bluuebunny/crossref_metadata_2025 huggingface dataset:
cAMP immunocytochemistry provides evidence for functional VIP receptors in trachea
Authors: Lazarus S. C. , Basbaum C. B. , Barnes P. J. , Gold W. M.
Published: December 2017

Vasoactive intestinal peptide (VIP), first isolated from porcine intestine (S. I. Said and V. Mutt, Science Wash. DC 169: 1217-1218, 1970), has been identified in postganglionic autonomic axons of many tissues. VIP has potent regulatory effects on the function of various cell types within these tissues, ranging from relaxation of smooth muscle to ion transport. Recently, VIP has been implicated in the regulation of mucus secretion in the respiratory tract, a process involving release of macromolecules from exocrine cells and transport of ions and water across the airway mucosa. However, because airway glands and mucosa both consist of mixed cell populations, it was unclear which specific cells contained VIP receptors and contributed to VIP-evoked responses. We identified these specific cells by using immunocytochemical techniques to monitor concentration changes in adenosine 3',5'-cyclic monophosphate (cAMP), the intracellular compound known to mediate VIP responses. Serous and mucous cells of ferret tracheal submucosal glands and ciliated and basal cells of dog tracheal mucosa all increased cAMP in response to VIP stimulation. We conclude that these cell types possess VIP receptors and thus participate in VIP-stimulated responses. In contrast, ferret tracheal epithelium and dog epithelial goblet cells showed little or no reactivity after VIP, and thus we believe that these cells lack VIP receptors.
Neuropeptides degranulate serous cells of ferret tracheal glands
Authors: Gashi A. A. , Borson D. B. , Finkbeiner W. E. , Nadel J. A. , Basbaum C. B.
Published: December 2017

To determine whether serous or mucous cells in tracheal submucosal glands respond to the neuropeptides substance P (SP) and vasoactive intestinal peptide (VIP), we studied the peptide-induced changes in gland cell morphology accompanying release of 35SO4-labeled macromolecules from tracheal explants of ferrets. Explants were labeled for 1 h in medium containing 35SO4 and washed for 3.5 additional hours. Base-line secretion in the absence of drugs declined between 1.5 and 3.5 h after the pulse. Between 2.5 and 3.5 h, the average percent change in counts per minute recovered per sample period was not significantly different from zero (P greater than 0.3; n = 6). Substance P (10(-5) M) and VIP (2 X 10(-6) M) added 4 h after labeling each increased greatly the release of 35SO4-labeled macromolecules (SP, 219%; VIP, 180%) above base line. Bethanechol, a muscarinic-cholinergic agonist (10(-5) M), increased secretion by an average of 142% above base line (each effect, P less than 0.05; n = 6 each). Light and electron microscopy of the control tissues showed glands with narrow lumens and numerous secretory granules. Glands treated with SP or VIP had enlarged lumens and the serous cells were markedly degranulated. These phenomena were documented by morphometry and suggest that SP and VIP cause secretion from glands at least partially by stimulating exocytosis from serous cells.
An established cell line from mouse kidney medullary thick ascending limb. II. Transepithelial electrophysiology
Authors: Valentich J. D. , Stokols M. F.
Published: December 2017

This study investigates the transepithelial electrophysiological properties of two mouse kidney medullary thick ascending limb cell lines developed in our laboratory. By using a modified Ussing chamber, the transepithelial voltage, transepithelial resistance, and short-circuit current of monolayer cultures were determined. Normal transport functions of the medullary thick ascending limb were not expressed in the cell lines. Instead, they exhibited electrogenic sodium absorption, which could be inhibited by the sodium channel blocker amiloride. Transplantation of the cell line M-mTAL-1C into allogeneic mice in diffusion chambers elicited reexpression of the medullary thick ascending limb transport phenotype, including development of a characteristic basal negative transepithelial voltage sensitive to the loop diuretic furosemide and to chloride removal. These results indicate that retention of renal tubule-specific transport properties is possible in long-term cell culture. However, their expression requires a special milieu not provided by current culture systems.
Phorbol ester-induced contraction in chemically skinned vascular smooth muscle
Authors: Chatterjee M. , Tejada M.
Published: December 2017

We studied the contractile response to phorbol esters and its relationship to myosin light chain phosphorylation in intact and Triton X-100-skinned porcine carotid preparations. Muscle contraction was activated by phorbol 12,13-dibutyrate (PDBu) and phorbol 12,13-didecanoate (PDD). Dose-dependent contractions to PDBu were obtained both in the intact and skinned preparations. The maximal values of stress in response to PDBu were 1.11 +/- 0.10 X 10(5) N/m2 (n = 7) in the intact and 5.72 +/- 0.59 X 10(4) N/m2 (n = 10) in the skinned muscles. The skinned tissues responded to PDD, which has been shown to activate protein kinase C, but not to the inactive isomer 4 alpha-PDD, thus ruling out nonspecific phorbol effects. The phorbol ester response exhibited a Ca2+ dependence. High stresses in the skinned muscles (5.53 +/- 0.69 X 10(4) N/m2, n = 8) were associated with low values of myosin light chain phosphorylation (0.18 +/- 0.01 mol Pi/mol light chain, n = 8). Thus phorbol esters can contract vascular smooth muscle by a mechanism that is not proportional to myosin light chain phosphorylation and that may involve activation of protein kinase C.
Isolation of smooth muscle cells from swine carotid artery by digestion with papain
Authors: Driska S. P. , Porter R.
Published: December 2017

A new method is described for the preparation of viable, elongated smooth muscle cells from the swine carotid artery. Cells were prepared by papain digestion of pressurized arteries in calcium-free solution. After digestion, the arteries were everted, and fine strips were teased from the intimal surface of the media in calcium-free solution, releasing single cells. Viability was assessed by exclusion of trypan blue and by appearance under phase-contrast microscopy. By these criteria, approximately 20% of the isolated cells were viable. The most distinguishing and unexpected characteristic of these cells was their length. Mean length of the relaxed viable cells was 240.4 +/- 47.4 microns (SD, n = 76), which is much longer than previously reported for arterial smooth muscle cells. Calcium (1.6 mM) caused most of the viable cells to contract slightly, and the mean cell length in calcium was 194.4 +/- 57.7 microns. Cells in 1.6 mM calcium contracted substantially in response to 10 microM histamine or the calcium ionophore A23187 (10 microM), demonstrating that histamine receptors and the contractile apparatus were still functional.
Pig coronary artery smooth muscle: substrate and pH dependence of the two calcium pumps
Authors: Grover A. K. , Samson S. E.
Published: December 2017

The properties of the pig coronary artery smooth muscle Ca transport mechanisms and the effects of pH were examined. The Ca pump in the plasma membrane requires less than 50 microM Mg-ATP2- for half-maximal activity, but the pump in the endoplasmic reticulum requires greater than 700 microM Mg-ATP2-. The Ca uptake by the various subcellular organelles occurs with the following affinity characteristics towards Ca2+: plasma membrane, Km = 0.91 +/- 0.06 microM, and Hill coefficient = 1.31 +/- 0.09; endoplasmic reticulum, Km = 0.58 +/- 0.05 microM, and Hill coefficient = 2.48 +/- 0.34; and mitochondria, Km = 7.1 +/- 1.16, and Hill coefficient = 0.93 +/- 0.10. The active Ca transport systems showed similar pH dependence in that the Ca uptake at pH 6.8 was greater than at 7.6. However, the pH effects were much larger on the Ca uptake by the endoplasmic reticulum and mitochondrial membranes than by the plasma membrane. The plasma membrane also showed a small amount of pH-dependent, high-affinity Ca binding. Ca efflux from passively loaded plasma membrane vesicles was similar at pH 6.4 and 7.4. From these data, it is concluded that the endoplasmic reticulum Ca pump may play a larger role in the vasodilation of large coronary artery, but the role of the plasma membrane may be more in maintenance of relaxation mechanism.
Water and nonelectrolyte permeability of isolated rat hepatocytes
Authors: Alpini G. , Garrick R. A. , Jones M. J. , Nunes R. , Tavoloni N.
Published: December 2017

We have measured the diffusive permeability coefficients of isolated rat hepatocytes to 3H2O, [14C]urea, [14C]erythritol, [14C]mannitol, [3H]sucrose, and [3H]inulin, employing a technique previously developed for erythrocytes (Redwood et al., J. Gen. Physiol 64:706-729, 1974). Diffusion coefficients for the tracer molecules were measured in packed hepatocytes, supernatant fluid, and intracellular medium (lysed hepatocytes) and were calculated assuming one-dimensional semi-infinite diffusion through a homogeneous medium. By applying the series-parallel pathway model, the following permeability coefficients (10(-5) cm/sec) for the hepatocyte plasma membrane were obtained. 3H2O, 98.6 +/- 18.4; [14C]urea, 18.2 +/- 5.3; [14C]erythritol, 4.8 +/- 1.6; [14C]mannitol, 3.1 +/- 1.4; [3H]sucrose, 0; [3H]inulin, 0. These results indicate that isolated rat hepatocytes are highly permeable to water and polar nonelectrolytes, when compared with other transporting epithelia. This relatively high cellular permeability is consistent with a model in which nonelectrolyte permeation is via an aqueous pathway of equivalent pore diameter of 8-12 A. The finding that [14C]erythritol and [14C]mannitol cross the hepatocyte plasma membrane indicates that these molecules enter the bile canaliculus through the transcellular route. Conversely, the failure of [3H]sucrose and [3H]inulin to permeate the hepatocyte in the isolated condition supports the concept that biliary entry of these large carbohydrates, at least that fraction which cannot be accounted for by a vesicular mechanism, must occur via the transjunctional shunt pathway.
Changes in plasma albumin concentration, synthesis rate, and mRNA level during acute inflammation
Authors: Liao W. S. , Jefferson L. S. , Taylor J. M.
Published: December 2017

Induction of acute inflammation in rats by the subcutaneous injection of turpentine resulted in a marked fall in the concentration of albumin in plasma. This fall, which reached a minimum of 45% of the control level at 36 h after injection of the inflammatory agent, occurred in the presence of no significant change in the concentration of total protein in plasma. It was accompanied by a corresponding decline in the relative abundance of albumin mRNA in liver, which reached a minimum of 25% of the control level at 36 h after initiation of the inflammatory response. Perfused livers from 36-h postinjection rats exhibited albumin secretion rates that were reduced to 38% of control values. In contrast, release of total secretory proteins, secretion of nonalbumin plasma proteins, and synthesis of nonexported proteins by perfused livers were elevated to 166, 266, and 117% of the control values, respectively, as a result of the inflammatory response. These results demonstrate that acute inflammation causes a relative reduction in hepatic albumin mRNA, which leads to a corresponding decrease in albumin synthesis and secretion by liver and a fall in the concentration of albumin in plasma. The concentration of total protein in plasma is maintained during acute inflammation in part by increased synthesis and secretion by liver of nonalbumin plasma proteins, e.g. the acute-phase reactants.
Inhibition and activation of Na+-Ca2+ exchange activity by quinacrine
Authors: de la Pena P. , Reeves J. P.
Published: December 2017

Quinacrine either inhibited or stimulated Na-Ca exchange in cardiac sarcolemmal vesicles, depending on the experimental conditions. When present in the assay medium for Na-Ca exchange, quinacrine inhibited both Nai-dependent Ca2+ uptake (Ki = 50 microM) and Nao-dependent Ca2+ efflux. Quinacrine's inhibition of Ca2+ efflux was attenuated by high concentrations of Na+. Quinacrine also blocked Na-Na and Ca-Ca exchange activities in the vesicles. The inhibitory effects of quinacrine on Na-Ca exchange activity are qualitatively similar to those reported previously for amiloride derivatives. When Na-loaded vesicles were preincubated with quinacrine and then assayed for Na-Ca exchange in a quinacrine-free medium, stimulation of exchange activity was observed. This stimulation was reversible on the removal of bound quinacrine and involved in a reduction in the apparent Km for extravesicular Ca2+. Stimulation of exchange activity under these conditions was also observed with the lipophilic cation tetraphenylphosphonium. Since Ca2+, quinacrine and tetraphenylphosphonium all bind strongly to sarcolemmal membranes, it is suggested that the observed stimulation of exchange activity involves a local electrostatic effect of the bound cations in accelerating a rate-limiting step in the reaction mechanism for Na-Ca exchange.
31P nuclear magnetic resonance measurements of intracellular pH in giant barnacle muscle
Authors: Hamm J. R. , Yue G. M.
Published: December 2017

The accuracy of intracellular pH (pHi) measurements by 31P nuclear magnetic resonance (NMR) spectroscopy was examined in single muscle fibers from the giant barnacle, Balanus nubilis. The pHi was derived from the chemical shifts of 2-deoxy-D-glucose-6-phosphate and inorganic phosphate. In fibers superfused with sea water at pH 7.7, pHi = 7.30 +/- 0.02 at 20 degrees C. Experimentally induced pHi changes were followed with a time resolution of 3 min. Intracellular alkalinization was induced by exposure to NH4Cl and intracellular acidification followed when NH3 was removed. Then acid extrusion was stimulated by exposure to bicarbonate containing sea water. In single muscle fibers 31P NMR results were in excellent agreement with microelectrode studies over the pH range of 6.5 to 8.0. The initial acid extrusion rate was 1.7 +/- 0.3 mmol X l-1 X min-1 at pHi 6.75. Our results showed that 31P NMR is a reliable in vivo pH probe.
Pretranslational regulation of myoglobin gene expression
Authors: Underwood L. E. , Williams R. S.
Published: December 2017

We have used blot hybridization techniques and a specific anti-sense RNA probe to determine whether variation in myoglobin gene expression among mammalian striated muscles is attributable to pretranslational regulatory events. We observed that myoglobin mRNA was expressed to approximately 10- and 5-fold greater levels, respectively, in cardiac and soleus (slow-twitch, oxidative, skeletal) muscles of adult rabbits than in tibialis anterior (fast-twitch, glycolytic, skeletal) muscles. Furthermore, when oxidative capacity of tibialis anterior muscles was increased by 21 days of indirect electrical stimulation, a model of exercise conditioning, myoglobin mRNA content increased approximately 15-fold. We conclude that pretranslational mechanisms are important in regulation of myoglobin gene expression in mammalian muscles.
Hormone 1 alpha,25-dihydroxyvitamin D3 modulates heat shock response in monocytes
Authors: Polla B. S. , Healy A. M. , Wojno W. C. , Krane S. M.
Published: December 2017

Monocytes, which play a central role in inflammatory reactions, are exposed to elevated temperatures in febrile states or to potential cellular toxins (e.g., reactive oxygen species, immune mediators). We analyzed the heat shock (HS) response in human peripheral blood monocytes and the monocytic line U937 compared with that in dermal fibroblasts. After HS, U937 cells and fibroblasts synthesized mainly the 70- and 83-kDa HS proteins (HSPs), whereas the spectrum of HSPs synthesized by monocytes varied over the range 41-45 degrees C. Incubation with hydrogen peroxide induced synthesis of the 70-kDa HSP in monocytes only. The hormone 1 alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] that protects monocytes from thermal injury had similar effects on the HS response in U937 cells, increasing synthesis of HSPs and levels of HSP 70 mRNA and partially preventing the decrease in normal protein synthesis, whereas interferon-gamma had no effect. 1,25-(OH)2D3 did not modify the HS response in fibroblasts, although these cells contained cellular receptors for 1,25-(OH)2D3. 1,25-(OH)2D3 could play a specific role in modulating the HS response in monocytes and related cells.
Mechanical properties of skinned single fibers of identified types from rat diaphragm
Authors: Eddinger T. J. , Moss R. L.
Published: December 2017

Maximum isometric tension (Po), maximum velocity of shortening (Vmax), and tension-pCa (i.e., -log[Ca2+]) relationships were determined in single skinned fibers from rat diaphragm. Histochemistry (myosin-ATPase) and sodium dodecyl sulfate (SDS) gel electrophoresis were performed on these same fibers to determine fiber type and protein composition. Physiologically fast fibers were found to have larger cross-sectional areas (CSA) and produced more tension per CSA and were less sensitive to [Ca2+] than physiologically slow fibers. Fast fibers were typed histochemically as type II and contained myosin heavy chains (MHC) and light chains (LC) of the fast type, whereas the slow fibers contained slow MHC and LC. There were also corresponding differences in the regulatory protein composition of these two fiber types. The histochemical sections confirmed a significant fiber size difference between the type IIa and IIb fibers. When fiber size was used to separate the fast fibers into two groups, type IIb fibers were found to have significantly greater Vmax and tension per CSA than the type IIa fibers. Although there were no noticeable differences in MHC composition between the type IIa and IIb fibers, there were some differences in the myosin LC and regulatory protein content.
Measurement of intracellular Ca2+ activity in Necturus gallbladder
Authors: Palant C. E. , Kurtz I.
Published: December 2017

To clarify the effects of Ca2+-free solutions on the electrophysiological properties of leaky epithelia, Necturus gallbladder was mounted in an Ussing-type chamber and its mucosal surface exposed to Ca2+-free EGTA (2 mM) Ringer. Lateral-space width was controlled by a -3-cmH2O pressure gradient on the serosal outflow. Transepithelial potential difference and resistance were monitored while cell membrane potential and intracellular Ca2+ activity (Ca2+i) were determined with conventional and Ca2+-sensitive microelectrodes. Ca2+i averaged 183 +/- 27 nM (n = 15). Reduction of mucosal Ca2+ activity to approximately 500 nM reversibly lowered transepithelial resistance while cell membrane potential remained unaltered and fractional membrane resistance increased from 0.77 +/- 0.01 to 0.83 +/- 0.02 (P less than 0.01, n = 5). In five gallbladders mucosal Ca2+ reduction induced a significant drop in Ca2+i from 133 +/- 26 to 77 +/- 20 nM (P less than 0.01, n = 5) while transepithelial resistance fell from 125 +/- 27 to 107 +/- 24 omega X cm2 (P less than 0.01). These results indicate that transepithelial resistance decrements observed during exposure to Ca2+-free solutions stem from a reversible increase in tight-junctional but not cell membrane permeability and that this effect is associated with a fall in intracellular Ca2+ activity.
Energy metabolism in contracting rat skeletal muscle: adaptation to exercise training
Authors: Constable S. H. , Favier R. J. , McLane J. A. , Fell R. D. , Chen M. , Holloszy J. O.
Published: December 2017

Rats were trained by means of a program of treadmill running. Hindlimb muscles were stimulated to contract in anesthetized rats. Measurements were made on the plantaris and the deep, predominantly fast-twitch red portion of the gastrocnemius. The concentration of ATP plus phosphocreatine (approximately P) decreased less and stabilized at a higher level, whereas inorganic phosphate (Pi) and AMP concentrations increased less and attained lower steady-state levels in trained than in untrained muscles at the same work rate. Similarly, when muscles were stimulated to contract in the perfused rat hindquarter preparation, phosphocreatine (PC) concentration decreased less in trained plantaris muscle during contractile activity that resulted in the same rate of oxygen uptake by trained and untrained muscles. In both preparations, glycogen concentration decreased less and lactate increased less in the trained muscle. From the changes that occurred in the PC-to-creatine ratio during contractile activity and from ATP concentration, it could be estimated that free ADP concentration increased less than one-half as much in trained as in untrained muscles. We conclude that, as a consequence of the adaptive increase in muscle mitochondria, approximately P concentration is higher and Pi, ADP, and AMP concentrations are lower in muscles of exercise-trained compared with untrained rats during the same contractile activity.
Isolated muscle cells as a physiological model
Authors: Lieberman M. , Hauschka S. D. , Hall Z. W. , Eisenberg B. R. , Horn R. , Walsh J. V. , Tsien R. W. , Jones A. W. , Walker J. L. , Poenie M. , et al.
Published: December 2017

Summary of a symposium presented by the American Physiological Society (Cell and General Physiology Section and Muscle Group) at the 70th Annual Meeting of the Federation of American Societies for Experimental Biology, St. Louis, Missouri, April 15, 1986, chaired by M. Lieberman and F. Fay. This symposium reflects a growing interest in seeking new technologies to study the basic physiological and biophysical properties of cardiac, smooth, and skeletal muscle cells. Recognizing that technical and analytical problems associated with multicellular preparations limit the physiological significance of many experiments, investigators have increasingly focused on efforts to isolate single, functional embryonic, and adult muscle cells. Progress in obtaining physiologically relevant preparations has been both rapid and significant even though problems regarding cell purification and viability are not fully resolved. The symposium draws attention to a broad, though incomplete, range of studies using isolated or cultured muscle cells. Based on the following reports, investigators should be convinced that a variety of experiments can be designed with preparations of isolated cells and those in tissue culture to resolve questions about fundamental physiological properties of muscle cells.
Evidence for a force-dependent component of calcium binding to cardiac troponin C
Authors: Hofmann P. A. , Fuchs F.
Published: December 2017

The duration of activation in cardiac muscle is a function of the load. On the basis of studies of Ca2+ transients in muscles subjected to quick release, it has been suggested that force or shortening-mediated changes in Ca2+-troponin C affinity may provide a mechanism for a contraction-activation feedback. This study was designed to test the hypothesis that the formation of force-generating complexes between actin and myosin enhances the affinity of cardiac troponin C for Ca2+. This was done by first establishing the normal relationship between Ca2+ binding and force development in chemically skinned bovine ventricular muscle bundles and then comparing the Ca2+-saturation curves obtained with relaxed and contracting muscle bundles. A double isotope technique was used to measure Ca2+ binding during ATP-induced force generation and during relaxation maintained by the phosphate analogue vanadate. The results showed that the generation of force was associated with an enhanced binding of Ca2+ to the Ca2+-specific regulatory site of cardiac troponin C. These data provide direct evidence that feedback between force and activation in the heart may be mediated by the Ca2+-regulatory site of troponin C.
Control of late G0/G1 progression and protein modification by SmC/IGF I
Authors: Olashaw N. E. , Van Wyk J. J. , Pledger W. J.
Published: December 2017

Somatomedin C/insulin-like growth factor I (SmC/IGF I) mediates traverse of late G0/G1 in density-arrested BALB/c-3T3 cells from a distinct growth arrest point in mid-G0/G1 (the V point) to the initiation of DNA synthesis. As a prelude to future studies aimed at defining the mechanism of action of SmC/IGF I, we investigated the level (e.g., transcriptional, translational) at which SmC/IGF I modulates V to S traverse. The post-V point progression of cells arrested at the V point by amino acid starvation and released into amino acid-replenished medium containing SmC/IGF I, insulin, or platelet-poor plasma (PPP) did not require either mRNA synthesis or an increase in the overall level of protein synthesis. Although two-dimensional gel analysis of proteins prepared from SmC/IGF I-treated cells did not reveal any preferentially synthesized proteins, several SmC/IGF I-induced protein modifications, which result in an increase in isoelectric point (pI) and occur in the absence of mRNA synthesis, were evident. These findings suggest that SmC/IGF I modulates late G0/G1 progression by a posttranscriptional process that may involve protein modification.
Protamine alters apical membrane K+ and Cl- permeability in gallbladder epithelium
Authors: Poler S. M. , Reuss L.
Published: December 2017

Protamine addition to the solution bathing the mucosal side of Necturus gallbladder epithelium (25-100 mg/l) caused depolarization of both cell membranes, a mucosa-negative change in transepithelial voltage, an increase in the apical membrane resistance (Ra) followed by a decrease, and a monotonic increase in transepithelial resistance (Rt). In protamine (25 mg/l), the change in apical membrane voltage elicited by elevating mucosal solution [K+] from 2.5 to 92.5 mM was reduced from 66 +/-2 to 38 +/- 5 mV (P less than 0.001). The K+-induced fall in Ra was also reduced in protamine. These effects could also be elicited by elevating mucosal solution [K+] simultaneously with the addition of protamine and by transient addition of protamine during exposure to the high K+ medium. The effect of protamine on the electrodiffusive Cl- permeability of the apical membrane (PCl) was studied both in control and forskolin-treated tissues. In the absence of forskolin, the hyperpolarization of Vmc produced by lowering mucosal [Cl-] to 10 mM was reversed to a small depolarization; in forskolin, the initial depolarization produced by lowering [Cl-] was significantly increased. Finally, exposure to protamine in the absence of forskolin produced an initial fall in intracellular Cl- activity. Our results indicate that protamine decreases apical membrane K+ permeability and increases apical membrane PCl. The time course of the effects of protamine suggests the possibility of an initial effect on surface potential, followed by secondary actions mediated by intracellular events.
Vasopressin-induced changes in the three-dimensional structure of toad bladder apical surface
Authors: Hartwig J. H. , Ausiello D. A. , Brown D.
Published: December 2017

The apical plasma membrane of toad bladder granular cells undergoes a rapid and dramatic increase in water permeability in response to vasopressin stimulation. Previous studies have shown that this permeability increase is accompanied by characteristic changes in the morphology of this membrane and that these changes may be involved in the hormonal response. In this report, we have used the technique of rapid freezing and freeze drying to obtain high resolution stereo images of the surface of the granular cell apical plasma membrane before and during vasopressin stimulation. Using this approach, we confirmed that vasopressin induces a ridge-to-villus transformation of the cell surface even in the absence of osmotic water flow, but now show that this transformation occurs at least in part via a retraction of segments of preexisting ridges, rather than by the growth of new microvilli from the apical cell surface. This is also demonstrated by the finding that vasopressin induces the ridge-to-villus transformation of the cell surface even in the presence of cytochalasin D. In addition, the rapid-freeze, freeze-dry technique reveals that the surface glycocalyx of the epithelial cells consists of a complex, three-dimensional network of filaments that is heterogeneous among different cells. Finally, vasopressin-induced tubular invaginations of the apical plasma membrane were visualized in stereomicrographs, and the number and size of such invaginations were altered in the presence of cytochalasin D. These may represent surface images of vasopressin-induced exo- and endocytotic events that are related to membrane permeability changes.
Interleukin 1 and tumor necrosis factor do not regulate protein balance in skeletal muscle
Authors: Moldawer L. L. , Svaninger G. , Gelin J. , Lundholm K. G.
Published: December 2017

Recent studies have claimed that interleukin 1-containing preparations increase skeletal protein degradation similar to that seen during infection and inflammation. However, preparations employed have contained other products of activated macrophages, including tumor necrosis factor-alpha. In the present report, we investigated the capability of recombinant-derived murine and human interleukins 1-alpha and 1-beta and human tumor necrosis factor-alpha to affect skeletal protein synthesis and degradation both in vitro and in vivo. Partially purified products of Staphylococcus albus-stimulated human blood monocytes increased skeletal protein degradation both in vivo and in vitro. However, none of the recombinant interleukin 1 nor the human tumor necrosis factor-alpha preparations had any impact on skeletal protein balance. Both recombinant interleukin 1 and tumor necrosis factor-alpha stimulated the production of prostaglandin E2 (PGE2). Furthermore, a polyclonal antibody to human interleukin 1 eliminated the lymphoproliferative response to partially purified monocyte preparations (interleukin 1 activity), but failed to abrogate the increased skeletal protein degradation in vitro. This study demonstrates that although interleukin 1 and tumor necrosis factor-alpha induce a PGE2 response by skeletal muscle in vitro, some macrophage product distinct from either interleukin 1 or tumor necrosis factor-alpha is responsible for the accelerated skeletal protein degradation seen with partially purified human blood monocyte products. Elevated PGE2 levels do not appear to regulate skeletal protein balance in vitro.
Regulation of the atrial natriuretic peptide receptor on a smooth muscle cell
Authors: Hughes R. J. , Struthers R. S. , Fong A. M. , Insel P. A.
Published: December 2017

We have used a smooth muscle cell line isolated from rabbit carotid artery (RCA) as a model system with which to study the ligand-mediated regulation of the atrial natriuretic peptide (ANP) receptor. RCA cells bind rat alpha-ANP-(1-28) semi-irreversibly at 4 degrees C, apparently to a single class of high-affinity binding sites (Kd approximately equal to 5 pM). In contrast, ANP initially bound to RCA cells at 37 degrees C is fully reversible after the cells have been cooled to 4 degrees C. Although the measured dissociation constant for binding rat alpha-ANP-(1-28) is 10-fold lower at 4 degrees C than at 37 degrees C, the maximum amount of specifically bound radioligand is equivalent at both temperatures. We infer this to be suggestive of ANP receptor recycling occurring at 37 degrees C. This hypothesis is substantiated by experiments demonstrating the rapid disappearance of ANP receptors (measured at 4 degrees C) after exposure of RCA cells to ANP at 37 degrees C. We believe this reflects a loss of cell-surface receptors that is reversible on removal of ANP from the incubation medium at 37 degrees C. The concentration dependence and rank order of potency of ANP in eliciting receptor redistribution resembles that for binding to the ANP receptor and not for eliciting cyclic GMP formation. In contrast to this striking loss of presumed cell-surface receptors, incubation of RCA cells with ANP elicits only limited desensitization of the cyclic GMP response.(ABSTRACT TRUNCATED AT 250 WORDS)
Alteration of intestinal tight junction structure and permeability by cytoskeletal contraction
Authors: Madara J. L. , Moore R. , Carlson S.
Published: December 2017

Permeabilized intestinal absorptive cell brush borders contain a perijunctional ring of actin and myosin (PAMR) that can be induced to contract. Recently, morphological changes suggestive of PAMR contraction were shown to occur in absorptive cells of ileal epithelium after exposure to cytochalasin D (CD) (J. Cell Biol. 102: 2125-2136, 1986). With this response, altered tight junction structure and enhanced tight junction permeability also occur. To further assess the relationship between PAMR contraction and enhanced tight junction permeability, we examined the effect of the uncoupler 2,4-dinitrophenol (DNP) on this CD response. Progressive depletion of functionally defined intraepithelial energy stores occurred with DNP concentrations of 0.1-1 mM. Such DNP concentrations did not independently impair tight junction barrier function. Depletion of energy stores before CD exposure ablated the ability of CD to induce abnormalities of tight junction permeability. Similarly, PAMR condensation and alterations in tight junction structure could be dissociated from CD exposure by prior depletion of functional energy reserves. These data tie CD elicited alterations in tight junction structure and permeability to an energy dependent event that appears to be PAMR contraction. We speculate that tensile forces within the PAMR regulate tight junction structure and function.
Quantitative determination of Na+-K+-ATPase and other sarcolemmal components in muscle cells
Authors: Hansen O. , Clausen T.
Published: December 2017

A recurring problem in the characterization of plasma membrane enzymes in tissues and cells is whether the samples tested are representative for the entire population of enzyme molecules present in the starting material. Measurements of [3H]-ouabain binding, enzyme activity, and maximum transport capacity all indicate that the concentration of Na+-K+ pumps in mammalian skeletal muscle is high (300-800 pmol/g wet wt). Studies on Na+-K+-ATPase activity in isolated sarcolemma, however, generally give little or no information on total cellular enzyme concentration. Due to the low and variable enzyme recovery (0.2-8.9%), such subcellular preparations may, therefore, give misleading data on factors regulating Na+-K+-ATPase in heart and skeletal muscle cells. As the same isolation and purification procedures are used for the study of other sarcolemmal components (lipids, hormone receptors, enzymes, and other transport systems), this inadequate recovery has general implications for statements on regulatory changes in the sarcolemmal composition of muscle cells. On the other hand, complete quantification of Na+-K+-ATPase in muscle tissue can now be achieved using simple procedures and the entire material (intact muscle fibers, biopsies, and whole homogenates). Recent studies have shown that regulatory changes in the entire population of Na+-K+ pumps in muscle can be quantified in measurements of [3H]-ouabain binding, K+-activated 3-O-methylfluorescein phosphatase activity, as well as maximum ouabain suppressible Na+-K+ transport capacity.
Cross-bridge phosphorylation and regulation of latch state in smooth muscle
Authors: Hai C. M. , Murphy R. A.
Published: December 2017

We have developed a minimum kinetic model for cross-bridge interactions with the thin filament in smooth muscle. The model hypothesizes two types of cross-bridge interactions: 1) cycling phosphorylated cross bridges and 2) noncycling dephosphorylated cross bridges ("latch bridges"). The major assumptions are that 1) Ca2+-dependent myosin phosphorylation is the only postulated regulatory mechanism, 2) each myosin head acts independently, and 3) latch bridges are formed by dephosphorylation of an attached cross bridge. Rate constants were resolved by fitting data on the time courses of myosin phosphorylation and stress development. Comparison of the rate constants indicates that latch-bridge detachment is the rate-limiting step. Model simulations predicted a hyperbolic dependence of steady-state stress on myosin phosphorylation, which corresponded with the experimental observation of high values of stress with low levels of phosphorylation in intact tissues. Model simulations also predicted the experimental observation that an initial phosphorylation transient only accelerates stress development, with no effect on the final steady-state levels of stress. Because the only Ca2+-dependent regulatory mechanism in this model was activation of myosin light chain kinase, these results are consistent with the hypothesis that myosin phosphorylation is both necessary and sufficient for the development of the latch state.
Depression of membrane-bound Na+-K+-ATPase activity induced by free radicals and by ischemia of kidney
Authors: Kako K. , Kato M. , Matsuoka T. , Mustapha A.
Published: December 2017

A partially purified, membrane-bound Na+-K+-ATPase fraction, prepared from the outer medulla of porcine kidney, was incubated in the presence of 0.1 mM FeCl3, 1 mM ADP, and 0.1-100 mM H2O2 for either 15 or 30 min at 37 degrees C. The activity of ouabain-sensitive Na+-K+-ATPase was reduced proportionally to the concentration of H2O2 and the duration of incubation. There were decreases in SH contents and turnover rates of the Na+-K+-ATPase preparation, while malondialdehyde (MDA) and conjugated dienes were generated from the membrane lipids in the course of the incubation. The concentrations of ethanolamine (E) plasmalogen and of arachidonic acid in the E glycerophospholipid molecules were reduced by the free radical reaction. Similarly, a reduction in Na+-K+-ATPase activity and the formation of MDA and conjugated dienes, together with a decrease in E glycerophospholipids, were observed when the membrane fraction was exposed to ultraviolet irradiation (254 nm) for 30 min at 4 degrees C. Administration of 10 mM dithiothreitol alleviated the reductions in enzyme activity, in turnover rate, and in SH content without suppressing MDA formation. Addition of 2 mM butylated hydroxytoluene to the incubation mixture prevented the lipid peroxidation without totally normalizing the enzyme activity in the H2O2 experiment, whereas this antioxidant restored the ATPase activity to normal in the ultraviolet experiment. Microsomal fractions, prepared from the outer medulla of canine kidney after 1 h of unilateral ischemia and 1 h of reperfusion, showed a decreased Na+-K+-ATPase activity, a reduced amount of SH groups, and an increased MDA.(ABSTRACT TRUNCATED AT 250 WORDS)
Receptors for and effects of insulin and IGF-I in rat glomerular mesangial cells
Authors: Arnqvist H. J. , Ballermann B. J. , King G. L.
Published: December 2017

Receptors for and biological effects of insulin and insulin-like growth factor I (IGF-I) were studied in cultured rat renal mesangial cells. Specific binding of 125I-IGF was over 200-fold greater (5.8%/0.2 mg cell protein) than the specific binding of 125I-insulin (0.2%/2 mg cell protein). Fifty percent inhibition of 125I-insulin binding was obtained with 8 x 10(-9) M unlabeled insulin. For 125I-IGF-I, 50% inhibition required 1.8 x 10(-9) M unlabeled IGF-I. 125I-IGF-I was also displaced by IGF-II and insulin but at 10-and 100-fold lower potencies, respectively, than IGF-I. Cross-linking of 125I-insulin and 125I-IGF-I to their receptors, using disuccinimidyl suberate (DSS), and identification of the receptor with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography showed a band with a molecular mass of 135 kDa, probably corresponding to the alpha-subunit of the insulin receptor and a major band with a molecular mass of 145 kDa for the alpha-subunit of the IGF-I receptor. Both insulin and IGF-I stimulated the incorporation of [3H]thymidine into DNA. A half-maximal effect was obtained at 1.6 x 10(-8) M for insulin and 1.2 x 10(-9) M for IGF-I. No additive effect on DNA synthesis was observed. Insulin at 8 x 10(-10) M increased the accumulation of [14C]glucose in mesangial cells, whereas IGF-I was 10-fold less potent.
Effect of a chloride channel blocker on bullfrog corneal epithelium
Authors: Scheide J. I. , Zadunaisky J. A.
Published: December 2017

The effect of the chloride channel blocker, diphenylamine-2-carboxylate (DPC), was investigated on the bullfrog cornea epithelium using microelectrodes. Perfusion of 2 X 10(-4) M DPC on the tear side resulted in a 44% reduction of the corneal transepithelial potential difference (TEP) and a 9% reduction in corneal conductance. The cornea epithelial cells hyperpolarized 7.2 +/- 1.5 mV, and the apical fractional resistance increased 35% (n = 8 tissues). The basolateral potential difference depolarized 5.5 +/- 1.8 mV. The relative apical conductance decreased 29%, whereas relative changes of basolateral membranes increased 56% with prolonged DPC exposure, indicating that the DPC inhibition was acting at the apical cell membrane. DPC-treated corneas could be stimulated with isoproterenol (10(-6) M), indicating that the DPC inhibition could be reversed with the stimulation of chloride conductance. The DPC inhibitory effect was dependent on basolateral chloride. Intracellular chloride activity increases with DPC, indicating that the integrity of the basolateral chloride entry mechanism was not altered. The DPC inhibitory action was due to a reduction in apical chloride conductance.
A linear model of muscle respiration explains monoexponential phosphocreatine changes
Authors: Meyer R. A.
Published: December 2017

Phosphocreatine (PCr) content was measured by phosphorus nuclear magnetic resonance spectroscopy in the gastrocnemius muscles of pentobarbital-anesthetized rats during and after twitch stimulation at rates up to 0.75 Hz. The monoexponential time constant for PCr changes was similar at the onset of vs. during recovery after stimulation and was not significantly different for different stimulation rates (mean time constant 1.44 min). Steady-state PCr level during stimulation was linearly related to the product of stimulation rate times peak twitch force. These results are shown to be consistent with a simple first-order electrical analog model of oxidative metabolism that is applicable at submaximal oxidative rates. The model assumes equilibrium of the creatine kinase reaction, which is modeled as a chemical capacitor, with capacitance proportional to the total creatine level, and PCr level proportional to the cytosolic free energy of ATP hydrolysis.
Functional significance of myosin transitions in single fibers of developing soleus muscle
Authors: Reiser P. J. , Kasper C. E. , Greaser M. L. , Moss R. L.
Published: December 2017

The maximal velocity of shortening and myosin heavy chain (MHC) composition of single, chemically skinned fibers from neonatal and adult rat soleus muscles were examined to determine the relationship between these parameters during slow muscle development in the rat. In addition, the MHC composition of bundles of fibers from soleus muscles at the same ages was studied. The MHC compositions were examined using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The results from the bundles of fibers indicate that from 3 days to 5 mo postnatal, the rat soleus contains predominantly MHCs that migrate in the vicinity of the MHC from adult slow muscle. From 14 days to 2 mo postnatal, there are also significant amounts of additional MHCs that comigrate on SDS gels with those characteristic of adult rat fast muscle. All the fibers studied at 3 and 7 days postnatal and at 5 mo and the majority of fibers from 14 days to 2 mo postnatal had relatively low shortening velocities. A few fibers from the latter group had significantly higher velocities. The faster fibers at each age had greater amounts of the MHCs that comigrate with the adult fast-type MHC on SDS gels. Thus the velocity of shortening of single fibers from the rat soleus muscle appears to be related to MHC composition during postnatal development.
Clenbuterol prevents or inhibits loss of specific mRNAs in atrophying rat skeletal muscle
Authors: Babij P. , Booth F. W.
Published: December 2017

It is known that denervation or hindlimb suspension both decrease the content of rRNA, alpha-actin mRNA, and cytochrome c mRNA in adult rat skeletal muscle. In the present study, the provision of clenbuterol (an anabolic agent) to adult female rats during a 7-day period of denervation of the soleus and gastrocnemius muscles prevented entirely the loss of rRNA, alpha-actin mRNA, and cytochrome c mRNA that normally occurs in denervated muscle. Although clenbuterol inhibited most of the loss of alpha-actin mRNA that occurred in the soleus and gastrocnemius muscles after 7 days of hindlimb suspension, clenbuterol administration had less effect on preventing the loss of rRNA and cytochrome c mRNA in hindlimb suspended skeletal muscle. Clenbuterol had no effect on protein content in atrophied muscle resulting from denervation or suspension. These data suggest that clenbuterol can maintain the expression of certain RNAs in atrophying adult rat skeletal muscle.
Ion transport in cultured fetal and adult rabbit tracheal epithelia
Authors: Zeitlin P. L. , Loughlin G. M. , Guggino W. B.
Published: December 2017

Transepithelial potential difference (Vte), short circuit current (Isc), and transepithelial resistance (Rte) were measured to assess Na+ and Cl- transport in cultured tracheal epithelial cells from fetal and adult rabbits. Cultured fetal and adult cells developed differentiated morphology as determined by electron microscopy. Base-line Vte, Isc, and Rte were equivalent in late gestation (28-30 days) and adult cultures. Amiloride (10(-4) M) (Na+ transport inhibitor) reduced Isc by 7.4 +/- 1.2 microA/cm2 (24%) in adult cultures but only by 1.8 +/- 0.7 microA/cm2 (7%) in fetal cultures (P less than 0.001). Furosemide (10(-4) M) (Cl- transport inhibitor) diminished Isc by 16.5 +/- 3.2 microA/cm2 (53%) in adult cultures and 18.8 +/- 2.8 microA/cm2 (74%) in fetal cultures (P less than 0.01). Epinephrine (10(-6) M) stimulated Isc by 24.2 +/- 5.8 microA/cm2 (72%) in adult cells but only by 7.0 +/- 1.1 microA/cm2 (32%) in fetal cells (P less than 0.05). After epinephrine exposure, furosemide-sensitive Isc increased to 41.5 +/- 7.7 microA/cm2 in adult cells but remained unchanged in fetal cells (P less than 0.001). Our results suggest that fetal cells absorb less Na+ and secrete more Cl- than adult cells and that the Cl- secretory response to epinephrine is greater in adult than in fetal cells.
(Na + K + 2Cl) cotransport in cultured embryonic chick heart cells
Authors: Liu Shi , Jacob Ron , Piwnica-Worms David , Lieberman Melvyn
Published: December 2017

Pages C721–C730: Shi Liu, Ron Jacob, David Piwnica-Worms, and Melvyn Lieberman.“(Na + K + 2Cl) cotransport in cultured embryonic chick heart cells.” Page C721: first and second sentences, second paragraph, second column, should read “In this study, coupled ion movements are investigated using Na-, K-, and Cl-selective microelectrodes, in contrast to previous work (30), in which we determined the contents of Na, K, and Cl by atomic absorption spectrophotometry (Nai and Ki) or coulometric titration (Cli). We find that Cl-HCO3 exchange does not appear to contribute to the maintenance of a high agrCli in the resting steady state.7#x201D; C722: Eq. 1 should read

(See PDF)