bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanisms underlying volume regulatory decrease by Necturus gallbladder epithelium
Authors: Furlong T. J. , Spring K. R.
Published: December 2017

Volume regulatory decrease (VRD) by Necturus gallbladder epithelial cells in Cl Ringer was unaffected by the addition of 5 mM BaCl2 to apical perfusates but was inhibited by the addition of 5 mM BaCl2 and 50 or 3 microM phencyclidine (PCP) to serosal perfusates, suggesting that K channels in the basolateral membrane were activated during VRD. VRD was unaffected by replacement of Cl with NO3 or SCN, suggesting that Cl-dependent Na-K-Cl and K-Cl cotransport were not involved. In SCN Ringer, VRD was inhibited by the addition of 0.1 mM bumetanide to serosal perfusates, suggesting that bumetanide-sensitive anion channels in the basolateral membrane were also activated. A transient 10-mV hyperpolarization of the membrane potential was associated with VRD. The channel blockers that inhibited VRD had little or no effect on the hyperpolarization, suggesting that the changes in membrane potential were unrelated to the changes in cell volume. Perfusion of the apical surface of the epithelium with isotonic solutions containing 10 mM D-glucose resulted in a variable increase in cell volume followed by a variable shrinkage to normal, suggesting that VRD was also activated during organic solute absorption. The increase in cell volume was blocked by the addition of 0.01 or 1 mM phlorizin to mucosal perfusates. The reduction in cell volume was inhibited by the addition of 0.1 mM bumetanide, but not BaCl2 or PCP, to serosal perfusates, indicating the the shrinkage mechanism secondary to glucose addition differed from that seen after exposure to hypotonic perfusates.
cAMP-binding proteins in epithelial cells cultured from human sweat glands
Authors: Pon D. J. , Wong M. , Riordan J. R. , Schimmer B. P.
Published: December 2017

This study characterized the various adenosine 3',5'-cyclic monophosphate (cAMP)-binding proteins in epithelial cells cultured from human sweat glands to identify potential pathways of cAMP action in this tissue. The cAMP-binding proteins were identified by specific labeling with the photoprobe 8-azido-[32P]-cAMP and visualized by autoradiography after electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Three cAMP-binding proteins, with molecular masses of 48, 52, and 54 kDa, were identified in subcellular fractions of the cultured sweat glands. On the basis of their relative electrophoretic mobilities and reactivity with specific antisera, the 48-kDa protein was identified as the regulatory subunit of the type 1 cAMP-dependent protein isozyme, and the 54-kDa protein was identified as the regulatory subunit of the type 2 cAMP-dependent protein kinase isozyme. The 52-kDa cAMP-binding protein appeared to be a distinct isoform of the regulatory subunit, possibly related to the subclass associated with neural tissue. As determined from the relative distributions of the photolabel, the 48-, 52-, and 54-kDa binding proteins were present in the cell cytosol at a ratio of 1:2:1. The 48- and 52-kDa proteins bound the photoprobe with apparent dissociation constants (Kd) of 20-40 nM, whereas the 54-kDa protein bound the photoprobe with much lower affinity (Kd 230 nM). Only the 48- and 52-kDa proteins were associated with cell membranes. They were present in the membrane fractions in approximately equal amounts and bound the photoprobe with apparent Kd values of greater than 100 nM.(ABSTRACT TRUNCATED AT 250 WORDS)
Human neutrophil stimulation by influenza virus: relationship of cytoplasmic pH changes to cell activation
Authors: Hartshorn K. L. , Wright J. , Collamer M. A. , White M. R. , Tauber A. I.
Published: December 2017

We have previously demonstrated that influenza A virus (IAV) stimulates the human neutrophil through phospholipase C activation. With the use of the fluorescent indicator 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF), cytoplasmic acidification and subsequent alkalinization are shown to accompany this activation. These responses are not inhibited by pertussis toxin (PT). The alkalinization is mediated largely *but not entirely) by the Na(+)-H+ antiporter and is not initiated, or modulated, by the IAV-induced cytosolic Ca2+ (Cai2+) rise. Rather, protein kinase C (PKC) is likely the mediator of cell alkalinization, based on studies using the PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7). The acidification can be dissociated from the alkalinization response, which is also independent of Cai2+ fluxes and of PKC. Both pHi responses can be dissociated from the respiratory burst. Cytosolic alkalinization and acidification seem to reflect two independently mediated responses of the activated neutrophil, the former resulting ultimately from phospholipase activation and the latter from other activities that are not yet fully characterized.
Determinants of the transmembrane distribution of chloride in rat lymphocytes: role of Cl(-)-HCO3- exchange
Authors: Garcia-Soto J. J. , Grinstein S.
Published: December 2017

The purpose of the present experiments was to establish the factors that determine the intracellular Cl- concentration ([Cl-]i) of lymphocytes. Coulometric and isotope equilibration determinations indicated that [Cl-]i was in the range of 70-85 mmol/l cells. Since the membrane potential (Em) of these cells approximates -55 mV, [Cl-]i is severalfold higher than the level expected at electrochemical equilibrium (approximately 16 mM). This suggests that conductive pathways contribute marginally to the distribution of Cl-. Accordingly, altering the force driving conductive Cl- fluxes by manipulating Em had little effect on [Cl-]i. The possible role of electroneutral cation-Cl- cotransport in the accumulation of intracellular Cl- was also assessed. 36Cl- uptake was largely unaffected by omission of extracellular Na+ and K+ or by addition of bumetanide, a potent cotransport inhibitor. Moreover, [Cl-]i remained unaltered for at least 1 h in cells incubated without Na+ or K+ or in the presence of loop diuretics. Thus it appears unlikely that Cl(-)-anion cotransport plays a major role in maintaining [Cl-]i. A vigorous stilbene disulfonate-sensitive anion exchanger was detected in thymocytes. This system constitutes a large fraction of the Cl- flux pathways and is possibly a major contributor to the establishment of [Cl-]i. Accordingly, modifying the force driving Cl- through the exchanger, by altering pH at constant PCO2, resulted in changes in cellular Cl- content and associated changes in cell volume. These effects were markedly reduced in the nominal absence of HCO3- or in the presence of disulfonic stilbene derivatives, suggesting that they are mediated by Cl(-)-HCO3- exchange.(ABSTRACT TRUNCATED AT 250 WORDS)
Local activation of contraction in isolated rat ventricular myocytes
Authors: O'Neill S. C. , Mill J. G. , Eisner D. A.
Published: December 2017

The aim of this paper was to examine whether contraction (which is thought to result from Ca-induced release of Ca2+ from the sarcoplasmic reticulum) can propagate along a cardiac cell. The experiments were performed on isolated rat ventricular cardiac myocytes. Two techniques were used to initiate contraction in a localized region of the cell. 1) The cells were loaded with the "caged" Ca-containing compound nitr-5. A region of the cell was illuminated with ultraviolet light to increase intracellular Ca2+ concentration ([Ca2+]i) in that area. 2) The cells were superfused with a low (0.1 mM) Ca solution to abolish contraction. Ca was applied to a region of the cell by iontophoresis, and the cell was then electrically stimulated. In both cases a local contraction was produced that did not spread to the rest of the cell. Like the normal twitch, the local contraction was abolished by ryanodine, showing that it is produced by Ca release from the sarcoplasmic reticulum. In the final series of experiments, the cell was stimulated to contract in a control (1 mM Ca2+) solution. Cd2+ was then iontophoresed to a region of the cell. Only the region of the cell exposed to Cd stopped contracting. In conclusion, the above results show that the systolic rise of [Ca2+]i cannot propagate through a cell. This lack of propagation is suggested to be important in stopping a rise of [Ca2+]i in one cell spreading to the rest of the heart.
Kinetics of Ca2(+)-ATPase activation in platelet membranes of essential hypertensives and normotensives
Authors: Takaya J. , Lasker N. , Bamforth R. , Gutkin M. , Byrd L. H. , Aviv A.
Published: December 2017

To explore the etiology of altered Ca metabolism in essential hypertension, we studied parameters, i.e., maximal initial reaction velocity (Vmax) and Michaelis constant (Km), of Ca activation kinetics of Ca2(+)-ATPase in membrane fractions (isolated by a sucrose gradient) from platelets of blacks and whites, 27 of whom were essential hypertensives, 17 of whom were normotensives with a family history of essential hypertension, and 10 of whom were normotensives without a family history of the disease. The Vmax of hypertensives was significantly lower than in normotensives without a family history of essential hypertension (hypertensives, 14.99 +/- 1.71 nmol Pi.mg protein-1.min-1; normotensives, positive family history, 22.67 +/- 3.17 nmol Pi.mg protein-1.min-1; normotensives, negative family history, 27.54 +/- 4.37 nmol Pi.mg protein-1.min-1; overall, P = 0.0078). The Km was lower in both hypertensives and normotensives with a positive family history of essential hypertension as compared with normotensives with a negative family history of the disease (hypertensives, 1.70 +/- 0.23 microM; normotensives, positive family history, 1.38 +/- 0.2 microM; normotensives, negative family history, 2.79 +/- 0.58 microM; overall, P = 0.0251). Furthermore, the Km in whites was inversely related to plasma renin activity (r = 0.50; P less than 0.005). We propose that a lower Vmax for Ca2(+)-ATPase may play a role in the higher level of free Ca in platelets of essential hypertensives and that a higher affinity of the enzyme to Ca may reflect a process compensating for the lower Vmax. We also suggest that a higher Km for Ca2(+)-ATPase in juxtaglomerular cells of whites would result in blunting the release of renin.
Synthesis of chromogranin A, dopamine beta-hydroxylase, and chromaffin vesicles
Authors: Corcoran J. J. , Kirshner N.
Published: December 2017

Primary cultures of bovine adrenal medullary cells synthesize chromogranin A (CgA) and dopamine beta-hydroxylase (DBH) and incorporate them into chromaffin vesicles. The incorporation of L-[35S]methionine into CgA, DBH, and total protein was approximately linear for 8 h at methionine concentrations of 12.5, 25, and 50 microM. Newly synthesized CgA and DBH were initially incorporated into vesicles of low buoyant density that matured over 24 h into vesicles having the greater buoyant density of chromaffin vesicles. Approximately 10% of the newly synthesized CgA is released constitutively within 4 h of formation, approximately 30-40% appears to be degraded, and the remainder is incorporated into chromaffin vesicles, which can secrete CgA in response to nicotinic stimulation. Newly synthesized DBH follows a similar course. Once incorporated into chromaffin vesicles, the newly synthesized CgA and DBH appear to be stable for 2-3 days and then decline with a half-time of 3-4 days. Primary cultures of bovine adrenal medullary cells are a good model system for studying factors regulating CgA and DBH synthesis and the formation of chromaffin vesicles.
Essential activation of Na(+)-H+ exchange by [H+]i in HL-60 cells
Authors: Restrepo D. , Cho D. S. , Kron M. J.
Published: December 2017

The intracellular pH (pHi) dependence of the rate of Na(+)-H+ exchange was determined in undifferentiated promyelocytic HL-60 cells by measuring alkalinization rates using the fluorescent pHi indicator 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). BCECF was calibrated in the pH range from 5 to 7 using the nigericin technique of Thomas and co-workers (J. A. Thomas, R. N. Buchsbaum, A. Zimniak, and E. Racker. Biochemistry 18: 2210-2218, 1979). Exchange rate increases as pHi is lowered below pH 7.00. At low pH (pH below 6.3), the dependence of Na(+)-H+ exchange rate on intracellular proton activity is well fitted by the Michaelis-Menten equation with a maximum exchange velocity of 33.7 +/- 2.4 mmol H(+).1 cell water-1.min-1 and a half-saturation constant of 1.35 +/- 0.28 microM (corresponding to a minus log of the Michaelis constant of 5.89). However, a Hill plot reveals that the Hill coefficient changes gradually from one to two when pH is changed from 5 to 7, ruling out Michaelian kinetics. The dependence of exchange flux on internal protons is well fit in the full pH range from 5 to 7 by a simple kinetic model (essential activation) with modifier and transport sites for internal proton binding. At low pH, failure to correct BCECF measurement of pHi for contribution to fluorescence signal from extracellular dye and for quenching of intracellular BCECF leads to an artifactual increase in the measured Hill coefficient. These two findings (increase in Hill coefficient as pHi is increased and artifactual increase in Hill coefficient because of methodological reasons) provide a good explanation for the wide range of Hill coefficients reported in the literature.
Chloride (or bicarbonate)-dependent copper uptake through the anion exchanger in human red blood cells
Authors: Alda J. O. , Garay R.
Published: December 2017

The initial rate of Cu2+ uptake in human red blood cells was measured by atomic absorption. About 80% of Cu2+ uptake was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) concentrations greater than 5-10 microM. DIDS-sensitive Cu2+ uptake required the presence of external HCO3- or external Cl-. Cl- strongly stimulated Cu2+ uptake following a Michaelis-like function, with apparent dissociation constant (KCl) of 72 +/- 9.4 (SD) mM (n = 6 experiments). HCO3- stimulated DIDS-sensitive Cu2+ uptake following a Michaelis-like function, with apparent dissociation constant (Kbic) of 10 +/- 1.9 (SD) mM (n = 4 experiments). Maximal rates (of Cl(-)- or HCO3(-)-stimulated Cu2+ uptake) were nonadditive. DIDS-sensitive Cu2+ uptake was not modified by physiological concentrations of phosphate or sulfate. Conversely, it was strongly inhibited by physiological concentrations of L-histidine and cysteine (at a Cu2+ concentration of 100 microM, these physiological ligands exhibited KHis and KCys of 50 and 80 microM, respectively). By using a copper-selective electrode, we found that at pH 7-7.4 copper is associated with OH-, particularly in the form of Cu(OH)2 complexes. In conclusion, the anion exchanger is the major transport mechanism for red blood cell Cu2+ uptake. The translocating species can be the monovalent anion complexes of copper with OH-, Cl-, and/or HCO3-.
ATP-dependent pHi recovery in lung macrophages: evidence for a plasma membrane H(+)-ATPase
Authors: Bidani A. , Brown S. E.
Published: December 2017

We have previously shown that cytoplasmic pH (pHi) recovery in pulmonary macrophages, under nominally HCO3(-)-free conditions, after acute intracellular acidification is Na+ and amiloride insensitive and is blocked by nonspecific proton adenosinetriphosphatase (ATPase) inhibitors N-ethyl-maleimide and N,N'-dicyclohexylcarbodiimide [Am. J. Physiol. 257 (Cell. Physiol. 26): C65-C76, 1989]. To further delineate the mechanism of H+ extrusion across plasma membranes of pulmonary macrophages, we investigated the effects of metabolic inhibitors of oxidative phosphorylation and glycolysis on cellular ATP content and pHi recovery from an intracellular acid load under nominally HCO3(-)-free conditions. Dose-dependent reductions in ATP levels and in the rate of pHi recovery were obtained in the presence of KCN (50% inhibition, 10(-4) M). Parallel reductions in ATP content and the rate of pHi recovery were noted in the presence of antimycin A, rotenone, oligomycin, and iodoacetate. However, inhibition by iodoacetate was reduced in the presence of pyruvate. The more specific vacuolar H(+)-ATPase inhibitors, bafilomycin A1 and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, resulted in no decrement in cellular ATP levels but significantly inhibited pHi recovery. These studies demonstrate that recovery from an acid load is ATP dependent and provide support for a plasmalemmal proton ATPase, perhaps of the vacuolar type, that participates in regulation of pHi in pulmonary macrophages.
Sequence homologies among intestinal and renal Na+/glucose cotransporters
Authors: Coady M. J. , Pajor A. M. , Wright E. M.
Published: December 2017

Sodium-dependent glucose transport occurs in the intestine and kidney of most animal species. The cDNA encoding the Na+/glucose cotransporter from rabbit jejunum was used to examine the distribution of homologous mRNA in other rabbit tissues and in the intestines of other species. Northern blots of mRNA extracted from various tissues were probed with radiolabeled cDNA of the cloned rabbit transporter. The probe hybridized with mRNA of approximately 2.2 kb from rabbit jejunum, renal cortex, and renal medulla, indicating that related mRNA of the same size is found in these tissues. With the use of the same cDNA probe, a 1.6-kb partial-length clone encoding 484 amino acids was isolated from a rabbit renal cortex cDNA library. There was greater than 99% identity between the cDNA sequences, and 100% identity between the amino acid sequences, of the renal clone and the rabbit intestinal Na+/glucose cotransporter. The 2.2-kb transcript was seen in mRNA from duodenum, jejunum, and ileum, with a distribution that matched the Na+/glucose transport capacity in these tissues. A faint signal at 2.2 kb was also seen in colon mRNA. There was no detectable hybridization to blots of stomach and heart mRNA. The rabbit probe also hybridized to intestinal mRNA from a number of species from trout to humans. We conclude that a Na+/glucose cotransporter of rabbit renal cortex is very similar to that of the intestine and that the intestinal transporter has been conserved during evolution.
Protein kinase C activation and myosin light chain phosphorylation in 32P-labeled arterial smooth muscle
Authors: Singer H. A.
Published: December 2017

Experiments using 32P-labeled strips of swine carotid artery medial smooth muscle were performed to define the relative contribution of myosin light chain (MLC) phosphorylation as an activation mechanism mediating contractile responses stimulated by phorbol dibutyrate (PDB). Tryptic phosphopeptide mapping of phosphorylated MLC indicated that near-maximal force responses were associated with increases in functional MLC phosphorylation of less than 10% of the total MLC content following tonic (45 min) stimulation by PDB. Significant phosphorylation of MLC residues, consistent with the specificity of protein kinase C, occurred in response to high concentrations of PDB (greater than 0.1 microM). Histamine (10 microM)-induced MLC phosphorylation after 2 min (72.5% of total MLC) or 45 min (61.7%) was restricted to serine residues on peptides thought to contain serine19. Although agonist (histamine)-induced responses were eliminated under conditions of Ca2+ depletion, near-maximal force in response to 10 microM PDB (89.4% of a standard KCl response) was associated with monophosphorylation of less than 9% of the total MLC on peptides interpreted as containing serine19. A substantial fraction of this was localized to threonine residues. The quantitative analysis of the relation between PDB-stimulated force and the residues in MLC phosphorylated supports the concept that PDB stimulation results in activation of arterial smooth muscle cross bridges by MLC-phosphorylation-independent mechanisms.
Vectorial secretion of a kallikrein-like enzyme by cultured renal cells. I. General properties
Authors: Jovov B. , Wills N. K. , Donaldson P. J. , Lewis S. A.
Published: December 2017

Urinary kallikreins are proteolytic enzymes known to be secreted by distal nephron tubules. In this study, we demonstrate (using the chromogenic tripeptide substrate S 2266) that the renal cell line A6 from Xenopus laevis secretes a kallikrein-like enzyme. Secretion is present only when the cells are grown on filters, and enzyme is secreted only into the apical membrane bathing solution. Enzyme secretion consists of two components, one soybean trypsin inhibitor (SBTI) sensitive (SSBTI) and the other insensitive to SBTI (ISBTI). Both enzymes were inhibited by aprotinin, a kallikrein-like enzyme inhibitor. Using a bioassay, only the ISBTI enzyme produced a hypotensive effect on blood pressure and is thus a kallikrein-like enzyme. The apical membrane of cells grown on filters contains both enzyme species, whereas the basolateral membrane contains only the ISBTI (kallikrein-like) enzyme. Both enzymes were present in the apical membrane of cells grown on plastic. Initiation of enzyme secretion occurred after the cells formed electrically tight monolayers and the increase in membrane activity always preceded enzyme secretion. Using an irreversible inhibitor of the apical membrane-bound enzymes, the turnover rate for the SSBTI and ISBTI enzymes (cells on filters) was 3 and 7 h, respectively. Because the recovery of enzyme secretion was proportional to the recovery of membrane-bound enzyme activities, this suggests that enzyme secretion is due to the release of membrane-bound enzyme.
Inhibitory influence of phosphate and arsenate on contraction of skinned skeletal and cardiac muscle
Authors: Nosek T. M. , Leal-Cardoso J. H. , McLaughlin M. , Godt R. E.
Published: December 2017

It has been widely observed that Pi decreases maximum calcium-activated force (Fmax) and calcium sensitivity of skinned skeletal and cardiac muscle. However, whether a particular ionic species of Pi (i.e., H2PO4-) is responsible for these effects is controversial. To clarify this issue, we examined the influence of Pi and its structural analogue arsenate (Asi) on contraction of skinned rabbit psoas (fast twitch), soleus (slow twitch), and cardiac papillary muscle. Asi decreased Fmax of all three muscles types to a greater extent than Pi. Both Pi and Asi decreased calcium sensitivity of psoas and cardiac muscles, with Asi having the greater effect. The effect of the protonated form of Pi and Asi on Fmax was evaluated by measuring the response to 30 mM total Pi or Asi at pH 7.4, 7.0, 6.6, and 6.2. In psoas fibers we found that both Pi and Asi were more effective in decreasing Fmax as the pH was lowered (i.e., as the concentration of the diprotonated forms increased). On the contrary, soleus and cardiac fibers did not exhibit this behavior. These differences in the effects of Pi and Asi on Fmax in psoas vs. cardiac and soleus muscles may be related to differences in their myosin heavy chains other than the binding site for the gamma-phosphate of ATP which appears to be conserved for all myosins.
Calcium channels and basal prolactin secretion in single male rat lactotropes
Authors: Cota G. , Hiriart M. , Horta J. , Torres-Escalante J. L.
Published: December 2017

Ba2+ currents through voltage-dependent Ca channels and basal prolactin secretion were measured in single, cultured lactotropes by the combined use of whole cell patch-clamp recording and the reverse hemolytic plaque assay. Measurements of plaque area, a cumulative index of the relative amount of prolactin released by a cell per unit time, indicate that lactotropes can be grouped in two main subpopulations that differ in basal secretory activity: small-plaque (SP) cells and large-plaque (LP) cells. Analysis of Ba2+ currents indicates that both SP and LP lactotropes express two types of Ca channels: low-threshold, inactivating, slowly deactivating (SD) channels and high-threshold, noninactivating, fast deactivating (FD) channels. Ba2+ current amplitude is smaller in SP cells than in LP cells. Plaque area, and thus prolactin release, is positively correlated with the density of Ba2+ current through FD channels, but not with that through SD channels. The results suggest that the surface density of functional FD Ca channels in the plasma membrane is a major factor that determines the rate of basal prolactin secretion in single lactotropes.
Reduction of ferrylmyoglobin in rat diaphragm
Authors: Eddy L. , Arduini A. , Hochstein P.
Published: December 2017

The oxidation of myoglobin was monitored by transmission spectroscopy in isolated, superfused preparations of rat diaphragms. In its deoxygenated form, during anoxia, myoglobin was oxidized by adding hydrogen peroxide (1.0 mM) to its ferryl form (FeIV). On the other hand, peroxide-induced formation of ferrylmyoglobin was not observed when the perfusate contained oxygen. Ferrylmyoglobin was visualized after its derivatization with Na2S to form sulfmyoglobin. Depending on the time of addition, ascorbate (4.0 mM) or ergothioneine (2.0 mM) either prevented the formation of or dissipated ferrylmyoglobin. These agents are known to be reductants of this hypervalent form of myoglobin. In addition to providing the first demonstration of ferrylmyoglobin in skeletal muscle, these observations are consistent with the concept that oxidation of myoglobin to hypervalent states might be an important event in the initiation of muscle damage associated with anoxia and reoxygenation. The rapid reduction of myoglobin would prevent peroxidatic alterations of essential cellular constituents by ferrylmyoglobin.
Responses of subcultured rat aortic smooth muscle myocytes to vasoactive agents and KCl-induced depolarization
Authors: Bodin P. , Richard S. , Travo C. , Berta P. , Stoclet J. C. , Papin S. , Travo P.
Published: December 2017

We have developed a culture system in which a single-mass primary culture can be used for as long as 6 wk as a source of subcultured smooth muscle myocytes for the study of the changes of their shape upon addition of vasoactive agents (angiotensin, vasopressin, norepinephrine, and serotonin) and KCl depolarization. Responses of subcultivated myocytes were shown to be reproducible with time in primary culture before subculture and consistent with responses of thoracic aorta to the same agents. Effect of KCl depolarization could be blocked with calcium antagonist PN 200-110. Consistently, the presence of calcium L-channels was shown using whole cell patch-clamp recordings. A comparative study of the responses of myocytes derived from two different segments of the thoracic aorta showed that these cells displayed responses with different maximal amplitudes and the same potencies according to their topological origin in the vessel.
Angiotensin II decreases cGMP levels in neuronal cultures from rat brain
Authors: Sumners C. , Myers L. M.
Published: December 2017

In previous studies we have determined that both cultured neuronal and astrocyte glial cells prepared from the hypothalamus and brain stem of 1-day-old rats contain specific receptors for angiotensin II (ANG II). Astrocyte glial receptors are coupled to inositol phospholipid hydrolysis, but there is little indication of the intracellular messengers or signal transduction mechanisms coupled to the neuronal ANG II receptors. In the present study, we have determined that ANG II decreases cellular guanosine 3',5'-cyclic monophosphate (cGMP) levels in neuronal but not in astrocyte glial cultures. This effect is both time and concentration dependent and is inhibited by the ANG II-receptor antagonist [Sar1,Ile8]ANG II, showing the involvement of specific ANG II receptors. ANG II has no effects on particulate or soluble guanylate cyclase activities or on efflux of cGMP from neuronal cultures. However, the effects of ANG II on cellular cGMP content are abolished by pretreatment with the calcium channel blockers cadmium and nifedipine, and by the nonselective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. These results suggest that calcium entry and possibly activation of a phosphodiesterase enzyme are involved in this ANG II-induced effect. This represents the first demonstration of a receptor-mediated effect of ANG II on an intracellular messenger in neuronal cultures. The functional role of cGMP as an intracellular messenger coupled to ANG II receptors in cultured neurons remains to be determined.
Myofilament overlap in swimming carp. II. Sarcomere length changes during swimming
Authors: Rome L. C. , Sosnicki A. A.
Published: December 2017

This study was performed to determine myofilament overlap during swimming in carp. By using frozen sections, we found that sarcomere lengths of the red and white muscle could be related to the curvature of the backbone. Sarcomere length (SL) during swimming was calculated from an analysis of backbone curvature in high-speed motion pictures. Because carp have the same myofilament lengths as frogs, we related force generation to SL using the frog SL-tension curve. At slow swimming speeds, the red fibers are used at a SL of 1.91-2.22 microns, where force generation is calculated to be no less than 96% maximal. If the red fibers powered the escape response they would have to shorten to 1.45-1.55 microns, where force generation would be reduced to approximately 50% maximal and the fibers damaged. Instead, the white muscle fibers are recruited and because of their helical orientation (resulting in a higher gear ratio), they shorten to only 1.75 microns, where they generate no less than 85% maximal tension. Thus, by recruitment of fiber types with different orientations, the full range of movements is powered by fibers operating at nearly maximal overlap. This suggests that myofilament overlap is an important design constraint of muscular systems.
Na(+)-independent Cl(-)-HCO3- exchange in sarcolemmal vesicles from vascular smooth muscle
Authors: Halligan R. D. , Shelat H. , Kahn A. M.
Published: December 2017

Intracellular pH (pHin) affects vascular smooth muscle function, but the mechanisms that control pHin in this tissue are not well understood. These studies were performed to determine whether sarcolemmal vesicles from bovine superior mesenteric artery (SMA) contain a Na(+)-independent Cl(-)-HCO3- exchanger and, if so, to determine its sensitivity to membrane voltage and inhibitors. 36Cl- was taken up by vesicles into an osmotically active intravesicular space. In Na(+)-free media, an outwardly or inwardly directed HCO3- gradient stimulated 36Cl- transport in the opposite direction. An outwardly directed unlabeled Cl- gradient stimulated 36Cl- uptake by a mechanism that was inhibited by external HCO3-. HCO3- or Cl- gradient-stimulated 36Cl- uptake was not due to voltage coupling between ions. In the nominal absence of HCO3-, a threefold outwardly directed OH- gradient did not affect 36Cl- uptake. Total 36Cl- uptake was stimulated by an inside-positive voltage, but the HCO3- gradient-stimulated component of 36Cl- uptake was insensitive to a change in membrane voltage. Finally, HCO3- gradient-stimulated 36Cl- uptake was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and furosemide, with 50% inhibitory concentration values equalling approximately 1.0 and 0.5 mM, respectively. These data indicate that sarcolemmal vesicles from bovine SMA contain a Na(+)-independent Cl(-)-HCO3- exchanger. This transport system is probably electroneutral and is inhibitable by DIDS and furosemide. A conductive pathway for Cl- is present in the vesicles, but Cl(-)-OH- exchange activity was not observed.
Response of isolated adult canine cardiac myocytes to prolonged hypoxia and reoxygenation
Authors: Hohl C. M. , Altschuld R. A.
Published: December 2017

Isolated adult canine ventricular myocytes incubated in the absence of glucose with the respiratory inhibitor rotenone retained 67% of ATP (control, 26.0 +/- 0.9 nmol/mg protein) during 3-h incubation, yet phosphocreatine fell to 23% of initial content. Lactate production proceeded at a constant rate of 5 nmol.mg-1.min-1 in rotenone-treated glucose-free myocytes. A 36% decline in rod-shaped cells and an increase in percent 22Na permeation from 37% in aerobic cells (approximately 13 mM intracellular sodium) to 68% in rotenone-treated glucose-free myocytes paralleled the loss of ATP. Total exchangeable calcium was maintained at control aerobic levels. Exposure of canine cells to 3-h hypoxia in the absence of glucose followed by 5-min reoxygenation resulted in a 73% decrease in ATP, a rise in calcium from 3.3 +/- 0.2 to 6.6 +/- 1.6 nmol/mg, and an increase in 22Na permeation to 111%. Under these conditions the number of rod-shaped myocytes declined by 77%, with corresponding increases in viable contracted and hypercontracted myocytes. The response of canine myocytes to severe hypoxia and reaeration contrasts greatly to earlier studies using adult rat cardiac myocytes [see Hohl et al. Am. J. Physiol. 242 (Heart Circ. Physiol. 11): H1022-H1030, 1982]. Species differences with respect to basal metabolism, rates of ATP production and degradation, and regulation of cation movements are most likely responsible for the observed differences.
Kinetic analysis of receptor-mediated endocytosis of epidermal growth factor by isolated rat hepatocytes
Authors: Yanai S. , Sugiyama Y. , Kim D. C. , Iga T. , Fuwa T. , Hanano M.
Published: December 2017

The interaction of epidermal growth factor (EGF) with cell surface receptors and their subsequent endocytosis in isolated rat hepatocytes were analyzed by measuring changes in the concentrations of cell surface-bound, internalized, and degraded EGF. The kinetic model proposed by Wiley and Cunningham (Cell 25: 433-440, 1981) and Gex-Fabry and Delisi [Am. J. Physiol. 247 (Regulatory Integrative Comp. Physiol. 16): R768-R779, 1984] was basically utilized for the model analysis. The following kinetic parameters were obtained: association and dissociation rate constants for EGF-receptor interaction, internalization rate constant for EGF-receptor complex (kappa e), internalization rate constant for free receptor (kappa t), sequestration rate constant (kappa s) of the complex from shallow (exchangeable) to deep (nonexchangeable) membraneous compartment, intracellular degradation rate constant and initial cell-surface receptor density. The kappa s value, which was obtained by analyzing the time profiles of EGF association with cells, was approximately 5-10 times larger than the kappa e value determined by directly measuring internalized EGF with the acid-washing technique. This suggests the necessary presence of deep (nonexchanging) compartment of the complex in the plasma membrane. The calculated kappa e value is at least several times larger than the kappa t value, yielding the kinetic basis for the occurrence of receptor downregulation induced by excess EGF. We conclude that, in the overall receptor-mediated processing of EGF after bound to the cell surface receptors, the dissociation process is rapid [half-time (t1/2) less than 1 min], the degradation process is much slower (t1/2 approximately equal to 3 h), and the receptor internalization process is intermediate (t1/2 approximately equal to 6-7 min). In addition, two pools for EGF-receptor complex in the plasma membrane seem to be present, although their identification cannot be made.
Contractile and biochemical properties of rat soleus and plantaris after hindlimb suspension
Authors: Diffee G. M. , Caiozzo V. J. , Herrick R. E. , Baldwin K. M.
Published: December 2017

This study examined the relationship between contractile and isomyosin changes occurring in rat soleus (SOL) and plantaris (PLAN) muscles after 28 days of hindlimb suspension. SOL muscles from suspended animals exhibited a 45% decline in muscle weight compared with controls (P less than 0.05) accompanied by a 49% decrease in peak twitch tension (Pt) and a 59% reduction in peak tetanic tension (Po). Smaller reductions were observed in muscle weight, Pt, and Po (12, 43, and 24%, respectively) for the suspended PLAN. Maximal shortening velocity (Vmax) of the suspended SOL and the velocity of unloaded shortening were increased by 36 and 35%, respectively, but there was no suspension-induced change in PLAN Vmax. Suspension induced a 22% increase in SOL myosin adenosinetriphosphatase (ATPase) activity that was accompanied by a shift in the native myosin isoform distribution characterized by an increase in the relative amounts of intermediate and fast myosin. The more modest changes in the contractile function of suspended PLAN were accompanied by a small (7%) increase in myosin ATPase activity but no significant changes in myosin isoform distribution. The results of this study confirm that hindlimb suspension results in significant speeding of SOL contractile properties and suggest that the shift toward faster myosin isoforms with a higher myosin ATPase activity likely accounts for these mechanical changes.
Tyrphostins inhibit PDGF-induced DNA synthesis and associated early events in smooth muscle cells
Authors: Bilder G. E. , Krawiec J. A. , McVety K. , Gazit A. , Gilon C. , Lyall R. , Zilberstein A. , Levitzki A. , Perrone M. H. , Schreiber A. B.
Published: December 2017

Tyrphostins are low-molecular-weight synthetic inhibitors of protein tyrosine kinase, which block cell proliferation. Since platelet-derived growth factor (PDGF) is thought to figure prominently in disorders of vascular smooth muscle cells (VSMC), such as atherosclerosis, hypertension, and restenosis, we examined whether tyrphostins would inhibit PDGF-induced mitogenesis in VSMC. In this communication, we demonstrate that tyrphostins with the benzenemalononitrile nucleus inhibited PDGF-dependent growth of VSMC as well as PDGF-dependent DNA synthesis in these cells, with the concentrations for 50% inhibition ranging from 0.04 to 9 microM. Up to 30-fold higher tyrphostin concentrations were required to inhibit serum-stimulated DNA synthesis of VSMC. The effect of the tyrphostins is reversible, since on their removal a normal proliferative response to PDGF was resumed. Tyrphostins also inhibited PDGF-receptor autophosphorylation and PDGF-induced phosphorylation of intracellular substrates, including the phosphorylation of phospholipase C-gamma, with a potency ratio similar to their antimitogenic activity. The expression of c-fos mRNA, a mitogenic nuclear signal, was also reduced in PDGF-stimulated VSMC treated with tyrphostins at concentrations which inhibit PDGF-induced mitogenesis. It is concluded that tyrphostins are potent reversible inhibitors of PDGF-induced mitogenesis which act by inhibiting the tyrosine kinase activity of the PDGF receptor and the subsequent signaling cascade. Tyrphostins may be useful in the study and treatment of VSMC proliferation disorders.
Urea transport deficiency in Jk(a-b-) erythrocytes
Authors: Frohlich O. , Macey R. I. , Edwards-Moulds J. , Gargus J. J. , Gunn R. B.
Published: December 2017

The activity of the urea transporter was determined in human erythrocytes of the Kidd blood type Jk(a-b-) by measuring unidirectional urea and thiourea fluxes in tracer flux experiments and urea net fluxes in light-scattering experiments. When compared with control cells, Jk(a-b-) cells exhibited diminished urea and thiourea fluxes and lacked the kinetic characteristics of mediated transport, suggesting that in these cells urea and thiourea moved only by simple diffusion through the lipid bilayer. Control experiments showed that anion, water, and ethylene glycol permeabilities were the same in Jk(a-b-) and control cells. These experiments thus demonstrate that Jk(a-b-) cells lack mediated urea transport and strongly support the notion that urea transport function is separate from the transport for anions, water, and ethylene glycol, probably because different proteins are responsible for these transport functions.
Cl- secretion by cultured shark rectal gland cells. I. Transepithelial transport
Authors: Valentich J. D. , Forrest J. N.
Published: December 2017

To facilitate analysis of the regulation of epithelial Cl- transport by hormones, neurotransmitters, and autocrine mediators, we have developed a primary monolayer culture system for shark rectal gland (SRG) epithelial cells. Cultures exhibit vigorous transcellular Cl- secretion which can be measured using short-circuit current or 36Cl flux methods. Transport is markedly reduced by bumetanide or barium, inhibitors of Na(+)-K(+)-2Cl- cotransport and K+ channels, respectively. This indicates that Cl- secretion by SRG monolayers occurs by a mechanism similar to that described in numerous native Cl- secretory epithelia. Forskolin, 10 microM 2-chloroadenosine, or vasoactive intestinal peptide, potent secretagogues in the isolated perfused SRG, stimulate Cl- secretion by SRG cultures. Submicromolar concentrations of 2-chloroadenosine, which inhibit Cl- secretion in the native SRG, reduce forskolin-stimulated short-circuit current in SRG cultures. Somatostatin, another inhibitor of Cl- secretion by the native SRG, reduces forskolin-stimulated adenosine 3',5'-cyclic monophosphate accumulation in SRG cultures. These results demonstrate that SRG cultures are fully responsive to mediators which activate or inhibit secretion by the native epithelium.
Effect of thin filament length on the force-sarcomere length relation of skeletal muscle
Authors: Granzier H. L. , Akster H. A. , Ter Keurs H. E.
Published: December 2017

We studied a slow- and a fast-twitch muscle fiber type of the perch that have different thin filament lengths. The force-sarcomere length relations were measured, and it was tested whether their descending limbs were predicted by the cross-bridge theory. To determine the predicted relations, filament lengths were measured by electron microscopy. Measurements were corrected for shrinkage with the use of I-band and H-zone periodicities. Thick filament lengths of the two fiber types were found to be similar (1.63 +/- 0.06 and 1.64 +/- 0.10 microns for slow- and fast-twitch fibers, respectively), whereas the thin filament lengths were clearly different: 1.24 +/- 0.10 microns (n = 86) for the slow-twitch type and 0.94 +/- 0.04 microns (n = 94) for the fast type. The descending limbs of the two fiber types are therefore predicted to be shifted along the sarcomere length axis by approximately 0.6 microns. Sarcomere length was measured on-line by laser diffraction in a single region in the center of the fibers. The passive force-sarcomere strain relation increased much more steeply in the slow-twitch fibers. The descending limb of the active force-sarcomere length relation of fast twitch fibers was linear (r = 0.92), but was found at sarcomere lengths approximately 0.1 micron greater than predicted. The descending limb of the slow-twitch fibers was also linear (r = 0.87) but was now found at sarcomere lengths approximately 0.05 microns less than predicted. The difference in position of the descending limbs of the two fiber types amounted to 0.5 microns, approximately 0.1 micron less than predicted. The difference between measured and predicted descending limbs was statistically insignificant.
Atriopeptin stimulates chloride secretion in cultured shark rectal gland cells
Authors: Karnaky K. J. , Valentich J. D. , Currie M. G. , Oehlenschlager W. F. , Kennedy M. P.
Published: December 2017

Monolayer cultures of shark rectal gland (SRG) epithelial cells were treated with atriopeptin (AP), and the effects on Cl- secretion and intracellular guanosine 3',5'-cyclic monophosphate (cGMP) accumulation were examined. Basolateral or apical exposure to 10(-7) M AP markedly stimulated (8-fold) Cl(-)-dependent, bumetanide-sensitive, short-circuit current (Isc). The AP-stimulated Isc exhibited transient oscillations before reaching a steady state. This behavior is not observed when Isc is activated by other secretagogues such as vasoactive intestinal peptide, 2-chloroadenosine, forskolin, or ionomycin. Intracellular cGMP was concomitantly elevated (10-fold) by 10(-7) M AP. Both Isc stimulation and cGMP accumulation responses exhibited a similar dose dependency beginning at an AP concentration of 1 nM. The bilateral response to AP suggests the presence of receptors on both apical and basolateral plasma membranes. These results are the first demonstration of a direct effect of AP on Cl(-)-secreting epithelial cells. These data also suggest a role for cGMP in mediating Cl- secretion in these cells.
Characterization of gap junction channels in A7r5 vascular smooth muscle cells
Authors: Moore L. K. , Beyer E. C. , Burt J. M.
Published: December 2017

Recent evidence suggest that coordination of blood flow in the microcirculation involves cell-to-cell coupling via gap junctions. In this study, using A7r5 cells as a model of vascular smooth muscle, we have characterized the gap junctions in terms of the unitary conductances of the observed channels, the responses to second messengers, and subunit protein composition. The cells were typically well coupled several hours after plating, with junctional conductances on the order 20-40 nS. Channels with mean conductances of 36 and 89 pS were observed in low-conductance cell pairs and in cell pairs whose macroscopic conductance was reduced by exposure to halothane. Connexin43 was the only known gap junction sequence detected by Northern blots (low and high stringency), immunoblots, or immunohistochemical studies. Junctional conductance was reduced 15% by 8-bromoadenosine 3',5'-cyclic monophosphate; 8-bromoguanosine 3',5'-cyclic monophosphate had no effect. The results suggest that connexin43 can form stable channels of at least two distinct conductances and gap junctions with differing responses to second messengers.
Na(+)-Ca2+ exchangers from rod outer segments and cardiac sarcolemma: comparison of properties
Authors: Nicoll D. A. , Barrios B. R. , Philipson K. D.
Published: December 2017

The properties of the Na(+)-Ca2+ exchangers from cardiac sarcolemma (SL) and rod outer segments (ROS) were studied in parallel by measuring the Na+ gradient-dependent Ca2+ uptake into SL or ROS vesicles. The ROS exchanger, but not the SL exchanger, has a striking specific dependence on K+. The ROS exchanger is stimulated by K+ with an apparent concentration at half-maximum of 1 mM. The addition of valinomycin, to produce an inside-positive membrane potential, stimulates the SL exchanger 1.8-fold and the ROS exchanger 1.2-fold. The Michaelis constant for half-maximal transport rate for Ca2+ and the maximal transport rate for the exchangers, in the absence of valinomycin, are estimated to be 20 microM and 8 nmol.mg-1.2.2 s-1 in SL and 5 microM and 1 nmol.mg-1.2.2 s-1 in ROS. Both exchangers are modulated by the same regulatory influences. For example, both are stimulated by proteases, phospholipase D, and intravesicular Ca2+.
cAMP-dependent protein kinase regulates renal epithelial cell properties
Authors: Amsler K. , Ghatani S. , Hemmings B. A.
Published: December 2017

Previous studies have implicated adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) in regulation of both growth and expression of differentiated function in the pig renal epithelial cell, LLC-PK1. To investigate this possible regulatory mechanism, we compared growth behavior, morphology, and appearance of two differentiated functions, Na-hexose symport (SYMP) and gamma-glutamyl transpeptidase (gamma-GT), in the LLC-PK1 line and two PKA-deficient mutants (FIB4 and FIB6). Compared with the wild-type cell line, the mutant lines continued to proliferate at higher population densities and exhibited altered cell morphology, poorer formation of the brush-border structure, and decreased or lack of expression of SYMP and gamma-GT activities. Wild-type and mutant cells exhibit an identical logarithmic growth rate. Both lines form cell-cell junctions and exhibit identical kinetic properties of expressed SYMP activity. These results strongly support the hypothesis that PKA modulates a defined subset of cellular processes, including aspects of growth control and expression of the differentiated phenotype, in this renal epithelial cell line.