bluuebunny/crossref_metadata_2025 huggingface dataset:
Location of major antibody binding domains on alpha-subunit of dog kidney Na+-K+-ATPase
Authors: Farley R. A. , Ochoa G. T. , Kudrow A.
Published: December 2017

The locations of binding sites on the alpha-subunit of dog kidney Na+-K+-ATPase for both monoclonal antibodies and antibodies from polyclonal antisera have been determined. Three distinct regions of the alpha-subunit, all located within the amino terminal half of the polypeptide, were recognized by the antibodies: a region near the amino terminus of the polypeptide and two regions that are separated by a site for trypsin cleavage of the ATPase in KCl. No significant binding of antibodies to the carboxy terminal region of the alpha-subunit was detected. The binding sites for the antibodies are located within regions of the polypeptide predicted to be exposed within the cytoplasm of the cell (P. L. Jorgensen, S. J. D. Karlish, and C. Gitler, J. Biol. Chem. 257: 7435-7442, 1982). This prediction was verified by the demonstration that the antibodies did not react with Na+-K+-ATPase in tight right-side-out vesicles but would bind to the protein after the vesicles had been disrupted with detergent. A model for the folding of the alpha-subunit through the membrane, based on these data, is presented.
Aortic pressure as a determinant of cardiac protein degradation
Authors: Gordon E. E. , Kira Y. , Demers L. M. , Morgan H. E.
Published: December 2017

Mechanical parameters and intracellular mediators that may control protein degradation were studied in isolated rat hearts subjected to increased aortic pressure. Elevation of aortic pressure from 60 to 120 mmHg in Langendorff preparations provided glucose or pyruvate as substrate decreased the rate of protein degradation during the second hour of perfusion. Intracellular contents of ATP or creatine phosphate or the creatine phosphate/creatine ratio did not indicate that energy depletion accounted for these effects. When ventricular pressure development was prevented by ventricular draining, and hearts were arrested with tetrodotoxin, protein degradation still decreased as aortic pressure was raised. The effect of elevated aortic pressure on proteolysis was unchanged when perfusate calcium concentrations were 0.6, 3.0, or 5.1 mM, or when indomethacin or meclofenamate was added to the perfusion buffer. These results provided no evidence to indicate that intraventricular pressure development or cardiac contraction was responsible for the inhibitory effect of increased aortic pressure on protein degradation. Instead, they suggested that stretch of the ventricular wall, as a consequence of increased aortic pressure, could be the mechanical parameter most closely related to the restraint on proteolysis. No evidence was obtained that the lower rate of degradation depended on energy or calcium availability or prostaglandin synthesis.
Ouabain binding to cultured vascular smooth muscle cells of the spontaneously hypertensive rat
Authors: Hopp L. , Khalil F. , Tamura H. , Kino M. , Searle B. M. , Tokushige A. , Aviv A.
Published: December 2017

The binding of ouabain and K+ to the Na+ pump were analyzed in serially passed cultured vascular smooth muscle cells (VSMCs) originating from spontaneously hypertensive (SH), Wistar-Kyoto (WKY), and American Wistar (W) rats. Our techniques have utilized analyses of displacement of [3H]ouabain by both unlabeled ouabain and K+ from specific binding sites on the VSMCs. We have found that each of the VSMC preparations from the three rat strains appeared to demonstrate one population of specific ouabain receptors (Na+ pumps); the number of Na+ pump units (mean +/- SE, expressed as 10(5) units/cell; number of observations indicated in parentheses) of both the SH and WKY rats was significantly lower than the number of Na+ pump units of W rat VSMCs [SH: 3.00 +/- 0.02 (231), WKY: 2.87 +/- 0.05 (245), and W: 3.62 +/- 0.04 (225)]; the equilibrium dissociation constant values (microM) for ouabain in VSMCs of SH and WKY rats were similar but were significantly higher than that of VSMCs derived from W rats [SH: 4.69 +/- 0.09 (231), WKY: 4.57 +/- 0.12 (245), and W: 3.69 +/- 0.17 (225)]; and among the VSMCs originating from the three rat strains, the apparent equilibrium dissociation constant value for K+ (mM) was the lowest in those of the SH rat [1.04 +/- 0.003 (143), compared with VSMCs of the WKY rat [1.54 +/- 0.006 (135)] and W rat [1.19 +/- 0.003 (136)]. Our previous studies have demonstrated increased passive Na+ and K+ transport rate constants of SH rat VSMCs compared with either W or WKY rat cells. These findings suggest the possibility of higher permeabilities of the SH cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Proton inhibition of chloride exchange: asynchrony of band 3 proton and anion transport sites?
Authors: Milanick M. A. , Gunn R. B.
Published: December 2017

The inhibition of chloride exchange at 0 degrees C by protons at the cytoplasmic and the extracellular surface of the band 3 protein of human erythrocytes was measured between pH 4.6 and 7.6. At constant external pH and chloride concentration, internal protons were a mixed inhibitor of chloride flux, with the apparent pK2 = 6.1 for protonation of the inward-facing empty transporter conformation and the apparent pK3 = 5.7 for protonation of the chloride-transporter complex. The activation of chloride exchange by external chloride was inhibited by internal protons, and internal protonation of the externally facing empty conformation had a pK1 = 6.1. External protons were also a mixed inhibitor of chloride exchange with the apparent pK1 = 5.0 for the empty outward-facing transporter conformation. Because of the pHo dependence of self-inhibition, the value of pK3 on the outside for chloride could not be accurately determined, but the apparent pK3 for protonation of the iodide-transporter complex on the extracellular surface was 4.9. The data support a mechanism with a single proton binding site that can alternatively have access to the cytoplasmic and extracellular solutions. It appears that this proton binding and transport site can be coupled to the single anion transport site for cotransport, but the two sites can be on opposite sides of the membrane at the same time and thus can be asynchronously transported by conformational changes of band 3.
Dexamethasone inhibits prostaglandin release from rabbit coronary microvessel endothelium
Authors: Rosenbaum R. M. , Cheli C. D. , Gerritsen M. E.
Published: December 2017

The effects of dexamethasone on prostaglandin secretion by cultivated rabbit coronary microvascular endothelial (RCME) cells were investigated. Incubation of RCME cells with dexamethasone resulted in a time- and concentration-dependent decrease in prostaglandin accumulation in the culture media and reduced basal and A23187-stimulated prostaglandin (PG) E2 and 6-keto-PGF1 alpha release. The maximal effects of dexamethasone (50-80% inhibition) were achieved after 16-18 h of incubation with the steroid at a final concentration of 10(-7) M. The effects of dexamethasone treatment were partially reversed 24 h after removal of the steroid from the culture media. Dexamethasone treatment did not reduce arachidonic acid-stimulated prostaglandin synthesis, indicating that the level of inhibition was proximal to that of cyclooxygenase. The inhibitory effects of dexamethasone could be prevented by pretreatment of the RCME cells with actinomycin D or cycloheximide, suggesting a requirement for protein synthesis in the inhibitory action of dexamethasone. Conditioned media from dexamethasone-treated cells contained a factor that inhibited porcine pancreatic phospholipase A2 (PLA2) in vitro. Transfer of conditioned media from dexamethasone-treated cells to untreated cells did not reduce basal or stimulated prostaglandin release; in contrast, a stimulatory action was consistently observed. Adherence of rabbit peripheral polymorphonuclear leukocytes (PMN) to RCME cells was reduced when the leukocytes were pretreated with 10(-7) M dexamethasone (4 h). However, dexamethasone pretreatment of the RCME cells did not significantly effect granulocyte adhesion. Thus coronary microvascular endothelial cell prostaglandin production is regulated by glucocorticoids, and glucocorticoid-pretreated microvascular endothelial cell release an inhibitor of PLA2 activity into the culture media.(ABSTRACT TRUNCATED AT 250 WORDS)
Influence of extracellular [Ca2+] on secretory and redox responses to CCK-8 in perfused rat pancreas
Authors: Kanno T. , Matsumoto T.
Published: December 2017

There is a distinct discrepancy between the dose-dependent secretory responses (pancreatic protein output and juice flow) and the dose-dependent redox responses of cytochromes aa3, b, and c+c1 to various concentrations of CCK-8: a bell-shaped relation for the secretory responses contrasts with a sigmoidal relation for the redox responses. Continuous stimulation with CCK-8 at physiological low concentrations (5-10 pM) induced an increase in pancreatic protein output with little if any reduction of cytochromes. Continuous stimulation with CCK-8 at a pharmacological intermediate concentration (50 pM), however, induced an increase in pancreatic protein output with delayed reduction of cytochromes. The secretory and redox responses were completely abolished when CaCl2 was removed from the perfusing and bathing solution. An almost linear relation was found between the magnitude of the protein output in response to 50 pM CCK-8 and extracellular [Ca2+] [( Ca2+]o) in the range of 0.5-2.5 mM. Similar relations were also found between the levels of reduction of cytochromes and [Ca2+]o. These results are compatible with the view that continuous stimulation with CCK-8 at a pharmacological or a pathological concentration may cause excess increase in intracellular [Ca2+] and intramitochondrial Ca2+ and in this way may accelerate the formation of reducing power for oxidative phosphorylation.
Creatine uptake, metabolism, and efflux in human monocytes and macrophages
Authors: Loike J. D. , Somes M. , Silverstein S. C.
Published: December 2017

The uptake of creatine in human monocytes and monocyte-derived macrophages can be divided into two components. One component of uptake is saturable (Km = 30 microM), specific for creatine, requires Na+ in the extracellular medium, and enables cells to take up creatine against a concentration gradient of creatine. The second component of uptake is nonsaturable and accounts for less than 10% of creatine uptake at physiological creatine concentrations (20-60 microM). Both monocytes and macrophages have similar Km values for creatine transport. However, macrophages exhibit a fivefold greater rate of creatine uptake than monocytes (3.6 vs. 0.7 pmol X micrograms protein-1 X h-1), indicating that monocyte differentiation into macrophages is associated with an increase in the number of creatine transporters in the membrane. Monocytes do not contain creatine kinase and do not phosphorylate creatine; monocyte-derived macrophages express creatine kinase and phosphorylate approximately 25% of intracellular creatine. These results and the finding that 2-deoxyglucose inhibits creatine phosphorylation by macrophages without affecting the transport of creatine indicate that phosphorylation of creatine is not required for net creatine accumulation.
Amiloride-sensitive sodium-hydrogen exchange in osmotically shrunken rabbit red blood cells
Authors: Jennings M. L. , Douglas S. M. , McAndrew P. E.
Published: December 2017

As part of a detailed study of cell volume regulation in high-potassium mammalian erythrocytes, we have characterized ouabain-insensitive sodium transport in normal and osmotically shrunken rabbit red cells. In cells of normal volume and physiological pH, there is no amiloride-inhibited component of the sodium efflux (into either sodium-containing or sodium-free media). Osmotic shrinkage activates an amiloride-sensitive (50% inhibitory concentration = 10(-5) M) sodium transport system that can catalyze net sodium movement in either direction. This system appears to be distinct from the sodium-sodium (sodium-lithium) counter-transporter that operates in cells of normal volume. Replacement of chloride with acetate does not inhibit the sodium flux, but replacement with either nitrate or thiocyanate is inhibitory. An inward sodium gradient in shrunken cells induces a net uphill efflux of acid equivalents, indicating that the sodium transport is a sodium-hydrogen exchange. However, a sevenfold inward gradient of hydrogen ions (pHo = 6.4; pHi = 7.2) does not stimulate net sodium efflux in shrunken cells. This suggests that the extracellular affinity of the transport site for hydrogen ions is high, and that there is an extracellular noncompetitive inhibitory site for proton binding. Bilateral pH reduction stimulates an amiloride-inhibitable sodium flux in cells of normal volume; this indicates that, as has been found in kidney, brain, and lymphocytes, there is an intracellular protonation site that can activate the transport. Shrinkage of the cells shifts the pH dependence of the transport, suggesting that part of the signal for the osmotic activation of the transport is a shift in the pKa of this modifier site.
Energetics and mechanics of frog skeletal muscle in hypotonic solution
Authors: Burchfield D. M. , Rall J. A.
Published: December 2017

Hypotonic solutions are known to potentiate muscle force production and increase actomyosin ATPase activity in solution. As such, both the rate and amount of ATP hydrolysis should increase during contraction. This was tested indirectly by measuring force and energy liberation in Rana pipiens semitendinosus muscles at 0 degrees C in hypotonic solution. Force and the amount and rate of energy liberation increased. This increase is consistent with the interpretation that the rate of ATP hydrolysis is increased in hypotonic solution. Muscles, stretched to beyond myofilament overlap, liberate a substantial fraction of the energy liberated at maximal myofilament overlap. This energy liberation, the activation heat, is thought to reflect the energy utilized to cycle Ca2+. Hypotonic solution decreased the amplitude and the rate of the activation heat, suggesting that the amount and rate of Ca2+ cycled by the sarcoplasmic reticulum is reduced. Thus in hypotonic solution, force production and the rate of ATP hydrolysis by the cross bridges are potentiated despite an apparent decrease in Ca2+ cycling.
Characteristics of rat hindlimbs perfused with erythrocyte- and albumin-free medium
Authors: Shiota M. , Sugano T.
Published: December 2017

The isolated rat hindlimb was perfused with Krebs-bicarbonate buffer without erythrocytes and albumin in a flow-through mode at 32 degrees C, and the viability and metabolic characteristics of perfused skeletal muscle were examined. 1) With the flow rate at 15 ml X min-1 X leg-1, glucose and O2 uptake, lactate release, lactate-to-pyruvate ratio in effluent, and tissue creatine phosphate and adenine nucleotides remained constant at rest during perfusion for 90 min. The twitch tension changed little over perfusion. 2) When the leg was stimulated at a frequency below 0.5 Hz, the standard flow rate adequately delivered O2 to the perfused leg. Sciatic nerve stimulation enhanced glucose uptake in the absence of insulin. 3) The stimulatory effect of insulin on glucose uptake was observed with a concentration as low as 0.1 mU/ml, and maximal effect was at approximately mU/ml, with a nearly eightfold increase in glucose uptake. 4) Epinephrine and isoproterenol at a concentration of 0.5 nM stimulated lactate release, with maximal effect at 5 nM. The response to catecholamines was reversible and reproducible with a single preparation during the perfusion period of 120 min. The results indicated that the perfusion of hindlimb with a hemoglobin- and albumin-free medium is a convenient and reliable tool for the biochemical investigations of the integral function of hindlimb skeletal muscle.
Effects of saxitoxin analogues and ligand competition on sodium currents of squid axons
Authors: Frace A. M. , Hall S. , Brodwick M. S. , Eaton D. C.
Published: December 2017

Saxitoxin (STX) and several STX analogues from dinoflagellates (genus Protogonyaulax) block sodium conductance in squid giant axons with variable potencies. Toxins, analyzed under voltage clamp, are 21-sulfosaxitoxin, 21-sulfosaxitoxin 11 alpha-hydroxysulfate, 21-sulfosaxitoxin 11 beta-hydroxysulfate, (B1, C1, C2, respectively) and gonyautoxins 2 and 3. The potency sequence for the toxins examined is STX greater than gonyautoxin 3 greater than B1 greater than C2 greater than gonyautoxin 2 much greater than C1. Guanidine, when substituted for sodium in external seawater, reduced the potency of STX to block inward current but did not affect tetrodotoxin activity. Methylguanidine also reduced the ability of STX to block outward sodium current. Inhibitory constants for guanidine and methylguanidine were 116 and 187 mM, respectively. Competition can be explained by binding at or near the toxin binding site but not by surface potential alteration.
Mucosal adenosine stimulates chloride secretion in canine tracheal epithelium
Authors: Pratt A. D. , Clancy G. , Welsh M. J.
Published: December 2017

Adenosine is a local regulator of a variety of physiological functions in many tissues and has been observed to stimulate secretion in several Cl-secreting epithelia. In canine tracheal epithelium we found that adenosine stimulates Cl secretion from both the mucosal and submucosal surfaces. Addition of adenosine, or its analogue 2-chloroadenosine, to the mucosal surface potently stimulated Cl secretion with no effect on the rate of Na absorption. Stimulation resulted from an interaction of adenosine with adenosine receptors, because it was blocked by the adenosine receptor blocker, 8-phenyltheophylline. The adenosine receptor was a stimulatory receptor as judged by the rank-order potency of adenosine and its analogues and by the increase in cellular adenosine 3',5'-cyclic monophosphate levels produced by 2-chloroadenosine. Adenosine also stimulated Cl secretion when it was added to the submucosal surface, although the maximal increase in secretion was less and it was much less potent. Part, but not all, of the lower potency of submucosal adenosine resulted from submucosal uptake and metabolism of the drug. The observation that mucosal 8-phenyltheophylline blocked the effect of submucosal 2-chloroadenosine, whereas submucosal 8-phenyltheophylline did not prevent a response to mucosal or submucosal 2-chloroadenosine, suggests that adenosine receptors are located on the mucosal surface. Thus submucosal adenosine may stimulate secretion by crossing the epithelium and interacting with receptors located on the mucosal surface. Because adenosine can be released from mast cells located in the airway lumen in response to inhaled material, and because adenosine stimulated secretion from the mucosal surface, it may be in a unique position to control the epithelium on a regional level.
Hormonal regulation of Na+-K+-ATPase in cultured epithelial cells
Authors: Johnson J. P. , Jones D. , Wiesmann W. P.
Published: December 2017

Aldosterone and insulin stimulate Na+ transport through mechanisms involving protein synthesis. Na+-K+-ATPase has been implicated in the action of both hormones. We examined the effect of aldosterone and insulin on Na+-K+-ATPase in epithelial cells in culture derived from toad urinary bladder (TB6C) and toad kidney (A6). Aldosterone, but not insulin, increases short-circuit current (ISC) in TB6C cells. Aldosterone increases Na+-K+-ATPase activity after 18 h of incubation, but no effect can be seen at 3 and 6 h. Amiloride, which inhibits aldosterone-induced increases in ISC, has no effect on either basal or aldosterone stimulated enzyme activity. Both aldosterone and insulin increase ISC in A6 cells and when added together are synergistic. Aldosterone stimulates enzyme activity in A6 cells, but insulin alone has no effect. However, aldosterone and insulin together stimulate enzyme activity more than aldosterone alone. It appears that stimulation of Na+-K+-ATPase activity is involved in aldosterone action in both cell lines but does not appear to be due to increased Na+ entry, since enhanced enzyme activity is not inhibited by amiloride. In contrast, insulin alone has no direct effect on Na+-K+-ATPase, although the increased enzyme activity following both agents in combination may explain their synergism on ISC.
Interaction of epinephrine with isolated rabbit tracheal epithelial cells
Authors: Liedtke C. M.
Published: December 2017

l-Epinephrine-mediated effects on intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels in rabbit surface tracheal epithelial cells (tracheocytes) were investigated to understand the difference in potency of epinephrine and isoproterenol compared with that observed in intact tracheal mucosa-submucosa. Epinephrine-mediated increases in cAMP levels reached a maximum in approximately 10 min then decreased; however, cAMP levels remained elevated even after 30 min of incubation with catecholamine. The beta-adrenergic antagonist propranolol partially blocked the dose-dependent increase in cAMP caused by epinephrine. Phentolamine, an alpha-adrenergic antagonist, and inhibitors of cyclooxygenase (ibuprofen, indomethacin, and acetylsalicylic acid) caused a shift to the right of the epinephrine dose-response curve, indicating antagonism of the response to epinephrine. Epinephrine, but not isoproterenol, stimulated the generation and release of prostaglandin (PG) E2, with 1.1 microM producing a half-maximal effect. This effect was antagonized by ibuprofen, phentolamine, and yohimbine, an alpha 2-adrenergic antagonist, but not by propranolol. The alpha 2-adrenergic effect of epinephrine was mimicked by clonidine, which stimulated PGE2 generation and release and caused an increase in cAMP levels in a dose-dependent manner; its effects on PGE2 release were blocked by ibuprofen and yohimbine. Methoxamine, an alpha 1-adrenergic agonist, did not stimulate PGE2 release. PGE2 alone caused a time- and dose-dependent increase in cAMP levels. In the presence of clonidine and isoproterenol, PGE2 was released, and the dose-dependent increase in cAMP levels mediated by isoproterenol was shifted to the left, indicating increased potency.(ABSTRACT TRUNCATED AT 250 WORDS)
Comparison of ouabain-sensitive 86Rb uptake of canine renal and femoral arteries
Authors: Navran S. S. , Allen J. C.
Published: December 2017

We have previously reported that various manifestations of the Na+ pump, such as ouabain binding, Na+-K+-ATPase, and 86Rb uptake, were similar in canine renal and femoral arteries [R.D. Bukoski, C.L. Seidel, and J.C. Allen. Am. J. Physiol. 245 (Heart Circ. Physiol. 14): H604-H609, 1983.]. In this report we compare ouabain-sensitive 86Rb uptake in renal and femoral arteries under two selected conditions of intracellular Na+:low Na+ and Na+ loaded. When intracellular Na+ was low, the renal artery dissociation constant for Rb+ was 1.13 mM compared with the Na+-loaded condition of 2.49 mM. The femoral artery dissociation constant remained the same at both Na+ levels. The maximal rate of 86Rb uptake (Vmax) of the renal artery was the same at both Na+ levels, but the femoral artery Vmax was 23.6 and 11.11 nmol X mg-1 X 20 min-1 in Na+-loaded and low-Na+ vessels, respectively. The reason for the difference in Na+ pump regulation in these two vessels is not clear, but these results suggest that there are heterogeneous mechanisms for controlling ionic movements in vascular smooth muscle.
Membrane currents recorded from a fragment of rabbit intestinal smooth muscle cell
Authors: Ohya Y. , Terada K. , Kitamura K. , Kuriyama H.
Published: December 2017

Properties of ionic currents in smooth muscle membranes of the longitudinal muscle layer of the rabbit ileum were investigated using the single electrode voltage clamp method. In the present experiments, this method was applicable only to the smooth muscle ball (fragment) and not for the dispersed whole cell, because of incompleteness of the voltage clamping. A voltage step elicited a transient inward current followed by an outward current. This outward current was partly inhibited by Mn2+ or nisoldipine or by a reduction in the extracellular [Ca2+] ([Ca2+]o). Tetraethylammonium (TEA) reduced the delayed outward current in a dose-dependent manner, but 50 mM TEA did not produce a complete block of a residual current. When the pipette contained K+-free (Cs+ with TEA+) solution, the residual outward current was abolished. The inward current was elicited at -30 mV (holding potential of -60 mV) and reached the maximal value at +10 mV; the polarity was reversed at +60 mV. This inward current depended on the [Ca2+]o and was blocked by Mn2+ or nisoldipine. Ba2+ also permeated the membrane, and the inward current evoked by Ba2+ was also blocked by Mn2+ or nisoldipine. Reduction of [Na+]o in a solution containing 2.4 mM Ca2+ neither modified the current-voltage relation nor the decay of the inward current, but when [Ca2+]o was reduced to below 1 microM, Na+ permeated the membrane and was blocked by nisoldipine. In conclusion, ionic currents were recordable from the fragmented ball of the longitudinal muscle of rabbit ileum. There were at least two K+ currents as the outward current (Ca2+-dependent K+ and delayed K+ currents) and a Ca2+ current as the inward current. The property of the Ca2+ channel was similar to that observed with other preparations.
Effects of mast cell-derived mediators on epithelial cells in canine trachea
Authors: Lazarus S. C. , McCabe L. J. , Nadel J. A. , Gold W. M. , Leikauf G. D.
Published: December 2017

We examined the interaction between mast cell-derived mediators and the electrical and ion transport properties of canine tracheal epithelium. We compared the effect of mediators released by immunologic challenge of sensitized lung parenchyma with that of mediators released from canine mastocytoma cells challenged with calcium ionophore A23187. Short-circuit current (Isc) increased by 19.2 +/- 3.0 microA/cm2 in response to mediators released from sensitized lung fragments challenged with ragweed antigen. This effect was not due to histamine. When the epithelial tissues were pretreated with indomethacin, the same mediator supernatant increased Isc by only 3.8 +/- 4.3 microA/cm2. The mediators released from 10(7) mastocytoma cells challenged with calcium ionophore increased Isc by 25.1 +/- 13.6 microA/cm2. In the presence of indomethacin, the Isc increased by 2.0 +/- 0.4 microA/cm2. Mastocytoma-derived mediators produced an increase in net chloride secretion without a significant effect on net sodium absorption. This study provides direct evidence that mast cell-derived mediators can stimulate epithelial ion transport in canine trachea and suggests that the effect is indirect and dependent on intact cyclooxygenase pathways in the tracheal epithelium.
Mitochondrial reticulum in limb skeletal muscle
Authors: Kirkwood S. P. , Munn E. A. , Brooks G. A.
Published: December 2017

High-voltage electron microscopy at 1,500 kV was used to examine mitochondrial morphology in three skeletal muscles of the rat. The soleus, deep portion of the vastus lateralis, and superficial portion of the vastus lateralis muscles were examined to represent slow-twitch oxidative, fast-twitch oxidative, glycolytic, and fast-twitch glycolytic skeletal muscle fiber types, respectively. Muscle samples were removed from six female Wistar rats. The tissues were fixed using standard electron microscopic techniques and were sectioned transversely with respect to muscle fiber orientation to approximately 0.5-micron thickness. The sections were stained on grids with uranyl acetate and Reynolds' lead citrate. Results revealed a mitochondrial reticulum in all three skeletal muscle fiber types. Stereological analyses of the electron micrographs were performed to measure volume densities and surface-to-volume ratios of mitochondria in the muscle samples. Cross-sectional volume densities of mitochondria in the soleus (15.5 +/- 1%) and deep portion of the vastus lateralis (16.1 +/- 2%) were significantly greater (P less than 0.05) than in the superficial portion of the vastus lateralis (8.7 +/- 1%). Surface-to-volume ratios of mitochondria were not significantly different between fiber types. It was concluded that the mitochondria in mammalian limb skeletal muscle are a reticulum, or network.
Subcellular distribution of calmodulin and its binding proteins within the rat submandibular gland
Authors: Singh J. , Brady R. C. , Dedman J. R. , Quissell D. O.
Published: December 2017

The involvement of calmodulin (CaM) in rat submandibular gland mucin secretion was investigated in vitro using a dispersed cell preparation. The CaM antagonist trifluoperazine (TFP) inhibited mucin secretion in response to both isoproterenol and dibutyryladenosine 3',5'-cyclic monophosphate. The inhibitory concentrations of TFP were between 10 and 100 microM. One millimolar TFP was toxic to submandibular cells and resulted in decreased levels of cellular ATP and a significant release of lactate dehydrogenase. Determination of CaM levels via radioimmunoassay within various subcellular fractions indicated that the majority of the CaM within the rat submandibular cell was located within the cytoplasm. CaM binding proteins were also identified within these subcellular fractions utilizing a gel overlay procedure. The two major, specific CaM binding proteins present within rat submandibular cells were a 59-kDa cytosolic protein and a 47-kDa membrane-associated protein.
Mechanical and histochemical characterization of skeletal muscles from senescent rats
Authors: Eddinger T. J. , Cassens R. G. , Moss R. L.
Published: December 2017

Maximal shortening velocity (Vmax) and isometric tension (Po) were measured in living fiber bundles and skinned fibers from extensor digitorum longus (EDL) and soleus (SOL) muscles of young adult (9 mo) and senescent (30 mo) Fisher 344 rats. The fiber type composition of each muscle preparation was determined using myosin (M)-ATPase histochemistry. Vmax, determined by the slack test method, was unchanged in the EDL but was increased in the SOL muscles of young adult vs. senescent rats. Velocities determined at intermediate loads using the load-stepping technique were slower for EDL bundles but were nearly identical for SOL bundles from senescent vs. young adult rats. Po was greater in SOL and was unchanged in EDL bundles and skinned fibers from senescent vs. young adult rats. M-ATPase histochemistry and Vmax were in agreement for fast and slow muscle bundles and fibers. The relationship between tension and pCa (i.e.,--log[Ca2+]) in skinned fibers from each muscle was similar in both age groups. Sodium dodecyl sulfate-polyacrylamide gels of the skinned fibers consistently showed fast light chains (LCs) in the EDL fibers and slow LCs in the SOL fibers, with no apparent age-related differences.
Velocity of shortening and myosin isozymes in two types of rabbit fast-twitch muscle fibers
Authors: Sweeney H. L. , Kushmerick M. J. , Mabuchi K. , Gergely J. , Sreter F. A.
Published: December 2017

The fast-twitch tibialis anterior muscle of the rabbit was stimulated (10 Hz, 8 h/day for 7 wk) to cause complete transformation of the fibers from type IIb to type IIa. The velocity of unloaded shortening of permeabilized single fiber segments dissected from control and chronically stimulated tibialis anterior muscles was measured by the slack test at 20 degrees C. The myosin isozymes in these segments were separated on pyrophosphate-containing polyacrylamide gels. Peptide mapping of the myosin chain was performed on the myosin bands cut from the gels. The velocity of unloaded shortening of the IIb fibers was significantly higher (2.50 +/- 0.09 fiber length/s; n = 6) than that of the IIa fibers (1.33 +/- 0.08 fiber lengths/s; n = 6). The two groups of fibers differed with respect to their alkali light chain complement, as assessed by nondenaturing gel analyses, and with respect to their myosin heavy chain complement, as demonstrated by peptide mapping. Thus two groups of fast-twitch muscle fibers that contain distinguishable myosin isozyme contents differ in their velocities of unloaded shortening by a factor of two.
Time dependence of shortening velocity in tracheal smooth muscle
Authors: Stephens N. L. , Kagan M. L. , Packer C. S.
Published: December 2017

It seems fairly well established that in the early phase of smooth muscle contraction cross bridges cycle at a relatively rapid rate. Later on these are replaced by very slowly cycling cross bridges or "latch bridges," operating with high economy. We describe a method to identify the time at which the transition occurs. By abruptly applying a light afterload at varying time intervals after stimulation of a canine tracheal smooth muscle, a point in time could be identified when cross-bridge cycling slowed. This was called the transition time. Because this transition was load dependent, the study was repeated with the preload abruptly reduced to zero. This permitted analysis of data in terms of cross-bridge activity. Maximum zero load velocity (Vo) of the contractile machinery was plotted against time and yielded a biphasic curve. The descending limb of the curve was fitted by a curve of the form Vo(t) = alpha e-K1t + beta e-K2t; K1 was almost three times greater than K2. We speculate that the faster rate constant represented activity of the early rapidly cycling cross bridges, and the slower constant reflected cycling rates in the latch state. These results are consistent with the latch bridge hypothesis put forward by Dillon et al. and enable us to provide a first approximation of the relative velocities of the two types of cross bridges.
Forskolin effects on frog and rabbit corneal epithelium ion transport
Authors: Candia O. A. , Grillone L. R. , Chu T. C.
Published: December 2017

The effects of forskolin on the electrophysiological parameters of the isolated corneal epithelium from bullfrog (Rana catesbeiana) were investigated. Forskolin stimulated the short-circuit current (SCC) and transepithelial potential difference (PDt), while reducing the transepithelial resistance. These effects were absent in Cl- -free bathing solutions. Furosemide, added either before or after forskolin, completely blocked the effects. Epinephrine and A23187, added after forskolin, produced only a small additional stimulation of the SCC. Propranolol neither blocked nor reduced the effect of forskolin. Forskolin increased the stroma to tear 36Cl flux by 61% and the tear to stroma 36Cl flux by 64%. Intracellular recordings showed that forskolin depolarized the potential difference across the apical membrane and reduced the apical/basolateral resistance ratio. Intracellular recordings in the isolated rabbit epithelium showed the same effects by forskolin except that there was only a brief stimulation of PDt, after which it stabilized slightly below the control level. These results are consistent with an increase in apical membrane permeability similar to that produced by adenosine 3',5'-cyclic monophosphate, epinephrine, and the Ca2+ ionophore A23187.
Effects of calcitonin on cytosolic Ca in a suspension of rabbit medullary thick ascending limb tubules
Authors: Murphy E. , Chamberlin M. E. , Mandel L. J.
Published: December 2017

A basal level of cytosolic free Ca (Caf) of 112 +/- 7 nM (n = 15) was measured in rabbit medullary thick ascending limb tubules that were loaded with the fluorescent Ca indicator quin 2. Calcitonin addition (10(-6) M) caused a greater than twofold increase in Caf. The KD for calcitonin stimulation of Caf was 8 X 10(-11) M. The rise in Caf had a half time of approximately 1.5 min. Neither parathyroid hormone (10(-6) M), dibutyryladenosine 3',5'-cyclic monophosphate (10(-4) M), nor forskolin (2 X 10(-5) M) had any significant effect on Caf levels, consistent with the rise in Caf being independent of an elevation in adenosine 3',5'-cyclic monophosphate. The effects of ouabain and the mitochondrial uncoupler, 1799, were also tested to obtain further information regarding the regulation of Caf. Ouabain (10(-4) M) caused no significant elevation in Caf; in contrast, 15 min of incubation with 1799 (10(-5) M) produced a twofold increase in Caf.
Maintenance of normal length improves protein balance and energy status in isolated rat skeletal muscles
Authors: Baracos V. E. , Goldberg A. L.
Published: December 2017

To clarify how muscle length or tension influences protein balance, we examined rates of protein synthesis and degradation in rat muscles incubated at different lengths. In soleus and extensor digitorum longus, protein degradation was lowest at resting length in situ (Lo), where the muscles are under slight tension. Muscles that were unrestrained shortened spontaneously and showed 25-45% greater net protein breakdown than muscles fixed at Lo. Protein synthesis did not differ in the two groups initially, but by 4 or 6 h was 25% greater in muscles at Lo. Maintenance of Lo improved overall protein balance largely through the reduction in proteolysis. In addition, muscles at Lo maintained high levels of ATP and phosphocreatine for 6 h, whereas the unrestrained muscles lost about half their energy stores. The acceleration of proteolysis in the shortened muscle appears independent of prostaglandin E2. However, the acceleration of protein breakdown in the unrestrained muscles could be prevented with inhibitors of thiol proteases (e.g., Ep-475 or leupeptin) or by incubation in Ca2+-free medium or with dantrolene (an inhibitor of Ca2+ release from sarcoplasmic reticulum). These treatments did not reduce the low level of proteolysis in muscles at Lo. Thus, if muscles are allowed to shorten in vitro, proteolysis is activated by a Ca2+-dependent mechanism, involving thiol proteases.
Effect of temperature on transmembrane potential of mouse liver cells
Authors: Wondergem R. , Castillo L. B.
Published: December 2017

Mouse liver transmembrane potential (Vm), measured under steady-state conditions with conventional microelectrodes, was -40 +/- 0.6 mV, and intracellular Na+ and K+ activities, measured with liquid ion-exchanger ion-sensitive microelectrodes, were 17 +/- 2 and 104 +/- 4 mM, respectively. The corresponding K+ and Na+ equilibrium potentials (EK and ENa) were -88 and 48 mV. Vm also varied as a linear function of temperature. In the range of 37-27 degrees C, the temperature coefficient (Q10) of 1.61 was greater than the Q10 of 1.033 predicted for a direct proportion of absolute temperature. A decrease in hepatocyte EK accounted for only a small portion of the total decrease in Vm resulting due to cooling from 37 to 25 degrees C. In contrast, slopes of the linear portion of Vm versus log10 external K+ activity were -24 and -14 mV/10-fold change in external K+ activity at 37 and 25 degrees C, respectively. This is consistent with an increase of membrane Na+- to -K+ permeability ratio (PNa/PK) with cooling. Ba2+ and quinine, which block membrane K+ channels, reversibly inhibited increases in hepatocyte Vm resulting due to heating from 37 to 40 degrees C. This suggests that membrane PK varies directly with temperature. We postulate that effects of temperature on liver Vm result from temperature effects on membrane K+ channel conductance and on the Na+-K+ pump. The results also are consistent with temperature effects on kinetic parameters for opening and closing of membrane K+ channels.
Transepithelial electrical potential of nonsensory region of gerbil utricle in vitro
Authors: Marcus D. C.
Published: December 2017

Transepithelial electrical potential difference (VT) was measured across the vestibular labyrinth of the inner ear in vitro by puncturing the epithelial wall of the utricle with a glass microelectrode. A region of nonsensory cells of the utricle was isolated from the sensory regions by introducing columns of liquid Sylgard 184. Under control conditions, the VT of this region was +7.5 +/- 0.3 mV (means +/- SE), lumen positive. This potential difference was rapidly reduced by either 1 mM ouabain, 10-100 microM bumetanide, 0.5-5.0 mM Ba (in the bathing solution), or cooling, but not by the disulfonic stilbene, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. Changes in VT due to reductions of Cl or Na or to increases of K in the bathing solution in exchange for presumably impermeant ions were observed in this region and were compared with those in a preparation in which the insulating seals were absent. The K-induced voltage change was significantly higher in the unblocked preparation, a finding consistent with a high K permeability of the sensory cells. The voltage change due to reduction of Cl was not inhibited by Cl channel blockers (9-anthracenecarboxylate and diphenylamine-2-carboxylate) in the bathing solution. These results represent the first direct demonstration that the nonsensory cells of the utricle produce a lumen-positive active-transport potential and characterize some of the properties of the cell membranes in terms of their pharmacological sensitivities and net voltage responses to changes in the bathing medium ions Na, K, and Cl.
Atriopeptin II decreases cytosolic free Ca in cultured vascular smooth muscle cells
Authors: Hassid A.
Published: December 2017

We have examined the hypothesis that atriopeptin II decreases cytosolic free Ca concentrations in cultured aortic smooth muscle cells. Ca levels were measured, using the Ca indicator fura-2, by dual wavelength fluorescence spectroscopy, in superfused primary cultures of smooth muscle cells. Basal Ca values were between 40 and 100 nM. Angiotensin II (10 nM) elicited a transient threefold increase in Ca, followed by return to a sustained Ca level that was 30% higher than the original basal values. Atriopeptin II dose-dependently decreased basal Ca concentrations by 10-40%. Atriopeptin II did not have a significant effect on the transient Ca response elicited by 10 nM angiotensin II, but the atrial peptide dose-dependently decreased the sustained increase that followed the transient response. Atriopeptin II also decreased cytosolic Ca levels that were elevated by 50 mM KCl. These results support the hypothesis that atriopeptins relax vascular smooth muscle by decreasing basal and vasoconstrictor-elevated cytosolic free Ca levels and that these peptides may function as endogenous antagonists of Ca-mediated processes.
Na-H exchange is a major pathway for Na influx in rat vascular smooth muscle
Authors: Little P. J. , Cragoe E. J. , Bobik A.
Published: December 2017

Rat aortic segments and aortic smooth muscle cells in primary culture were used to examine the importance of the Na-H exchange system in transporting Na into smooth muscle. Ethylisopropylamiloride was approximately 100 times more potent than amiloride at inhibiting Na influx into smooth muscle. In a 135 mM Na-containing medium approximately 80% of the Na influx rate could be inhibited by 100 microM ethylisopropylamiloride. The rate of Na entry into cells was markedly influenced by extracellular and intracellular pH. Elevating extracellular pH from 6.0 to 8.0 increased the Na influx rate. The dependence of the Na influx rate on intracellular pH was demonstrated by acidification of cells with nigericin or preincubation with ammonium chloride. These two procedures increased 22Na+ influx rate by about 3.5-fold. In both instances the increases in 22Na+ influx rate could be completely attenuated by ethylisopropylamiloride. Increases in Na influx rate via the Na-H exchange also increased the activity of the Na-K pump, thereby maintaining intracellular Na content approximately constant. These results indicate that Na-H exchange is a major influx pathway for Na in rat vascular smooth muscle. Activation of this system activates the Na-K pump, which maintains intracellular Na constant.
Cardiac protein synthesis and degradation during thyroxine-induced left ventricular hypertrophy
Authors: Parmacek M. S. , Magid N. M. , Lesch M. , Decker R. S. , Samarel A. M.
Published: December 2017

Assessment of cardiac protein metabolism in thyroxine-induced left ventricular hypertrophy requires measurements of both protein synthesis and degradation. In vivo protein degradative rates can best be measured as the difference between rates of protein synthesis and growth. Accordingly, rates of left ventricular protein accumulation were determined in growing rabbits, and in animals administered intravenous L-thyroxine (200 micrograms X kg-1 X day-1) for up to 15 days. Left ventricular protein fractional synthetic rates in euthyroid and thyroxine-treated rabbits were measured by continuous infusion of [3H]leucine (200 mu Ci/h X 6 h), and results converted to milligrams protein synthesized and degraded per day. Thyroxine administration produced left ventricular hypertrophy by increasing the rate of total protein synthesis (35.7 +/- 2.0, 71.0 +/- 7.0, and 62.6 +/- 4.0 mg of left ventricular protein synthesized per day for 0-, 3-, and 9-day, thyroxine-treated rabbits, respectively). However, the increased rate of total protein synthesis was greater than the measured rate of total protein accumulation (8.1 vs. 15.9 mg protein/day for euthyroid and thyroxine-treated animals), indicating that left ventricular protein degradative rates were increased as well. These studies indicate that accelerated proteolysis may be important in the molecular and architectural remodeling of the rapidly hypertrophying heart during thyrotoxicosis.
Lysosomal changes during thyroxine-induced left ventricular hypertrophy in rabbits
Authors: Parmacek M. S. , Decker M. L. , Lesch M. , Samarel A. M. , Decker R. S.
Published: December 2017

By use of a combined morphologic, immunocytochemical, and biochemical approach, this study demonstrates the changes in the lysosomal vacuolar apparatus that accompany thyroxine-induced cardiac hypertrophy. During the 1st wk of thyroxine administration, immunocytochemical studies revealed a decline in cathepsin D within many myocytes, but an increase within interstitial cells. These events transpired with only a modest rise in cathepsin D activity. During the 2nd wk, cathepsin D reappeared within most myocytes and continued to increase within the interstitial population and was associated with a general rise in lysosomal enzyme activity. These data demonstrate the importance of employing both immunocytochemical and biochemical approaches to evaluate the status of the lysosomal vacuolar apparatus, and suggests that the lysosomal vacuolar apparatus may be involved in the apparent remodeling that attends rapid cardiac growth, which may represent one component of the enhanced rates of proteolysis documented in this model of cardiac hypertrophy.