bluuebunny/crossref_metadata_2025 huggingface dataset:
G-1,6-P2, glycolysis, and energy metabolism during circulatory occlusion in human skeletal muscle
Authors: Katz A.
Published: December 2017

The effect of circulatory occlusion on the content of glucose 1,6-bisphosphate (G-1,6-P2), glycogenolytic intermediates, and high-energy phosphates in the quadriceps femoris muscles of eight men was investigated. Needle biopsies were obtained at rest, after 30 min of circulatory occlusion, and 15 min after the occlusion was released. G-1,6-P2 averaged 75 +/- 8 (SE) mumol/kg dry wt at rest and did not change significantly after occlusion (82 +/- 10; P greater than 0.05) but was slightly elevated after 15 min recovery (88 +/- 12; P less than 0.05 vs. rest). Phosphocreatine (PCr) decreased in all subjects after occlusion (from 80.4 +/- 2.6 to 66.2 +/- 4.7 mmol/kg dry wt; P less than 0.001) and was completely resynthesized after recovery (80.9 +/- 2.4). Fructose 1,6-bisphosphate (F-1,6-P2) was doubled after occlusion (P less than 0.05). During occlusion, the average glycolytic and anaerobic ATP turnover rates were 0.08 +/- 0.02 mmol.kg dry wt-1.min-1 (approximately 4 times the calculated rate at rest) and 0.7 +/- 0.2 mmol.kg dry wt-1.min-1 (less than 20% of the calculated rate at rest), respectively. Total glycolysis was strongly related to the calculated increase in inorganic phosphate (Pi, r = 0.93; P less than 0.01), the decrease in PCr/Cr (reflects an increase in free ADP and AMP) (r = 0.92; P less than 0.01), and the increase in hexosemonophosphates (r = 0.77; P less than 0.05). It is concluded that short-term ischemia in human skeletal muscle results in no change in the content of G-1,6-P2.(ABSTRACT TRUNCATED AT 250 WORDS)
Cell uncoupling and protein kinase C: correlation in a cell line but not in a differentiated tissue
Authors: Chanson M. , Bruzzone R. , Spray D. C. , Regazzi R. , Meda P.
Published: December 2017

Second messengers have been implicated in the control of communication between cells of various tissues and of a number of cell lines. To assess whether protein kinase C (PKC) is involved in the regulation of gap junctions between primary differentiated cells, we studied the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on PKC translocation and junctional conductance of rat pancreatic exocrine cells. Our results show that although TPA induced the translocation of PKC from a "cytosolic" to a "microsomal" fraction within minutes, it failed to block the junctional conductance of acinar cell pairs up to 30 min after application. By contrast, analogous experiments on a liver-derived cell line (WB cells) showed that TPA-induced PKC translocation was paralleled by a marked and irreversible inhibition of intercellular coupling. These results indicate that, in contrast to the effects on transformed or dedifferentiated permanent cell lines, PKC is not involved in gating gap junctional channels between primary differentiated secretory cells of the pancreas.
Potassium transport by pancreatic and parotid zymogen granule membranes
Authors: Gasser K. W. , DiDomenico J. , Hopfer U.
Published: December 2017

Zymogen granules that were stable at physiological conditions of pH, ionic strength, and temperature were isolated from the rat pancreas and parotid. The cation permeability of these granules was evaluated to characterize the mechanism of secretagogue-stimulated fluid secretion by acinar cells. Granule swelling and lysis provide a measure of the rate of cation transport, since the use of ionophore combinations such as tripropyltin and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) will render cation conductance the rate-limiting step for salt influx. This technique supplies evidence for the existence of K+ conductance in the granule membrane. The pancreatic and parotid granules have a K+-selective conductance that is not inhibited by the K+ channel blockers barium, tetraethylammonium, quinidine, cesium, or 4-aminopyridine. Furthermore, the intragranular pH of pancreatic zymogen granules was measured to be approximately 6.5 and was identified as a factor that modulates the K+ conductance. Although the pancreatic and parotid granules were qualitatively identical, quantitatively the relative K+ transport rate constant was over twofold higher for the parotid than for the pancreatic granules. The zymogen granule K+ conductance may have an important role in active K+ secretion by exocrine glands, which is prominent in the parotid after stimulation with beta-adrenergic agents.
5-HETE: uptake, distribution, and metabolism in MDCK cells
Authors: Gordon J. A. , Figard P. H. , Quinby G. E. , Spector A. A.
Published: December 2017

The interaction of (S)-5-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid (5-HETE) with Madin-Darby canine kidney (MDCK) cells was investigated to determine whether this lipoxygenase product might influence tubular epithelial function. When incubated with arachidonic acid, MDCK cells failed to synthesize any 5-HETE. However, MDCK cells can take up 5-HETE to a much greater extent than either 12- or 15-HETE. 5-HETE uptake occurred from both the apical and basolateral surfaces and was not saturated at concentrations up to 10 microM. Much of the 5-HETE was incorporated into phospholipids, primarily phosphatidylcholine and phosphatidylethanolamine. After a 1-h incubation 5-HETE was found to be localized in either the microsomal and/or plasma membrane of MDCK cells. After pulse labeling for 1 h, MDCK cells released 35% of 5-HETE compared with 10% of the incorporated arachidonate during the next 24 h, indicating a much more rapid turnover of newly incorporated 5-HETE. When MDCK cells were incubated with 5.0 microM 5-HETE, their capacity to produce prostaglandin E2 was reduced greater than 50% in as little as 5.0 min. Since 5-HETE enters epithelial phospholipids and reduces prostaglandin production, it apparently has the capacity to modulate renal function if it is released in the proximity of the tubular epithelium during inflammatory reactions.
Regulation of bovine glomerular endothelial cell growth in vitro
Authors: Ballermann B. J.
Published: December 2017

To enable the study of glomerular endothelial cell functions and interactions with other glomerular cells, bovine glomerular capillary endothelial cells were established in culture. Selective media were used to facilitate endothelial cell proliferation and to suppress glomerular mesangial cell growth. Glomerular endothelial cells were separated from other cell types by fluorescence-activated cell sorting or, alternatively, by cloning. Glomerular endothelial cells expressed angiotensin I-converting enzyme and factor VIII activity and acetylated LDL uptake, properties generally held to be specific for endothelial cells. Proliferation of subconfluent glomerular endothelial cells was stimulated by basic fibroblast growth factor and, in the presence of heparin sodium, by acidic fibroblast growth factor. Platelet-derived growth factor was without effect on glomerular endothelial cell proliferation. Coculture with mesangial cells markedly inhibited proliferation of subconfluent glomerular endothelial cells. By contrast, medium conditioned by confluent glomerular endothelial cells markedly enhanced proliferation of subconfluent glomerular endothelial cells. These findings suggest that glomerular endothelial cell growth is under autocrine and paracrine control.
Activation of an apical Cl- conductance by Ca2+ ionophores in cystic fibrosis airway epithelia
Authors: Willumsen N. J. , Boucher R. C.
Published: December 2017

Cystic fibrosis (CF) airway epithelia express a defect in adenosine 3',5'-cyclic monophosphate (cAMP)-dependent regulation of apical membrane Cl- channels. Recent patch-clamp studies have raised the possibility that Ca2+ -dependent mechanisms for the activation of Cl- secretion may be preserved in CF airway epithelia. To determine 1) whether intact normal (N1) and CF airway epithelia exhibit a Ca2+ -dependent mechanism for activation of Cl- secretion and 2) whether Ca2+ -dependent mechanism for activation of Cl- secretion and 2) whether Ca2+ -dependent mechanisms initiate Cl- secretion via activation of an apical membrane Cl- conductance (GCl-), nasal epithelia from N1 and CF subjects were cultured on collagen membranes, and responses to isoproterenol or Ca2- ionophores [A23187 10(-6) M; ionomycin (10(-5)M)] were measured with transepithelial and intracellular techniques. Isoproterenol induced activation of an apical membrane GCl- in N1 cultures but was ineffective in CF. In contrast, in both N1 and CF amiloride-pretreated cultures, A23187 induced an increase in the equivalent short-circuit current that was associated with an activation of an apical membrane Gc1- and was bumetanide inhibitable. A23187 addition during superfusion of the lumen with a low Cl- (3 mM) solution reduced intracellular Cl- activity of CF cells. A Ca2+ ionophore of different selectivity properties, ionomycin, was also an effective Cl- secretagogue in both N1 and CF cultures. We conclude that 1) the A23187 induced Cl- secretion via activation of an apical GCl- in N1 human nasal epithelium, and 2) in contrast to an isoproterenol-dependent path, a Ca2+ -dependent path for GCl- activation is preserved in CF epithelia.
Inhibition of N-glycosylation affects transepithelial Na+ but not Na+-K+-ATPase transport
Authors: Zamofing D. , Rossier B. C. , Geering K.
Published: December 2017

Tunicamycin (TM) was used in toad urinary bladder (TBM) cells to study the role of N-glycosylation of the beta-subunit of Na+-K+-ATPase. Inhibition of the beta-subunit core glycosylation was dose dependent and coincided with a specific 70% decrease in newly synthesized beta- and alpha-subunits. Na+-K+-ATPase activity paralleled the decrease in the cellular content of the alpha-subunit, although the cellular and cell surface-expressed Na+-K+-ATPase pool was progressively filled up with nonglycosylated beta-subunits. In addition, the decrease in maximal Na+ transport capacity of the Na+-K+-ATPase as assessed by short-circuit current (SCC) measurements in the presence of amphotericin B correlated with the decrease in the total cell surface-expressed beta-subunit population despite the fact that it was composed of 47% nonglycosylated beta-subunits after 42 h of TM treatment. These results are consistent with the interpretation that beta-subunit glycosylation is not important either for the enzyme's intracellular sorting to the plasma membrane or its hydrolytic and transport properties. Finally, TM produced effects on basal SCC and electrical resistance that differed in their times of onset and time periods needed for recovery. Thus, in addition to the Na+-K+-ATPase, other glycoproteins in the apical membrane and the tight junctions must be implicated in the maintenance of transepithelial Na+ transport.
Oxygen diffusion and mitochondrial respiration in neuroblastoma cells
Authors: Robiolio M. , Rumsey W. L. , Wilson D. F.
Published: December 2017

Suspensions of human neuroblastoma cells consume oxygen at a constant rate when the oxygen pressure is greater than approximately 11 Torr. The rate of oxygen consumption, however, becomes dependent on the oxygen pressure below this level. The falling respiratory rate at the lower pressures gives rise to an oxygen pressure for half-maximal respiration (P50) of approximately 0.8 Torr, which is consistent with the 0.5 Torr value for suspensions of isolated mitochondria in the presence of ATP (J. Biol. Chem. 263: 2712-2718, 1988). When the cellular metabolic energy state is lowered by addition of an uncoupler of mitochondrial oxidative phosphorylation, the respiratory rate increases up to fivefold, but the P50 decreases to approximately 0.6 Torr. In the cells treated with uncoupler, the P50 decreases further when the mitochondrial respiratory rate is inhibited with amobarbital (amytal), an inhibitor of the respiratory chain. The additional decrease in P50 is proportional to the decrease in respiratory rate. Thus, for cells treated with uncoupler, the P50 appears to be limited by oxygen diffusion from the external medium to the mitochondria. When the respiratory rate of the uncoupled cells is inhibited to the level of coupled cells, the P50 for the former is less than 0.15 Torr. This indicates that for coupled cells the difference in oxygen pressure from the external medium to the mitochondria is less than 0.15 Torr at half-maximal respiratory rate and does not significantly affect the P50 for oxygen that occurs at 0.8 Torr.(ABSTRACT TRUNCATED AT 250 WORDS)
Anisosmotic cell volume regulation: a comparative view
Authors: Chamberlin M. E. , Strange K.
Published: December 2017

A variety of organisms and cell types spanning the five taxonomic kingdoms are exposed, either naturally or through experimental means, to osmotic stresses. A common physiological response to these challenges is maintenance of cell volume through changes in the concentration of intracellular inorganic and organic solutes, collectively termed osmolytes. Research on the mechanisms by which the concentration of these solutes is regulated has proceeded along several experimental lines. Extensive studies on osmotically activated ion transport pathways have been carried out in vertebrate cells and tissues. Much of our knowledge on organic osmolytes has come from investigations on invertebrates, bacteria, and protists. The relative simplicity of bacterial genetics has provided a powerful and elegant tool to explore the modifications of gene expression during volume regulation. An implication of this diverse experimental approach is that phylogenetically divergent organisms employ uniquely adapted mechanisms of cell volume regulation. Given the probability that changes in extracellular osmolality were physiological stresses faced by the earliest organisms, it is more likely that cell volume regulation proceeds by highly conserved physiological processes. We review volume regulation from a comparative perspective, drawing examples from all five taxonomic kingdoms. Specifically, we discuss the role of inorganic and organic solutes in volume maintenance and the mechanisms by which the concentrations of these osmolytes are regulated. In addition, the processes that may transduce volume perturbations into regulatory responses, such as stretch activation of ion channels, intracellular signaling, and genomic regulation, are discussed. Throughout this review we emphasize areas we feel are important for future research.
D 600 block of L-type Ca2+ channel in vascular smooth muscle cells: comparison with permanently charged derivative, D 890
Authors: Leblanc N. , Hume J. R.
Published: December 2017

It has been reported that D 600 blocks the high-threshold Ca2+ channel (L-type) from the outside in isolated vascular and ileal smooth muscle cells of the rabbit (Y. Ohya, K. Terada, K. Kitamura, and H. Kuriyama. Pfluegers Arch. 408: 80-82, 1987). We have reinvestigated this hypothesis by comparing the effects of external and internal applications of D 600 and the permanently charged quaternary derivative D 890 on the whole cell Ca2+ current (Ica) recorded in vascular smooth muscle cells isolated from the rabbit portal vein. At low frequencies of stimulation (0.05 Hz), externally applied D 600 inhibited Ica in a dose-dependent fashion, with a complete block occurring at 10(-4) M. D 600 was approximately 1,000 times more potent than D 890 for causing inhibition of Ica using this protocol. During a train of stimulations at 0.5 Hz, D 600 (10(-6) M) produced a minor additional frequency-dependent block of Ica, as shown in other preparations. During superfusion with D 890 (10(-4) M), a similar protocol produced little if any decline in the amplitude of Ica. No evidence of block could be detected during intracellular dialysis of D 600 (10(-4) M). At the same concentration, intracellular application of D 890 produced a slow block of Ica. To test whether D 600 could be effectively dialysed using a patch micropipette, similar experiments were performed in cardiac ventricular myocytes. In this preparation, intracellular dialysis of D 600 induced a rapid inhibition of the Ica.(ABSTRACT TRUNCATED AT 250 WORDS)
Altered electrical potential profile of human reabsorptive sweat duct cells in cystic fibrosis
Authors: Reddy M. M. , Quinton P. M.
Published: December 2017

The electrophysiological properties of reabsorptive sweat duct (RSD) cells from normal and cystic fibrosis (CF) subjects were studied using intracellular microelectrodes. The apical membrane potential (Va) of CF duct cells was reversed in "polarity" (+28.0 +/- 2.4 mV, n = 46) compared with normal duct cells (-24.9 +/- 0.4 mV, n = 145), and the basolateral membrane potential (Vb) of CF cells was hyperpolarized significantly (-50.1 +/- 1.2 mV, n = 46) in comparison to normal cells (-34.6 +/- 0.4 mV, n = 145). The substitution of the impermeant anion gluconate for Cl- in the lumen of the normal duct depolarized Va from -24.9 +/- 1.1 to 8.9 +/- 3.1 mV (n = 18) and hyperpolarized Vb from -34.3 +/- 1.1 to -55.6 +/- 3.7 mV (n = 18), which mimicked the cell electrical potential profile of CF ducts even in the presence of Cl-. Cl- substitution in the bath depolarized Vb of normal ducts by 22.5 +/- 2.6 mV (n = 24), while hyperpolarizing Va by -3.4 +/- 1.6 mV (n = 24). The response of the electrical profiles of CF cells to Cl- substitution in either the lumen or the bath was significantly reduced compared with normal cells. The effect of the Na+ conductance blocker amiloride (10(-4) M) on Vb was not significantly different in CF (delta Vb = -26.4 +/- 3.8 mV, n = 9) vs. normal (delta Vb = -27.6 +/- 2.5 mV, n = 30) cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Anionic amino acid uptake by microvillous membrane vesicles from human placenta
Authors: Moe A. J. , Smith C. H.
Published: December 2017

The transport mechanisms for anionic amino acids in trophoblast microvillous (maternal facing) membrane were investigated by characterization of L-[3H]aspartate and L-[3H]glutamate uptake in membrane vesicles. Uptake of the anionic amino acids was by a single high-affinity Na+-dependent K+-stimulated cotransporter that is pH sensitive and electrogenic. A second Na+-dependent transporter could not be discriminated, and there was no observable Na+-independent uptake. An outwardly directed K+ gradient (100 mM KCl inside) resulted in a 5- to 10-fold stimulation in glutamate uptake in the presence of Na+. Intravesicular KCl had no effect on transporter affinity but increased transporter velocity in a concentration-dependent manner. Inhibition of Na+-K+-dependent uptake of L-aspartate and L-glutamate (20 mM, 30 s) by 2 mM unlabeled amino acids demonstrated stereoselectivity for L-glutamate but not for L-aspartate. The neutral amino acids (L-alanine, L-threonine, L-serine, L-cysteine, L-phenylalanine) were not effective inhibitors. These data are consistent with an anionic amino acid transporter in the microvillous membrane of the trophoblast, which has characteristics qualitatively similar to the X-AG system found in other epithelia. This system may mediate the concentrative placental uptake of anionic amino acids from maternal blood in utero.
Volume-activated K+ and Cl- pathways of dissociated epithelial cells (MDCK): role of Ca2+
Authors: Rothstein A. , Mack E.
Published: December 2017

Osmotic swelling of dissociated Madin-Darby canine kidney (MDCK) cells in NaCl medium is followed by shrinking (regulatory volume decrease, or RVD) or in KCl medium by secondary swelling. The cation ionophore gramicidin has little effect on volumes of isotonic cells but accelerates volume-activated changes in either medium. Immediately after hypotonic exposure, the membrane becomes transiently hyperpolarized followed by depolarization. The depolarization phase is diminished by the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Swelling is also associated with an almost immediate increase in Ca2+ influx and elevation of cytoplasmic Ca2+ ([Ca2+]i) preceding RVD. In Ca2(+)-free medium, [Ca2+]i rapidly declines to a low level. Osmotic swelling, under these circumstances, is associated with a small transient increase in [Ca2+]i, but RVD or secondary swelling (in KCl) are minimal. Under these conditions, addition of gramicidin or the Ca2(+)-ionophore A23187 induces significant volume changes, although not as large as those found in the presence of Ca2+. Quinine inhibits RVD in the absence of gramicidin, but not in its presence; oligomycin C, DIDS, and trifluoperazine, on the other hand, inhibit in the presence of the ionophore. These findings suggest that in MDCK cells RVD involves activation of distinct conductive K+ and Cl- pathways which allow escape of KCl and osmotically obligated water and that activation of both pathways is associated with elevated [Ca2+]i derived largely from volume activation of a Ca2(+)-influx pathway.
K depletion alters angiotensin II receptor expression in vascular smooth muscle cells
Authors: Linas S. L. , Marzec-Calvert R. , Ullian M. E.
Published: December 2017

Dietary K depletion (KD) results in increases in the number of angiotensin II (ANG II) receptors and prevents ANG II-induced downregulation of ANG II receptors in membrane preparations of vessels from KD animals. Because dietary KD results in changes in factors other than K, we K depleted vascular smooth muscle cells (VSMC) in culture to determine the specific effects of KD on ANG II receptor expression and processing. Scatchard analysis of ANG II uptake at 4 degrees C revealed that the number of surface receptors was increased by 37% in cells in which K had been reduced by 45%. This increase also occurred in the presence of cycloheximide. To determine the effect of KD on receptor processing, we measured the number of surface receptors after exposure to ANG II in concentrations sufficient to cause down-regulation. After 30-min exposure to ANG II, the number of surface receptors was reduced by 63% in control cells but only 33% in KD cells. Thirty minutes after withdrawing ANG II, surface binding returned to basal levels in control cells but was still reduced by 20% in KD cells. To determine the functional significance of impaired receptor processing, we measured ANG II uptake at 21 degrees C. Uptake at 21 degrees C depends on the functional number of receptors, i.e., the absolute number of surface receptors and the rate at which receptors are recycled to the surface after ANG II binding. ANG II uptake at 21 degrees C was reduced by 50% in KD cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Calcium channels, potassium channels, and voltage dependence of arterial smooth muscle tone
Authors: Nelson M. T. , Patlak J. B. , Worley J. F. , Standen N. B.
Published: December 2017

Resistance arteries exist in a maintained contracted state from which they can dilate or constrict depending on need. In many cases, these arteries constrict to membrane depolarization and dilate to membrane hyperpolarization and Ca-channel blockers. We discuss recent information on the regulation of arterial smooth muscle voltage-dependent Ca channels by membrane potential and vasoconstrictors and on the regulation of membrane potential and K channels by vasodilators. We show that voltage-dependent Ca channels in the steady state can be open and very sensitive to membrane potential changes in a range that occurs in resistance arteries with tone. Many synthetic and endogenous vasodilators act, at least in part, through membrane hyperpolarization caused by opening K channels. We discuss evidence that these vasodilators act on a common target, the ATP-sensitive K (KATP) channel that is inhibited by sulfonylurea drugs. We propose the following hypotheses that presently explain these findings: 1) arterial smooth muscle tone is regulated by membrane potential primarily through the voltage dependence of Ca channels; 2) many vasoconstrictors act, in part, by opening voltage-dependent Ca channels through membrane depolarization and activation by second messengers; and 3) many vasodilators work, in part, through membrane hyperpolarization caused by KATP channel activation.
Regulation of Na-K-Cl cotransport in cultured canine airway epithelia: a [3H]bumetanide binding study
Authors: Haas M. , Johnson L. G. , Boucher R. C.
Published: December 2017

We examined [3H]bumetanide binding to membranes isolated from canine tracheal and bronchial epithelia and to confluent primary cultures of these cells. Crude plasma membranes from trachea and bronchus bind [3H]bumetanide in a saturable manner; tracheal membranes have a higher affinity but lower maximal binding (K1/2 approximately equal to 0.7 microM; Bmax approximately equal to 2.5 pmol/mg protein) than do bronchial membranes (K1/2 approximately equal to 3.5 microM; B(max) approximately equal to 7.5 pmol/mg). In both cases, saturable binding is reduced by greater than 65% when either Na, K, or Cl is removed from the medium. In primary cultures, saturable [3H]bumetanide binding (inhibited by a 30-fold excess of unlabeled bumetanide) occurs when [3H]bumetanide (1.0 microM) is added to the solution bathing the basolateral side of tracheal (1.20 +/- 0.10 pmol bound/mg total cell protein) and bronchial (1.79 +/- 0.52 pmol/mg) cultures; minimal binding is seen with apical [3H]bumetanide. Isoproterenol (10(-5) M; basolateral exposure) produces approximately 100% increase in saturable basolateral [3H]bumetanide binding to tracheal cultures and approximately 30% increase in bronchial cultures. Similar augmentation of binding is seen when apical Cl is reduced from 134 to 4 mM and when both apical and basolateral media are made hypertonic by addition of 100 mM sucrose. Under these latter two conditions, isoproterenol produces little or no additional increase in binding. Our results indicate that the increase in basolateral Cl influx via Na-K-Cl cotransport that must occur during beta-adrenergic stimulation of net salt secretion in canine airway epithelia is related to an actual increase in the number of functioning cotransporters in the basolateral membrane and is not simply due to a change in ion gradients. The increase in cotransport sites, however, may be secondary to initial stimulation of apical Cl channels, with resultant cell shrinkage.
Sulfhydryls on frog skeletal muscle membrane participate in contraction
Authors: Oba T. , Yamaguchi M.
Published: December 2017

To examine the molecular mechanism underlying contractile activation, we studied effects of a sulfhydryl reagent, N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM), on twitch, Ag(+)-induced contraction, and K+ and caffeine contractures in single toe muscle fibers of frog. DACM suppressed twitch and Ag(+)-induced contraction, dose dependently, but not caffeine contracture. K+ contracture also was decreased appreciably by exposure to 40 microM DACM for 10 min. DACM elicited no shift of the mechanical threshold or inhibition of resting potential but slightly inhibited action potential. Increase of the fluorescence intensity produced by binding of 10 microM DACM to sulfhydryl groups was depressed by brief pretreatment with 100 microM Ag+. When exposed to 1 mM dithiothreitol (DTT) within 5 s of the rising phase of 5 microM Ag(+)-induced contraction, the fiber rapidly decreased the tension to the resting level. In this case, reapplication of 5 microM Ag+ after washing out DTT elicited a new contraction similar to the first Ag(+)-induced contraction. The second contraction amplitude depended on the time between the onset of the first Ag(+)-induced contraction and DTT application. If DTT was applied after more than 16 s, tension no longer developed on the second exposure to Ag+ or K+. The experiments provide evidence that crucial sulfhydryl groups participate in muscle activation. The possible role of the sulfhydryl group on the transverse tubular membrane in tension development is discussed.
Cell fractionation and electron microscope studies of kidney folate-binding protein
Authors: Hjelle J. T. , Christensen E. I. , Carone F. A. , Selhub J.
Published: December 2017

The subcellular distribution of folate-binding protein (FBP) and [3H]folate in the proximal tubule was examined using cell fractionation and different electron microscope (EM) techniques. Cell fractionation of rabbit proximal tubules revealed that FBP distributed into two modes: 50% of FBP distributed with alanylaminopeptidase activity (brush border), and the remaining FBP distributed with organelles of lower density that did not show a large digitonin-induced shift to greater density. Infusion of [3H]folate into the kidney followed by isolation and fractionation of the proximal tubules revealed a time-dependent shift of [3H]folate from the heavy (brush border) mode to the lighter organelle mode. By EM immunocytochemistry, rat kidney FBP locates in the brush border, endocytic invaginations, endocytic vacuoles, and dense apical tubules of proximal tubule cells. EM autoradiography of rat kidney 10 min after intravenous infusion of [3H]folate revealed that the label was significantly concentrated only in the brush border, endocytic vesicles, and lysosomes. These data support a mechanism of receptor-mediated endocytosis for the process of FBP-mediated folate transport in the kidney.
Calcium stimulates glucose transport in skeletal muscle by a pathway independent of contraction
Authors: Youn J. H. , Gulve E. A. , Holloszy J. O.
Published: December 2017

In this study we investigated the possibility that an increase in cytoplasmic Ca2+ concentration that is too low to cause muscle contraction can induce an increase in glucose transport activity in skeletal muscle. The compound N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), which induces Ca2+ release from the sarcoplasmic reticulum (SR), caused a dose-dependent increase in tension in rat epitrochlearis muscles at concentrations more than approximately 200 microM. Although 100 microM W-7 did not increase muscle tension, it accelerated loss of preloaded 45Ca2+. Glucose transport activity, measured with the nonmetabolizable glucose analogue 3-O-methylglucose, increased sixfold in muscles treated for 100 min with 50 microM W-7 (P less than 0.001) and eightfold in response to 100 microM W-7 (P less than 0.001). The increase in glucose transport activity was completely blocked with 25 microM cytochalasin B. There was no decrease in ATP or creatine phosphate concentrations ([approximately P]) in muscles incubated with 50 microM W-7. Dantrolene (25 microM), which blocks Ca2+ release from the SR, blocked the effects of W-7 both on 45Ca2+ release and on glucose transport activity. 9-Aminoacridine, another inhibitor of Ca2+ release from the SR, also blocked the stimulation of hexose transport by W-7. Caffeine, a compound structurally unrelated to W-7 that also releases Ca2+ from the SR, also increased glucose transport activity. Incubation of muscles with 3 mM caffeine for 30 min, which did not cause contraction or lower [approximately P], induced a threefold increase in 3-O-methylglucose transport (P less than 0.001). These results provide evidence suggesting that an increase in cytoplasmic Ca2+ too low to cause contraction or [approximately P] depletion can bring about an increase in glucose transport activity in skeletal muscle.
Clear zone in osteoclast function: role of podosomes in regulation of bone-resorbing activity
Authors: Teti A. , Marchisio P. C. , Zallone A. Z.
Published: December 2017

The adhesion of osteoclasts to the bone matrix is mandatory for bone resorption. Contact of the osteoclast with bone surface induces, in fact, cell polarization and organization of the resorbing apparatus, the so-called “ruffled border.” Cell-matrix interaction in osteoclasts is a complex phenomenon resulting from formation of the “clear zone,” a cytoplasmic area presenting the adhering plasma membrane, or “sealing membrane.” The sealing membrane surrounds the ruffled border and seals the resorbing compartment, namely the extracellular space in which bone resorption takes place. Adhesion at this level occurs via specialized discrete structures, the “podosomes.” Podosomes present most of the protein commonly found in focal adhesions, but with a peculiar organization. They are dynamic elements suitable for regulation, according with the functional demand of the cell. Their assembly increases during bone resorption and is regulated by the cytosolic free calcium concentration and the activity of protein kinase C.
Integration of embryonic nephrogenic cells carrying a reporter gene into functioning nephrons
Authors: Koseki C. , Herzlinger D. , al-Awqati Q.
Published: December 2017

We developed a procedure to introduce and stably express foreign genes into the kidney. The Lac Z reporter gene encoding the bacterial protein beta-galactosidase was introduced by retrovirus-mediated gene transfer into rat nephrogenic mesenchymal cells, which were induced for 24 h with embryonic spinal cord in vitro. The Lac Z-tagged mesenchymal cells were subsequently transplanted underneath the capsule of the neonatal kidney. Two weeks after transplantation, the Lac Z-tagged cells derived from transplants were identified by their beta-galactosidase expression. Well-differentiated Lac Z positive cells were observed in glomerulus and proximal and distal nephron segments. To determine if the tagged mesenchymal cells developed into functional nephrons, fluorescein isothiocyanate-labeled dextran was infused into transplanted animals before death. We observed that fluorescent apical vesicles were colocalized to beta-galactosidase positive proximal tubular cells, indicating that the transplanted mesenchymal cells were integrated into reabsorbing nephrons. These results show the feasibility of introducing foreign genes into epithelia of functioning nephron segments.
Calcium-permeable channels in rat hepatoma cells are activated by extracellular nucleotides
Authors: Bear C. E. , Li C. H.
Published: December 2017

Extracellular ATP is known to cause uptake of Ca2+ by rat liver cells. The specific pathway permitting influx of Ca2+ has not yet been identified. In the present investigations, we studied the properties of ATP-evoked 45Ca2+ uptake in rat hepatoma cell monolayers and then used patch-clamp electrophysiology to identify the channel that may account for this uptake. The results suggest that ATP-stimulated 45Ca2+ uptake occurs as a result of P2-purinergic receptor interaction because uptake was inhibited by Reactive Blue (100 microM), a blocker of this type of receptor. Furthermore, the ability of other adenine nucleotides to stimulate 45Ca2+ uptake was related to the selectivity sequence for binding to the P2-purinergic receptor. ATP-stimulated 45Ca2+ uptake occurs primarily through a conductance pore since it was inhibited by 70% upon dissipation of the membrane potential using the K+ ionophore valinomycin. The calcium channel blockers nifedipine and verapamil failed to inhibit 45Ca2+ uptake, but gadolinium (GdCl3) was an effective blocker. In cell-attached patch-clamp experiments, a single type of channel was activated with ATP (100 microM) addition to the bath in 18 of 32 trials. The current-voltage relationship of the ATP-activated channel is identical to that of the stretch-activated channel previously characterized in this laboratory as a calcium-permeable cation-nonselective channel [Am. J. Physiol. 258 (Cell Physiol. 27): C421-C428, 1990]. There are several lines of evidence which suggest that this cation-nonselective channel may account for ATP-stimulated 45Ca2+ uptake.(ABSTRACT TRUNCATED AT 250 WORDS)
Control of myosin heavy chain expression: interaction of hypothyroidism and hindlimb suspension
Authors: Diffee G. M. , Haddad F. , Herrick R. E. , Baldwin K. M.
Published: December 2017

The aim of this study was to contrast competing influences, hypothyroidism and hindlimb suspension, on myosin heavy chain (MHC) expression studied at the protein level and mRNA level. Female Sprague-Dawley rats were assigned to either normal control (NC), normal suspended (NS), or hypothyroid (thyroidectomized) control (TC) and suspended (TS) groups. NS and TS animals were suspended for 14 days following which myofibrils and total RNA were purified from the hindlimb muscles. In the soleus and vastus intermedius (VI), there was an increase in type I MHC and a decrease in type IIa MHC in both the TC and TS groups and a decrease in type I and increase in type IIa MHC in the NS group. At the mRNA level, similar shifts were observed with the exception that 1) the increased type IIa MHC seen in the soleus and VI of the NS animals was not accompanied by an increase in IIa mRNA and 2) type IIb mRNA was increased in the NS soleus without concomitant changes in IIb protein levels. These data suggest the following: 1) a hypothyroid state predominates over mechanical unweighting factors in the control of MHC distribution in slow muscles; and 2) translational or posttranslational factors may be important in the regulation of type IIa and IIb MHC expression during hindlimb suspension.
Acute cell volume changes in anisotonic media affect F-actin content of HL-60 cells
Authors: Hallows K. R. , Packman C. H. , Knauf P. A.
Published: December 2017

To investigate the possible role of the cytoskeleton in volume regulatory responses of human promyelocytic leukemic (HL-60) cells, we monitored and modulated the F-actin content of these cells undergoing volume regulation in anisotonic media. Initial volume changes of HL-60 cells suspended in hypertonic media followed a Van't Hoff relationship, and intracellular F-actin content during volume regulatory responses in anisotonic media changed concomitantly as an inverse function of the volume shifts. These F-actin changes were shown to be an explicit function of cell volume and not tonicity of the medium. The data fit with the idea that changes in affinity of actin-binding proteins (ABPs) for actin and/or changes in the overall effective critical concentration of actin occur during acute cell volume changes, producing shifts in the relative amounts of G- and F-actin. Treatment of HL-60 cells with dihydrocytochalasin B (DHB), which perturbs cellular actin assembly, lowered resting levels of intracellular F-actin but did not prevent volume-associated F-actin changes in anisotonic media. Despite the lowered F-actin levels, HL-60 cells in the presence of DHB still undergo normal volume regulatory responses. Thus the absolute amount of intracellular F-actin does not appear to be critical for volume regulation in HL-60 cells.
ATP-dependent multispecific organic anion transport system in rat erythrocyte membrane vesicles
Authors: Heijn M. , Oude Elferink R. P. , Jansen P. L.
Published: December 2017

The uptake of oxidized glutathione (GSSG) into inside-out membrane vesicles of Wistar rat erythrocytes was studied. Uptake was ATP dependent, into an osmotically active space, and saturable. Analysis of saturable ATP-dependent GSSG uptake showed two affinities for GSSG [concentration for half-maximal velocity (K1/2 1), 26 microM; K 1/2 2, 4 mM; maximum transport rate (Vmax 1), 100 pmol.mg-1.min-1; Vmax 2, 360 pmol.mg-1.min-1]. Interactions of the high-affinity system with different organic compounds were studied. Leukotriene C4, bromosulfophthalein-S-glutathione, and 2,4-dinitrophenyl-S-glutathione were effective inhibitors. In addition, anionic nonglutathione conjugates, like indocyanine green, rose bengal, dibromosulfophthalein, and sulfated or glucuronidated (divalent) bile acids inhibited GSSG transport. Monovalent bile acids had no influence on GSSG transport. Inhibition by 2,4-dinitrophenyl-S-glutathione [inhibition constant (Ki) = 2.6 microM] and sulfated glycolithocholic acid (Ki = 2.9 microM) was purely competitive. The use of adenosinetriphosphatase (ATPase) inhibitors suggested a resemblance with E1E2-type ATPase. Vesicles of erythrocytes isolated from the TR- rat, a mutant rat strain with a defective biliary secretion of organic anions, have an impaired uptake of GSSG (Vmax was decreased 2-fold). In conclusion, erythrocytes have an ATP-dependent organic anion transport system that can be inhibited by a broad range of organic anions. This system is very similar if not identical to the hepatocanalicular ATP-dependent organic anion transporter.
Modulation of the delayed rectifier, IK, by cadmium in cat ventricular myocytes
Authors: Follmer C. H. , Lodge N. J. , Cullinan C. A. , Colatsky T. J.
Published: December 2017

The effects of cadmium on the delayed outward potassium current (IK) were investigated in isolated cat ventricular myocytes using the single suction pipette voltage-clamp technique. IK activation was examined using peak tail currents elicited after 750-ms voltage-clamp steps to selected membrane potentials from a holding potential of -40 mV. In the presence of Cd2+ (0.2 mM), peak tail currents increased from a control value of 85 +/- 12 to 125 +/- 18 pA (n = 4). Activation curves constructed from the average peak tail-current measurements in all experiments showed that Cd2+ shifted the voltage dependence of activation to more positive potentials by 16.4 +/- 2.0 mV and increased the slope factor of the activation curve from 6.1 +/- 0.2 to 6.9 +/- 0.2 mV. In the absence of Cd2+, increases in holding potential from -30 to -70 mV had no effect on the magnitude of the peak tail currents, suggesting that the Cd(2+)-induced increase was not the result of a voltage-dependent increase in the number of available K+ channels at the holding potential. Slow voltage ramps from -70 to +70 mV revealed that Cd2+ increased the outward current at membrane potentials positive to +20 mV and shifted the voltage range in which IK inwardly rectified to more positive potentials. The fully activated current-voltage relationship was also shifted to more positive potentials by Cd2+. Cd2+ did not alter channel selectivity for K+.(ABSTRACT TRUNCATED AT 250 WORDS)
Characterization of myocardial Na(+)-Ca2+ exchange in rainbow trout
Authors: Tibbits G. F. , Philipson K. D. , Kashihara H.
Published: December 2017

This study compared Na(+)-Ca2+ exchange from the hearts of rainbow trout with that from canines. In several respects, trout cardiac Na(+)-Ca2+ exchange is functionally similar to that from dogs and other mammals. Trout cardiac Na(+)-Ca2+ exchange is stimulated approximately 200% after 30-min incubation with 10 micrograms/ml chymotrypsin at 21 degrees C, similar to mammals. On the other hand, both the temperature and pH dependencies are strikingly different between the trout and canine myocardial Na(+)-Ca2+ exchange. While canine heart Na(+)-Ca2+ exchange exhibits a Q10 of greater than 2 (similar to values observed in other mammals), that from trout is relatively insensitive to temperature with a Q10 of approximately 1.2. The absolute rates of Na(+)-Ca2+ exchange in trout heart are four- to sixfold higher than that in mammals when measured at 7 degrees C. Furthermore, the temperature insensitivity of trout myocardial Na(+)-Ca2+ exchange is retained when the exchanger is reconstituted into an asolectin bilayer, suggesting that this property is intrinsic to the protein and not dependent on species differences in lipid bilayer composition. Trout Na(+)-Ca2+ exchange is not markedly stimulated by alkaline pH, in contrast to mammals, and this characteristic is also maintained after reconstitution. Western blots of trout cardiac sarcolemma run on 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis react with antibodies raised against the canine Na(+)-Ca2+ exchanger with a similar pattern of bands (70, 120, and 160 kDa). Furthermore, a cDNA probe from canine Na(+)-Ca2+ exchanger hybridizes on Northern blots of trout heart mRNA to a 7-kb band, similar to that in mammals. Thus, while important functional differences in Na(+)-Ca2+ exchange exist between trout and mammalian hearts, the molecular basis is not yet known.
Thyroid hormone induction of rat myocardial Na(+)-K(+)-ATPase: alpha 1-, alpha 2-, and beta 1-mRNA and -protein levels at steady state
Authors: Hensley C. B. , Azuma K. K. , Tang M. J. , McDonough A. A.
Published: December 2017

In this study, we measured the time courses of change in Na(+)-K(+)-ATPase alpha 1-, alpha 2-, and beta 1-subunit mRNA and protein abundance in cardiac myocytes isolated from euthyroid, hypothyroid, and hyperthyroid (hypothyroids injected daily with 1 microgram T3/g body wt) rats. In hypothyroids, alpha 1-, alpha 2-, and beta 1-protein levels were decreased to 0.55, 0.42, and 0.57 of euthyroids, predicting the decrease in Na(+)-K(+)-ATPase activity to 0.53 of control. There was no change in these subunits' mRNA levels, indicating that the decreases in protein levels were not due to decreased subunit transcription rates. In hyperthyroids, the alpha 1-mRNA increased to a steady state of threefold over hypothyroid by 1 day of T3 treatment, and the alpha 1-protein levels increased to twofold over hypothyroid by 4 days of T3. alpha 2-mRNA increased to 5-fold over hypothyroid by 2 days, whereas the alpha 2-protein levels increased to 14-fold above hypothyroid by 4 days of T3. Beta 1-mRNA increased to 12-fold above hypothyroid by 1 day of T3 treatment, whereas beta 1-protein increased to only 2.5-fold over hypothyroid by 4 days of T3. The discoordinate changes in alpha 2- and beta 1-mRNA vs. protein can be reconciled with the hypothesis that beta 1 is rate limiting for assembly in eu- and hypothyroids, and favors assembly with alpha 1, while excess unassembled alpha 2 is degraded. In the hyperthyroids we predict beta 1 is not rate limiting and there is increased alpha 2 beta 1-assembly. We calculate that T3 decreases the alpha 1-to-alpha 2 ratio from 24:1 in hypothyroid to 3.4:1 in hyperthyroid cardiomyocytes.
Enhancement of glucose transport in response to inhibition of oxidative metabolism: pre- and posttranslational mechanisms
Authors: Shetty M. , Loeb J. N. , Ismail-Beigi F.
Published: December 2017

Addition of 5 mM sodium azide to Clone 9 cells, a rat liver cell line characterized by intracellular glucose concentrations of less than 10% that of the external medium and limited glycogen stores, results in a 50-80% reduction in cell ATP content within 20 min which then recovers to near-basal levels within 1 h and is subsequently maintained at normal levels for 24 h despite continuing the presence of the inhibitor. Associated with this adaptive response is a striking stimulation of facilitated glucose transport, mediated by the GLUT-1 transporter, that exhibits "early" and "late" phases that appear to be mechanistically different. During the early phase of the response (0-2 h), glucose transport rate is enhanced 12-fold in the absence of any change in cell GLUT-1 or GLUT-1 mRNA content. In contrast, the late phase of the response (8-24 h) is characterized by a further large stimulation of glucose transport (to 1.6 times the 2-h value) that is associated with 2- to 3- and 6- to 10-fold increments in cell GLUT-1 and GLUT-1 mRNA content, respectively. In time course studies an increase in GLUT-1 mRNA content was observed at 4 h and preceded the increment in GLUT-1 which became detectable after 8 h of exposure to azide. A marked induction of GLUT-1 mRNA by azide was also demonstrable in cells incubated in medium containing higher concentrations of glucose (10.6 mM), although the increment was approximately 20% less than when cells were incubated in standard medium (containing 5.6 mM glucose).(ABSTRACT TRUNCATED AT 250 WORDS)