bluuebunny/crossref_metadata_2025 huggingface dataset:
Biochemical and subcellular distribution of arachidonic acid in rat myocardium
Authors: Miyazaki Y. , Gross R. W. , Sobel B. E. , Saffitz J. E.
Published: December 2017

Selective release of arachidonic acid from prelabeled phospholipid pools has been observed following exposure of neonatal rat cardiac myocytes to metabolic inhibitors in vitro and has been correlated temporally with the development of irreversible sarcolemmal damage. Hydrolysis of phospholipids with release of arachidonic acid may be an important mechanism in the pathogenesis of sarcolemmal damage induced by ischemia. To elucidate potential subcellular loci of arachidonic acid release in ischemic myocardium, we characterized the phospholipid composition of adult rat myocardial sarcolemma and delineated the biochemical and subcellular distribution of radiolabeled arachidonic acid in neonatal rat myocytes incubated with [3H]-arachidonic acid for selected intervals. Although arachidonic acid comprised 13 +/- 3% of total aliphatic moieties in combined choline and ethanolamine glycerophospholipids isolated from purified adult rat myocardial sarcolemma, radiolabeled arachidonic acid could not be detected in sarcolemma of cultured neonatal rat myocytes incubated with 5 nM [3H]arachidonic acid for 24 h. Radioactivity was located almost exclusively in mitochondria and internal cytoplasmic membranes (primarily sarcoplasmic reticulum), which collectively contained 90% of myocyte radioactivity. These results indicate that radiolabeled arachidonic acid released from prelabeled phospholipid pools on exposure of neonatal rat myocytes to oxidative inhibitors is derived from mitochondria and internal cell membranes. The diminutive labeling of the sarcolemma suggests that turnover of arachidonoyl phospholipids is slower in the sarcolemma than in other membranous organelles.
Effects of inhibition of mitochondrial protein synthesis in skeletal muscle
Authors: Williams R. S. , Harlan W.
Published: December 2017

To evaluate the participation of proteins derived from mitochondrial genes in the adaptive response of skeletal muscle to increased contractile activity, we administered chloramphenicol (CAP; 200-1,000 mg.kg-1.day-1), an inhibitor of translation from mitochondrial ribosomes, to adult rabbits undergoing electrical stimulation of the tibialis anterior muscle of one hind limb. In unmedicated animals, 10 days of electrical stimulation increased maximum velocity (Vmax) of cytochrome oxidase and citrate synthase by 214 +/- 17 and 201 +/- 16% (P less than 0.01). In a dose-dependent manner, CAP abolished activity-induced increases in cytochrome oxidase Vmax, suggesting that augmented mitochondrial protein synthesis is necessary for the adaptive response of enzymes that require protein subunits encoded by mitochondrial genes. However, CAP failed to inhibit activity-induced changes in Vmax of enzymes derived exclusively from nuclear genes (citrate synthase and aldolase). CAP also failed to inhibit activity-induced increases in mRNA transcribed from the nuclear genes encoding beta-F1 ATPase or myoglobin, or from the mitochondrial genes encoding 12S rRNA, 16S rRNA, or cytochrome b. These latter findings suggest that mitochondrial translation products do not participate in pretranslational regulation of these nuclear or mitochondrial genes in response to changes in contractile activity of skeletal muscle.
Interaction of angiotensin II with functional smooth muscle cells in culture
Authors: Paglin S. , Stukenbrok H. , Joyce N. C. , Jamieson J. D.
Published: December 2017

In this study we report on the characterization of a highly enriched population of cultured vascular smooth muscle cells (SMC) prepared from collagenase-treated medial layer explant outgrowths of rabbit aortae. Studies done on cells from first passage explant outgrowths showed that the cells retain the fine structural features of vascular SMC in situ, can be immunostained with anti-smooth muscle myosin IgG, and bind [125I]angiotensin II (ANG II) in a specific and saturable manner with an apparent Kd of 1 nM. Addition of ANG II (0.1 microM) to the cultures causes obvious shape changes and retraction of cell processes. Electron microscopic autoradiography of cells labeled with [125I]ANG II show that the initial site of interaction of ANG II with the SMC is the plasma membrane. The distribution of ANG II receptors among cells in the population was studied using light microscopic autoradiography. The autoradiographical grain density varied among cells in the population ranging from cells that were heavily labeled to those that possessed virtually no label. These data imply that the expression of ANG II receptors may be limited to a certain progeny within the cell population or is a function of their stage within the cell cycle.
Cyclic nucleotide-dependent protein kinase inhibition by H-8: effects on ion transport
Authors: O'Grady S. M. , DeJonge H. R. , Vaandrager A. B. , Field M.
Published: December 2017

We explored the potential role of cyclic nucleotide-dependent protein phosphorylation in regulating ion transport across flounder intestinal mucosa by studying the effects of N-[2(methylamino)-ethyl]-s-isoquinolinesulfonamide (H-8), a selective inhibitor of cyclic nucleotide-dependent protein kinase in vitro. Addition of H-8 reversed the inhibitory effects of 8-bromoguanosine 3',5'-cyclic-monophosphate (8-BrcGMP), 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), atriopeptin III (AP III), and vasoactive intestinal peptide (VIP) on the short-circuit current (Isc) and transepithelial potential difference (PD). Flux measurements established that these changes in Isc and PD directly reflected changes in Na and Cl absorption by the intestine. H-8 was unable, however, to reverse the inhibitory effects on Isc and PD of the Ca ionophore ionomycin and of substance P at dosages exceeding those needed to reverse the effects of AP III, VIP, and the cyclic nucleotides. We conclude that 1) H-8 (100 microM or less) does not exert toxic effects, 2) exogenously added cyclic nucleotide analogues inhibit ion transport through activation of cyclic nucleotide-dependent kinases resulting in protein phosphorylation, 3) activation of these kinases is an essential intermediate step in the inhibitory action of AP III and VIP on ion transport, and 4) the Ca ionophore ionomycin and substance P appear to inhibit ion transport by a mechanism that is independent of cyclic nucleotide-dependent protein phosphorylation.
Purification of PRL receptors from toad kidney: comparisons with rabbit mammary PRL receptors
Authors: Dunand M. , Kraehenbuhl J. P. , Rossier B. C. , Aubert M. L.
Published: December 2017

The binding characteristics of the prolactin (PRL) receptors present in toad (Bufo marinus) kidneys were investigated and compared to those of PRL receptors present in rabbit mammary glands. The molecular characteristics of the Triton X-100 solubilized renal and mammary PRL receptors were assessed by gel filtration and by migration analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after affinity labeling of the binding sites with 125I-human growth hormone. Similar results were obtained for both receptors. Partial purification of the toad PRL receptor could be achieved by affinity chromatography. The molecular weight of this purified receptor could be determined by analysis on SDS-PAGE. With the use of a polyclonal antiserum raised against a purified preparation of rabbit mammary PRL receptor, one or several antigenic epitope(s) could be identified on the core of the toad renal PRL receptor. In conclusion, although the structure and the biological role(s) of PRL have substantially changed during evolution, the receptor for this hormone has retained many of its structural features as could be assessed between an amphibian and a mammalian species on functionally different target tissues.
A simple hydraulic drive for remote control of the Huxley-style micromanipulator
Authors: Krueger J. W.
Published: December 2017

Electrodeposited metal bellows permit the construction of simple, reliable, and inexpensive hydraulic drives for the Huxley-style micromanipulator. This approach enables precise remote control of micromanipulation without introducing conductive leads or other potential sources of electrical noise near the experimental system. Moreover, the bellows arrangement preserves a stability and reproducibility in the positioning of the heavier transducers that is not matched by other hydraulic micromanipulators. The small size of the hydraulic coupler enables easy installation, and it can be adapted to preexisting manipulators. Importantly, the manipulator's original micrometer control is preserved, a feature that allows the investigator to tailor the apparatus for novel arrangements for micromanipulation.
Potassium-induced cell swelling in Necturus gallbladder epithelium
Authors: Davis C. W. , Finn A. L.
Published: December 2017

In Necturus gallbladder epithelium, elevation of mucosal K+ to 95 mM in the presence of 10 mM Na+ resulted in cell swelling at a rate of 3.2% original volume per minute, followed by volume-regulatory shrinking. When Na+ was completely removed from or when amiloride (10(-4) M) was added to the mucosal medium, K+-induced cell swelling was abolished. In the presence of 10 mM Na+, 1 mM Ba2+ abolished and substitution of mucosal Cl- by NO-3 had no effect on K+-induced swelling. Thus solute entry following elevation of mucosal K+ is effected by separate K+ and Cl- pathways. Furthermore, substitution of 95 mM K+ for Na+ in the mucosal bathing medium leads to the development of a Cl- conductance in the basolateral membrane as long as some Na+ remains in the medium. However, cell swelling induced by mucosal dilution does not lead to the appearance of a Cl- conductance. Thus the activation of this conductance requires both swelling and membrane depolarization. These results show that 1) high mucosal K+ leads to cell swelling due to the entry of Cl- along with K+ and the Cl- can enter across either membrane, 2) the Cl- pathways require the presence of mucosal Na+, and 3) cell volume regulation is activated by an increase in volume per se, i.e., a hyposmotic exposure is not required for volume regulation to occur.
Oxidative potential in developing rat diaphragm, EDL, and soleus muscle fibers
Authors: Smith D. , Green H. , Thomson J. , Sharratt M.
Published: December 2017

To examine the effect of postnatal development on changes in oxidative potential of fibers of specific types (I, IIa, IIb, and IIc) in the rat diaphragm, determinations of succinate dehydrogenase (SDH) activity were made using microphotometric measures of optical density. Samples of the costal region of the diaphragm were extracted from 56 male Wistar rats ranging in age from 8 to 85 days and subgrouped into seven developmental periods (1, 2, 3, 4, 6, 9, and 12 wk). For type I fibers, increases of 17% (P less than 0.05) in SDH activity occurred during 2nd wk, remained elevated through 4th wk, and increased further (P less than 0.05) to 137% of 1-wk values by the end of 6th wk. No further increases were noted between 6 and 12 wk. A similar maturational trend was evident for type IIa fibers, although SDH activities remained higher throughout development when compared with type I fibers. In contrast, SDH in type IIb fibers, although increasing by 14% during the first two measurement weeks (P less than 0.05), declined from 6 to 9 wk before ultimately reaching a value similar to 3 wk. SDH activity was also assessed in a typical slow- (soleus) and fast-twitch (extensor digitorum longus, EDL) muscle of the hindlimb to contrast their development with that of the diaphragm. Generally, SDH in type I and IIa fibers was approximately 40 and 20% higher, respectively, in the diaphragm than in matched fiber types in the other muscles throughout development (diaphragm greater than EDL greater than soleus).(ABSTRACT TRUNCATED AT 250 WORDS)
Uptake and intracellular fate of [14C]sucrose-insulin in perfused rat livers
Authors: Surmacz C. A. , Wert J. J. , Ward W. F. , Mortimore G. E.
Published: December 2017

Insulin was covalently linked to [14C]sucrose by means of cyanuric chloride to provide a label that would remain entrapped within the vacuolar system. The uptake of the conjugate by the perfused rat liver was rapid (half-life = 2.9 min), competitively inhibited by native insulin, and abolished by alkali denaturation. As assessed by its distribution on self-generating gradients of colloidal silica-povidone, label in lysosome-enriched samples of liver taken at different times after the addition of the conjugate moved progressively during 15 min from the plasma membrane into an intermediate peak and then to dense lysosomal fractions. After 30-60 min, the label had equilibrated throughout the lysosomal-vacuolar system. The initial movement from the plasma membrane to the intermediate peak occurred between 2 and 5 min. Because label in the peak could be physically separated from the lysosomal marker, beta-acetylglucosaminidase, by dispersing the sample through the gradient mixture before centrifugation rather than layering it, we concluded that the intermediate particles in question were not lysosomal in nature. On gel-filtration chromatography, label extracted from the intermediate peak did not move with insulin but rather as a broad band of lower molecular weight products, suggesting that insulin is subject to early proteolytic attack within a nonlysosomal compartment.
Inactivation of amino acid transport in rat hepatocytes and hepatoma cells by PCMBS
Authors: Chiles T. C. , Dudeck-Collart K. L. , Kilberg M. S.
Published: December 2017

the transport of amino acids by both normal rat hepatocytes and rat H4 hepatoma cells has been tested for inactivation by sulfhydryl-preferring, protein-modifying reagents. Amino acid transport by systems A, ASC, N, L, and y+ in the H4 hepatoma cells was relatively resistant to inactivation by the alkylating reagent N-ethylmaleimide (NEM), whereas uptake mediated by systems A, ASC, and L was decreased in normal rat hepatocytes. In contrast, nearly all of the amino acid transport systems in both cell types were inhibited strongly by p-chloromercuribenzene sulfonate (PCMBS). The exceptions were the H4 hepatoma system y+ activity (72% of control) and system L-mediated uptake (121% of control) in normal hepatocytes. Although transport via system A was equally sensitive to inhibition by PCMBS in both cell types, substrate-dependent protection from this inactivation was observed only in the H4 hepatoma cells. These results illustrate the significant differences that exist between normal and transformed liver cells in respect to amino acid transport inactivation by sulfhydryl reagents.
Intracellular pathways of insulin transport across vascular endothelial cells
Authors: Hachiya H. L. , Halban P. A. , King G. L.
Published: December 2017

Processing and transport of hormones across vascular endothelial cells may modulate hormone action at subendothelial tissue sites. Insulin was transported across cultured rat capillary and bovine aortic endothelial cells, after a delay of 5-10 min, at a constant rate for 60 min at 37 degrees C. 125I-labeled insulin transport was inhibited by 88 +/- 11% (SE, n = 4) and 75 +/- 18% (SE, n = 4) in the presence of anti-insulin receptor antibody and unlabeled insulin (at 10(-7) M), respectively. Reverse phase high-performance liquid chromatography showed 88% of the 125I-insulin transported over 60 min was indistinguishable from the 125I-insulin added to the cells at 4 degrees C. In aortic endothelial cells preincubated with 2.3 x 10(-9) M of insulin for 24 h, insulin receptor binding was downregulated by 67%, and 125I-insulin transport was decreased by 52 +/- 11%. The proton ionophore monensin (0.05 mM) increased the internalized insulin in bovine aortic endothelial cells by 78%, with a corresponding decrease in 125I-insulin released by 76 +/- 2% (SE, n = 4). 125I-insulin transport across the aortic endothelial cell monolayer was similarly decreased (54 +/- 12%, SE, n = 4) by monensin. In contrast, the lysosomal protease inhibitor leupeptin had no effect. Degradation and transport were similarly dissociated by low temperature. At 15 degrees C, no significant insulin degradation was detected, whereas 125I-insulin release from the cells continued at 30 +/- 3% of the rate at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
Na+-coupled sugar transport: membrane potential-dependent Km and Ki for Na+
Authors: Kimmich G. A. , Randles J.
Published: December 2017

Kinetic analysis of the characteristics of phlorizin binding and of the Na+, sugar, and potential dependence of alpha-methylglucoside (alpha-MG) influx into isolated avian intestinal cells has pointed toward two alternative models for the transport mechanism (D. Restrepo and G. A. Kimmich, J. Membr. Biol. 89: 269-280, 1986). One of these models envisions a potential-dependent Na+ binding event (Na+ well concept) as a part of the molecular mechanism. The data reported here show that the apparent Km for Na+ for sugar transport is sharply dependent on the magnitude of the membrane potential. When intracellular Na+ is absent, the maximal velocity (Vmax) achieved for sugar influx is the same with or without a potential, although Vmax is obtained at a lower Na+ concentration when a potential is imposed (interior negative). Intracellular Na+ severely inhibits the influx of sugar in the absence of a potential, but this effect is largely overcome when a potential is present. The Vmax obtained when intracellular Na+ is present is a function of the potential. These results are consistent with a transport model in which Na+ binding to the Na+-dependent sugar carrier at the extracellular surface of the membrane and debinding at the inner surface of the membrane are both potential-dependent events.
Cell cycle dependence of inositol phosphate levels in neuroblastoma cells
Authors: Fleischman L. F. , Cantley L.
Published: December 2017

To investigate the timing of inositol lipid turnover in relation to the cell cycle, inositol phosphates and lipids were measured in neuroblastoma (Neuro-2A) cells that were prelabeled with [3H]inositol and synchronized by a mitotic shakeoff technique. Distinct early and late phases of inositol phosphate production were identified. The early peak occurs between the 2nd and 4th hour after mitosis near the G1/S transition. A later peak occurs around the peak of S phase (DNA synthesis) at 7-8 h after mitosis. These findings suggest that activation of phosphatidylinositol turnover generates signals that play a role in cell cycle progression.
Chronic stimulation of mammalian muscle: changes in metabolite concentrations in individual fibers
Authors: Henriksson J. , Salmons S. , Chi M. Y. , Hintz C. S. , Lowry O. H.
Published: December 2017

Single fibers were analyzed from rabbit fast-twitch tibialis anterior muscles freeze-clamped during continuous stimulation at 10 Hz for up to 8 wk. ATP declined after 1 wk to a stable level approximately 30% below controls. Phosphocreatine decreased earlier and to a greater extent (approximately 50%). Glycogen varied considerably among stimulated fibers and decreased on average approximately 75% by 8 wk. Glucose, lactate, citrate, and malate had changed little in the first 30 h and then increased four-, two-, four-, and sevenfold, respectively, over the next 5 wk. Glucose 6-phosphate showed the most unexpected behavior: with an overall upward trend, it descended to extremely low values (10% of control) after approximately 1 wk of stimulation. As long as high- and low-oxidative fibers were present, the former showed slightly higher levels of ATP, lactate, and malate; other metabolites did not differ in a consistent way. These unexpected observations, which differ strikingly from data for acute stimulation, shed light on adaptations that enable a chronically stimulated muscle to sustain a continuous high level of ATP utilization.
Na+-sugar cotransport system as a polarization marker during organization of epithelial membrane
Authors: Lasheras C. , Scott J. A. , Rabito C. A.
Published: December 2017

The present study analyzed the changes in Na+-dependent sugar transport and transepithelial electrical resistance as LLC-PK1 cells reorganize into epithelial membranes. Sugar influx increased to reach a maximum 9 h after plating. The increase in the transepithelial electrical resistance, however, showed a significant delay, reaching steady state 15 h after plating. No changes in the electrochemical Na+ gradient were observed during the reorganization of the epithelial membranes. Kinetic analysis and [3H]phlorizin-binding studies showed that the increase in sugar influx resulted from an increase in the number of carriers. Unidirectional sugar influx measurements indicated that the sugar transporters were primarily located at the apical surface of the epithelial cells. These observations are consistent with the hypothesis that the sorting of native proteins occurs intracellularly before their insertion in the apical membrane, or as an alternative that they are randomly inserted, but then immediately sorted such as any carrier could be detected in the basolateral side during the reorganization process. In addition, the results suggest that the functional development of the apical membrane may occur before the complete sealing of the intercellular space during the development of the occluding junctions. Furthermore, development of the sugar transport system and occluding junctions was inhibited by cycloheximide and puromycin but not by actinomycin D, suggesting that the expression of epithelial cell polarization is probably a posttranslational event in the protein synthesis.
Increased trehalase expression after glucose limitation of LLC-PK1 clones
Authors: Yoneyama Y. , Lever J. E.
Published: December 2017

Expression of the apical membrane disaccharidase trehalase correlates with the development of a proximal tubule-specific differentiated phenotype in the LLC-PK1 cell line derived from renal epithelia. Substitution of galactose for glucose in the culture medium of confluent cultures resulted in a significant trehalase induction; a smaller induction was observed after replacement by fructose, mannose, or the nonmetabolizable sugar alpha-methyl-D-glucose. Induction was specific for trehalase among other microvillar hydrolases, reversible after replacement of glucose, and was increased as a function of decreasing glucose concentration. Stable clonal cell lines that were inducible by glucose limitation and noninducible clones were isolated nonselectively. Both types exhibited trehalase induction after exposure to the differentiation inducer N,N'-hexamethylene-bis-acetamide (HMBA). These results suggest that trehalase induction after glucose deprivation occurs by a different mechanism than that mediated by HMBA.
Carrier-mediated transport of pyroglutamyl-histidine in renal brush border membrane vesicles
Authors: Skopicki H. A. , Fisher K. , Zikos D. , Flouret G. , Bloch R. , Kubillus S. , Peterson D. R.
Published: December 2017

These studies were performed to determine if a transmembrane carrier for pyroglutamyl-histidine (pGlu-His) is present in the luminal membrane of renal proximal tubular cells. Previous studies have suggested the intact transepithelial transport of pGlu-His, a dipeptide formed by the hydrolysis of luteinizing hormone-releasing hormone by enzymes associated with the brush border in the proximal nephron. With the use of a renal brush border membrane vesicle preparation, pGlu-His showed H+-stimulated, Na-independent, saturable transport into an osmotically active space. High-pressure liquid chromatographic analysis of both the intravesicular and extravesicular fluids indicated intact uptake of the dipeptide. The transport constant (Kt) and Vmax for pGlu-His transport were 9.3 X 10(-8) M and 6.1 X 10(-12) mol.mg-1.min-1, respectively. Transport of pGlu-His was not inhibited by the dipeptides glycyl-proline, glycyl-sarcosine, and N-beta-alanyl-L-histidine, which have been previously shown to be transported into renal brush border vesicles via a single, low-affinity, high-capacity, Na-independent, and H+-stimulated peptide carrier. In addition, the gamma-glutamyl-containing peptides gamma-glutamyl-histidine and N(N-L-gamma-glutamyl-L-cysteinyl)glycine and the tripeptide pyroglutamyl-histidyl-prolinamide were without an inhibitory effect. In contrast, transport of pGlu-His was inhibited by the dipeptide pyroglutamyl-alanine. This study demonstrates the existence of a high-affinity, low-capacity H+ cotransport system for pGlu-His in the proximal tubular luminal plasmalemma, which appears to be specific for pyroglutamyl-containing dipeptides. The data indicate that multiple dipeptide carriers are present in the proximal nephron.
Capillary and size interrelationships in developing rat diaphragm, EDL, and soleus muscle fiber types
Authors: Smith D. , Green H. , Thomson J. , Sharratt M.
Published: December 2017

The effects of maturation on the interrelationship between skeletal muscle fiber area and capillarization was investigated in specific fiber types (I, IIa, IIb, IIc) of male Wistar rats at seven developmental periods ranging from 8 to 85 days postnatal. Fiber type specific developmental properties were compared in three different muscles, the diaphragm (DIA), extensor digitorum longus (EDL), and soleus (SOL), which are known to differ widely in function. All fiber types in each of the three muscles examined exhibited large increases in area (FA), the magnitude and time course of the increase being related to both the type of fiber and the muscle in which the fiber was located. For type I fibers, areas increased from 3- to 18-fold (SOL greater than EDL greater than DIA), whereas in type IIa fibers, area increased ranged between 5- to 11-fold (SOL greater than EDL greater than DIA). Growth rates in IIb fibers were more homogeneous between muscles ranging from 11- to 14-fold. Capillarization, as indicated by the capillary contacts per fiber (CC), increased in all fiber types regardless of muscle origin. These increases ranged between 1.7- and 2.2-fold for type I fibers, between 2.4- and 2.5-fold for type IIa fibers, and between 2.0- and 3.0-fold for type IIb fibers. In general, capillary density expressed as the ratio of the number of capillary contacts divided by the fiber area (CC/FA) progressively declined in all fiber types with age. The rate of the decline in CC/FA was mediated in large part by the changes in fiber area.(ABSTRACT TRUNCATED AT 250 WORDS)
Effect of ANP on sustained aldosterone secretion stimulated by angiotensin II
Authors: Isales C. M. , Bollag W. B. , Kiernan L. C. , Barrett P. Q.
Published: December 2017

The sustained aldosterone secretory response to angiotensin II (ANG II) depends on receptor-mediated increases in membrane diglyceride (DG) and an increase in calcium influx rate. These signals serve to activate membrane-associated protein kinase C (PKC) and result in enhanced phosphorylation of a unique set of proteins. These events can be mimicked by the addition of a phorbol ester, 12-O-tetra decanoyl phorbol 13-acetate (TPA), and a calcium ionophore, A23187, that bypass the initial receptor-associated events. We studied the inhibitory action of atrial natriuretic peptide (4-28 hANP) on the sustained secretory response to ANG II in isolated bovine adrenal glomerulosa cells. Although 10 nM ANP inhibited aldosterone secretion, it did not significantly alter the ANG II-elicited rise in 45Ca2+ influx rate [control (CON): 0.44 +/- 0.06; ANG II: 1.11 +/- 0.12 (P less than 0.001); ANG II + ANP: 1.18 +/- 0.14], the steady-state level of aequorin luminescence [intracellular [Ca2+] ([Ca2+]i)], or the rise in cellular DG content [CON: 0.132 +/- 0.01; ANG II: 0.194 +/- 0.01 (P less than 0.005); ANG II + ANP: 0.202 +/- 0.01 nmol/10(6) cells]. IN addition, ANP was able to inhibit aldosterone secretion stimulated by the combined addition of A23187 + TPA. When protein phosphorylation in the ANP-inhibited cells was evaluated, ANG II-induced protein phosphorylation events were preserved. In contrast to the effect of ANP, the calcium channel blocker nitrendipine abolished the ANG II-induced rise in 45Ca2+ influx rate, reduced the steady-state level of [Ca2+]i, and returned the phosphoproteins to their control states.(ABSTRACT TRUNCATED AT 250 WORDS)
Regulatory volume decrease by cultured renal cells
Authors: Knoblauch C. , Montrose M. H. , Murer H.
Published: December 2017

Volume regulatory responses of OK cells (a continuous epithelioid cell line from opossum kidney) are examined by electronic cell sizing and measurements of intracellular pH in cell suspensions. In response to a 40% reduction in osmolality, the cells swell and then subsequently shrink toward their starting volume. This regulatory volume decrease (RVD) is reduced by replacement of Cl- in the medium with acetate. Replacement of Cl- with NO3- accelerates the RVD. The RVD response is inhibited by 1 mM quinine or 100 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) in the medium. The inhibitory effect of 100 microM DIDS (but not 1 mM quinine) is altered by replacement of Cl- by NO3- in the medium. Hypotonic challenge does not induce a DIDS-sensitive net flux of acid-base equivalents. Addition of (9 microM) valinomycin also inhibits the RVD response. It is suggested that the RVD response of OK cells involves activation of separate K+ and Cl- channels.
Stimulation of K-C1 cotransport in rat red cells by a hemolytic anemia-producing metabolite of dapsone
Authors: Haas M. , Harrison J. H.
Published: December 2017

Dapsone, a sulfone compound used in the treatment of leprosy and, more recently, Pneumocystis carinii pneumonia, produces as a major side effect a hemolytic anemia. This anemia is characterized by oxidation of hemoglobin to methemoglobin and increased splenic uptake of red blood cells. Using a rat model, Grossman and Jollow (J. Pharmacol. Exp. Ther. 244: 118-125, 1988) found that dapsone hydroxylamine (DDS-NOH), a dapsone metabolite, is responsible for its hemolytic effect in vivo. DDS-NOH also promotes hemoglobin binding to SH groups on rat red cell membrane proteins (Budinsky et al., FASEB J. 2: A801, 1988). Since the binding of hemoglobin and other reagents (e.g., N-ethylmaleimide) to membrane SH groups has been associated with increased K transport in red blood cells, we examined the effect of DDS-NOH on K efflux from rat red blood cells in vitro. Cells shrink when exposed to DDS-NOH (100 microM) in media with plasma-like ionic composition. This shrinkage is prevented if extracellular K is raised to 110 mM or if intra- and extracellular Cl are replaced by methylsulfate (MeSO4), suggesting involvement of a K-Cl cotransport pathway. Indeed, 100 microM DDS-NOH produces a 4- to 5-fold increase in K efflux in cells containing Cl but less than a 2-fold increase in cells containing MeSO4. This stimulatory effect is specific for K; Na efflux is slightly inhibited by 100 microM DDS-NOH. The concentrations of DDS-NOH required for half-maximal stimulation of Cl-dependent K efflux (53 microM) is similar to its half-maximal hemolytic concentration in rats (approximately 100 microM). Furthermore, the stimulation of Cl-dependent K efflux by DDS-NOH is greater than 80% reversed by subsequent treatment of the cells with dithiothreitol, suggesting involvement of SH groups. Our results indicate that DDS-NOH exposure stimulates an apparent K-Cl cotransport in rat red blood cells, resulting in cell shrinkage under physiological ionic conditions. Since shrinkage of red blood cells renders them less deformable (Mohandas et al., J. Clin. Invest. 66: 563-573, 1980), this suggests a pathophysiological mechanism whereby DDS-NOH exposure in vivo could promote increased splenic uptake of red blood cells and hemolytic anemia.
Effects of general anesthetics on current flow across membranes in guinea pig myocytes
Authors: Niggli E. , Rudisuli A. , Maurer P. , Weingart R.
Published: December 2017

Myocytes were isolated from adult guinea pig ventricles. Whole cell, tight-seal recording was employed to investigate the electrical properties of the junctional (nexal membrane) and nonjunctional membrane (sarcolemma) under the influence of n-alkanols (heptanol, octanol) and halothane. Studies of cell pairs with a double voltage-clamp approach showed that these agents give rise to a reversible electrical uncoupling. Examination of single myocytes with a single voltage-clamp method showed that these substances modify several sarcolemmal current systems. The slope conductance was reduced over the entire voltage range examined (-90 to +50 mV). The Ca2+ inward current (Isi) showed a decreased amplitude and an accelerated inactivation. The repriming of Isi remained unchanged. The steady-state inactivation of Isi was shifted by 2-3 mV toward more negative potentials. Optical measurements demonstrated an increase in sarcomere spacing at rest and a decrease during peak systolic shortening. The results suggest that n-alkanols and halothane exert their effects on membrane currents via incorporation into the lipid bilayer.
Acetylcholine-induced intracellular acidosis in rabbit salivary gland acinar cells
Authors: Lau K. R. , Elliott A. C. , Brown P. D.
Published: December 2017

Intracellular pH (pHi) was measured in acini isolated from rabbit mandibular salivary glands using the fluorescent pH-sensitive probe 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Resting pHi was estimated to be 7.13 +/- 0.01 (mean +/- SE of 29 experiments). Stimulation with acetylcholine (ACh) caused an intracellular acidosis followed by a return of pHi toward the control value with a half time of approximately 3 min. The intracellular acidosis was dose dependent and could be abolished by pretreatment of the acini with atropine (10 microM), suggesting that it was due to a receptor-mediated event. Incubation of the acini in HCO3- -free solutions or treatment of the acini with the carbonic anhydrase inhibitor acetazolamide (1 mM) abolished the acidosis, suggesting that the acidosis might be caused by loss of HCO3- from the cell. The acidosis was not affected by either 1) pretreatment of the acini with the anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), or 2) equilibration of the acini in Cl- -free solution (Cl- substituted with glucuronate). These results suggest that the postulated HCO3- efflux does not occur by Cl- -HCO3- exchange. However, Cl- -HCO3- exchange did appear to be present because replacement of Cl- caused a large DIDS-sensitive alkalinization of pHi, presumably caused by HCO3- uptake in exchange for Cl-. The recovery of pHi after the initial acidosis on stimulation with ACh could be blocked by 1 mM amiloride, suggesting that the recovery phase was mediated by Na+-H+ exchange.
Ca-induced Ca release in malignant hyperthermia-susceptible pig skeletal muscle
Authors: Ohta T. , Endo M. , Nakano T. , Morohoshi Y. , Wanikawa K. , Ohga A.
Published: December 2017

To provide information regarding the cause of the muscle rigidity in malignant hyperthermia-susceptible (MHS) pigs, the Ca-induced Ca-release mechanism of the sarcoplasmic reticulum (SR), the Ca uptake by the SR, and the Ca-activated tension production of the contractile system were examined in skinned skeletal muscle fibers from MHS and normal pigs. In muscles of MHS pigs, the rate of Ca-induced Ca release was significantly higher than in normal muscle. The potentiation effect on Ca-induced Ca release by halothane and caffeine did not differ appreciably between MHS and normal fibers. The rate of Ca uptake by the SR and the Ca sensitivity of the contractile system of MHS fibers were not different from those of normal fibers, and halothane in an anesthetic concentration exerted no effect on them. Dantrolene inhibited the Ca-induced Ca release at 38 degrees C. These results suggest that the principal cause of malignant hyperthermia (MH) in MHS pigs is due to the enhancement of the Ca-induced Ca-release mechanism of the SR of the skeletal muscle.
A simple device for rapidly exchanging solution surrounding a single cardiac cell
Authors: Spitzer K. W. , Bridge J. H.
Published: December 2017

This study describes the design and various physiological applications of a simple device to rapidly change the solution surrounding a single intact cardiac cell. It consists of a short length of double-barreled glass tubing (theta-tubing) attached to a miniature solenoid. A cell is positioned in one of two parallel streams of solution that simultaneously flow from each barrel. Rapid solution switching is achieved by activation of the solenoid that directs the adjacent stream over the cell, changing the bulk solution within 7 ms. Approximately 150 ms were required to change the solution at the membrane surface of guinea pig ventricular cells, judging from potassium-induced changes in resting membrane potential. This delayed response was probably due to, in part, restricted diffusion in the transverse tubular system. The switching speed of this device makes it possible to change extracellular solutions during action potentials and voltage-clamp pulses.
Sulfate transport in human lung fibroblasts (IMR-90): effect of pH and anions
Authors: Elgavish A. , Meezan E.
Published: December 2017

We previously reported the presence of a carrier-mediated sulfate transport system in human lung fibroblasts (IMR-90) (A. Elgavish, J. B. Smith, D. J. Pillion, and E. Meezan. J. Cell. Physiol. 125: 243-250, 1985). Kinetic studies carried out in the lung fibroblasts show that Cl- inhibits SO4(2-) uptake in a competitive manner. Taken together with the fact that high extracellular Cl- stimulates SO4(2-) efflux, these results suggest that SO4(2-) uptake into lung fibroblasts occurs via a SO4(2-)-Cl- exchange mechanism. Extracellular HCO3- inhibits sulfate influx in a competitive manner (pH 7.5) but has no marked effect on sulfate efflux. SO4(2-) and HCO3- may therefore have the ability to bind to a common extracellular anion binding site, but they do not appear to exchange for one another. Lowering extracellular pH has a stimulatory effect on the initial rate of sulfate uptake. The pK of the extracellular pH effect is around pH 7.0, indicating that small changes in the extracellular pH around the ambient levels encountered under physiological conditions will markedly affect sulfate influx into the cell. Kinetic studies suggest that lowering extracellular pH increases the initial rate of sulfate influx by increasing the affinity of the carrier for sulfate twofold. Lowering intracellular pH inhibits the initial rate of sulfate influx into the cell. The pK of this intracellular pH effect is also around pH 7.0, indicating that physiological levels of intracellular protons are necessary for the normal activity of the anion exchanger.(ABSTRACT TRUNCATED AT 250 WORDS)
ANP relaxes bovine tracheal smooth muscle and increases cGMP
Authors: Ishii K. , Murad F.
Published: December 2017

Effects of atrial natriuretic peptides (ANP) on the tension, content of guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP) and activity of particulate and soluble forms of guanylate cyclase were examined in bovine tracheal smooth muscle. Atrial natriuretic factor (ANF), atriopeptin II, and atriopeptin III were found to induce relaxation of tracheal smooth muscle precontracted with 5 x 10(-8) M carbachol (an approximate median effective concentration) in a concentration-dependent manner. However, atriopeptin I failed to induce significant relaxation of the muscle. Similar results were obtained when 3 x 10(-6) M histamine or 5 x 10(-7) M serotonin was used as the contracting agent. However, the relaxant effects of ANF, atriopeptin II, and atriopeptin III were much less when a higher concentration of carbachol or 30 mM K+ was used as the contractile agent. Maximally inhibitory concentration (IC50) values of ANF, atriopeptin II, and atriopeptin III for inhibition of muscle contraction induced by 5 x 10(-8) M carbachol ranged from 3.8 to 8.3 x 10(-9) M, indicating that these peptides have intermediate potency between isoproterenol (IC50, 2.0 x 10(-9) M) and sodium nitroprusside (IC50, 2.0 x 10(-8) M). Treatment of the muscle with 3 x 10(-7) M ANF slowed the rate of tension development of the muscle by 10(-7) M carbachol. Tissue levels of cAMP were not influenced by any of the atrial peptides at concentrations of 10(-9)-10(-6) M; however, cGMP levels were increased about five- to ninefold.(ABSTRACT TRUNCATED AT 250 WORDS)
Inhibition of ICa in single frog cardiac cells by quinidine, flecainide, ethmozin, and ethacizin
Authors: Scamps F. , Undrovinas A. , Vassort G.
Published: December 2017

The effects of four class I antiarrhythmic compounds on the Ca current (ICa), recorded with whole cell patch clamp in single isolated frog ventricular cells, were compared. Na and K currents were blocked by tetrodotoxin and Cs. Quinidine and flecainide induced an apparent tonic block with a 50% effective dose (ED50) at 10 and 20 microM, respectively; there was no clear use-dependent inhibition. Ethmozin and ethacizin, two phenothiazine derivatives, exhibited both tonic and use-dependent inhibition. Ethacizin was at least 10 times more potent than ethmozin; at 1 microM, it induced a 15% tonic block and 5, 35, and 42% use-dependent block at 0.125, 1, and 2 Hz, respectively. These compounds appeared only 3- to 10-fold less efficient on the ICa than on the Na current recorded in parallel experiments, with flecainide showing the largest different potency. All four compounds shifted the availability curves by a few millivolts toward hyperpolarization, had a clear voltage-dependent inhibition, and slowed reactivation, the latter effect being more marked with less negative holding potential. Consequently, the absence of use-dependence inhibition with quinidine and flecainide could be the consequence of a very fast association of the two compounds with the open channels that would be complete during the 200-ms depolarizing pulse. Since arrhythmias are frequently associated with tissue depolarization and can be related to Ca-dependent action potential and slow conduction, the inhibition of the ICa reported above should account, in part, both for the antiarrhythmic and the negative inotropic effects of these compounds.
Water movement: does thermodynamic interpretation distort reality?
Authors: Essig A. , Caplan S. R.
Published: December 2017

In a recent theoretical analysis of water flow, Finkelstein (Water Movement Through Lipid Bilayers, Pores, and Plasma Membranes: Theory and Reality, 1987) has attacked the contributions of irreversible thermodynamics, stating that "the thermodynamic treatment of uphill water flow completely distorts reality." Instead he presents a mechanistic formulation. For a porous membrane, water flow is attributed to convection generated by a favorable hydrostatic pressure gradient within pores, even when in the presence of permeant solutes water moves against its chemical potential gradient; water flow may "drag", solute, to an extent determined by the solute partition coefficient, but the possibility that solute flow may drag water is excluded. We argue that this formulation violates the second law of thermodynamics. Water cannot move against its chemical potential gradient because of the influence of only part of the chemical potential gradient. Furthermore, the proposed mechanism requires that at one of the membrane-solution interfaces water must move against both its concentration gradient and the hydrostatic pressure gradient. Also considered by Finkelstein is the nature of the reflection coefficient sigma, a kinetic variable, which he concludes can be evaluated (in a porous membrane) by measurement of the (equilibrium) solute partition coefficient. We claim that in general it is not possible to evaluate a kinetic variable from measurements of equilibrium parameters alone. A valid kinetic analysis must incorporate the contribution of all coupled flows.
Electrogenic bicarbonate secretion by guinea pig gallbladder epithelium: apical membrane exit
Authors: Stewart C. P. , Winterhager J. M. , Heintze K. , Petersen K. U.
Published: December 2017

Guinea pig gallbladder epithelium secretes HCO3- by electroneutral mechanisms, resulting in transepithelial Cl- -HCO3- exchange. Adenosine 3',5'-cyclic monophosphate (cAMP) converts HCO3- secretion into an electrogenic process. This transformation was examined using voltage-clamp, pH-stat, and microelectrode techniques. Prostaglandin E1 (PGE1; 10(-6) M) was used to raise intracellular cAMP levels. It increased short-circuit current (Isc) by approximately 1.8 mumol.cm-2.h-1, an effect dependent on serosal HCO3- and, partly, on mucosal Cl-. Mucosal 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS; 10(-3) M) halved Isc, but only in Cl- containing solutions. PGE1 increased the secretory HCO3- flux from approximately 2.0 to approximately 2.7 mumol.cm-2.h-1 and reduced the absorptive HCO3- flux from approximately 1.1 to approximately 0.5 mumol.cm-2.h-1, with net HCO3- secretion accounting for the increase in Isc. During single-cell impalements, PGE1 depolarized the apical membrane by greater than 10 mV (transiently in the absence of HCO3-) and decreased the apparent ratio of membrane resistances (Ra/Rb) from 5-8 to a value close to zero. These effects were largely reduced in magnitude and rapidity by removing Cl- and HCO3- from both sides of the epithelium. Ion substitutions in the luminal perfusate revealed substantial Cl- and HCO3- permeabilities at the apical membrane under PGE1 conditions. Our results indicate that, in the presence of PGE1 (cAMP), HCO3- crosses the apical membrane by two different routes. A SITS-sensitive fraction leaves the cell in exchange for luminal Cl-, which, in turn, recycles into the lumen by electrodiffusion. The remaining HCO3- exits through a HCO3- conductive pathway.
Vascular myosin expression during cytokinesis, attachment, and hypertrophy
Authors: Seidel C. L. , Wallace C. L. , Dennison D. K. , Allen J. C.
Published: December 2017

Primary cultures of canine saphenous vein smooth muscle cells downregulate the expression of the two muscle myosin heavy chains (mMHC) and upregulate the expression of a nonmuscle myosin heavy chain (nmMHC) when maintained in medium containing 10% fetal calf serum (FCS). The cellular function and control of these changes in contractile protein expression are not known. The purposes of these experiments were to determine whether the expression of nmMHC was required for cytokinesis, whether cell attachment stimulated nmMHC expression, and whether during changes in total myosin production the relative amounts of the two mMHCs remained constant. Primary cultures were maintained in FCS, in a serum-free defined medium (SFM), or after 2 days in SFM switched to FCS to induce proliferation and changes in myosin expression. nmMHC expression occurred before cytokinesis if cells were placed directly in FCS, whereas it occurred after cytokinesis if growth-arrested cells were exposed to FCS. The position of the cell in the cell cycle was responsible for these differences, and the temporal correlation of cell-cycle progression and nmMHC expression indicated that expression occurred during G1. Cells that remained unattached in the presence of FCS or attached in SFM did not express nmMHC. During net production or loss of mMHC by growth in SFM or FCS, respectively, the relative amounts of the two mMHC remained constant. These results suggest that 1) the expression of nmMHC requires the combined effect of FCS and attachment, 2) it occurs in cells progressing through G1 but is not required for cytokinesis, and 3) during changes in net myosin production, the two mMHC are coregulated.