bluuebunny/crossref_metadata_2025 huggingface dataset:
Magnesium increases rate of onset of desensitization in frog muscle
Authors: Manthey A. A.
Published: December 2017

The effect of changes in extracellular and intracellular magnesium content on desensitization rate in frog (Rana pipiens) sartorius muscle was studied using measurements of input conductance of single fibers during local superperfusion of the postjunctional region with carbamylcholine chloride (0.27–2.7 mM). Two intracellular KCl-filled glass capillary microelectrodes were used for current injection and recording in fibers equilibrated in high-K+ media (33–165 mM). In low-ionic strength solutions (33 mM K), time to half-decline of conductance during desensitization to carbamylcholine (T1/2) was decreased by increasing extracellular Mg2+ concentration throughout range 0-100 mM. Equivalent effects on T1/2 were produced by increased extracellular Ca2+ in lower concentrations (0–10 mM). Increase in K+ concentration or decrease of carbamylcholine concentration resulted in increased T1/2 in both Mg2+ and Ca2+ media. Increase of intracellular Mg2+ by soaking in high-Mg2+ solutions (100 mM) or by intracellular iontophoresis caused decrease in T1/2. It is concluded that Mg2+, when introduced either in the extracellular or intracellular phase, can promote increased desensitization rate and that this action is similar to but weaker than that of calcium.
Macrophage phagocytosis: analysis of particle binding and internalization
Authors: Besterman J. M. , Airhart J. A. , Low R. B.
Published: December 2017

An improved radioassay for analysis of phagocytosis has been used to quantitate and characterize the binding and internalization of zymosan by monolayers of rabbit pulmonary alveolar macrophages. This method distinguishes zymosan particles reversibly bound to the cell surface from those internalized. The zymosan was radiolabeled with technetium 99m(99mTc), a gamma-emitter with a 6-h half-life. Use of 99mTc as the radiolabel also permitted simultaneous determinations of pinocytosis and cellular protein content using [14C]sucrose and [3H]amino acids, respectively. All endocytic data were normalized per adherent cell based on this latter measurement. A significant fraction of the cell-associated particles was bound to the cell surface but not internalized. Failure to correct for this compartment would have resulted in overestimation of phagocytic rate and total cellular capacity. Both binding and ingestion of functionally unopsonized zymosan were found to be saturable, temperature sensitive, dependent on glycolytic energy, dependent on a trypsin-sensitive membrane component, described by a maximal rate, and limited by a finite capacity. The time courses of both processes were found to be similar. These results led us to conclude that, in our system, both binding and internalization were active processes and that the limited capacity to ingest zymosan was not explained by a concomitant reduction in binding of the particle to the cell membrane. Furthermore, it was found that phagocytosis did not change the rate of fluid-phase pinocytosis, consistent with the concept that the cell membrane is a functional mosaic as has been previously found by others for phagocytic and transport sites in this cell type.
Transepithelial potential during strontium-induced spontaneous sweating
Authors: Sato K. , Sato F.
Published: December 2017

Transepithelial electrical potential difference (PD) of the secretory portion of the rhesus monkey palm eccrine sweat gland was studied in both Ca2+-containing (control and Ca2+-free Sr2+ media. In the control medium, the luminal PD was a saturation function of the methacholine (MCH) concentration, reaching a plateau at about -6 mV (lumen negative) at 10(-5) M MCH. In the Sr2+ medium, the luminal PD increased spontaneously with a time course similar to that the spontaneous sweat secretion (SSS). Like SSS, the spontaneous luminal PD in the Sr2+ medium is a saturation function of the Sr2+ concentration in the bath, reaching a plateau at -9 mV at 8 mM Sr2+. Addition of MCH to the Sr2+ medium caused a rapid increase in the lumen-negative PD as seen in the control medium. The spontaneous lumen-negative PD was abolished by subsequent addition of Ca2+ to the bath, as was the SSS [K. Sato, Am. J. Physiol. 239 (Cell Physiol. 8): C90–C97, 1980]. The data further reinforce the notion that Sr2+-induced SSS represents true secretory processes which are triggered by Sr2+ leaking into the cell bypassing the receptor-agonist interaction.
Influence of presynaptic receptors on neuromuscular transmission in rat
Authors: Wilson D. F.
Published: December 2017

The presence and physiological significance of acetylcholine (ACh) receptors on motor nerve terminals was examined at the rat diaphragm neuromuscular junction. Intracellular recording techniques were used to monitor end-plate potentials (EPP), miniature end-plate potentials (MEPP), and resting potentials of the muscle fibers. Muscle action potentials were blocked by the cut-muscle technique. Quantal release was determined by the ratio EPP/MEPP, after correcting for nonlinear summation. Blockade of acetylcholinesterase with eserine and neostigmine was tested to determine the influence of residual ACh on transmitter release. Partial blockade of ACh receptors with curare was examined to further clarify the role of these presynaptic receptors. The experiments demonstrate that residual ACh inhibits transmitter release and that blockade of ACh receptors enhances transmitter release. It is concluded that presynaptic ACh receptors exist and that they serve an important physiological function. It is suggested that the presynaptic ACh receptors normally serve to limit transmitter release in a negative feedback pathway.
Release of creatine kinase from frog muscle by osmotic changes
Authors: Suarez-Kurtz G.
Published: December 2017

The rate of creatine kinase (CK) release from isolated frog extensor longus digiti IV (,toe”) muscle increased on transfer from the control Ringer solution (referred to as 1.0 T) to a hyperosmotic solution (1.0 T plus 400 mM glycerol) or to hypertonic solutions (1.5–2.5 T) prepared by addition of NaCl or sucrose to 1.0 T. On return to 1.0 T, CK release rate was further elevated, reaching peak values (14.4- and 12-fold above the basal value, respectively, with the glycerol treatment and with solution 2.5 T) within 10–15 min and declining subsequently toward base line. Although the stimulation of CK release by changes in external tonicity increased with the magnitude of the osmotic step change, there was no enhancement of release when the tonicity changes were performed gradually in 0.1-T steps at 1-min intervals. The stimulation of CK release by tonicity changes cannot be ascribed to irreversible loss of functional integrity of the surface or tubular membranes or to CK diffusion through aqueous pores. The possibility that an exocytosislike process is involved is discussed.
2-Deoxy-D-glucose uptake by rat granular pneumocytes in primary culture
Authors: Kerr J. S. , Reicherter J. , Fisher A. B.
Published: December 2017

Uptake of 2-deoxy-D-glucose (DG) was investigated with rat granular pneumocytes isolated in primary culture. Cells attached to flasks were incubated in Minimal Essential Medium usually containing 5 mM DG in place of glucose. Uptake of DG increased progressively with time of incubation and approached a plateau value of 35-40 mumol/10(6) cells at 60 min. Uptake increased as a function of external DG concentration with half-maximal uptake at approximately 2.0 mM DG. DG uptake was inhibited by the presence of glucose, alpha-methylglucoside, phlorizin, ouabain, or sodium-free medium. After 60 min incubation, approximately 20% of total intracellular DG was in the free form, and the calculated mean intracellular concentration of free DG (n = 4) was approximately twice the external concentration. Phosphatase activity was indicated by increase in free DG and efflux from cells after removal of external DG. In comparison with pneumocytes, uptake of DG by alveolar macrophages showed different kinetics, and intracellular free DG did not exceed the extracellular concentration. These findings indicate that type II cells take up DG by a sodium-dependent, carrier-mediated transport process that results in accumulation of free sugar against a concentration gradient.
Sodium tracer kinetics and transmembrane flux in tissue-cultured chick heart cells
Authors: Wheeler D. M. , Horres C. R. , Lieberman M.
Published: December 2017

Considerable difficulty has been encountered in defining the physiological significance of sodium tracer kinetic measurements in cardiac muscle. In this study, 24Na+ efflux experiments were performed by directly monitoring tissue radioactivity during the superfusion of growth-oriented embryonic chick heart cells in tissue cultured. The cellular 24Na+ efflux from contractile preparations exhibited at least two exponential components whereas noncontractile, fibroblastlike preparations had a single efflux component similar in rate to the slower component of the contractile preparations. We concluded that the slow component represents efflux from nonmuscle cells, whereas the faster component reflects the muscle cell compartment. The mean Na+ efflux rate constants for contractile preparations (beating 150 min-1) were 3.1 and 0.35 min-1. Intracellular Na+ concentrations, as determined by isotope uptake and by flame photometry, were 18 and 16 mM for contractile and nonmuscle preparations, respectively. The steady-state, transmembrane fluxes are 98 and 5 pmol . cm-2 . s-1 for muscle and nonmuscle cells, respectively. The Na+ efflux kinetics in 10(-4) M ouabain were reduced by approximately 16% from the control value. These findings indicate that the greater part of the steady-state Na+ efflux in cultured heart cells is due to mechanisms other than the Na+-K+ pump.
Regulation of purine metabolism by plasma membrane and cytoplasmic 5'-nucleotidases
Authors: Edwards N. L. , Recker D. , Manfredi J. , Rembecki R. , Fox I. H.
Published: December 2017

The contribution of plasma membrane 5'-nucleotidase (E.C. 3.1.3.5) to intracellular purine degradation and release was evaluated in cultured human lymphoblasts. B-lymphoblasts and T-lymphoblasts are characterized by high and low levels of plasma membrane 5'-nucleotidase activity, respectively. After radiolabeling of the cellular adenine nucleotide pools with [8-14C]adenine, deoxyglucose-induced purine nucleotide degradation resulted in a 2-2.5 times greater release of cellular radioactivity from the B-lymphoblasts than from the T-lymphoblasts. Specific inhibition of plasma membrane 5'-nucleotidase with 50 microM alpha, beta-methylene adenosine diphosphate (AMPCP) did not decrease purine release during deoxyglucose-induced nucleotide degradation. Similarly, the inhibition of B-lymphoblast membrane 5-nucleotidase did not alter the incorporation of [8-14C]adenine into the nucleotide pool. Therefore, to explain the relatively high release of purine nucleotide degradation products in B-lymphoblasts when compared with T-lymphoblasts, cytoplasmic 5'-nucleotidase activity was investigated in these cell lines. B-lymphoblasts have seven times more cytoplasmic 5'-nucleotidase activity for dAMP and two to three times more activity for other purine nucleoside 5'-monophosphates than do T-lymphoblasts at pH 7.4. Membrane and cytoplasmic nucleotidase activities are produced by different enzymes that can be distinguished by differences in pH optima, Michaelis constants for purine substrates, divalent cation requirements, and susceptibilities to AMPCP inhibition. The data suggest that plasma membrane 5'-nucleotidase hydrolyzes extracellular nucleoside 5'-monophosphates only. Cytoplasmic 5'-nucleotidase most likely regulates the degradation of intracellular nucleoside 5'-monophosphates and may be responsible for the increased purine release observed in B-lymphoblasts.
Na+-dependent hexose transport in vesicles from cultured renal epithelial cell line
Authors: Moran A. , Handler J. S. , Turner R. J.
Published: December 2017

Apical membrane vesicles were prepared from cultured epithelia formed by LLC-PK1 cells using a calcium precipitation technique. alpha-Methylglucoside uptake into this vesicle preparation was markedly stimulated by sodium and inhibited by phlorizin. In addition, a transient "overshoot" of intravesicular alpha-methylglucoside concentration above its equilibrium value was observed under initial sodium gradient conditions. The specificity of this sodium-dependent hexose transporter closely resembled that found in the mammalian kidney brush border membrane, e.g., alpha-methylglucoside, D-glucose, and D-galactose apparently share the transporter while 2-deoxy-D-glucose, mannose, and fructose do not. Kinetic analysis of the sodium-dependent component of alpha-methylglucoside flux into LLC-PK1 apical membrane vesicles indicates the existence of single transporter with Km congruent to 2 mM and Vmax congruent to 3 nmol.min-1.mg protein-1. Measurement of alpha-methylglucoside uptake as a function of sodium concentration is consistent with a sodium:sugar stoichiometry of approximately 2:1.l There is a good correlation over time between the development of the concentrating capacity of the intact epithelium for alpha-methylglucoside and the transport properties of the vesicle preparation.
Energetics of isometric contractions as a function of muscle temperature
Authors: Rome L. C. , Kushmerick M. J.
Published: December 2017

The energetic cost of generating isometric force in isolated frog muscle was examined at 10, 20, and 30 degrees C. Recovery O2 consumption (delta O2) and recovery lactate production (delta Lact) were measured under conditions in which O2 was not limiting metabolism. Both increased linearly with the force-time integral (integral of Fdt) generated by the muscle. The slopes of the regression equations for both delta O2 and delta Lact as a function of integral of Fdt increased with increasing temperature with a temperature coefficient (Q10) near 3. Total high-energy phosphate resynthesis from recovery metabolism was calculated by scaling the delta O2 regression equation and the delta Lact regression equation into equivalent ATP units and summing them. This total recovery metabolism was modeled as the sum of two components, a "cost of maintaining force" (slope of the equation) and a saturable "start up cost" (intercept of the equation). The cost of maintaining force increased with temperature with a Q10 near 3 over the whole temperature range, whereas the start up cost was nearly independent of temperature between 0 and 20 degrees C and fell to near zero at 30 degrees C. Delta O2 measurements from a series of tetani given in rapid succession showed that for contractions subsequent to the first, no start up cost was incurred and that the "cost of generating force" for these contractions was equal to the slope of the regression line for single tetani. The practical consequence of these facts is that, in series of tetani, the cost of generating force increases with a Q10 of 3.
Antiserum to liver insulin receptor hyperpolarizes rat muscle
Authors: Zierler K. , Rogus E. M.
Published: December 2017

Antibodies to insulin receptors have been reported to have some insulinlike metabolic effects. If insulin-induced electrical hyperpolarization of skeletal muscle is part of the transduction chain between receptor and certain metabolic responses, then receptor antiserum should hyperpolarize. The Jacobs antiserum (rabbit antiserum against rat liver insulin receptor) hyperpolarized rat caudofemoralis muscle. Maximum effect, about 4.5 mV, occurred at 1:10,000 dilution, half maximum at about 1:40,000. Maximum effect of antiserum was only as great as half maximum hyperpolarization by insulin on this muscle. 2-Deoxyglucose uptake was also stimulated by antiserum but required greater concentration than for hyperpolarization, and the stimulation was only by about one-third the maximum effect of insulin.
Na-Ca exchange in a smooth muscle plasma membrane-enriched fraction
Authors: Grover A. K. , Kwan C. Y. , Rangachari P. K. , Daniel E. E.
Published: December 2017

Na-Ca exchange in the plasma membrane-enriched fraction N1 isolated from rat myometrium has been demonstrated as Ca gain or loss depending on the direction of the [Na+] gradient [Grover et al., Am. J. Physiol. 240 (Cell Physiol. 9): C175-C182, 1981]. In this study, the [Na+]gradient-dependent Ca uptake by this membrane fraction was demonstrated to occur optimally at pH 6.67; and 7.0 +/- 2.2 microMs Ca2+ was required for the half-maximal uptake. Such Ca uptake occurred in vesicles previously loaded with a [Ca] that was the same as or higher than the one present in the Ca-uptake medium, i.e., as an uphill Ca-transport process. The Na-Ca exchange-dependent Ca uptake was abolished by the Ca ionophore A23187 or by the monovalent cation (Na+) ionophore monensin. It was not influenced by the presence of KC1, KC1 plus valinomycin, or the proton uncouplers carbonyl cyanine p-trifluoromethoxyphenyl hydrazone or 5'-chloro-3-t-butyl-2'-chloronitrosalicylanilide in the Ca-uptake medium. The properties of the Na-Ca exchange are compared with those of passive and ATP-dependent Ca uptake by this membrane fraction, and physiological implications of this process are discussed.
Effect of alloxan diabetes on a Ca2+-activated proteinase in rat skeletal muscle
Authors: Brooks B. A. , Goll D. E. , Peng Y. S. , Greweling J. A. , Hennecke G.
Published: December 2017

The effect of making rats diabetic by alloxan injection on activity of the muscle Ca2+-activated proteinase (CAF) was investigated. Groups of four to seven control or alloxan-injected rats were killed 10 min (0 day) and 10, 17, and 24 days after a second alloxan injection. The second alloxan injection was given 3 days after the first. CAF activity was assayed in fractions precipitated between 0 and 45% ammonium sulfate saturation (P0-45 crude CAF fractions) that had been prepared so as to remove the protein inhibitor of CAF. Gel permeation, followed by DEAE-cellulose chromatography of pooled P0-45 crude CAF fractions from each time-treatment group, demonstrated that the assays used in this study were specific for CAF activity. Muscle CAF activity was up to 50% higher in alloxan-injected rats than in control rats, regardless of whether activity was expressed per gram sarcoplasmic protein, per gram contractile protein, or per gram skeletal muscle fresh weight. Alloxan injection diminished rate of muscle mass accumulation but did not change the proportion of sarcoplasmic or contractile protein in skeletal muscle. Hence, alloxan injection decreased the rate of contractile protein deposition. The elevation of muscle CAF activity in alloxan-injected rats is consistent with the proposed role of CAF in initiating metabolic turnover of myofibrillar proteins but does not prove this role nor exclude participation of other proteinases.
Myosin phosphorylation and regulation of cross-bridge cycle in tracheal smooth muscle
Authors: Gerthoffer W. T. , Murphy R. A.
Published: December 2017

We have tested the hypothesis that phosphorylation of the 20,000-dalton myosin light chains (LC 20) in rabbit tracheal smooth muscle modulates cross-bridge kinetics and isotonic shortening velocity. The thin muscle [190 +/- 10 (SE) microns] allowed detection of rapid changes in carbachol-induced active stress development, LC 20 phosphorylation, and isotonic shortening velocities. Phosphorylation of the LC 20 in resting muscle was 0.12 +/- 0.04 mol Pi/mol LC 20. Carbachol (10(-5) M) increased the level of phosphorylation to 0.46 +/- 0.03 mol Pi/mol LC 20 within 30 s. Phosphorylation then declined significantly as steady-state active stress was reached. A positive correlation was always found between LC 20 phosphorylation and shortening velocity. This result supports the hypothesis that the level of myosin phosphorylation was related to the mean cross-bridge cycling rate rather than the number of cross bridges contributing to the developed stress. Dephosphorylation of LC 20 occurred at about the same rate as the decline in shortening velocity and stress upon stimulus washout.
Increased phosphate efflux from acinar cell during protein secretion
Authors: Melese T. , Rothman S. S.
Published: December 2017

The permeability of the pancreatic epithelium to two water soluble molecules, sucrose and inulin, increases when protein secretion is augmented by a cholinergic agonist. An increase in the permeability of passive paracellular shunts (3) has been proposed to account for these observations. In the present experiments we have measured the distribution of another molecule, phosphate ion, across the epithelium by following its secretion from the cannulated duct of whole-rabbit pancreas in short-term organ culture. A cholinergic stimulant increases phosphate ion concentration in secretion in a similar fashion to that seen for the watersoluble nonelectrolytes. However, both the unstimulated rate of phosphate secretion and the increase observed with cholinergic stimulation were not dependent on the presence of the ion in the medium, and therefore its secretion in both cases reflects phosphate efflux from the cell and not its paracellular transport. The results indicate that either the phosphate ion is excluded from paracellular shunts or that such shunts do not contribute substantially to the transpancreatic passage of molecules of this size.
Insulin effects on protein synthesis are independent of glucose and energy metabolism
Authors: Flaim K. E. , Kochel P. J. , Kira Y. , Kobayashi K. , Fossel E. T. , Jefferson L. S. , Morgan H. E.
Published: December 2017

Protein synthesis was accelerated in rat hearts that were provided insulin compared with provision of glucose or pyruvate alone or a mixture of glucose and pyruvate. The faster synthetic rates were accompanied by a reduction in numbers of ribosomal subunits, indicating that peptide chain initiation was accelerated relative to elongation/termination. In hearts supplied glucose, 65% of the maximal effect on protein synthesis was achieved by addition of 1.7 X 10(-10) M insulin, but significant effects on glucose uptake as well as on tissue contents of glucose 6-phosphate and creatine phosphate were obtained only with 7 X 10(-10) M insulin. Addition of glucose to perfusates containing pyruvate did not accelerate protein synthesis, although the glucose 6-phosphate content was raised. Similarly, the stimulatory effects of insulin on protein synthesis in hearts supplied pyruvate did not depend on changes in glucose 6-phosphate content, creatine phosphate/creatine, ATP/ADP, or adenylate energy charge. These studies indicate that insulin accelerated peptide-chain initiation and protein synthesis in rat heart by mechanisms independent of the hormone's effect on glucose or energy metabolism.
Metabolic alterations and distribution of rat liver lysosomes in colloidal silica
Authors: Surmacz C. A. , Wert J. J. , Mortimore G. E.
Published: December 2017

Lysosome-enriched samples from normal fed rat livers have been shown to separate into dense and buoyant bands when layered on self-generating gradients of colloidal silica. The hypothesis that the buoyant peak is comprised largely of autophagic vacuoles that are selectively restrained by sedimenting mitochondria was tested in livers subjected to conditions known to accelerate autophagy or suppress it. In all cases, a sharp peak of beta-N-acetyl-D-glucosaminidase activity was evident when autophagy was present but was undetectable when it was eliminated or virtually eliminated. Moreover, the sensitivity to osmotic shock of buoyant peak particles, but not those of the dense band, was increased after autophagic stimulation. The total amount of enzyme that moved to buoyant fractions with the induction of autophagy generally correlated with the degree of autophagic acceleration. However, with maximal autophagy, the buoyant peak was broad, and only 70% was capable of being dislodged and returned to the dense peak by dispersing samples in the gradient medium and/or reducing the sample size. On the other hand, at roughly half-maximal stimulation and lower, the buoyant distribution was sharply defined and completely reversible. These findings support the above hypothesis and suggest that the buoyant peak could be used as a measure of autophagy in the physiological range of regulation.
Apical sodium uptake in toad kidney epithelial cell line A6
Authors: Sariban-Sohraby S. , Burg M. B. , Turner R. J.
Published: December 2017

The characteristics of the apical entry pathway for sodium into the cultured toad kidney epithelial cell line A6 are studied. Unidirectional apical sodium fluxes were determined by measuring the uptake of 22Na into confluent A6 epithelia growing in filter-bottomed cups. Apical sodium uptake was found to be a saturable function of sodium concentration with a Michaelis constant of 18 mM and a maximum velocity of 2.5 nmol X min-1 X cm-2. Amiloride competitively inhibits this sodium entry pathway with an inhibitor dissociation constant of 5 X 10(-8) M. Incubation of the epithelium with 10(-7) M aldosterone leads to a threefold increase in apical sodium uptake after 4 h. Both the aldosterone-stimulated and base-line sodium fluxes are completely inhibited by 10(-4) M amiloride. The similarity of these results to those from other tissues such as toad bladder and frog skin indicate that the A6 cells provide a useful model system for studying the apical entry pathway for sodium in tight epithelia.
Direct anabolic effects of thyroid hormone on isolated mouse heart
Authors: Crie J. S. , Wakeland J. R. , Mayhew B. A. , Wildenthal K.
Published: December 2017

The direct effects of L-and D-triiodothyronine (T3) on cardiac protein metabolism were investigated using fetal mouse hearts in organ culture. This model allowed the production of "thyrotoxicosis" in isolated hearts in vitro in the absence of the usual systemic metabolic and hemodynamic effects of thyroid hormones. Hearts were studied during the first 24 h of T3 exposure in culture, before changes in beating rate due to T3 occurred. Phenylalanine release was decreased by 26 +/- 2.3% (P less than 0.001) by the optimal concentrations of T3 (10(-7) to 10(-6) M). Changes were similar in the presence or absence of insulin. D-T3 was also anabolic, decreasing phenylalanine release by 24 +/- 2.5% (P less than 0.001) at concentrations of 10(-6) to 10(-5) M. The L-isomer increased protein synthesis by 23 +/- 6.8% (P less than 0.05) and decreased protein degradation, as measured by phenylalanine release in the presence of cycloheximide, by 5 +/- 1.6% (P less than 0.01). The D-isomer also increased protein synthesis but had no measurable effect on protein degradation. We conclude that thyroid hormones can exert direct anabolic effects on heart in the absence of systemic hemodynamic and metabolic changes. These effects are mediated primarily through an acceleration of the rate of protein synthesis; in the case of L-T3, a small inhibition of proteolysis may also occur.
Nitrogen-14 nuclear magnetic resonance spectroscopy of mammalian tissues
Authors: Balaban R. S. , Knepper M. A.
Published: December 2017

The use of 14N nuclear magnetic resonance (NMR) to monitor the concentrations of nitrogenous compounds in biological tissues was investigated. 14N-NMR spectra were collected in vivo and in vitro from several tissues of the rat and rabbit. Many nitrogen-containing compounds were detected, including urea, NH+4, trimethylamines, and several amino acids. In general, the compounds detected had approximately tetrahedral symmetry about the 14N nucleus and concentrations in the millimolar range. A problem that may limit the usefulness of 14N-NMR in intact tissue is the rapid exchange of nitrogen compounds in solution with those bound to sites on macromolecules. Such interactions tend to broaden the 14N resonance signal considerably. The binding of urea to intra- and extracellular proteins is presented as a specific example of this phenomenon. A particularly interesting finding was the high concentration (approximately 90 mM) of trimethylamine compounds in the renal inner medulla. We propose that 14N-NMR is a potentially useful technique for noninvasive detection of specific nitrogen-containing compounds in intact biological tissues.
Effects of hypertonic solutions on contraction of frog tonic muscle fibers
Authors: Godt R. E. , Kirby A. C. , Gordon A. M.
Published: December 2017

The influence of solution hypertonicity on contraction was studied in small bundles of tonic muscle fibers from the iliofibularis muscle of the frog Rana pipiens. Muscles were activated with high-K+ solutions that had osmolalities which were increased with tris(hydroxymethyl)aminomethanepropionate. Peak potassium contracture force decreased monotonically with tonicity and was zero in solutions with 2.5 or 3 times the osmolality of control Ringer. Contracture force at all tonicities studied (less than or equal to 3 X Ringer) was increased by increasing Ca2+ in the media 10-fold (to 20 mM) and/or by adding caffeine (10-20 mM). Nevertheless, this potentiated force also was diminished as tonicity increased. Force of single, mechanically skinned tonic fibers taken from these bundles was activated by Ca2+ over the same concentration range as that of twitch fibers. Moreover, maximal Ca2+-activated force, normalized per cross-sectional area, was similar in skinned tonic and twitch fibers. As was shown previously in twitch fibers, maximal Ca2+-activated force was decreased when ionic strength was increased. These data suggest that, as with twitch fibers, increased tonicity depresses contraction of tonic fibers by increasing the intracellular ionic strength, which in turn inhibits the ability of the contractile apparatus to generate force. Unlike twitch fibers, however, disruption of the excitation-contraction coupling process probably plays a more significant role in the action of hypertonicity on tonic fibers.
Effects of cellular acidification on ADH-induced intramembrane particle aggregates
Authors: Parisi M. , Bourguet J.
Published: December 2017

8-Bromoadenosine 3',5'-cyclic monophosphate [an analogue of adenosine 3',5'-cyclic monophosphate (cAMP), the intracellular mediator for antidiuretic hormone (ADH) action] induces, in frog urinary bladder, an increase in water permeability that is rapidly and reversibly inhibited by cellular acidification. The effect of CO2 bubbling on the simultaneously observed intramembranous particle aggregates, which probably represent water channels, depended on the time that elapsed after changing medium pH: 3 min of CO2 bubbling depressed the water flux by 70%, whereas the membrane surface occupied by the aggregates remained unchanged. On the contrary, after 9-15 min of CO2 bubbling, both the water flux and the surface area occupied by the aggregates were strongly reduced. These results can be interpreted by accepting two post-cAMP levels of action for cellular acidification: 1) the channels themselves that, as previously suggested by ADH experiments at low temperature, would shift their structure from an "open" to a "closed" state, and 2) the mechanism that controlled the aggregates' plug in and removal.
Amiloride and diltiazem inhibition of microsomal and mitochondrial Na+ and Ca2+ transport
Authors: Sordahl L. A. , LaBelle E. F. , Rex K. A.
Published: December 2017

Amiloride, a K+-sparing diuretic, and diltiazem, a Ca2+ channel antagonist, have both been reported to inhibit Na+ transport-associated processes in different subcellular membrane systems. In this report, similar inhibitory effects of both agents are demonstrated on Na+-induced Ca2+ release from rabbit heart mitochondria and on Na+ uptake in a kidney medulla microsomal preparation. Both amiloride and diltiazem produce 50% inhibition of Na+ uptake in kidney microsomes at the same concentrations. Heart mitochondrial Na+-induced Ca2+ release was 50% inhibited by 6 microM diltiazem and 200 microM amiloride. No effects of either agent on mitochondrial respiratory activity were observed. The results suggest a specific effect of both drugs on a Na+-binding site associated with an antiport exchange process. These data also extend previous observations suggesting the use of these agents as tools to define further ion transport mechanisms in biological membranes.
Cobalt inhibition of insulin release: evidence for an action not related to Ca2+ uptake
Authors: Wollheim C. B. , Janjic D.
Published: December 2017

Insulin release, Ca2+ fluxes, and adenosine 3',5'-cyclic monophosphate (cAMP) levels were measured under various conditions to test the selectivity of cobalt (Co2+) actions in pancreatic islets. Insulin release was measured simultaneously either with 45Ca2+ uptake or 45Ca2+ efflux from rat islets maintained 2 days in tissue culture. Glucose (16.7 mM)-stimulated insulin release was inhibited by Co2+ (0.25, 0.5 mM) without concomitant inhibition of glucose-stimulated 45Ca2+ uptake. At higher concentrations of Co2+ (1.0, 2.5 mM) glucose- as well as K+-stimulated insulin release were inhibited in parallel with 45Ca2+ uptake. In the presence or the absence of external Ca2+, Co2+ (0.5 mM) also decreased 3-isobutyl-1-methylxanthine-stimulated insulin release, which does not depend on the stimulation of Ca2+ uptake. Co2+ did not alter islet cAMP levels. Co2+ also did not affect the unidirectional basal 45Ca2+ efflux measured in the absence of Ca2+ but completely overcame the stimulated 45Ca2+ efflux evoked by the removal of external Mg2+. Under the latter conditions, 50, but not 5 microM verapamil, decreased the stimulated 45Ca2+ efflux. It is concluded that Co2+ is able to inhibit insulin release and lower Ca2+ efflux without apparent interference with Ca2+ uptake. This suggests that Co2+, in addition to its well-known inhibition of Ca2+ uptake, may exert its action at a distal step in the insulin release process.
Stimulation of cAMP accumulation and lipolysis in hamster adipocytes with forskolin
Authors: Schimmel R. J.
Published: December 2017

This study compares the effects of forskolin and isoproterenol on lipolysis and adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in hamster white adipocytes. Rates of lipolysis in forskolin-stimulated cells were equivalent to those in cells incubated with isoproterenol, but cAMP levels were more than 10-fold greater in the presence of forskolin. The stimulatory effects of forskolin were partially inhibited by N6-phenylisopropyl adenosine but not by 2',5'-dideoxyadenosine. In other experiments, cells were exposed to forskolin in combination with either isoproterenol or adenosine deaminase. A concentration of forskolin that caused only a small increase in lipolysis was used. When isoproterenol or adenosine deaminase were added with forskolin, lipolysis increased dramatically, but cAMP content either did not change, as occurred with isoproterenol, or increased only slightly with adenosine deaminase. Isoproterenol potentiation of forskolin's lipolytic action persisted in the absence of extracellular K+, even though the lipolytic response to isoproterenol alone was absent in K+-free media. These data demonstrate that the lipolytic responses of adipose tissue are more complex than are responses simply in proportion to cellular concentration of cAMP. Such complexity could arise if lipolytic regulatory factors other than cAMP existed or if cAMP and protein kinase were functionally segregated within adipocytes.
Flounder intestinal absorptive cells have abundant gap junctions and may be coupled
Authors: Curtis R. L. , Trier J. S. , Frizzell R. A. , Lindem N. M. , Madara J. L.
Published: December 2017

We noted that, unlike mammalian intestinal absorptive cells, cells of the winter flounder (Pseudopleuronectes americanus) displayed abundant gap junctions on the lateral plasma membrane. We compared the distribution of gap junctions in winter flounder to that in rabbit intestinal epithelium. We also examined for evidence of gap junction-mediated intercellular coupling by comparing the cell-to-cell variation of electrical potential difference across winter flounder intestinal cell apical membranes with that in rabbit small intestinal epithelium in which gap junctions are rare. Gap junctions were seen in 95% of flounder absorptive cells and were localized largely to the apical third of the lateral membrane. Individual gap junctions often contained several hundred uniform 9-nm intramembrane particles. Gap junction size and structure was independent of the position of individual absorptive cells on mucosal folds. These findings sharply contrasted flounder intestinal absorptive cells with rabbit small intestinal absorptive cells, in which gap junctions were rarely detected and when present consisted of few intramembrane particles. Correlating with this distribution of morphologically detectable gap junctions, rabbit small intestinal epithelial cells demonstrated marked variability in potential difference across their apical membranes, whereas those in flounder small intestine showed little variation in apical membrane potential difference. Thus, in contrast to intestinal epithelium of rabbits, flounder intestinal epithelium demonstrates morphological and functional characteristics, suggesting a substantial degree of electrical coupling.
Cytosolic lysophospholipase in cardiac myocytes and its inhibition by L-palmitoyl carnitine
Authors: Gross R. W. , Ahumada G. G. , Sobel B. E.
Published: December 2017

Since lysophosphatides have been implicated as arrhythmogenic metabolites, modulation of their catabolism in cardiac myocytes has been characterized. Rat cardiac myocytes and mesenchymal cells grown in culture were found to contain cytosolic lysophospholipase with specific activities of 1.3 +/- 0.1 and 0.9 +/- 0.1 nmol X mg-1 X min-1, respectively. Rat myocytic lysophospholipase had a molecular mass of approximately 20,000 daltons, estimated by gel filtration chromatography. Kinetic analysis of cytosolic myocytic lysophospholipase demonstrated a Michaelis constant of 11 microM, a pH optimum of 8.0, and competitive inhibition by L-palmitoyl carnitine (inhibitory constant of 12 microM). Although lysophospholipase-transacylase activity could not be detected in rat myocyte or mesenchymal cell cultures, rabbit myocytes isolated by perfusion of isolated hearts with collagenase contained lysophospholipase-transacylase in cytosolic extracts with a specific activity of 0.2 nmol X mg-1 X min-1. These results demonstrate the presence of lysophospholipase in cardiac myocytes and suggest that the increase in long-chain acyl carnitine, which occurs during myocardial ischemia, may contribute to accumulation of lysophosphatides within cardiac myocytes.
Beta-adrenergic stimulation of 42K influx in isolated smooth muscle cells
Authors: Scheid C. R. , Fay F. S.
Published: December 2017

The present studies were designed to examine the effect of the beta-adrenergic agent isoproterenol (ISO) on 42K influx into isolated smooth muscle cells. Unidirectional 42K influx studies were carried out as described previously using suspensions of smooth muscle cells derived from the toad stomach. Results from these studies indicated that ISO (10(-4) M) produces an approximate threefold increase in 42K influx within 1 min after drug addition. This response is transient such that the flux rate returns to near basal levels within 5 min after drug addition. The stimulatory effect of ISO on 42K influx is blocked by pretreatment with ouabain (1–7.5 X 10(-4) M). ISO thus appears to increase 42K influx by stimulating the Na+-K+ pump in the smooth muscle cells. Other findings indicate that the stimulatory effect of ISO on Na+-K+ pumping is dose dependent, blocked by the beta-antagonist pindolol, and mimicked by dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP). These findings support our hypothesis that ISO elicits a beta-adrenergic, cAMP-dependent activation of the Na+-K+ pump in isolated smooth muscle cells.
Beta-adrenergic effects on transmembrane 45Ca fluxes in isolated smooth muscle cells
Authors: Scheid C. R. , Fay F. S.
Published: December 2017

The effect of the beta-adrenergic agent isoproterenol (ISO) on transmembrane fluxes of 45Ca was assessed in smooth muscle cells in suspension. Exposure of the cells to this smooth muscle relaxant provoked a large (13-fold) transient increase in 45Ca efflux from the isolated cells. This effect was blocked by the beta-adrenergic antagonist pindolol and mimicked by exposure of the cells to dibutyryl adenosine 3',5'-cyclic monophosphate. ISO had no significant effect on 45Ca binding or on 45Ca influx in those cells, thus the ISO-induced increase in 45Ca efflux appears to reflect increased Ca2+ extrusion from the smooth muscle cells. Because the ISO-induced increase in 45Ca efflux was abolished by pretreatment of the cells with the Na+-K+ pump inhibitor ouabain and by lowering extracellular Na+ levels, we propose that the increased Ca2+ extrusion in response to ISO is coupled to increased activity of the plasmalemmal Na+-K+ pump via increased Na+-Ca2+ exchange.
Changes in actin synthesis and alpha-actin-mRNA content in rat muscle during immobilization
Authors: Watson P. A. , Stein J. P. , Booth F. W.
Published: December 2017

The fractional rates of actin synthesis in adult rat gastrocnemius muscle from control and 6-h hindlimb-immobilized animals were determined by the constant-infusion technique. The rate of actin synthesis in gastrocnemius muscle was significantly decreased from control values during the 6th h of hindlimb immobilization. The content of alpha-actin-specific mRNA was then estimated in adult rat gastrocnemius muscle from control, 6-h, 72-h, and 7-day immobilized animals by “dot blot” hybridization. RNA extracted from control and immobilized animals was secured on nitrocellulose filters and hybridized to 32P-labeled plasmid p749 (containing a cDNA sequence produced from rat alpha-actin mRNA). The relative content of alpha-actin-specific mRNA in gastrocnemius muscle was significantly decreased at 7 days of immobilization but not at 6 or 72 h of immobilization. It is concluded that a change in the content of alpha-actin mRNA does not contribute significantly to the rapid onset of the decrease in actin synthesis rate observed after 6 h of immobilization. An alteration in the translation of alpha-actin-specific mRNA must occur to account for the early decline in actin synthesis during immobilization.
Parallel stimulation by Ca2+ of inotropism and pyruvate dehydrogenase in perfused heart
Authors: Bunger R. , Permanetter B.
Published: December 2017

The effects of extracellular Ca2+ (0.375-;3.75 mM) on pyruvate oxidation and active form of the pyruvate dehydrogenase complex (PDCa) were quantitated in perfused guinea pig hearts in relation to inotropism, hydraulic work performance, and myocardial oxygen uptake (MVO2). The effects of afterload and norepinephrine (NE), alone or combined with the Ca2+ channel blocker D 600, were also examined. Hearts utilized 1-5 mM pyruvate in presence of 5 mM DL-3-hydroxybutyrate as substrates. Pyruvate oxidation and MVO2 increased essentially in parallel regardless of whether inotropism and energy metabolism were stimulated by increasing the Ca2+ concentration [( Ca2+]), the NE concentration [( NE]), or the afterload. PDCa activity was also directly related to [Ca2+], [NE], and afterload, respectively. Elevated [Ca2+] failed, however, to stimulate pyruvate oxidation and PDCa activity when MVO2 was held constant by an appropriate decrease in afterload at constant preload. Compound D 600, theophylline, and dibutyryl adenosine 3',5'-cyclic monophosphate also produced parallel alterations in cardiac mechanics, pyruvate oxidation, and MVO2. The striking proportionality between PDCa parameters, MVO2, and cardiac mechanics during the various alterations in cellular Ca2+ metabolism seemed to suggest that the observed Ca2+ stimulation of the PDC might be mainly secondary to increased myocardial energy utilization and myocyte respiration. Evidence for an additional direct effect of Ca2+ on the intact PDC system was not obtained.
A spectrin-like protein from mouse brain membranes: phosphorylation of the 235,000-dalton subunit
Authors: Goodman S. R. , Zagon I. S. , Whitfield C. F. , Casoria L. A. , Shohet S. B. , Bernstein S. E. , McLaughlin P. J. , Laskiewicz T. L.
Published: December 2017

A mouse brain spectrin-like protein, which was an immunoreactive analogue of erythrocyte spectrin, has been isolated from demyelinated membranes. This spectrin analogue was a 10.5 S, 972,000 molecular weight (Mr) (alpha beta)2 tetramer containing subunits of 240,000 (alpha) and 235,000 (beta) Mr. We demonstrated that in vivo only the 235,000 Mr beta subunit of the mouse brain spectrin-like protein was phosphorylated, which was an analogous situation to mouse erythrocyte spectrin in which only the 220,000 Mr beta subunit was phosphorylated. Incubation of isolated membrane fractions with [gamma-32P]ATP +/- adenosine 3',5'-cyclic monophosphate (cAMP) indicated that mouse brain spectrin-like protein, mouse erythrocyte spectrin, and human erythrocyte spectrin's beta subunits were all phosphorylated in vitro by membrane-associated cAMP-independent protein kinases.
Effect of thyroid hormone on carbohydrate content of Na+-K+-adenosine triphosphatase
Authors: Lo C. S. , Klein L. E. , Lo T. N.
Published: December 2017

The effect of L-3,5,3'-triiodothyronine (T3) (50 micrograms/100 body wt) on the incorporation of labeled glucosamine and fucose into the subunits of Na+-K+-ATPase was examined by gel electrophoresis in sodium dodecyl sulfate. T3 augmented the incorporation of glucosamine into the alpha- and beta-subunits by 51 and 58%, respectively, in the 22-h chase experiments. Similarly T3 augmented the incorporation of fucose into the alpha- and beta-subunits by 58 and 43%, respectively. Reverse T3 did not alter the incorporation of labeled fucose in either subunit. The effect of T3 on the rate constant of degradation of renal cortical Na+-K+-ATPase was assessed. The rate constant of degradation (Kd) of the [3H]fucose labeled alpha- and beta-subunits for the hypothyroid rats were both 0.20, and for T3-treated rats, the Kd of the alpha- and beta-subunits were 0.23 and 0.18, respectively, suggesting that T3 enhanced fucose incorporation into the subunits of Na+-K+-ATPase rather than retarding the degradation of this enzyme.
Age-associated changes in neuromuscular transmission in the rat
Authors: Wilson D. F. , Cardaman R. C.
Published: December 2017

The physiological changes in neuromuscular transmission associated with age were examined in rats between the ages of 1 mo (28 days) and 1 yr (364 days). Intracellular recording techniques were used to monitor end-plate potentials and miniature end-plate potentials at the rat diaphragm neuromuscular junction. Muscle action potentials were blocked by cutting the muscle fibers. Neuromuscular transmission was significantly different in 28-day-old rats compared with the older rats (42-364 days). The 28-day-old rats released fewer quanta. The statistical store in the immature rats was also significantly smaller than in the older age groups. The statistical probability of release, however, was not significantly different from the older animals. Examination of the presynaptic parameters in rats between the ages of 42 and 364 days revealed no significant increases in quantal release. It is concluded that the neuromuscular junction matures physiologically by 6 wk of age in rats and remains stable through the 1st yr of life.