventricle
Authors: Brommundt G. , Kavaler F.
Published: December 2017

45Ca washout from ventricles of the bullfrog was followed through an automated technique previously described. Lanthanum decreased the fractional rate of loss of 45Ca (by 67% at the saturating concentration, 1 mM, for this effect of the trivalent cation). In contrast, both manganous and nickel ions decreased the fractional rate of loss of 45Ca in 21 of the 30 ventricles studied. In the remainder of the experiments these ions increased this rate. This augmentation of the rate of 45Ca efflux by manganese and nickel could be converted consistently to an inhibition by experimental maneuvers known to increase the intracellular concentration of sodium-potassium-free washout solution, dihydroouabain. These results are consistent with the hypothesis that manganese and nickel increase 45Ca efflux through a process of sarcolemmal manganese-calcium (or nickel-calcium) exchange, operating through the sodium-calcium exchanger. Support for this interpretation was obtained in the further observation that the rate of loss of radioactive nickel from 63Ni-loaded ventricles was monotonically related to the extracellular concentration of calcium. Thus the augmenting effect of the divalent cations on 45Ca efflux appears to be mediated by the sodium-calcium exchanger. That the inhibitory effect arises from an action of the cations on the ATP-dependent component of 45Ca efflux is indicated by the fact that it disappears after vigorous ATP depletion in the presence of cyanide and 2-deoxy-D-glucose.
Differences in CuZn superoxide dismutase induction in lungs of neonatal and adult rats
Authors: Hass M. A. , Massaro D.
Published: December 2017

The failure of adult rats to survive prolonged exposure to greater than 95% O2 is generally ascribed to the inability of their lungs to increase antioxidant enzyme synthesis in response to the oxidant challenge. We studied the synthesis rate of the antioxidant enzyme CuZn superoxide dismutase (CuZn SOD) in lungs of adult and neonatal rats exposed to conditions that alter the lung's oxidant-to-antioxidant balance. Lung CuZn SOD synthesis in the adult was significantly increased after 24 h of hyperoxia but fell to control levels after further exposure, whereas in neonatal lungs an increased rate of synthesis of CuZn SOD was found only after 72 h of hyperoxia. The adult lung responded to two in vitro oxidant stresses, [diethyldithiocarbamate exposure and heat (42 degrees C)] with increases in CuZn SOD synthesis twice the magnitude of those in the neonatal lung. These data indicate that the adult lung is at least as capable as the neonatal lung of increasing its synthesis of CuZn SOD in response to an oxidative stress. However, the inability of the adult lung to maintain an increased rate of CuZn SOD synthesis during in vivo hyperoxia may contribute to the poor tolerance of the adult lung to greater than 95% O2.
Effect of N-ethylmaleimide on K transport in density-separated human red blood cells
Authors: Berkowitz L. R. , Walstad D. , Orringer E. P.
Published: December 2017

N-ethylmaleimide (NEM) is a sulfhydryl-reacting agent known to stimulate chloride-dependent K transport in a variety of red cells. In high K sheep red cells, NEM-induced K movements are greater in magnitude in young cells compared with old cells. We hypothesized that human red cells might respond to NEM like high K sheep red cells. To test this idea, cells of various age were exposed to 0.5 mM NEM. We found that, after a 4-h incubation, young cells lost 50% of cell K, compared with 10% K loss in older cells. K loss in all fractions was inhibited by chloride replacement or furosemide.
Diphenylamine-2-carboxylate blocks Cl(-)-HCO3- exchange in Necturus gallbladder epithelium
Authors: Reuss L. , Costantin J. L. , Bazile J. E.
Published: December 2017

Intracellular microelectrode techniques were employed to study the effects of diphenylamine-2-carboxylate (DPC) on ion transport in Necturus gallbladder epithelium. Under control conditions, addition of DPC to the mucosal bathing solution caused a concentration-dependent, reversible hyperpolarization of both cell membranes with no measurable resistance changes. In addition, DPC caused the following effects, all consistent with inhibition of apical membrane Cl(-)-HCO3- exchange: fall in intracellular Cl- activity (aCli), increase in intracellular pH (pHi), reduction of the changes in aCli and pHi produced by lowering mucosal solution [Cl-], and reduction of the change in pHi produced by lowering mucosal solution [HCO3-]. Similar studies in theophylline-treated preparations indicate that DPC also inhibits anion exchange under these conditions, but has no effect on the apical membrane electrodiffusive Cl- permeability induced by cyclic AMP. Under these conditions, DPC caused cell membrane hyperpolarization but had no effect on the apparent ratio of membrane resistances. In addition, DPC had no effects on the rapid changes in apical membrane voltage elicited by altering mucosal [Cl-], but caused significant reductions of the slower, secondary voltage changes observed in response to changes in mucosal [Cl-], and the changes in aCli and pHi produced by lowering mucosal [Cl-]. Because others have demonstrated that DPC blocks Cl- channels in other epithelia (Distefano, A., M. Wittner, E. Schlatter, H. J. Lang, H. Englert, and R. Greger. Diphenylamine-2-carboxylate, a blocker of the Cl(-)-conductive pathway in Cl(-)-transporting epithelia. Pfluegers++ Arch. 405: S95-S100, 1985), it is possible that the structures of those channels and that induced by cyclic AMP in Necturus gallbladder are different. Because of its relatively high affinity and rapid reversibility, DPC may become useful in studies of anion exchange in other cells.
Muscarinic cholinergic inhibition of adenylate cyclase in airway smooth muscle
Authors: Jones C. A. , Madison J. M. , Tom-Moy M. , Brown J. K.
Published: December 2017

The goal of our study was to test for an inhibitory effect of acetylcholine on adenylate cyclase activity in canine trachealis muscle. Therefore, cells were dispersed from the muscle enzymatically and lysed, and adenylate cyclase activity was assayed in a membrane suspension isolated from the lysates. Maximal beta-adrenergic stimulation, in the presence of GTP (10(-4) M), increased the activity of adenylate cyclase twofold above the activity induced by GTP alone. In the presence of GTP, acetylcholine (10(-4) M) decreased activity from 97 +/- 21 to 55 +/- 13 pmol cyclic AMP X min-1 X mg protein-1 (means +/- SE; n = 5; P less than 0.05); in the presence of GTP plus isoproterenol (10(-4) M), the acetylcholine-induced decreases were from 163 +/- 29 to 101 +/- 15 pmol cyclic AMP X min-1 X mg protein-1 (P less than 0.05). These decreases were dose dependent and they were altered by a series of cholinergic agents in a pattern consistent with a muscarinic effect. Our results suggest that one biochemical effect of vagal stimulation in the central airways of dogs may be attenuated adenylate cyclase activity in the smooth muscle.
Inhibition by amiloride analogues of Na+-dependent hexose uptake in LLC-PK1/Cl4 cells
Authors: Cook J. S. , Shaffer C. , Cragoe E. J.
Published: December 2017

Amiloride and four analogues of amiloride were shown to inhibit Na+-dependent, phlorizin-sensitive hexose uptake by a clone of pig kidney cells, LLC-PK1/Cl4. The analogues tested were: 5-(N-ethyl-N-isopropyl)amiloride (EIPA), 5-(N-methyl-N-isobutyl)amiloride (MIBA), 3',4'-dichlorobenzamil, and phenamil. The transport substrate was the nonmetabolizable glucose analogue alpha-methyl-D-glucoside. Blockade of Na+-K+ transport at the basolateral membranes or removal of divalent cations from the assay medium had little effect on the initial rate of hexose uptake, whereas MIBA remained an effective inhibitor under both conditions. The inhibitions by EIPA of Na+-H+ exchange and hexose-dependent Na+ uptake could be distinguished by appropriate choice of concentrations of the inhibitor. Hexose transport inhibition does not appear to be secondary to other known effects of the amilorides. Inhibition by all analogues is enhanced when they are tested in low (2 mM) Na+ medium, where they show half-maximal inhibition in the range of 100-300 microM. More detailed kinetic analysis of inhibition by EIPA shows it to be competitive with Na+ with a Ki of 73-107 microM. It is concluded that the amilorides are acting directly on the hexose transporter.
Photolabeling of a 150-kDa (Na + K + Cl) cotransport protein from dog kidney with a bumetanide analogue
Authors: Haas M. , Forbush B.
Published: December 2017

(Na + K + Cl) cotransport is the major mechanism of salt transport across the apical membrane of the epithelial cells of the thick ascending limb of Henle's loop of mammalian kidney and the site of action of "loop" diuretics such as furosemide and bumetanide. We have identified a 150-kDa protein in membranes from dog kidney cortex that is photolabeled by a radiolabeled, benzophenone analogue of bumetanide, [3H]4-benzoyl-5-sulfamoyl-3-(3-thenyloxy)benzoic acid ([3H]BSTBA). Several pieces of evidence strongly suggest that this 150-kDa protein is at least part of the (Na + K + Cl) cotransport system. 1) Photoincorporation of [3H]BSTBA into this protein is completely blocked by inclusion of 10 microM unlabeled bumetanide in the photolysis medium. 2) Photoincorporation of [3H]BSTBA into this protein shows a saturable dependence on [3H]BSTBA concentration, with a K 1/2 (approximately 0.1 microM) very similar to that for reversible [3H]BSTBA binding to kidney membranes. 3) Photolabeling of this protein by [3H]BSTBA requires the simultaneous presence of Na, K, and Cl in the photolysis medium. 4) When crude membranes from dog kidney cortex are centrifuged on sucrose density gradients, saturable [3H]bumetanide binding and photoincorporation of [3H]BSTBA in the 150-kDa region show a very similar distribution among the 15 gradient fractions collected. [3H]BSTBA is also photoincorporated into at least two lower molecular mass proteins, the largest of which is approximately 50 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)
Coupling of aerobic glycolysis and Na+-K+-ATPase in renal cell line MDCK
Authors: Lynch R. M. , Balaban R. S.
Published: December 2017

The relation between the activity of the Na+-K+-ATPase and the metabolic source of ATP was investigated in suspensions of MDCK cells. The pump activity of Na+-K+-ATPase was estimated from the initial rate of ouabain-sensitive K+ uptake into K+-depleted cells. Uptake was initiated by the reintroduction of K+ to the medium in which the cells were suspended. The metabolic source of ATP was varied by changing the substrates supplied to the suspension. Cells respiring on glutamine produced ATP from oxidative metabolism alone, whereas cells incubated with glucose and glutamine produced ATP via glycolysis and oxidative phosphorylation. Over a wide range of extracellular K+ concentrations, the initial rate of K+ uptake was faster in cells incubated with glucose and glutamine when compared with cells incubated with glutamine alone. Kinetic analysis together with ouabain-binding data demonstrated that this increase in K+ uptake was due to an increase in maximal velocity (Vmax) at a constant number of Na+-K+-ATPase transport sites. In addition, steady-state studies revealed that the addition of glucose to K+-depleted cells respiring on glutamine alone resulted in a net ouabain-sensitive influx of K+. These data demonstrate that in MDCK cells the maximal capacity for transport via the Na+-K+-ATPase is greater when ATP is produced from both glycolysis and oxidative phosphorylation than when ATP is produced from oxidative phosphorylation alone.(ABSTRACT TRUNCATED AT 250 WORDS)
Effects of 12-HETE on renal tubular epithelial cells
Authors: Gordon J. A. , Spector A. A.
Published: December 2017

The interaction of 12-HETE with MDCK cells was investigated to determine whether this lipoxygenase product might influence tubular epithelial function. MDCK cells took up 12-HETE from both the luminal and interstitial surfaces. Much of the 12-HETE was incorporated into phospholipids but, unlike arachidonic acid, there was no specificity for the sn-2 position. 12-HETE was incorporated without chemical modification, suggesting that it might perturb tight packing in the lipid bilayer. 12-HETE uptake was reduced by the presence of arachidonic acid, but not by oleic acid. 12-HETE was removed from the cell lipids more rapidly than arachidonic acid and it was released into the medium in the form of both more and less polar metabolites. When MDCK cells were incubated with 12-HETE, their capacity to produce prostaglandin E2 was reduced. Since 12-HETE enters epithelial phospholipids and is converted to metabolic products and reduces prostaglandin production, it apparently has the capacity to modulate renal function if it is released in the proximity of the tubular epithelium.
Distinction between mechanisms underlying alpha 1- and beta-adrenergic respiratory stimulation in brown fat cells
Authors: Mohell N. , Connolly E. , Nedergaard J.
Published: December 2017

Experimental conditions are described for selective alpha 1- and beta-adrenergic stimulation of the respiration of brown fat cells. The dual agonist norepinephrine was unsuitable as a selective alpha 1-agonist, since unacceptably high concentrations of propranolol were needed to abolish the beta-response. Phenylephrine at 50 microM, in the presence of 5 microM dl-propranolol, was shown to lead to a maximal, selective alpha 1-stimulation, whereas maximal, selective beta-stimulation was achieved with 1 microM isoproterenol in the presence of 5 microM prazosin. The mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) was able to further increase respiration that was already maximally alpha 1-stimulated, but when added before the alpha 1-stimulation, FCCP totally abolished the response. In contrast, FCCP had no effect on the beta-stimulated response. Similarly, oligomycin (an inhibitor of mitochondrial ATP synthesis) inhibited alpha 1-respiration but had a much smaller effect on beta-respiration. Ouabain (an inhibitor of the Na+-K+-ATPase) halved alpha 1-respiration but only induced a small inhibition of beta-respiration. It is concluded that only a small fraction of thermogenesis from beta-adrenergic processes is due to oxidative phosphorylation, whereas alpha 1-respiration is largely due to the oxygen cost of mitochondrial ATP synthesis, and a large fraction of this ATP is apparently used for the restoration of ion gradients.
Reversible MM-creatine kinase binding to cardiac myofibrils
Authors: Ventura-Clapier R. , Saks V. A. , Vassort G. , Lauer C. , Elizarova G. V.
Published: December 2017

Skinned rat papillary muscles and purified preparations of rat cardiac myofibrils were used to study the nature of the interaction of creatine kinase with cardiac myofibrils. High activity of creatine kinase (2 IU/mg protein in fibers and 0.9 IU/mg in purified myofibrils) was due mostly to reversibly bound enzyme. This activity could be removed and rebound. The process of creatine kinase rebinding was characterized by apparent Km value of 0.14 mg/ml (approximately equal to 2 X 10(6) M). Rebinding of creatine kinase to cardiac myofibrils restored the phenomenon of functional compartmentation of adenine nucleotides in myofibrillar space and restored the ability of phosphocreatine to decrease the rigor tension in the presence of MgADP. The physiological experiments with quick length changes showed that rebinding of creatine kinase to skinned papillary muscle also restored Ca sensitivity, increased maximal tension development, decreased stiffness, and restored the tension recovery after quick length changes in muscle under condition of inhibition of endogenous creatine kinase by 1-fluoro-2,4-dinitrobenzene. It is concluded that creatine kinase reversibly bound to cardiac myofibrils is involved in the energy supply for cardiac contraction.
Stiffness of active smooth muscle during forced elongation
Authors: Meiss R. A.
Published: December 2017

The stiffness of isometrically contracting mesotubarium superius and ovarian ligament smooth muscle from estrous female rabbits was measured continuously by using sinusoidal length perturbations (at 80 Hz, less than 15 microns peak to peak). Muscles were stimulated with alternating current fields, and all records were digitized using a microcomputer system. Phase-angle data were used to resolve computed stiffness into elastic and viscous components. Stiffness measurements were continued during long ramp-type stretches (up to 25% of muscle length) delivered as soon as force was maximal. To use the period of isometric tension development as a standard for comparison, the expected stiffness was computed during the long stretch. Stiffness was reduced in approximate proportion to the ramp stretch rate, and the reduction was confined largely to the elastic component. Cooling the muscle increased the stiffness deviation at a given stretch rate. It is proposed that the long stretch detaches cross bridges that can reattach to new sites as myofilaments shear past one another. At higher shearing speeds, less time is available for reattachment and stiffness is further reduced.