bluuebunny/crossref_metadata_2025 huggingface dataset:
Human orbital fibroblasts in culture bind and respond to endothelin
Authors: Smith T. J. , Kottke R. J. , Lum H. , Andersen T. T.
Published: December 2017

Human fibroblasts in primary cell culture were studied for their ability to bind to endothelin (ET), a 21-amino acid peptide with profound vasoconstricting properties. When 125I-labeled ET-1 was incubated with confluent orbital fibroblasts in the presence of increasing concentrations of unlabeled ligand, a single class of binding site was defined with a dissociation constant of 1.42 x 10(-8) M and a maximal binding capacity of 9.1 x 10(-10) mol/micrograms protein. ET-3 was a substantially less potent competitor for 125I-ET-1 binding sites than was unlabeled ET-1. Dermal fibroblasts demonstrated approximately 75% less ET-1 saturation binding activity, on a cellular protein basis, than did those from the orbit. Orbital fibroblasts responded to ET-1 (10(-9) M) with a rapid and transient increase in the free concentration of intracellular Ca2+ ([Ca2+]i) as assessed by monitoring acetoxymethyl ester of fura 2 fluorescence intensity. Rechallenge with the peptide elicited a substantially attenuated response than that seen after the initial treatment. There was no consistent effect of ET-1 on [Ca2+]i in dermal cultures. ET-3 failed to influence [Ca2+]i in either type of fibroblast. It would appear that orbital fibroblasts bind and respond to ET in a manner distinct from that observed in dermal fibroblasts, raising the possibility that the peptide may have site-specific actions in orbital connective tissue.
IIx and slow myosin expression follow mitochondrial increases in transforming muscle fibers
Authors: Jacobs-El J. , Ashley W. , Russell B.
Published: December 2017

Metabolic profile and contractile isoform expression commonly define classic fiber types in skeletal muscle. Little is known about how metabolic requirements determine expression of fast IIx and slow myosin isoforms in muscles undergoing fiber type conversion. Tibialis anterior muscles from female New Zealand White rabbits were stimulated continuously at 10 Hz for 4-21 days. Quantitative fiber analysis was made for oxidative potential by histochemistry and for fast IIx and slow myosin mRNA content by in situ hybridization. In control muscle we found 3 +/- 0.27% fibers coexpress both fast IIx and slow myosin mRNA and so were not assignable to a classic fiber type. After stimulation, increase in fiber oxidative potential was detectable by 4 days and preceded IIx mRNA increases on a fiber-by-fiber basis. Slow myosin transcripts were detected by 7 days in fibers with higher oxidative levels. Coexpression of IIx and slow transcripts peaked at 22 +/- 2.5% of fibers by 7 days. IIx then declined, leaving slow myosin expressed in 62 +/- 0.45% of fibers by 3 wk. We conclude that during fiber type transformation individual fibers can transcribe two myosin mRNAs synchronously. Metabolic demand precedes and may be linked to IIx and slow myosin isoform expression.
Regulation of renin processing and secretion: chemiosmotic control and novel secretory pathway
Authors: King J. A. , Lush D. J. , Fray J. C.
Published: December 2017

The renin-angiotensin-aldosterone system (RAAS) plays an important role in cardiovascular and electrolyte regulation in health and disease. Juxtaglomerular cells in the kidney regulate endocrine RAAS by physiologically controlling conversion of prorenin and secretion of renin. The classical baroceptor, neurogenic, and macula densa mechanisms regulate renin expression at the cellular level by Ca2+, adenosine 3',5'-cyclic monophosphate (cAMP), and chemiosmotic forces (K+, Cl-, and water flux coupled to H+ movement). The baroceptor mechanism (through Ca2+) activates K+ and Cl- channels in the surface membrane and deactivates a KCl-H+ exchange chemiosmotic transporter in the secretory granular membrane. The neurogenic mechanism (through cAMP) promotes prorenin processing to renin. The macula densa mechanism (through K+ and Cl-) involves the processing of prorenin to renin. Ca2+, by inhibiting the KCl-H+ exchange transporter, prevents secretory granules from engaging in chemiosmotically mediated exocytosis. cAMP, on the other hand, by stimulating H+ influx, provides the acidic granular environment for prorenin processing to renin. It is concluded that, in the presence of a favorable chemiosmotic environment, prorenin is processed to renin, which may then be secreted by regulative degranulation or divergence translocation, a novel secretory pathway used by several secretory proteins, including renin.
Phosphorylation-dependent regulation of nonselective cation channels in guinea pig chromaffin cells
Authors: Inoue M. , Imanaga I.
Published: December 2017

In guinea pig chromaffin cells, acetylcholine induces a nonselective cation current by activating a muscarinic receptor, probably m4. We investigated activation and deactivation processes of this current. The calmodulin antagonists trifluoperazine and calmidazolium reversibly suppressed the muscarinic activation of the nonselective cation current in a dose-dependent manner; the former was about six times less potent than the latter. H-7, a kinase inhibitor, also reversibly inhibited the muscarinic current with a concentration eliciting 50% of maximal inhibition of 277 microM. Internal application of vanadate, a nonspecific phosphatase inhibitor, enhanced the muscarinic current and markedly slowed the time course of its deactivation, but the phosphatase 1 and 2A inhibitors okadaic acid and calyculin A had no effects. Lowering Mg2+ concentration in the patch solution mimicked the effects of vanadate on the muscarinic current. Internal dialysis with vanadate or low-Mg2+ solution gradually led to development of an inward current, and the related voltage dependence was similar to that seen with the muscarinic cation current. These results suggest that kinase and Mg(2+)-dependent phosphatase are responsible for activation and deactivation of nonselective cation channels. The channels seem to be under the influence of both enzymes, even in the absence of external signals.
Transtrophoblast and microvillus membrane potential difference in mature intermediate human placental villi
Authors: Greenwood S. L. , Boyd R. D. , Sibley C. P.
Published: December 2017

Single mature intermediate villi from term placentas after normal gestation and vaginal or cesarian delivery were identified microscopically and mounted in a bath for conventional micro-electrode studies. With the application of strict selection criteria, the following observations on electrical potential difference (PD) were made. 1) With the tissue bathed in Earle's medium (37 degrees C) the PD across the syncytiotrophoblast microvillus membrane with respect to the bath was not normally distributed. The median PD was -22 mV (range -12 to -60 mV, n = 200). This fell to -6 mV after prior incubation for 4 h with cyanide (3 mM) or iodoacetate (2 mM) but was not altered by short-term application of these agents or by 0.1 mM ouabain. Substituting Na+ in Earle's medium with choline had no effect on PD, but replacing Cl- with gluconate caused a depolarization of 6 mV (P < 0.002). Increasing KCl in the bath fluid revealed an apparently low microvillus membrane K+ conductance. The low microvillus membrane PD may reflect a low Na(+)-K(+)-ATPase activity and/or a low membrane permeability to K+. 2) The transtrophoblast PD measured by insertion and withdrawal of the electrode was significantly different from zero [P < 0.003, median -3 mV (range 0 to -15 mV), n = 11]; and PD measured by insertion of the electrode into the villus core and beyond was -6 mV (significantly different from zero P < 0.003, range -2.5 to -10 mV, n = 6). If a similar PD were to exist in vivo, it could have a significant influence on ion transport across the placenta at term.
Cross-bridge kinetics during shortening in early and sustained contraction of intestinal smooth muscle
Authors: Hellstrand P. , Nordstrom I.
Published: December 2017

Mechanisms responsible for the decrease in shortening velocity after prolonged contraction ("latch" state) were investigated at identical force during early (20 s, "phasic") and sustained (5 min, "tonic") phases of high-K+ (25-30 mM) contractions in smooth muscle of guinea pig taenia coli. Cytoplasmic Ca2+ concentration, myosin light-chain phosphorylation, and maximum shortening velocity all declined from 20 s to 5 min of contraction. The time course of shortening following isotonic quick release was biexponential, with a fastest rate constant of approximately 80 s-1 in both phasic and tonic contractions. Stiffness was identical in phasic and tonic contraction; however, after a release to slack length and unloaded shortening, stiffness during restretch was greater in tonic contraction (51 vs. 43% of isometric stiffness after 16 ms of unloaded shortening). Stiffness decreased after release with a rate constant of approximately 200 s-1, slightly greater in phasic than in tonic contraction. The results indicate that the number of attached cross bridges during unloaded shortening, while substantially reduced relative to the isometric value, is higher in latch than in nonlatch, consistent with a lower detachment relative to attachment rate.
ATP-sensitive K+ channels in rat aorta and brain microvascular endothelial cells
Authors: Janigro D. , West G. A. , Gordon E. L. , Winn H. R.
Published: December 2017

The endothelium plays an important role in the modulation of vascular tone and blood cell activation. Extensive work has demonstrated that the release of endothelium-derived relaxing factor (EDRF) from the endothelium is evoked by a number of physical and chemical stimuli requiring Ca2+. Because endothelial cells do not express voltage-dependent Ca2+ channels, Ca2+ influxes following receptor activation may be facilitated by cell hyperpolarizations mediated by the activation of K+ conductances. There has been recent interest in the role of ATP-sensitive K+ channels (KATP) suggesting that KATP may play a role in the regulation of blood flow. We have investigated the electrophysiological properties of an ATP-sensitive K+ conductance in whole cell and membrane patches from rat aorta and brain microvascular endothelial cells. Whole cell as well as single-channel currents were increased by either intracellular dialysis of ATP or application of glucose-free/NaCN (2 mM) solutions. Both currents were reversibly blocked by glibenclamide (1-100 microM). The KATP channel opener pinacidil (30 microM) caused activation of an outward current in the presence of physiological intracellular ATP concentrations. In inside-out patches, 10 microM-1 mM ATP invariably caused a dramatic decrease in channel activity. We conclude that both rat aorta and brain microvascular endothelial cells express KATP channels. KATP may play a role in the regulation of endothelial cell resting potential during impaired energy supply and therefore modulate EDRF release and thus cerebral blood flow.
Angiotensin II type 1 receptor-mediated stimulation of c-fos gene expression in astroglial cultures
Authors: Raizada M. K. , Rydzewski B. , Lu D. , Sumners C.
Published: December 2017

Angiotensin II (ANG II) stimulates plasminogen activator inhibitor 1 (PAI-1) gene expression in astroglial cells prepared from rat brains. In this study, we investigated whether c-fos gene expression may be involved in this cellular action of ANG II. Incubation of astroglial cultures with ANG II caused a time- and dose-dependent transient stimulation of the steady-state levels of c-fos mRNA, with a maximal stimulation of 50-fold observed with 100 nM ANG II within 30-45 min. This stimulation was completely abolished by the presence of the type 1 ANG II (AT1) receptor antagonist losartan but not by the type 2 ANG II receptor blocker PD-123177. Depolarization of brain cell cultures with 50 mM K+ also caused a 100-fold increase in c-fos mRNA levels, an effect partially blocked by losartan. These observations show that AT1 receptor activation stimulates expression of the c-fos gene, which may act as a third messenger in the regulation of cellular actions of ANG II, including PAI-1 gene expression in astroglial cells.
Adenosine A1 receptors and erythropoietin production
Authors: Ohigashi T. , Brookins J. , Fisher J. W.
Published: December 2017

N6-cyclopentyladenosine (CPA), a selective adenosine A1 receptor agonist, in a concentration range of 10(-9) to 10(-7) M, produced a significant decrease in erythropoietin (EPO) levels in a human hepatocellular carcinoma (Hep G2) cell culture (medium levels of EPO, 91.81 +/- 1.61 and 94.36 +/- 0.97% of control, respectively) after 24 h incubation in a hypoxic atmosphere. CPA, at a concentration of 10(-9) M, also produced a significant decrease in Hep G2 cell levels of adenosine 3',5'-cyclic monophosphate (cAMP; 78.13 +/- 3.89% of control) after 2 h incubation. CPA (10(-9) M) also significantly inhibited forskolin-stimulated increases in EPO production and cAMP accumulation in Hep G2 cells. On the other hand, 2-[p-(2-carboxyethyl)phenethyl-amino]-5'-N-ethylcarboxamidoadenosine (CGS-21680), a selective adenosine A2-receptor agonist, produced no significant change in EPO production in a dose range of 10(-10) to 10(-6) M but increased cAMP accumulation at 10(-6) M. A1-receptor binding assays using N6-[3H]cyclohexyladenosine revealed a single type of adenosine receptor binding site on Hep G2 cell membranes with a dissociation constant of 71.4 nM and a binding capacity of 1,530 fmol/mg protein. These results indicate that Hep G2 cells contain high-affinity adenosine A1 receptors that are linked to decreased cAMP accumulation and EPO production.
Effect of osmotic swelling and shrinkage on Na(+)-K+ pump activity in mammalian cardiac myocytes
Authors: Whalley D. W. , Hool L. C. , Ten Eick R. E. , Rasmussen H. H.
Published: December 2017

The effect on the sarcolemmal Na(+)-K+ pump of exposure to anisosmolar solutions was examined using whole cell patch clamping and ion-selective microelectrodes. Na(+)-K+ pump currents were measured in single ventricular myocytes by using pipette Na+ concentrations ([Na]pip) of 0-70 mM. The relationship between [Na]pip and pump current was well described by the Hill equation. The [Na]pip for half-maximal pump current (K0.5) was 21.4 mM in isosmolar (310 mosM) solution. K0.5 was 12.8 mM during cell swelling in hyposmolar solution (240 mosM) and 39.0 mM during cell shrinkage in hyperosmolar solution (464 mosM). The maximal pump currents, derived from the best fit of the Hill equation, and the Hill coefficients were similar in isosmolar, hyposmolar, and hyperosmolar solutions. A sustained (> 20 min) decrease in the intracellular Na+ activity developed during exposure of intact papillary muscles to hyposmolar solutions, and a sustained increase developed during exposure to hyperosmolar solutions. We conclude that osmotic myocyte swelling stimulates the sarcolemmal Na(+)-K+ pump at near-physiological levels of intracellular Na+, whereas shrinkage inhibits the pump. These changes are due to increases and decreases, respectively, in the apparent affinity of the pump for Na+.
Characterization and confocal imaging of calponin in gastrointestinal smooth muscle
Authors: Walsh M. P. , Carmichael J. D. , Kargacin G. J.
Published: December 2017

Calponin isolated from chicken gizzard smooth muscle binds in vitro to actin in a Ca(2+)-independent manner and thereby inhibits the actin-activated Mg(2+)-adenosinetriphosphatase of smooth muscle myosin. This inhibition is relieved when calponin is phosphorylated by protein kinase C or Ca2+/calmodulin-dependent protein kinase II, suggesting that calponin is involved in thin filament-associated regulation of smooth muscle contraction. To further examine this possibility, calponin was isolated from toad stomach smooth muscle, characterized biochemically, and localized in intact isolated cells. Toad stomach calponin had the same basic biochemical properties as calponin from other sources. Confocal immunofluorescence microscopy revealed that calponin in intact smooth muscle cells was localized to long filamentous structures that were colabeled by antibodies to actin or tropomyosin. Preservation of the basic biochemical properties of calponin from species to species suggests that these properties are relevant for its in vivo function. Its colocalization with actin and tropomyosin indicates that calponin is associated with the thin filament in intact smooth muscle cells.
Neutrophil-activating intercrine secreted by porcine platelets is active without proteolytic processing
Authors: Yan Z. , Holt J. C. , Stewart G. J. , Niewiarowski S.
Published: December 2017

A new member of the cytokine intercrine alpha-subfamily, porcine neutrophil-activating peptide 2 (pNAP-2), was isolated to homogeneity. Amino acid sequencing analysis showed two species of pNAP-2, a long form (pNAP-2-L) and a short form (pNAP-2-S). pNAP-2-L had seven more amino acids at the NH2-terminus than pNAP-2-S. The remaining amino acid sequences of the two molecules were identical. pNAP-2-S shared 65% homology with human neutrophil-activating peptide 2 (hNAP-2) including four cysteines in identical positions. Moreover, the NH2-terminal sequence Glu-Leu-Arg (E-L-R) was conserved in both molecules. Both pNAP-2-L and pNAP-2-S induced mobilization of cytosolic calcium in neutrophils and caused release of granulocyte elastase in a dose-dependent manner, although pNAP-2-L was less active. A desensitization study suggested that both hNAP-2 and pNAP-2-S may act on the same receptor. Whereas human platelets release inactive precursors that can be converted to hNAP-2 by cathepsin G from activated neutrophils, porcine platelets, upon stimulation with thrombin, appear to secrete active forms of pNAP-2. The activated neutrophils are not involved in the generation of pNAP-2.
Secretagogue-induced RVD in HSY cells is due to K+ channels activated by Ca2+ and protein kinase C
Authors: Moran A. , Turner R. J.
Published: December 2017

To maintain cell volume, absorptive epithelia must coordinate ion fluxes associated with transcellular transport with those required for volume regulation. K+ channels are thought to play a central regulatory role in this process. Electronic cell sizing was used to study the regulatory volume decrease (RVD) response of the human salivary ductal cell line HSY. Following a hypotonic challenge, RVD was markedly enhanced by the muscarinic agonist carbachol (half-maximal effect approximately 1.4 microM). Carbachol-induced RVD was mimicked by the K+ ionophore valinomycin and inhibited by K+ channel blockers, indicating that it is due to the activation of K+ channels. Carbachol-induced RVD was blocked by maneuvers that blunted the carbachol-induced rise in intracellular Ca2+ concentration ([Ca2+]i) and mimicked by ionomycin-induced increases in [Ca2+]i, but concentrations of ionomycin (100 nM) yielding [Ca2+]i well above carbachol-induced levels were required. However, when treatment with 2.5 nM ionomycin, which produced [Ca2+]i in the carbachol-generated range, was combined with treatment with low concentrations of an active phorbol ester, an enhancement of RVD similar to that observed with carbachol alone was observed. These data provide strong evidence that carbachol-induced RVD in the HSY cells involves K+ channels that are tightly regulated by both [Ca2+]i and protein kinase C.
Regulation of renal cell organic osmolyte transport by tonicity
Authors: Handler J. S. , Kwon H. M.
Published: December 2017

Madin-Darby canine kidney cells accumulate several nonperturbing organic osmolytes when cultured in a hypertonic medium. Myo-inositol, betaine, and taurine are accumulated secondary to an increase in uptake, the first coupled to sodium entry, the latter two coupled to sodium and chloride entry. The transport rates increase as the result of an increase in maximum velocity for each cotransporter, with peak activity 24 h after the increase in tonicity. The cDNA for each cotransporter has been cloned. Their sequences indicate that the myo-inositol cotransporter belongs to the gene family that includes the sodium-coupled glucose transporter (SGLT1); the betaine and taurine cotransporters belong to the gene family of sodium- and chloride-coupled transporters that are responsible for neuronal uptake of many neurotransmitters. Assays of mRNA abundance and nuclear run-on assays reveal that shifts in tonicity have a major effect on transcription of the genes for the sodium-myo-inositol (SMIT) and sodium-chloride-betaine (BGT1) cotransporters. The ensuing increase in mRNA abundance for the two cotransporters and presumed increase in synthesis of the cotransporter proteins can explain the increase in transport activity in response to changes in tonicity.
Muscarinic stimulation of gallbladder epithelium. II. Fluid transport, cell volume, and ion permeabilities
Authors: Altenberg G. A. , Subramanyam M. , Reuss L.
Published: December 2017

Activation of muscarinic receptors in the fluid-absorptive epithelium of the Necturus gallbladder elevates cytosolic Ca2+ concentration, transiently hyperpolarizes the cell membrane voltages, and decreases the apparent fractional resistance of the apical membrane [G. A. Altenberg, M. Subramanyam, J. S. Bergmann, K. M. Johnson, and L. Reuss. Am. J. Physiol. 265 (Cell Physiol. 34): C1604-C1612, 1993]. In these studies, we show that at the peak of the hyperpolarization both apical and basolateral membrane resistances (Ra and Rb, respectively) decreased, but in 2-3 min Ra returned to control values while Rb rose to a level approximately 60% higher than control. The acetylcholine (ACh)-induced decrease in Ra is caused by activation of apical membrane maxi K+ channels secondary to elevation of cytosolic Ca2+ concentration. The increase in Rb is due to decreases in K+ and Cl- conductances. ACh had no effects on cell KCl content or water volume, although K+ conductance transiently increased. These results can be explained by the changes in basolateral membrane conductances. ACh did not alter fluid absorption. In conclusion, ACh has complex time-dependent effects on K+ and Cl- electrodiffusive permeabilities without measurable changes in cell volume or in the rate of transepithelial fluid transport.
Beat-to-beat measurements of [Ca2+]i and force in ferret cardiac muscle after chemical loading of aequorin
Authors: Urthaler F. , Walker A. A. , Reeves R. C. , Hefner L. L.
Published: December 2017

This communication reports the development of a modified procedure for chemical loading of aequorin in small multicellular cardiac preparations, with special emphasis directed toward the implementation of a new method for computer-controlled low-photon counting and digital processing and analysis of the data to obtain intracellular Ca2+ concentration ([Ca2+]i). In eight ferret right ventricular trabeculae, we measured the mechanical performance and found that, at 1.25 mM extracellular Ca2+ concentration ([Ca2+]o), resting tension, developed tension, and time to peak tension were unchanged by the loading procedure. Estimated resting and peak systolic [Ca2+]i were 299 +/- 65 and 766 +/- 131 nM, respectively. Thirty minutes after raising the [Ca2+]o to 5 mM, there was a robust increase in mechanical performance, with peak systolic [Ca2+]i averaging 1,218 +/- 222 nM. The diastolic [Ca2+]i remained unchanged. In four other trabeculae, exposure to a low-Na(+)-containing superfusate demonstrated a remarkable beat-to-beat correspondence of increases in diastolic [Ca2+]i and resting tensions. The same beat-to-beat concordance was also observed between the rapidly changing amplitudes of peak [Ca2+]i and developed tension. In additional experiments, simultaneous recordings of [Ca2+]i and force transients were obtained during rapid pace pause maneuvers. These studies showed distinct and quantifiable fluctuations of [Ca2+]i in a 1:1 relation to the mechanical record to a frequency of at approximately 300 beats/min. These results demonstrate that beat-to-beat measurements of [Ca2+]i and tension transients can be obtained with good resolution in multicellular cardiac preparations.
K-Cl cotransport, pH, and role of Mg in volume-clamped low-K sheep erythrocytes: three equilibrium states
Authors: Lauf P. K. , Erdmann A. , Adragna N. C.
Published: December 2017

Ouabain-resistant K efflux and Rb influx in Cl and NO3 media were studied in volume-clamped low-K (LK) sheep red blood cells (SRBC) with normal and experimentally reduced cytoplasmic Mg (Mgi) levels as function of pH and at 37 degrees C. Sucrose was added to solutions with constant ionic strength and variable pH to maintain normal cell volume. Cl-dependent ouabain-resistant K(Rb) fluxes (K-Cl cotransport) at unity relative cell volume exhibited a maximum at pH approximately 7 in normal-Mgi LK cells consistent with the apparent acid pH activation reported for human erythrocytes. However, in LK SRBC with Mgi lowered by A-23187 and an external Mg chelator, K(Rb)-Cl cotransport was reversibly activated as the pH was raised from 6.5 to 9. The alkaline pH effect on Cl-dependent Rb influx in low-Mgi LK SRBC was due to a 10-fold rise in the maximum velocity values without a major change in the Km values. The pH dependence of the experimental flux reversal point, i.e., the extracellular Rb concentration at which no net K-Cl cotransport occurs, approximately paralleled that of the flux reversal point predicted from the ratio of the ion products, in both control and low-Mgi LK cells, albeit with a small displacement to higher extracellular Rb concentration at all pH values. The kinetic data can be explained by a general minimum three-state equilibrium in which deprotonation recruits transporters from a resting R state into the active A state modified by Mgi to an inactive I state.(ABSTRACT TRUNCATED AT 250 WORDS)
19F-NMR study of primary human T lymphocyte activation: effects of mitogen on intracellular pH
Authors: Bental M. , Deutsch C.
Published: December 2017

Intracellular pH of purified human T lymphocytes was studied using nuclear magnetic resonance (NMR) spectroscopy under physiological conditions. In this paper we introduce a new improved 19F-NMR pH probe, 2-amino-3,3'-difluoroisobutyric acid (vic-difluoro-alpha-methylalanine), which has a pKa of approximately 7.0, sensitivity of 0.83 ppm/pH, is noncytotoxic, and provides better signal-to-noise ratio for intracellular pH determinations. Quiescent and stimulated lymphocytes display different homeostatic intracellular pH values. Quiescent cells maintain intracellular pH of 7.04 +/- 0.03 at extracellular pH values between 6.9 and 7.3, and stimulated cells maintain intracellular pH of 7.25 +/- 0.05 at extracellular pH values between 7.0 and 7.5. Stimulation with ionomycin plus phorbol 12-myristate 13-acetate leads to intracellular alkalinization within 90 min, reaching the more alkaline steady-state value of 7.25 within 7-10 h. Proliferation, but not viability, of lymphocytes is dependent on extracellular pH in the range of 6.4-8.0, and this dependence is not due to limiting interleukin-2 elaboration. The mechanisms of pH regulation and the possible implications of a permissive pH for T lymphocyte proliferation are discussed.
Angiotensin II-dependent proximal tubule sodium transport requires receptor-mediated endocytosis
Authors: Schelling J. R. , Linas S. L.
Published: December 2017

Angiotensin II (ANG II) receptors are present on apical and basolateral surfaces of proximal tubule cells. To determine the cellular mechanisms of proximal tubule ANG II receptor-mediated Na transport, apical-to-basolateral 22Na flux was measured in cultured proximal tubule cells. Apical ANG II caused increases in 22Na flux (maximum response: 100 nM, 30 min). Basolateral ANG II resulted in 22Na flux that was 23-56% greater than 22Na flux observed with equimolar apical ANG II. Apical ANG II-induced 22Na flux was prevented by preincubation with amiloride, ouabain, and the AT1 receptor antagonist losartan. Because apical ANG II signaling was previously shown to be endocytosis dependent, we questioned whether endocytosis was required for ANG II-stimulated proximal tubule Na transport as well. Apical (but not basolateral) ANG II-dependent 22Na flux was inhibited by phenylarsine oxide, an agent which prevents ANG II receptor internalization. In conclusion, apical and basolateral ANG II caused proximal tubule Na transport. Apical ANG II-dependent Na flux was mediated by AT1 receptors, transcellular transport pathways, and receptor-mediated endocytosis.
Mineralocorticoid and glucocorticoid receptor steroid binding and localization in colonic cells
Authors: Schulman G. , Robertson N. M. , Elfenbein I. B. , Eneanya D. , Litwack G. , Bastl C. P.
Published: December 2017

In rat colon epithelium glucocorticoids and mineralocorticoids regulate Na transport by binding to distinct receptors and stimulating different pathways. The distribution and intracellular localization of mineralocorticoid (MR) and glucocorticoid (GR) receptors in colonic Na-absorbing surface cells and Cl-secreting crypt cells is unknown. Surface and crypt cells were sequentially isolated from rat distal colon by EDTA chelation and mechanical dissociation. Cell viability was confirmed by trypan blue exclusion and low rates of 2',7'-bis(2-carboxyethyl)-5(6)-carboxylfluorescein leak. Histologic examination, alkaline phosphatase activity, and rates of [3H]leucine incorporation confirmed separation of surface from crypt cells. Scatchard analysis of [3H]aldosterone and [3H]triamcinolone acetonide binding demonstrated that the number of MR decreased from 7,228 +/- 1,067 in surface to 2,299 +/- 434 receptors/cell in crypt cells, whereas the number of GR increased from 20,857 +/- 4,241 in surface to 58,598 +/- 8,207 receptors/cell in crypt cells. The dissociation constants were 2.8 +/- 0.4 nM for the MR and 12 +/- 3 nM for the GR. Indirect immunofluorescence using the specific anti-MR antibody hMRsN and the anti-GR antibody BuGR-2 demonstrated that both unliganded receptors were cytoplasmic and translocated to the nucleus after hormone binding. These data indicate that both surface and crypt cells are potentially responsive to mineralocorticoids and glucocorticoids and that both the MR and GR require hormone for nuclear translocation.
Role of protein kinase C in insulin activation of the Na-K pump in cultured skeletal muscle
Authors: Sampson S. R. , Brodie C. , Alboim S. V.
Published: December 2017

Administration of insulin to preparations of skeletal muscle causes an increase in Na(+)-K+ pump activity within 15-30 min. Although several mechanisms have been proposed, such as promotion of Na+ influx and translocation of pumps from intracellular to membrane sites, the early events involved in this effect remain unknown. We have investigated the possibility that activation of protein kinase C (PKC) may be an initial event in Na(+)-K+ pump activation in primary cultures of rat skeletal muscle. Insulin (80-100 mU/ml) and tumor-promoting phorbol esters (10-100 nM) increased Na(+)-K+ pump activity as determined by measurements of ouabain-suppressible 86Rb uptake, electrogenic pump component of membrane potential, and specific [3H]ouabain binding. These effects were not reduced by treatment of myotubes with amiloride, which blocks Na(+)-H+ exchange, or with tetrodotoxin, which blocks voltage-dependent Na+ channels. Effects of insulin and phorbol esters were not additive, suggestive of a common mechanism. Effects of both phorbol esters and insulin were significantly reduced by staurosporine (50-100 nM) and by downregulation of PKC (by pretreatment of myotubes with phorbol ester for 24 h). The findings suggest that insulin may stimulate Na(+)-K+ pump activity in skeletal muscle by activation of PKC.
Regulation of alpha 1B-adrenergic receptor half-life: protein synthesis dependence and effect of norepinephrine
Authors: Izzo N. J. , Colucci W. S.
Published: December 2017

Agonist-mediated down-regulation of alpha 1B-adrenergic receptors (AAR) may involve a decrease in synthesis, an increase in degradation, or a combination of the two mechanisms. Norepinephrine (NE) causes downregulation of AAR in rabbit aortic smooth muscle cells (RbSMC). To study the role of receptor degradation in this phenomenon, we studied the half-life (t1/2) of the AAR under basal conditions and during exposure to NE. We first determined the disappearance rate of AAR in the presence of cycloheximide, a commonly used method for measuring receptor t1/2. By this approach, the basal t1/2 was surprisingly long, 172 +/- 20 h. In contrast, the t1/2 in NE-treated cells was 11.8 +/- 0.3 h, suggesting that either NE decreased the t1/2 of AAR or cycloheximide increased the basal t1/2. To distinguish these possibilities, basal receptor t1/2 was determined by a second, independent method based on the repopulation of AAR after irreversible alkylation with chloroethylclonidine. This approach indicated a basal t1/2 (7.4 +/- 0.2 h) that was similar to the t1/2 with NE but more than 20-fold shorter than the t1/2 with cycloheximide. We conclude that in RbSMC NE-induced downregulation of AAR occurs without a decrease in receptor t1/2. The unexpected finding that cycloheximide markedly increases the basal t1/2 of the AAR further indicates that the t1/2 of the AAR is regulated, at least in part, by a short-lived protein that may regulate and/or mediate receptor degradation.
Effect of brefeldin A on ADH-induced transport responses of toad bladder
Authors: Weng K. , Wade J. B.
Published: December 2017

We have used brefeldin A (BFA) to examine the role of membrane traffic in the short-circuit current (ISC) and water permeability responses of the toad urinary bladder. BFA treatment of 1 or 5 micrograms/ml had a complex effect on the response of the ISC to antidiuretic hormone (ADH) or forskolin stimulation. Although the responses to initial challenges by ADH were not impaired by BFA, subsequent ISC responses were progressively reduced. Similarly, while the response to an initial challenge by forskolin was modestly reduced by BFA, subsequent responses were markedly reduced. Inhibition of protein synthesis with cycloheximide (CHM) affected ISC responses similarly. Neither BFA nor CHM had an effect on water permeability responses. These observations show that although the membrane traffic responsible for the water permeability response is insensitive to inhibition by BFA or CHM, the stimulation of Na+ transport becomes increasingly sensitive to these inhibitors with successive challenges by ADH or forskolin. Although initial increases in Na+ transport utilize preexisting components, subsequent responses appear to require an intact system for membrane biogenesis.
Identification and characterization of the rat intestinal epithelial cell (IEC-18) interleukin-1 receptor
Authors: Sutherland D. B. , Varilek G. W. , Neil G. A.
Published: December 2017

Interleukin-1 (IL-1), an important mediator of inflammation, may act in chronic inflammatory disorders of the intestine such as idiopathic inflammatory bowel diseases. Although the IL-1 receptor (IL-1R) has been studied extensively in many cell lines, including T cells, B cells, and fibroblasts, it has not been demonstrated on intestinal epithelial cells. IL-1 affects intestinal epithelial cell proliferation in vitro, suggesting the presence of IL-1Rs on intestinal epithelium. This paper demonstrates and further characterizes an IL-1R in a rat intestinal epithelial cell. Cells from rat intestinal epithelial cell line IEC-18 were grown to confluence in six-well plates, and association studies with 125I-labeled IL-1 beta to determine specific binding and equilibrium conditions were performed. A competitive-inhibition curve verified the IL-1 concentration required to saturate the IL-1R, and a Scatchard plot revealed a dissociation constant (Kd) of 1.2 x 10(-10) M, with 1,000 receptors per epithelial cell. Binding studies using increasing concentrations of 125I IL-1 beta alone confirmed the receptor density. Cross-linking 125I-IL-1 beta to the IEC-18 IL-1R demonstrated an IL-1R of approximately 80.5 kDa. cDNA transcribed from IEC-18 mRNA was used as a template for amplification of a segment of the IL-1R using complementary oligonucleotides. The resulting sequence of the IL-1R demonstrated a high degree of homology with both the human and mouse type I IL-1R.(ABSTRACT TRUNCATED AT 250 WORDS)
Gene expression during phorbol ester-induced differentiation of cultured human megakaryoblastic cells
Authors: Dorn G. W. , Davis M. G. , D'Angelo D. D.
Published: December 2017

Platelet protein makeup is determined during transformation of megakaryoblasts to mature megakaryocytes, the immediate precursor of circulating platelets. To better understand the molecular mechanisms of megakaryocyte formation, gene expression was characterized by Northern analysis and RNA fingerprinting of cultured human CHRF-288 megakaryoblastic cells undergoing phorbol ester-stimulated megakaryocytic differentiation or serum-stimulated megakaryoblast proliferation. Protooncogenes c-fos and c-jun were coordinately upregulated in both proliferating and differentiating cells, whereas c-myc transcripts were upregulated during proliferation only. In contrast, mRNAs for transforming growth factor-beta 1 (TGF-beta 1) and thromboxane receptors were coordinately upregulated during differentiation but differentially regulated during proliferation. RNA fingerprinting revealed multiple transcripts specific to either proliferating or differentiated cells. Three of these were identified by homology to known DNA sequence: CDw44 adhesion molecule (upregulated during differentiation), glutathione sulfhydryl peroxidase (downregulated during differentiation), and plectin cytoskeletal protein (upregulated during differentiation). Thus, although megakaryoblast proliferation and megakaryocyte differentiation both involve DNA and protein synthesis, each growth response is characterized by a distinct pattern of gene expression.
Shifts in type I fiber proportion in rat hindlimb muscle are accompanied by changes in HSP72 content
Authors: Locke M. , Atkinson B. G. , Tanguay R. M. , Noble E. G.
Published: December 2017

Heat-shock protein 72 (HSP72), the inducible isoform of the HSP70 family, is constitutively expressed in rat hindlimb muscles in proportion to the content of type I muscle fibers. To determine whether this relationship was maintained after fiber transformation, male Sprague-Dawley rats were treated with 3,5,3'-triiodo-DL-thyronine (T3) for 40 days or underwent surgical removal of the left gastrocnemius muscle, after which the left plantaris muscle was allowed to hypertrophy for 30 days. Hypertrophied plantaris muscles exhibited an increased number of type I fibers, type I myosin heavy-chain (MHC) protein, and HSP72 content compared with contralateral muscles. Soleus muscles from rats administered T3 exhibited an increased number of type II fibers, citrate synthase activity, and decreased HSP72 content compared with soleus muscles from controls. These results indicate that the relationship between HSP72 content and type I muscle fiber-MHC composition is maintained when muscles undergo fiber transformation and substantiate that HSP72 content in rat skeletal muscle is not directly linked to a muscle's oxidative capacity.
Water channel vesicles from toad urinary bladder contain a family of proteins present in other tissues
Authors: Paredes A. , Briscoe D. M. , Williams C. K. , Garcia-Perez A. , Wade J. B. , Harris H. W.
Published: December 2017

Antidiuretic hormone (ADH) stimulation causes the fusion and subsequent retrieval of cytoplasmic vesicles containing water channels (WCV) with the apical membrane of toad bladder granular cells. Previously, we showed that purified WCV contain 12 major protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. To identify various WCV proteins, we screened a panel of mouse monoclonal antibodies and characterized an immunoglobulin G1 monoclonal antibody, 5E5, that recognizes integral membrane WCV proteins of 38, 33, and 31 kDa. Immunocytochemistry and Western blot analyses show that 5E5 binds to multivesicular body endosomes shown previously to contain ADH water channels. In addition, 5E5 recognizes these proteins in selected cells of the skin, intestine, liver, kidney, spleen, and lung. However, 5E5 does not appear to recognize components of the water channel itself. We conclude that WCV contain several membrane proteins recognized by 5E5 that are present in certain cells of the other organs. Monoclonal 5E5 provides a probe to determine the structure and function of these endosomal proteins as well as their role in the ADH water permeability response.
Bradykinin and angiotensin II: activation of protein kinase C in arterial smooth muscle
Authors: Dixon B. S. , Sharma R. V. , Dickerson T. , Fortune J.
Published: December 2017

The effects of bradykinin (BK) and angiotensin II (ANG II) were compared in cultured rat mesenteric arterial smooth muscle cells. BK and ANG II activated a phosphoinositide-specific phospholipase C, leading to the rapid release of [3H]inositol phosphates, an increase in intracellular calcium, and formation of sn-1,2-diacylglycerol (DAG). DAG formation was biphasic with a transient peak at 5 s followed by a sustained increase from 60 to 600 s. The BK-mediated increases in inositol triphosphate and DAG were dose dependent with half-maximal increases at concentrations of 5 and 2 nM, respectively. Both hormones were found to activate protein kinase C (PKC) as assessed by phosphorylation of the 68- to 72-kDa intracellular PKC substrate myristoylated alanine-rich C kinase substrate. However, despite similar phosphorylation of this substrate, only ANG II produced a significant increase in membrane-bound PKC activity. The mechanism accounting for the inability of BK to increase membrane-bound PKC activity is unclear. Our studies excluded differential translocation of PKC to the nuclear membrane, production of an inhibitor of membrane-bound PKC activity, and expression of BK and ANG II receptors on different cells as the mechanism. Vascular smooth muscle cells were found to express at least four different PKC isozymes: alpha, delta, zeta, and a faint band for epsilon. All of the isozymes except zeta-PKC were translocated by treatment with the phorbol ester 4 beta-phorbol 12-myristate 13-acetate. However, neither ANG II nor BK produced significant translocation of any measured isozyme; therefore, we could not exclude the possibility that ANG II and BK activate different isozymes of PKC. Both hormones were found to have a similar small and inconsistent effect in stimulating [3H]thymidine incorporation. These observations demonstrate that BK and ANG II have similar biochemical effects on vascular smooth muscle cells and imply that, in selected vessels, the vasodilatory effects of BK mediated by the endothelium may be partially counterbalanced by a vasoconstrictor effect on the underlying vascular smooth muscle cells.
Modulation by L-type Ca2+ channels and apamin-sensitive K+ channels of muscarinic responses in cat chromaffin cells
Authors: Uceda G. , Artalejo A. R. , de la Fuente M. T. , Lopez M. G. , Albillos A. , Michelena P. , Garcia A. G. , Montiel C.
Published: December 2017

In the perfused cat adrenal gland stimulated with the muscarinic agonist methacholine chloride (100 microM for 3 min), two components were detected in the catecholamine secretory response: 1) an early phasic component that peaked at 300 ng/5 s catecholamine release and 2) a tonic component whose peak was transient and declined to a plateau of about 140 ng/5 s. Apamin (0.1 microM) increased the phasic component to 1,200 ng/5 s and the tonic component to approximately 350 ng/5 s. In single fura 2-loaded cat adrenal chromaffin cells, the cytosolic Ca2+ concentration ([Ca2+]i) also followed a biphasic pattern after stimulation with methacholine. Depletion of extracellular Ca2+ reduced the phasic [Ca2+]i peak by > 50% and the phasic secretory peak by approximately 90%; both the tonic components of [Ca2+]i and secretion were abolished. Depletion of intracellular Ca2+ pools decreased the phasic and tonic components of [Ca2+]i and secretion with respect to control values; however, the phasic components diminished more than the tonic components of [Ca2+]i and secretion. Although 3 microM furnidipine (a dihydropyridine L-type Ca2+ channel blocker) inhibited the phasic component of [Ca2+]i and secretion, its effects were more pronounced on the tonic component. omega-Conotoxin GVIA (1 microM, an N-type Ca2+ channel blocker) did not affect the [Ca2+]i or the methacholine secretory responses. The secretion peak seems to depend on both extracellular free Ca2+ (Cao2+) entry through L-type Ca2+ channels as well as on the mobilization of Ca2+ from intracellular stores; the plateau depends only on Cao2+ entry through L-type Ca2+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)
Antidiuretic hormone-responding nonselective cation channel in distal nephron epithelium (A6)
Authors: Marunaka Y. , Tohda H. , Hagiwara N. , Nakahari T.
Published: December 2017

Arginine vasotocin (AVT, 70 mU/ml) added from the basolateral side transiently activated a nonselective cation (NSC) channel with a single-channel conductance of 28.5 pS and almost identical selectivity for Na+ and K+ in the apical membrane of distal nephron cells (A6) cultured on permeable supports for 10-12 days in media containing 10% fetal bovine serum without supplemental aldosterone. The open probability (Po) of the NSC channel at the apical resting membrane potential in cell-attached patches was approximately 0.09 and increased when the apical membrane depolarized. The Po of the NSC channel was decreased by a rise in cytosolic Ca2+ concentration within a range of 30 nM-1 microM but not affected by cytosolic pH within a range of 6-8. The channel was activated by the application of negative pressure (10-60 cmH2O) into the patch pipette. Gadolinium (2 microM), an inhibitor of stretch-activated channels, decreased the Po by 40%. This blocking action of gadolinium was more effective after the channel was activated by stretch, i.e., 2 microM gadolinium decreased the Po by 70% when a negative pressure (60 cmH2O) was applied into the patch pipette. Amiloride (10 and 100 microM) showed a blocking action on the channel only when the NSC channel was activated by stretch.
Regulation of endothelin-induced Ca2+ mobilization in smooth muscle cells by protein kinase C
Authors: Xuan Y. T. , Wang O. L. , Whorton A. R.
Published: December 2017

We have investigated the role of protein kinase C (PKC) in regulating vascular smooth muscle cell responses to endothelin (ET). During the initial phase of the response, ET stimulated rapid formation of diacylglycerol due to rapid and transient activation of phosphatidyl inositol-specific phospholipase C and to rapid and prolonged activation of phospholipase D. Concurrently, ET stimulated translocation of PKC activity that reached a peak at 1 min and remained elevated for at least 20 min. Activation of PKC produced early inhibitory effects. Treatment of cells with phorbol 12-myristate 13-acetate (PMA) 5 min before stimulation with ET inhibited total inositol phosphate formation by > 50%. Because each inositol phosphate isomer was equally affected, the target appears to be either phospholipase C or some upstream component of the receptor coupling mechanism. Activation of PKC was important for sustained response to ET. Treatment of cells with staurosporine significantly reduced sustained elevation of cytosolic free Ca2+ concentration ([Ca2+]i) normally seen with ET. We had previously shown that sustained elevation of [Ca2+]i initiated by ET was due to continued activity of L-type Ca2+ channels. Our current data suggest that PKC is important in this response. For example, staurosporine inhibited both ET-induced 45Ca2+ and Mn2+ entry occurring 10 min after stimulation of influx mechanisms by the agonist. Similarly, pretreatment of cells for 18 h with phorbol dibutyrate depleted the cells of PKC and blocked the sustained activity of Ca2+ entry mechanisms stimulated by ET. Finally, PMA initiated a slowly developing, sustained elevation of [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)