bluuebunny/crossref_metadata_2025 huggingface dataset:
A visual aid for teaching ventilation-perfusion relationships.
Authors: Norton J M
Published: December 2017

To help students understand the concept of the ventilation-perfusion ratio (VA/Q) and the effects that VA/Q mismatching has on pulmonary gas exchange, a "sliding rectangles" visual aid was developed to teach VA/Q relationships. Adjacent rectangles representing "ventilation" and "perfusion" are slid past one another so that portions of the ventilation and perfusion rectangles are not touching, illustrating the concepts of dead-space ventilation (VD) and shunt flow (QS). The portion of the ventilation bar representing VD is further subdivided into anatomical and alveolar VD and used to show the effects of alveolar dead space on the PO2 (PAO2) and PCO2 of alveolar air (PACO2); movement away from the "ideal" point). Similarly, the portion of the perfusion bar representing QS is used to define anatomical and physiological shunts and the effect of shunts on the PO2 (PaO2) and PCO2 of arterial blood (PaCO2). The genesis of the PAO2-PaO2 (A-a) PO2 difference as well as the effects of VA/Q mismatching and diffusion abnormalities can all be discussed with this visual aid. This approach has greatly assisted some students in mastering this traditionally difficult area of respiratory physiology.
DETERMINANTS OF CARDIAC FUNCTION: SIMULATION OF A DYNAMIC CARDIAC PUMP FOR PHYSIOLOGY INSTRUCTION
Authors: Davis Michael J. , Gore Robert W.
Published: December 2017

A computer model is described that simulates the cardiac cycle of a mammalian heart. The model emphasizes the pressure-volume plot as a teaching tool to explain the behavior of the heart as a pump. It exhibits realistic responses to changes in preload, afterload, contractility, and heart rate while displaying time-dependent changes in pressure and volume in addition to the pressure versus volume plot. It differs from previous models by graphing these parameters on a beat-to-beat basis, allowing visualization of the dynamic adaptation of the pumping heart to various stimuli. A system diagram is also included to further promote student understanding of the physiology of cardiac function. The model is useful for teaching this topic to medical, graduate, or undergraduate students. It may also be used as a self-directed computer laboratory exercise.
SLEEP AS A TEACHING TOOL FOR INTEGRATING RESPIRATORY PHYSIOLOGY AND MOTOR CONTROL
Authors: Thompson Stephen R. , Ackermann Uwe , Horner Richard L.
Published: December 2017

Sleep exerts major effects on most fundamental homeostatic mechanisms. Current data suggest, however, that students of physiology and medicine typically receive little or no formal teaching in sleep. Because sleep takes up a significant component of our life span, it is proposed that current teaching in systems and integrative physiology is not representative if it is confined to functions describing wakefulness only. We propose that sleep can be readily integrated into various components of physiology and medical curricula simply by emphasizing how commonly taught physiological processes are importantly affected by sleep mechanisms. In our experience, this approach can be used to reinforce basic physiological principles while simultaneously introducing sleep physiology into the students’ training. We find that students have a general and inherent interest in sleep and related clinical disorders, and this proves useful as an effective means to teach the material. In this paper, examples of how sleep influences motor control and the respiratory system will illustrate these points. These considerations also highlight some important gaps in traditional teaching of respiratory physiology.
ENDOCRINE PHYSIOLOGY IN A PATIENT-CENTERED LEARNING CURRICULUM
Authors: Vari Richard C. , Borg Kurt E. , McCleary Vikki L. , McCormack John T. , Ruit Kenneth G. , Sukalski Katherine A. , Olson Linda M.
Published: December 2017

The medical curriculum at the University of North Dakota School of Medicine and Health Sciences has recently been redesigned into a problem-based/traditional hybrid model that utilizes an integrated organ systems-based approach to teach basic and clinical sciences. The number of lecture hours in general has been greatly reduced, and, in particular, lecture hours in physiology have been reduced by 65%. Students learn basic science in small groups led by a faculty facilitator, and students are responsible for a great deal of their own teaching and learning. The curriculum is centered around patient cases and is called patient-centered learning (PCL). The curriculum includes traditional lectures and laboratories supporting faculty-generated learning objectives. Endocrine physiology is taught in year one, utilizing four weeks of patient cases that emphasize normal structure and function of endocrine systems. Endocrine physiology is revisited in year two, which is primarily focused on pathobiology. The PCL curriculum, with emphasis on the endocrine component, is described in detail along with key portions of an endocrine case.
Reorientation of myofilaments during contraction of a vertebrate smooth muscle
Authors: Fisher B. A. , Bagby R. M.
Published: December 2017

The purpose of the investigation was to determine whether filaments within smooth muscle cells changed their orientation (with respect to the main axis of the cell) during contraction. The stomach muscle of Bufo marinus was used, since its cells may be easily isolated, enabling direct observation in living cells. In addition to still micrography, cinemicrography was used to record continuously during contraction. Polarization microscopy revealed a change in birefringence after contraction, with relaxed cells exhibiting uniform birefringence while contracted cells displayed a discontinuous pattern. Movies revealed a progressive change in orientation of birefringent elements from nearly parallel to the cell's main axis in relaxed cells to increasingly larger angles to the cell's axis as contraction progressed. Phase-contrast microscopy revealed a change in filamentous components, from being parallel to the cell's axis in relaxed cells to being in an undulating or helical pattern during concentration. Cell shape tended to follow the configuration of the filamentous component. Electron microscopy of muscle strips corroborated the observations of living cells and substantiated the conclusion that filaments change their orientation from parallel to oblique (with respect to the cell's axis) during shortening with an undulating or helical pattern of filaments in shortened muscles.
Response of mitochondria of different types of skeletal muscle to thyrotoxicosis
Authors: Winder W. W. , Holloszy J. O.
Published: December 2017

To determine the effect of long-term thyrotoxicosis on muscle mitochondria, we measured representative mitochondrial enzymes from three different types of skeletal muscle (fast-twitch red and fast-twitch white from the quadriceps, and slow-twitch red from the soleus) in rats given 3 mg L-thyroxine and 1 mg triiodo-L-thyronine per kilogram of diet for 12 wk. Marker enzymes of the electron transport chain and citric acid cycle (cytochrome oxidase, cytochrome c, and citrate synthase) increase approximately twofold in soleus muscle in response to this treatment. The fast-twitch muscles exhibit no more than 44% increases in these enzymes in response to the same treatment. Relative to initial concentration, 3-hydroxybutyrate dehydrogenase increased to the same extent in fast-twitch red muscle as it did in the soleus (70%). Mitochondrial alpha-glycerophosphate dehydrogenase increased 76% in red quadriceps and 170% in soleus, but did not change in white muscle in the thyrotoxic rats. This differential sensitivity of the three types of muscle provides a tool for studying the mechanisms underlying the action of thyroid hormones on muscle mitochondria.
Regional distribution of autonomic nerves in guinea pig uterus
Authors: Thorbert G. , Alm P. , Owman C. , Sjoberg N. O.
Published: December 2017

Uterine adrenergic and cholinergic nerves were examined histochemically and chemically in combination with surgical denervation. There was a clear regional variation with a greater number of axon terminals in the tubal end of the uterine horn and in the cervix compared to the remainder (major part) of the uterine horn. No cholinergic nerves were found in the uterus but were abundant around the uterine artery and its primary ramifications. Clusters of ganglion cells were located near the cervix: the majority were acetylcholinesterase positive, whereas the remainder stored catecholamines. Unexpectedly, the suspensory ligament (connecting the tubal end of the uterine horn with the two lower ribs) was found to contain an extensive number of adrenergic nerves. The major part of the uterine horns, as well as the cervix, was innervated mainly from two sources contributing almost equally: the paracervical ganglia (short adrenergic neurons) and postganglionic fibers running in the hypogastric nerves. A restricted area at the tubal end of the uterus received most of its via the suspensory ligament.
Chemical energetics of single isometric tetani in mammalian smooth muscle
Authors: Butler T. M. , Siegman M. J. , Mooers S. U. , Davies R. E.
Published: December 2017

A rabbit taenia coli preparation has been used to study the chemical energetics of smooth muscle contraction. Under the experimental conditions, the muscle had no spontaneous mechanical activity, but could be fully activated with the use of electrical field stimulation. ATP resynthesis from glycolysis and respiration was stopped with a procedure that involved treatment with metabolic inhibitors at 5 degrees C followed by rewarming to 18 degrees C. This procedure did not alter the high-energy phosphate contents of mechanical responses of the muscle. Use of this preparation to determine ATP and phosphorylcreatine changes during isometric tetani at 18 degrees C and a length of 86% LO showed that there was net ATP breakdown initially with no significant phosphorylcreatine splitting. This was followed by an increase in the rate of phosphorylcreatine splitting with net ATP synthesis. The average rate of total high-energy phosphate utilization was about 0.01 mumol/g.s for up to 60 s or about 150 times less than that of frog sartorius at the same temperature.
Modification of slow sodium inactivation in nerve after internal perfusion with trypsin
Authors: Starkus J. G. , Shrager P.
Published: December 2017

Crayfish axons, internally perfused and held at depolarized membrane potentials, exhibit an inactivation of sodium conductance with slow kinetics. Restoration of maximum peak early currents requires prepulse hyperpolarizations of up to 1 s duration. Addition of trypsin to the internal perfusate at low concentrations (0.02 mg/ml) causes a rapid and irreversible loss of slow inactivation at the resting potential and a corresponding increase in Na currents to maximum values. After trypsin action, steady-state slow Na inactivation is shifted by 20--25 mV in the depolarizing direction, with no change in fast (h) inactivation. N-ethylmaleimide (NEM), a reagent with a high specificity for sulfhydryl groups, has been shown to induce slow inactivation, modify fast inactivation, and block a fraction of the Na conductance. After trypsin action NEM no longer increases slow Na inactivation but other effects remain. Prior exposure to NEM does not protect axons against the loss of slow inactivation caused by trypsin.
Effects of thyroid deficiency and sympathectomy on cardiac enzymes
Authors: Baldwin K. M. , Campbell P. J. , Hooker A. M. , Lewis R. E.
Published: December 2017

The effects of thyroid deficiency (Td) and of chemical sympathectomy (Sx) were studied on marker enzymes of energy metabolism in cardiac muscle of neonatal and of adult rats. Td prevented the normal development of neonatal body weight, relative heart mass, and cardiac levels of cytochrome c (-22%), citrate synthase (-27%), phosphofructokinase (-20%) and Mg2+- and Ca2+-ATPase activity of purified myofibrils (-33%, -44%). Exogenous thyroxin replacement restored those parameters studied to normal with the exception that it persistently elevated citrate synthase activity significantly above normal control levels. Responses similar to those of Td neonates occurred when adult rats were similarly treated. Sx produced no consistent effects on respiratory and glycogenolytic marker enzymes, but caused a 20% reduction in Ca2+-ATPase activity of both neonatal and adult cardiac myofibrils. These findings suggest that cardiac muscle cells require thyroxin for normal growth and enzyme development. Also, Sx may impair cardiac functional capacity by altering Ca2+ activity of actomyosin ATPase.
Substrate oxidation specificity in different types of mammalian muscle
Authors: Hooker A. M. , Baldwin K. M.
Published: December 2017

The maximal capacity of homogenates prepared from cardiac, fast-oxidative-glycogenolytic (FOG) vastus lateralis, fast-glycogenolytic (FG) vastus lateralis, and slow-oxidative (SO) soleus muscle to oxidize pyruvate, palmitate, alpha-glycerophosphate, and acetoacetate was assessed by measuring oxygen consumption under conditions of nonlimiting substrate and cofactors. Pyruvate oxidation varied eight-fold among the muscle types and was highest in cardiac, followed by FOG, SO, and FG muscle. Palmitate was oxidized at 97%, 85%, 77%, and 57% of the relative rate for pyruvate in cardiac, FOG, SO, and FG muscle, respectively. In contrast, alpha-glycerophosphate oxidation rates were highest in FG muscle, followed by cardiac, FOG, and SO muscle. Although cardiac muscle possessed the highest absolute rate for acetoacetate oxidation, it had the lowest capacity relative to pyruvate (19%), whereas SO muscle possessed the highest (61%). FOG and FG muscle had similar relative capacities for this substrate (30%). These results provide further evidence to suggest that mammalian muscle types are differentiated in terms of both mitochondrial mass and substrate oxidation specificity.
Thyroid hormone-aldosterone interaction on Na+ transport in toad bladder
Authors: Rossier B. C. , Gaggeler H. P. , Brunner D. B. , Keller I. , Rossier M.
Published: December 2017

Repeated administration of thyroxine (T4) in vivo (2 microgram/100 g body wt for 6 days) lowered by 2.3 times (P less than 0.025, df = 18) the basal rate of Na+ transport measured by the short-circuit current (SCC) in vitro in the urinary bladder of the toad (Bufo marinus). This difference was not accounted for by a change in the plasma aldosterone concentration. Moreover the response of the SCC to aldosterone in vitro was markedly inhibited in bladders from T4-treated animals (P less than 0.001, df = 18). These findings raised the possibility of a direct interaction between thyroid hormone and aldosterone in the target cell. The effects of L-triiodothyronine (T3) and aldosterone were examined in vitro. T3 alone (60 nM) had no significant effect on the base-line SCC (deltamuA = -14 +/- 11 (SE) muA per hemibladder; P greater than 0.3, n = 10). By contrast, T3 (60 nM) inhibited the response of the SCC to aldosterone from 6 to 8 h after its addition (deltamuA = -98 +/- 19 muA per hemibladder; P less than 0.001, n = 10). The inhibition by T3 was present at 6 nM (P less than 0.01, n = 10) and became not significant at 0.6 nM. T3 had no significant effect on base-line or aldosterone-stimulated H+ transport. Thyroid hormone might therefore regulate the late response of the SCC to aldosterone at the level of its target cell.
Cadmium transport in isolated perfused rat liver: zinc-cadmium competition
Authors: Kingsley B. S. , Frazier J. M.
Published: December 2017

The hypothesis that one component of cadmium uptake by rat hepatocytes involves a mediated transport pathway normally operative for zinc transport was tested in the isolated perfused rat liver preparation. Excess zinc in the perfusion medium suppressed cadmium uptake as indicated by the decrease in the normalized clearance (initial clearance divided by liver weight) from 0.340 +/- 0.019 (ml/min)/g in the presence of normal zinc concentrations (Zn:Cd molar ratio, 1.6) to 0.138 +/- 0.017 (ml/min)/g (Zn:Cd molar ratio, 13.0). In excess-zinc control experiments (no cadmium present) little zinc is accumulated by the liver, apparently due to competition between intrahepatic and extracellular binding. Exposure to cadmium increases both zinc secretion into the perfusion medium and biliary excretion of zinc. The effect at the sinusoidal membrane is probably a result of both the blockage of zinc resorption during cadmium uptake and the displacement of intrahepatic zinc. The effect on biliary excretion of zinc is due solely to displacement of intrahepatic zinc. These results are consistent with the proposed hypothesis for cadmium transport.
Calcium mediation of cholinergic-stimulated amylase release from mouse parotid gland
Authors: Watson E. L. , Williams J. A. , Siegel I. A.
Published: December 2017

Amylase release from mouse parotid fragments was stimulated independently by cholinergic and beta-adrenergic agents. The cholinergic agonist, carbachol, significantly increased release of amylase only in Ca2+ containing medium whereas isoproterenol-stimulated amylase release was unaffected by Ca2+ removal. The ionophore, A23187, mimicked the effect of cholinergic stimulation when Ca2+ was present in the medium. Uptake of 45Ca2+ into tissue fragments was enhanced by carbachol and A23187 but not by isoproterenol; atropine blocked the effect of carbachol. Diphenylhydantoin (DPH) and verapamil partially inhibited carbachol-stimulated amylase release and 45Ca2+ uptake, whereas diazoxide potentiated these effects; in all cases there was good parallelism between 45Ca2+ uptake and amylase release. It was concluded that the primary step in the release of amylase from mouse parotid gland in response to cholinergic agents is an increased influx of Ca2+.
Effect of chemotactic factors on calcium levels of rabbit neutrophils
Authors: Petroski R. J. , Naccache P. H. , Becker E. L. , Sha'afi R. I.
Published: December 2017

Studies were undertaken to provide a systematic investigation of the effect of the chemotactic factor, formyl-methionyl-leucyl-phenylalanine (F-Met-Leu-Phe), on the levels of total and exchangeable calcium in rabbit neutrophils. The 45Ca2+ specific activity of the cells was thus determined before and after stimulation by chemotactic factors. Total cell calcium was found to be constant in neutrophils equilibrated with 50–1,000 microM extracellular calcium but decreased by 30% at an extracellular calcium concentration of 5 microM. F-Met-Leu-Phe had little, if any, statistically significant effect on the level of total cell calcium. In contrast, F-Met-Leu-Phe affects greatly the steady-state levels of radioactive calcium in the neutrophils. The primary effect is an initial loss followed by an increase of cell-associated radiolabeled calcium. The extent and even the direction of the effect depends on the level of extracellular calcium as well as the concentration of the chemotactic factor and the duration of its interaction with the cell. The results are discussed in terms of the hypothesis that the binding of the chemotactic factor to its receptor leads to, among other things, a graded displacement of previously bound Ca2+.
Interstitial anion distribution in striated muscle determined with [35S]sulfate and [3H]sucrose
Authors: Macchia D. D. , Page E. , Polimeni P. I.
Published: December 2017

The distributions of a charged and an uncharged extracellular tracer in the interstitial spaces of skeletal and heart muscles were examined in vivo by a double-labeling technique. 35SO4(2-) and [3H]sucrose were simultaneously injected intraperitoneally into rats and toads, and extracellular volume was determined in the rat gastrocnemius and left ventricle and in the toad semitendinosus. In nephrectomized rats and in toads with intact kidneys, sucrose and SO4(2-) spaces were constant for several hours. Sucrose and SO4(2-) spaces did not significantly differ in rat ventricle (P greater than 0.80); in rat gastrocnemius the sucrose space was much larger than SO4(2-) space (2P less than 0.0005), while in toad semitendinosus sucrose space was somewhat smaller than SO4(2-) space (2P less than 0.005). These observations suggest that fixed charges in the interstitial compartment can lead to extracellular anion exclusion in some tissues and perhaps to accumulation in others. The magnitude and direction of these effects differ for different striated muscles.
Secretory response of dispersed rat submandibular cells. I. Potassium release
Authors: Quissell D. O.
Published: December 2017

The secretory response of dispersed rat submandibular cells as it relates to the net efflux of K+ following sympathomimetic and parasympathomimetic stimulation was evaluated. The cholinergic agonists acetylcholine and carbamylcholine were found to have equal efficacy but dissimilar receptor affinity. Median effective concentrations (EC50's) were 1.7 x 10(-7) M and 7.1 x 10(-7) M, respectively. Pilocarpine was found to be acting as a "partial" agonist and had an EC50 at 5.6 x 10(-7) M. (-)-Norepinephrine and (-)-epinephrine were found to have similar efficacy and potency with EC50's at 5.6 x 10(-7) M and 3.8 x 10(-7) M, respectively. (-)-Phenylephrine was found to act as a partial agonist with an EC50 at 3.8 x 10(-6) M, whereas (-)-isoproterenol stimulation resulted in no net K+ efflux within the concentrations tested (10(-8) M--10(-3) M). Extracellular Ca2+, but not Mg2+, was shown to be required to elicit a net K efflux following either cholinergic or alpha-adrenergic stimulation.
Extremes of urine osmolality: lack of effect on red blood cell survival
Authors: Leon H. A. , Fleming J. E.
Published: December 2017

Rats were allowed a third of normal water intake for 20 days, and food consumption decreased. The reticulocyte count indicated a suppression of erythropoiesis. Urine osmolality increased from 2,000 mosmol/kg to 3,390 mosmol/kg. Random hemolysis and senescence of a cohort of red blood cell (RBC) previously labeled with [2-14C]glycine was monitored via the production of 14CO. Neither hemolysis nor senescence was affected. Following water restriction, the polydipsic rats generated a hypotonic urine. Urine osmolality decreased to 1,300 mosmol/kg for at least 6 days; a reticulocytosis occurred, but RBC survival was unaffected. These results contradict those previously reported, which suggest that RBC survival is influenced by the osmotic stress imposed on the RBC by extremes of urine tonicity. This discrepancy, we conclude, is due to differences in the methods employed for measuring RBC survival. The random-labeling technique employed previously assumes a steady state between RBC production and destruction. The cohort-labeling technique used here measures hemolysis and senescence independent of changes in RBC production, which is known to be depressed by fasting.
Stretch-induced growth in chicken wing muscles: biochemical and morphological characterization
Authors: Barnett J. G. , Holly R. G. , Ashmore C. R.
Published: December 2017

Skeletal muscle growth induced by passive stretch was characterized in chicken wing muscles. Within 24 h after installing the stretch apparatus on the birds, the sarcomere length of the stretched patagialis was 40% greater than control. This was accompanied by increasing muscle wet weight, protein content, DNA and RNA concentrations. Sarcomere length returned to near control values by 3 days, but muscle wet weight and protein content continued to increase through 10 days. DNA and RNA concentrations reached a peak after 5-7 days and began to decline. Histological examination after 7 days revealed no change in the concentration of nuclei inside the basement membrane surrounding the muscle fibers, but the concentration of nuclei outside the basement membrane had increased. Therefore, the ratio of protein to DNA can be a poor index of muscle fiber DNA unit size. Additionally, no evidence of new muscle fiber formation was found. Electron microscopy demonstrated that passive stretch destroyed sarcomere registration between adjacent myofibrils. We concluded that passive stretch is a powerful inducer of muscle growth.
Enzyme levels in individual rat muscle fibers
Authors: Hintz C. S. , Lowry C. V. , Kaiser K. K. , McKee D. , Lowry O. H.
Published: December 2017

Individual muscle fibers from the rat anterior tibialis and soleus muscles were each analyzed in duplicate for lactate dehydrogenase (LDH, EC 1.1.1.27), malate dehydrogenase (MDH, EC 1.1.1.37), 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35), fumarate hydrotase (EC 4.2.1.2), glycogen phosphorylase (EC 2.4.1.1), 6-phosphofructokinase (EC 2.7.1.11), pyruvate kinase (EC 2.7.1.40), fructose-bisphosphatase (EC 3.1.3.11), and creatine kinase (EC 2.7.3.2). A few fibers were also analyzed for adenylate kinase (EC 2.7.4.3). In general, there was a wide and almost continuous spectrum of coordinated enzyme activities. In the tibialis muscle, two fiber groups could be clearly distinguished on the basis of MDH activity. The high MDH group had on the average lower LDH activity, but there was a great deal of overlap in LDH between the two groups. Less overlap was observed for phosphorylase and fructose-bisphosphatase, both inversely related to MDH. Only one main group of fibers (presumably slow twitch) was found in the soleus muscle, although enzyme activities also covered a wide range. These soleus fibers were clearly distinguished from the high MDH tibialis group by much lower activities of LDH, pyruvate kinase, and fructose-bisphosphatase.
Prolactin effects on ion transport across cultured mouse mammary epithelium
Authors: Bisbee C. A.
Published: December 2017

Prolactin is a known osmoregulatory hormone in lower vertebrates, and recent evidence indicates that this hormone modulates ionic concentrations in milk. In an ultrastructurally and biochemically differentiated primary cell culture system in which mouse mammary epithelium is maintained on floating collagen gels, prolactin causes an increase in short-circuit current (Isc) of monolayers of cells derived from midpregnant (24.6 to 48.0 microA . cm-2) and lactating (10.4 to 16.1 microA . cm-2) glands. Transepithelial potential differences (basal side ground) average about -12 mV and are similar to those seen in vivo. Prelactating mammary epithelial cell cultures have transepithelial resistances ranging from 374 omega . cm2 (prolactin present) to 507 omega . cm2 (prolactin absent), and lactating cell cultures have resistances averaging almost 1,000 omega . cm2. Prolactin effects require at most one day of culture maintenance in prolactin-containing medium, and the effects are not due to known contamination of prolactin preparations with arginine vasopressin or growth hormone. Medium concentrations of prolactin as low as 1 ng/ml can elicit these effects. In prelactating cell cultures not treated with prolactin, the Isc is equal to the rate of sodium absorption. Prolactin increases sodium absorption fourfold but increases Isc only twofold. Clearly, prolactin induces other active transport; neither potassium nor chloride movements can account for this additional transport. Resistance values, current-voltage plots, and permeability coefficients indicate that in vitro mammary epithelium is a moderately “tight” tissue. Comparisons with intact glands indicate that in vitro mammary epithelium closely resembles its in vivo counterpart. Floating collagen gel cultures appear suitable for elucidating transport properties in cellularly heterogeneous and structurally complex mammalian tissues.
Enzymatic isolation of cells from bone: cytotoxic enzymes of bacterial collagenase
Authors: Hefley T. , Cushing J. , Brand J. S.
Published: December 2017

The enzymatic isolation of cells from fetal rat calvaria is most effectively achieved with crude Clostridium histolyticum collagenase. However, this bacterial collagenase damages the cells during the digestion of the tissue. We have used cell density, as measured by isopycnic centrifugation on polysucrose gradients, as an indicator of cell damage. There are at least two enzymes in crude bacterial collagenase capable of damaging the cells in this tissue. One of these is clostripain that has been well characterized. The other cytotoxic enzyme is uncharacterized, and its effects are not evident until the clostripain activity has been inhibited by alpha-tosyl-lysyl chloromethane. The apparent activity of this second enzyme can be inhibited by withholding magnesium from the digestion medium and by increasing the potassium concentration of the digestion medium.
Unidirectional flux ratio for potassium ions in depolarized frog skeletal muscle
Authors: Spalding B. C. , Senyk O. , Swift J. G. , Horowicz P.
Published: December 2017

Small bundles of frog skeletal muscle fibers were loaded with 305 mM K+ and 120 mM Cl-, and 42K+ tracer efflux and influx were measured as a function of external K+ concentration ([K+]o) at a resting potential of -2 mV. As [K+]o was lowered from 305 mM, efflux decreased along a markedly sigmoidal curve, reaching a constant nonzero value at low [K+]o. Influx varied linearly with [K+]o at low [K+]o and more steeply at higher [K+]o. The ratio of influx to efflux was described by the equation: influx/efflux = exp[-n(V - VK)F/RT] with n = 2 at high [K+]o, but the ratio approached this equation with n = 1 at low [K+]o. Efflux did not depend on [K+]o when the membrane potential was raised to +36 mV, whereas at low [K+]o decreasing the membrane potential to -19 mV further activated the efflux. The results are discussed in terms of an inwardly rectifying potassium channel with two or more activating sites within the membrane that bind K+ and are accessible from the external solution.
Hormone-induced changes in NADH fluorescence and O2 consumption of rat hepatocytes
Authors: Balaban R. S. , Blum J. J.
Published: December 2017

Hepatocytes isolated from fasted male rats were incubated with a mixture of glucose, ribose, mannose, glycerol, and acetate and then treated with vasopressin (ADH), glucagon, or vasoactive intestinal polypeptide (VIP). Each of these hormones causes a rapid transient increase in the fluorescence signal arising from NADH and a sustained increase in the rate of metabolic oxygen consumption (QO2). The NADH transient was largest in response to glucagon, followed by ADH and VIP, respectively. For each hormone the responses were prevented by addition of a calcium-chelating agent. These results show that a transient, Ca2+-dependent redox shift in NADH and stimulation of QO2, perhaps resulting from an increase in the rate of delivery of reducing equivalents to the mitochondrion, occur early in the sequence of events by which several hormones that increase gluconeogenesis act.
Maximal force potential of tetanized mammalian smooth muscle
Authors: Stephens N. L. , Brutsaert D. L.
Published: December 2017

By applying abrupt load clamps of varying magnitude at a given moment during the course of an isotonic tetanus in smooth muscle of canine trachea (TSM) and saphenous vein (CSV), it is possible to obtain a measure of the maximal load-bearing capacity of the force-producing sites in these muscles. This capacity has been termed maximal force potential (MFP). By applying such load clamps at differing times throughout the course of the tetanus and beyond, we have been able to obtain MFP curves for TSM and CSV. MFP curves for these smooth muscles differ from those for striated muscle. First, although MFP in striated cardiac muscle is only about 20% greater than tetanic force, it is up to nearly 200% greater in smooth muscle. Second and more importantly, although in striated muscle MFP curves return to zero along with the tetanic force curves, in smooth muscle MFP is maintained for long after the isometric tetanus has returned to zero.
Peripheral nerve as an osmometer: role of the perineurium in frog sciatic nerve
Authors: Ask P. , Levitan H. , Robinson P. J. , Rapoport S. I.
Published: December 2017

Measurements of volume and hydrostatic pressure in the frog sciatic nerve in vitro demonstrate that the nerve acts as an osmometer, in large part because the perineurium is a semipermeable membrane for water flow. Endoneurial hydrostatic pressure in nerves in isotonic Ringer exceeds bath pressure by about 7 mmHg. In Ringer made hypertonic by addition of sucrose, nerve volume and endoneurial pressure fall linearly in relation to 1/osmolality. The slope of the plot of pressure against volume provides a value for nerve compliance equal to 0.006 mm2/mmHg. Calculations based on the model of the nerve as an osmometer indicate that the nerve has an osmotically "inactive" volume equal to 0.19 mm3/mm, which is about 75% of the total volume of a nerve segment of unit length in normal Ringer. Perineurial hydraulic conductivity (Lp) equals 7.5 x 10(-13) cm3.s-1.dyn-1, a value characteristic of nonleaky epithelia. The perineurium is an elastic tissue with a constant modulus of elasticity equal to 3 x 10(6) dyn/cm2 when not markedly stretched and may limit nerve swelling under pathological conditions of nerve edema.
Energy metabolism of preimplantation mammalian blastocysts
Authors: Benos D. J. , Balaban R. S.
Published: December 2017

We have measured the relative contributions of glycolytic and oxidative energy production pathways of metabolism in rabbit and mouse preimplantation blastocysts. We have further studied the relationship between these pathways and active transepithelial Na+ transport. Our results show that over 85% of all ATP production arises from mitochondrial respiration. By using amphotericin B to increase the Na+ permeability of the apical (or uterine-facing) membrane of the blastocyst, we have determined that the ratio of ouabain-sensitive Na+ influx to ATP consumption is 3. Based on the measurements of ouabain-sensitive Na+ influx across blastocysts incubated in glucose-containing medium, only 6% of the total energy consumption of the embryo is used for active transepithelial Na+ transport.
Effects of ATP on lysosomes: protection against hyperosmolar KCl
Authors: Ruth R. C. , Weglicki W. B.
Published: December 2017

After incubation at 37 degrees C in isosmolar (200 mM) KCl, rat liver lysosomes are susceptible to damage caused by brief exposure to hyperosmolar (greater than 200 mM) KCl. Lysosomes that are exposed to hyperosmolar KCl do not undergo significant lysis as long as they are maintained at hyperosmolar conditions; however, they will lyse on being returned to lower osmolarities. If the hyperosmolar KCl-treated lysosomes are intermittently transferred into equally hyperosmolar sucrose, they no longer undergo lysis on subsequent exposure to lower osmolarities; this confirms the reversible nature of the hyperosmolar KCl-induced damage. Thus the hypothesis that the hyperosmolar KCl damage involves an isosmotic permeating of the lysosome by KCl appears reasonable. The increase caused by the hyperosmolar KCl in free activity of beta-N-acetyl-D-glucosaminidase is reduced by about 50% by ATP but not by ATP analogues. ATP protects provided that it is added either before, or simultaneously with, exposure of the lysosomes to hyperosmolar KCl. However, if the ATP is not added until the lysosomes are already in the presence of the hyperosmolar KCl, it does not reverse the damaging effects of the KCl even though actual lysis has not yet occurred at the time of the ATP addition. The protective effect is established very rapidly, because ATP added simultaneously with the addition of the hyperosmolar KCl protects to the same extent as does ATP added any time prior to the KCl addition. The protective effect requires Mg2+ and is not supported by Ca2+. Maximal protection is provided by 5 X 10(-4) M ATP. It is postulated that ATP protects lysosomes by reducing an increase in intralysosomal concentration of KCl, which occurs when incubated lysosomes are exposed to hyperosmolar KCl.
Uptake of liposomal phosphatidylcholine by granular pneumocytes in primary culture
Authors: Chander A. , Claypool W. D. , Strauss J. F. , Fisher A. B.
Published: December 2017

Reuptake of pulmonary surfactant phospholipids was investigated with rat granular pneumocytes in primary culture and L-alpha-[2-palmitoyl-9,10-3H]dipalmitoyl phosphatidylcholine:egg phosphatidylcholine:phosphatidylglycerol:cholesterol (10:5:2:3, mol/mol) liposomes. Uptake of liposomal phosphatidylcholine by granular pneumocytes increased with time and concentration of phosphatidylcholine in the medium. With 150 microM phosphatidylcholine, uptake was about 5 nmol/mg cell protein in 2 h. Phosphatidylcholine uptake was in large part due to net transfer of vesicles as indicated by uptake of [14C]sucrose encapsulated in the aqueous compartment of liposomes. Using dipalmitoyl phosphatidylcholine:cholesterol (1:1) liposomes, uptake was inhibited significantly at 26 degrees C and completely at 4 degrees C. Inhibitors and uncouplers of oxidative phosphorylation had no effect on uptake although uptake was somewhat inhibited in the presence of either 5.6 mM 2-deoxy-D-glucose or 10 mM sodium fluoride. Cell-associated lipid radioactivity decreased after treatment with 0.25% trypsin. The percent radioactivity that was trypsin releasable decreased with increasing time and phosphatidylcholine concentration. The results suggest that uptake of phospholipids by these cells is by surface binding followed by internalization. After 2 h of incubation, 65.3 +/- 3.1% of the cell-associated radioactivity was present in phosphatidylcholine, a small fraction in phosphatidylglycerol, and the remainder in lysophosphatidylcholine, free fatty acids, and other neutral lipids, suggesting metabolic degradation of internalized lipids. This process of phospholipid uptake and degradation may have a physiological role in metabolism of surfactant phospholipids in the lung.
Heterogeneity in regard to enzymes and metabolites within individual muscle fibers
Authors: Hintz C. S. , Chi M. M. , Lowry O. H.
Published: December 2017

The possibility of variability along individual rat and human muscle fibers was assessed for four enzymes and four substances responsive to stimulation. Heterogeneity was determined by the differences between ends of fiber segments several millimeters long and by fluctuations in successive samples along the entire length of such segments. Among the enzymes, a cytosolic enzyme, lactate dehydrogenase, varied the least: average coefficient of variation (CV) of 5%. This is only a little greater than the analytic error. On the other hand, the CV for a mitochondrial enzyme, fumarase, was 13%. A mixed cytosolic-mitochondrial enzyme, malate dehydrogenase, was intermediate (CV of 9%). The CV for glycogen phosphorylase, which is normally bound to glycogen particles, was 34% along one fiber segment. Among the nonenzyme components, average CVs in stimulated fibers were 34, 20, 7, and 7%, respectively, for glucose 6-phosphate, phosphocreatine, ATP, and malate. Major differences were not random but developed gradually over distances of 0.5-2 mm along the fibers, and in some cases significant correlations between enzyme and metabolite levels were demonstrable.
Mechanism of protection by verapamil and nifedipine from anoxic injury in isolated cardiac myocytes
Authors: Cheung J. Y. , Leaf A. , Bonventre J. V.
Published: December 2017

This study was designed to determine whether slow Ca2+ channel blocking agents exert a direct protective effect on the anoxic myocardial cell and, if so, what the mechanism of protection is. Isolated Ca2+-tolerant rat cardiac myocytes were incubated under aerobic or anaerobic conditions, with or without verapamil or nifedipine, in the resting and contractile state. Protection against cell injury was assessed by preservation of rod-shaped morphology, cellular ATP levels, intracellular ionic composition, and lactate dehydrogenase release. Resting myocytes incubated anaerobically lost their rod-shaped appearance, accumulated Na+ and lost K+, and suffered a significant loss of cellular ATP. The release of lactate dehydrogenase into the medium was increased twofold, indicating significant membrane injury. Verapamil (1 microM) or nifedipine (1 microM) did not afford any protection against anoxic injury as measured by these parameters. Furthermore, on reoxygenation, anoxic verapamil- and nifedipine-treated myocytes had significantly higher cellular Ca2+ levels than control aerobic cells. When anoxic myocytes were paced at a rate of 300/min for 10 min, there were marked decreases in the number of rod-shaped cells and cellular ATP levels, whereas identically paced aerobic cells sustained no significant injury. Verapamil (1 microM) or nifedipine (1 microM) protected cells paced at 300/min from anoxic injury, but the cells were unable to sustain contraction rates at the frequency of the imposed pacing.(ABSTRACT TRUNCATED AT 250 WORDS)
Fluid composition of basolateral space of kidney cells in culture and its modification by intracellular cAMP
Authors: Corman B. , Roy C. , Roinel N. , de Rouffignac C.
Published: December 2017

LLC-PK1 kidney cells, like other epithelial cells, form domes due to Na+ and water movements from the apical to the basolateral side of the epithelium when cultured on a nonpermeable support. The composition of the fluid trapped under these domes can therefore be considered to reflect the nature of the fluid reabsorbed through the epithelium. Collecting the basolateral fluid by micropuncture and analyzing it by electron microprobe revealed that it was isosmotic with the external medium and that the concentrations of Na+, Cl-, Ca2+, Mg2+, and phosphate were nearly the same in both. In contrast, the K+ concentration was found to be 40% lower under the domes than in the bath. Changing the osmolality of the apical medium showed that the epithelium was leaky for water, although it maintained an ionic gradient for at least 20 min. Fifteen minutes after addition of dibutyryl adenosine 3',5'-cyclic monophosphate or 3-isobutyl-1-methylxanthine (5 X 10(-4) M) to the external medium, the phosphate concentration in the basolateral fluid was increased. This rise peaked at 1 h and diminished thereafter. The significance of these observations is discussed in regard to transepithelial permeability and to the transport properties of LLC-PK1 cells.
Transport and proteolytic processing of rabbit cardiac cathepsin D
Authors: Samarel A. M. , Ferguson A. G. , Worobec S. W. , Lesch M.
Published: December 2017

Rabbit cardiac cathepsin D is synthesized as a 53,000-mol wt precursor that undergoes limited proteolysis at an unknown intracellular site to a 48,000-mol wt active form. To examine the site of proteolytic processing, isolated perfused rabbit hearts were fractionated by differential centrifugation 150 or 300 min after pulse labeling with [35S]methionine. Newly synthesized precursor and processed cathepsin D were quantitatively isolated from each fraction by extraction, immunoadsorption, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After 30-min pulse perfusions, all of the 35S-labeled cathepsin D was present as precursor, with the greatest amounts found in low-density subcellular fractions. Proteolytic processing of cathepsin D precursor occurred after chase perfusions that were coincident with the subcellular redistribution of newly synthesized enzyme from sites of synthesis to heavier subcellular structures. Pulse-chase perfusions with chloroquine (10 microM) inhibited precursor proteolytic processing and the time-dependent subcellular redistribution of newly synthesized cathepsin D. The data are consistent with a model for cardiac lysosomal enzyme maturation in which limited proteolytic processing occurs coincident with or soon after the transport of precursors to an acidic intracellular compartment. The results thus suggest that cathepsin D proteolytic processing occurs within cardiac lysosomes.
Hydrogen transport in papillary collecting duct of rabbit kidney
Authors: Prigent A. , Bichara M. , Paillard M.
Published: December 2017

To examine the cellular mechanisms of H+ transfer in rabbit papillary collecting duct (PCD), the 5,5-[14C]dimethyloxazolidine-2,4-dione-derived cell pH (pHi), the [3H]triphenylmethylphosphonium-derived membrane potential (Em), the lumen-to-cell Na+ concentration gradient [( Na+]o/[Na+]i), and cell potassium and chloride concentrations were studied at 37 degrees C in separated PCD from rabbits pretreated with deoxycorticosterone acetate. The variations in cell pH values were used as an index of changes in H+ secretion. Under standard conditions pHi was 7.30 +/- 0.04, [Na+]o/[Na+]i was 2.46 +/- 0.43, Em was 78 +/- 7 mV (cell negative), [K+]i was 105 +/- 10 mM, and [Cl-]i was 33 +/- 6 mM; the value of pHi thus remained higher than expected if H+ ions were passively distributed (6.13). Acetazolamide, 10(-4) M, alkalinized the cells. When [Na+]o/[Na+]i was reduced (low-Na+ medium or 10(-3) M ouabain), the cells did not acidify, suggesting that net H+ secretion did not decrease; also, pHi was not linked to the variations in the transmembrane chloride concentration gradients. When the cells were depolarized (low-Na+ medium), they became more alkaline; when the cells were hyperpolarized (10(-4) M amiloride), they became more acid; minor change in Em (ouabain) was associated with no change in pHi. It is concluded that: 1) H+ is actively secreted into the lumen; 2) active H+ secretion may not be secondary, via electroneutral Na+:H+ countertransport or HCl cotransport, but probably occurs via a primary H+ pump; 3) variations in Em probably affect pHi by acting on both the active H+ transport system and passive movements of HCO-3 (or its equivalent).
Effect of duration of stimulation and pH on energetics of rabbit rectococcygeus muscle
Authors: Gibbs C. L. , Kotsanas G. , McCance I.
Published: December 2017

The mechanical and energetic performance of the rabbit rectococcygeus preparation have been studied at 27 degrees C. Energy flux has been measured either myothermically (heat production) or by recording the O2 consumption and lactate production of the muscle. The maximal active-to-basal O2 consumption ratio was 2.42. The peak oxidative energy flux was 3.5 mW/g, and the peak initial rate was about 5.0 mW/g. We could find no evidence for an increment in lactate production associated with mechanical activity. The effects of increasing the stimulus duration on the “economy” of force maintenance was assessed by plotting the heat-to-tension ratio against duration. For stimulus periods up to 20 s there was no evidence of a change in the economy: the slope of the relationship was similar in myothermic and O2 consumption experiments with split muscles. When total heat production was plotted against the stress-time integral for stimulus periods up to 60 s at both pH 7.4 and 6.7, there was no change in energy usage in the longer duration tetani (2–60 s). However, the reduction in pH, which had minimal mechanical effects other than prolonging relaxation, did slow the rate of evolution of recovery heat (time constant doubled) and reduced the rate of O2 consumption. It is concluded that the rectococcygeus preparation cannot make use of the energy-saving mechanisms that operate in arterial and several invertebrate smooth muscles.