bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of pHi in Saos-2 cells by thrombin: roles of proteolytic activity and cytosolic calcium transients
Authors: Ofori-Darko E. , Tashjian A. H.
Published: December 2017

Some, if not all, of the cellular actions of alpha-thrombin are now believed to be mediated by proteolytic cleavage of the cell surface thrombin receptor to yield a tethered ligand that initiates signal transduction via the receptor. We have investigated the actions of alpha-thrombin on the regulation of cytosolic free Ca2+ concentration ([Ca2+]i) and intracellular pH (pHi) in human osteoblast-like Saos-2 cells. After acidification with nigericin, thrombin induced an acute increase of [Ca2+]i and a rise in pHi. The action of thrombin on pHi was dependent on activation of the Na(+)-H+ antiporter. Thrombin elicited parallel concentration-dependent increases in both [Ca2+]i and pHi, and the rise in [Ca2+]i was a prerequisite for the increase in pHi. Preincubation of thrombin with the active site proteolytic inhibitor, BOC-D-Phe-L-Pro-D,L-Lys-CF3, prevented the alkalinization response to thrombin but had little or no effect on the thrombin-induced rise in [Ca2+]i. Hirudin, a natural inhibitor of thrombin, acts by tight binding to several discrete regions on the thrombin molecule. Preincubation of thrombin with hirudin completely blocked the rise in both [Ca2+]i and pHi. These results demonstrate that the thrombin-induced rise in [Ca2+]i alone is not sufficient to cause alkalinization in Saos-2 cells. More importantly, our findings reveal that not all of the cellular actions of thrombin can be explained by proteolytic cleavage of the thrombin receptor and suggest that different domains on the thrombin molecule may be required for eliciting signals that raise [Ca2+]i and pHi in Saos-2 cells.
Uterine contraction and physiological mechanisms of modulation
Authors: Wray S.
Published: December 2017

Control of the smooth muscle in the uterus (the myometrium), is of vital importance during pregnancy and parturition. It is therefore understandable that several physiological mechanisms (neuronal, hormonal, metabolic, and mechanical) play a role in the control of myometrial activity. As our knowledge of the mechanism of uterine contraction has increased much in recent years, it is now possible to begin to explain, in some detail, how the contractile activity may be modulated. A detailed account of the mechanism of contraction in the uterus is therefore given, followed by examples of modulation of this process for each of the four physiological methods listed above. Examples have been chosen to illustrate either general or particular mechanisms of modulation. The goal of many working in this field is to understand these processes and thus prevent preterm labor and uterine dysfunction in term labor, which are still significant clinical problems.
Na+ influx is mediated by Na(+)-K(+)-2Cl- cotransport and Na(+)-H+ exchange in sublingual mucous acini
Authors: Zhang G. H. , Cragoe E. J. , Melvin J. E.
Published: December 2017

The intracellular free Na+ concentration ([Na+]i) was studied using dual-wavelength microfluorometry of the fluorescent Na+ indicator sodium-binding benzofuran isophthalate (SBFI) to determine the mechanism(s) by which muscarinic stimulation increases the Na+ content in rat sublingual mucous acini. [Na+]i was 15.5 +/- 0.7 mM in acini superfused with a Na(+)-containing medium (135 mM Na+). Application of ouabain, a Na(+)-K(+)-adenosinetriphosphatase inhibitor, resulted in an increase in [Na+]i (approximately 75% in 10 min), whereas replacement of extracellular Na+ with N-methyl-D-glucamine induced a gradual decrease in [Na+]i (approximately 55% decrease in 5 min). The recovery of [Na+]i in Na(+)-depleted acini was K+ and Cl- dependent and was inhibited by bumetanide (Bum), a specific Na(+)-K(+)-2Cl- cotransport inhibitor, and by 5-(N-methyl-N-isobutyl)amiloride (MIBA), an amiloride derivative that specifically blocks Na(+)-H+ exchange. Stimulation with a muscarinic agonist (10 microM carbachol) resulted in a dramatic increase in the [Na+]i [approximately 180%, half time (t1/2) approximately 1 min] and a net increase in Na+ content, as measured with 22Na+ (approximately 110%, t1/2 approximately 1 min). Both the initial rate of the increase in [Na+]i and the magnitude of the net increase in Na+ content were dramatically blunted by Bum and MIBA. Increasing the intracellular free Ca2+ concentration ([Ca2+]i) with ionomycin, a Ca2+ ionophore, resulted in an increase in [Na+]i. Preventing the [Ca2+]i increase by chelating cytosolic Ca2+ with bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid completely abolished the agonist-induced evaluation in [Na+]i.(ABSTRACT TRUNCATED AT 250 WORDS)
Dual effect of phosphatase inhibitors on calcium channels in intestinal smooth muscle cells
Authors: Obara K. , Yabu H.
Published: December 2017

The effects of okadaic acid (OA) and calyculin A (CL-A), potent inhibitors of protein phosphatases type 1 (PP1) and type 2A (PP2A), on inward current carried by Ba2+ through voltage-dependent Ca2+ channel in guinea pig teniae coli smooth muscle cells were investigated using whole-cell patch-clamp technique. High concentrations of OA (5 x 10(-8)-5 x 10(-6) M) and CL-A (10(-9)-10(-7) M) dose dependently increased the inward current. The concentration producing apparent half-maximum enhancing effect values for OA and CL-A were 1.12 x 10(-7) and 1.78 x 10(-9) M, respectively. CL-A appeared to be approximately 100-fold more potent in increasing the inward current than OA. Lower concentrations of OA (10(-10)-2 x 10(-8) M) and CL-A (10(-11)-10(-9) M) decreased the inward current. The maximum inhibitory effects of OA and CL-A were observed at 10(-8) M OA and 5 x 10(-10) M CL-A, respectively. CL-A is approximately 100 times more effective inhibitor of PP1 than OA, and lower concentrations of OA and CL-A used in the present study inhibit PP2A activity, but they have no or little effect on PP1 activity (Ishihara, H., B. L. Martin, D. L. Brautigan, H. Karaki, H. Ozaki, Y. Kato, N. Fusetani, S. Watabe, K. Hashimoto, D. Uemura and D. J. Hartshorne. Biochem. Biophys. Res. Commun. 159: 871-877, 1989). In the absence of ATP in pipette solution, OA and CL-A did not affect the inward current.(ABSTRACT TRUNCATED AT 250 WORDS)
Electrical resistance method for measuring volume changes in monolayer cultures applied to primary astrocyte cultures
Authors: O'Connor E. R. , Kimelberg H. K. , Keese C. R. , Giaever I.
Published: December 2017

An electrical resistance method was developed to measure volume changes in substratum-attached monolayer cultures. Astrocytes in primary monolayer cultures prepared from neonatal rat cerebral cortex were placed in a confined channel containing a balanced salt solution, and the electrical resistance of the channel was measured using an applied alternating current. If the volume of the cells increases, then the volume of the solution within the channel available for current flow decreases by the same amount, resulting in an increase in the measured resistance through the channel. If the volume of the cells decreases, a decrease in resistance would be recorded. This method allows continuous measurements of volume changes in real time. When primary astrocyte monolayers were exposed to hyposmotic solutions (93-193 mosmol/kgH2O), they showed a rapid initial swelling and, in the continued presence of hyposmotic media, a characteristic regulatory volume decrease (RVD) in which there was a return to normal cell volume within approximately 20 min. Astrocytes exposed to hyperosmotic media (343-493 mosmol/kgH2O) gave a decrease in electrical resistance, indicating shrinkage. Putative endogenous effectors of astrocytic swelling, such as high extracellular K+ and glutamate, resulted in a much slower onset of swelling and no sign of RVD. This system can reliably measure the average change in cell monolayer volume to 1-2% and thus provides a sensitive means of continuous measurements of changes in cell volume in monolayer cultures.
Differential effects of cytokines on long-term mitogenic and secretory responses of fetal rat pancreatic βcells
Authors: Sjöholmöholm A.
Published: December 2017

Pages C116 and C118: Å. Sjőholm. “Differential effects of cytokines on long-term mitogenic and secretory responses of fetal rat pancreatic betacells.” Page C116, Table 1, the units for [Glucose] should read mM, and page C118, Table 3, the units for [IFN-ggr should read U/ml, as shown in the corrected tables. (The errors were inadvertentlv introduced by the printer at the final stage of proof.)

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Exogenous ATP induces a limited cell cycle progression of arterial smooth muscle cells
Authors: Malam-Souley R. , Campan M. , Gadeau A. P. , Desgranges C.
Published: December 2017

Because exogenous ATP is suspected to influence the proliferative process, its effects on the cell cycle progression of arterial smooth muscle cells were studied by investigating changes in the mRNA steady-state level of cell cycle-dependent genes. Stimulation of cultured quiescent smooth muscle cells by exogenous ATP induced chronological activation not only of immediate-early but also of delayed-early cell cycle-dependent genes, which were usually expressed after a mitogenic stimulation. In contrast, ATP did not increase late G1 gene mRNA level, demonstrating that this nucleotide induces a limited cell cycle progression of arterial smooth muscle cells through the G1 phase but is not able by itself to induce crossing over the G1-S boundary and consequently DNA synthesis. An increase in c-fos mRNA level was also induced by ADP but not by AMP or adenosine. Moreover, 2-methylthioadenosine 5'-triphosphate but not alpha, beta-methyleneadenosine 5'-triphosphate mediated this kind of response. Taken together, these results demonstrate that extracellular ATP induces the limited progression of arterial smooth muscle cells through the G1 phase via its fixation on P2 gamma receptors.
Characterization of endogenous sodium channel gene expressed in Chinese hamster ovary cells
Authors: Lalik P. H. , Krafte D. S. , Volberg W. A. , Ciccarelli R. B.
Published: December 2017

Chinese hamster ovary (CHO-K1) cells were observed to display transient inward Na+ currents of average amplitude (-92 +/- 20 pA), which activated at voltages more than -40 mV, and peak inward currents were observed at potentials equal to or more than +10 mV. Inward Na+ currents in these cells were eliminated after treatment with 500 or 50 nM tetrodotoxin (TTX), whereas 5 nM TTX resulted in 64 +/- 10% inhibition of Na+ current. Using DNA primers designed to bind to the rat brain IIA Na+ channel subtype, we amplified specific polymerase chain reaction (PCR) fragments from CHO-K1 poly-(A)+RNA. The cloning and sequencing of two of these fragments confirmed the presence of an endogenously expressed Na+ channel gene in these cells, which we have termed cho 1. Comparison of the DNA sequence of cho 1 PCR fragments with other known Na+ channel genes indicated a high degree of homology with rat brain Na+ channel subtypes. Northern blots using riboprobes generated from the cho 1 PCR fragments revealed the presence of a specific 9-kb mRNA in these cells. The molecular and electrophysiological data suggest that the cho 1 Na+ channel gene from CHO-K1 cells is closely related to brain-type Na+ channels.
Muscle contraction and inward current induced by silver and effect of Ca2+ channel blockers
Authors: Oba T. , Aoki T. , Koshita M. , Nihonyanagi K. , Yamaguchi M.
Published: December 2017

Single fibers from toe or anterior tibialis muscle contracted transiently and then tonically in the presence of 1.8 mM Ca2+ on addition of 10 microM Ag+. Exposure of fibers to Cd2+ completely inhibited tonic contraction and modified phasic contraction to some extent. Nifedipine at 10 microM initially potentiated and then completely inhibited twitch tension; subsequently, fibers no longer contracted phasically in response to 20 microM Ag+, whereas slight tonic contraction still occurred. Fibers with membrane potential clamped at -90 mV produced maintained inward current on application of Ag+. Simultaneous administration of 1 mM Cd2+ and 10 microM Ag+ to fibers voltage clamped with the double mannitol gap technique almost completely blocked the inward current. Removal of Cd2+ elicited a rapid and large inward current. Ag(+)-induced inward current was inhibited when 1 mM Cd2+ was applied to fibers during development of the inward current. In fibers paralyzed with 10 microM nifedipine, the inward current induced by 10 microM Ag+ was partially inhibited. These results suggest that phasic contraction induced by Ag+ is controlled by L-type Ca2+ channels (probably voltage sensors) located in the T-tubular membrane, whereas tonic contraction involves Ca2+ channels sensitive and/or insensitive to dihydropyridine in the surface and T-tubular membranes.
Long-term cAMP activation of Na(+)-K(+)-2Cl- cotransporter activity in HT-29 human adenocarcinoma cells
Authors: Slotki I. N. , Breuer W. V. , Greger R. , Cabantchik Z. I.
Published: December 2017

Cl- channel and Na(+)-K(+)-2Cl- cotransport activities were studied in various cystic fibrosis transmembrane conductance regulator (CFTR)-expressing cells with the aim of assessing integrative patterns of regulation of Cl- secretion. Human colonic HT-29 cells express relatively high levels of CFTR and cotransporter but relatively low Cl- channel activity. These cells showed commensurate activations of both transport systems evoked by short-term (minutes) or long-term (hours) exposures to adenosine 3',5'-cyclic monophosphate (cAMP). However, unlike in the case of CFTR and Cl- channels, long-term induction of cotransporter did not depend on de novo protein synthesis or changes in number of transporters. The patterns of activation of both transporters were also examined in CFTR-deficient cell lines (CFPAC and the viral-transfected CFPAC-PLJ) and in the viral CFTR-transfected derivative (CFPAC-4.7). All these cells displayed relatively low basal cotransport activity and a correspondingly low number of transporters, whereas only CFPAC-4.7 cells showed short-term (but not long-term) activatable Cl- channels. However, irrespective of the presence or absence of CFTR in CFPAC cells, neither short- nor long-term cAMP exposures induced significant cotransporter activation. Our studies with the various epithelial cell lines indicate that expression of CFTR activity per se is not sufficient for stimulation of cotransporter activity. Moreover, despite apparent correction of CFTR levels in CFPAC cells by gene transfer, the apparent Cl- secretory capacity might be limited by the low cotransport activity, such as that found in CFPAC cells, with obvious implications for proposed gene therapy of cystic fibrosis.
Steroid hormone stimulation of Na+ transport in A6 cells is mediated via glucocorticoid receptors
Authors: Schmidt T. J. , Husted R. F. , Stokes J. B.
Published: December 2017

The A6 cell line derived from the toad kidney forms polarized, highly differentiated epithelial monolayers in culture and has been utilized as an experimental model for studying regulation of transepithelial Na+ transport by aldosterone. In the present study we evaluated the specific role(s) of glucocorticoid and mineralocorticoid receptors in mediating this enhanced electrogenic Na+ transport, which was measured experimentally as an increase in short-circuit current (Isc). Our data demonstrate that specific glucocorticoid agonists (100 nM), including RU 28362 and RU 26988, elicit “mineralocorticoid-like” increases in Isc that are blocked by the glucocorticoid antagonist RU 38486 but are unaffected by mineralocorticoid antagonists including RU 28318 and RU 26752. The stimulatory effects of aldosterone (100 nM) were also blocked by RU 38486 and not by mineralocorticoid antagonists. These data extend earlier studies suggesting that in this cell line aldosterone mediates its physiological effects via binding with relatively low affinity (dissociation constant Kd congruent to 25-50 nM) to glucocorticoid receptors, despite the presence of apparently normal mineralocorticoid receptors. Our in vitro biochemical studies also demonstrate that A6 glucocorticoid receptor complexes can be thermally activated or transformed to DNA binding forms which exhibitaltered elution profiles from anion-exchange resins. Thus, based on several criteria, these amphibian glucocorticoid receptors appear very similar to classical mammalian receptors and are capable of mediating all of the stimulatory effects of aldosterone on net Na+ transport.
Regulation of CFTR Cl- conductance in secretion by cellular energy levels
Authors: Bell C. L. , Quinton P. M.
Published: December 2017

Recent studies suggested dual regulation of the Cl- conductance (GCl) affected in cystic fibrosis, one by protein kinase A-dependent phosphorylation and a second by low-affinity ATP binding. We proposed that ATP binding may couple the transport demands to the energy level of the cell. In the present study we examined this hypothesis further in a purely secretory function using the epithelial cell line T84. We used a depletion-permeabilization protocol on cells grown on permeable supports to deplete the cells of endogenous ATP and to provide access to the intracellular compartment for the impermeable nucleotides adenosine 3',5'-cyclic monophosphate (cAMP) and ATP. In contrast to non-depleted permeabilized cells, which responded to 0.1 mM cAMP with an increase in transepithelial potential (delta Vt = 29.8 +/- 3.0 mV, n = 4) and conductance (delta Gt = 1.23 +/- 0.54 mS/cm2, n = 4), addition of cAMP to ATP-depleted cells resulted in insignificant changes in Vt (delta Vt = 0.7 +/- 0.2 mV, n = 26; P < 0.05) and Gt (delta Gt = 0.020 +/- 0.003 mS/cm2, n = 26; P < 0.05). However, the cAMP response was restored by addition of 5 mM ATP (delta Vt = 21.7 +/- 1.5 mV, n = 26; delta Gt = 0.59 +/- 0.06 mS/cm2, n = 26). ATP dose-response experiments, taken together with the effect of cAMP with and without ATP, suggest that phosphorylation is necessary, but not sufficient, for activation. The data provide evidence for a second level of regulation of GCl, which requires high concentrations of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)
Extracellular ATP-induced regulation of epidermal growth factor signaling in cultured renal LLC-PK1 cells
Authors: Harada H. , Tai H. , Motomura A. , Suzuki S. , Suketa Y.
Published: December 2017

We investigated the effect of extracellular ATP on the interaction of epidermal growth factor (EGF) with its receptor in cultured renal epithelial cells, LLC-PK1. Pretreatment with ATP, but not adenosine, inhibited the binding of 125I-labeled EGF. The inhibition demonstrated by ATP resulted from a decrease in the affinity of EGF receptors for its ligand, with no change in the number of EGF receptors. Incubation of phorbol 12-myristate 13-acetate (PMA) for 30 min mimicked the ATP-mediated inhibition. On the other hand, prolonged pretreatment with PMA, which leads to disappearance of protein kinase C activity, reversed the inhibition. In addition, pretreatment with the protein kinase C inhibitor 1-(5-isoquinoline sulfonyl)-2-methylpiperazine prevented the ATP-mediated inhibition. ATP triggered an increase in inositol 1,4,5-trisphosphate levels and translocation of protein kinase C from cytosol to membranes, consist with the stimulation of phospholipase C and the activation of protein kinase C. These results demonstrate that extracellular ATP attenuates the ligand binding affinity of EGF receptor via the stimulation of phospholipase C, leading to the activation of protein kinase C in the LLC-PK1 cells.
Phosphatidic acid modulates Cl- secretion in T84 cells: varying effects depending on mode of stimulation
Authors: Vajanaphanich M. , Kachintorn U. , Barrett K. E. , Cohn J. A. , Dharmsathaphorn K. , Traynor-Kaplan A.
Published: December 2017

Cl- secretion in T84 cells evoked by a stimulus that activates protein kinase C, carbachol, was associated with elevated levels of 32P-labeled phosphatidic acid (PA). PA's role in the regulation of Cl- secretion was explored by examining the effect of exogenous PA (10(-4) M) on Cl- secretion and intracellular Ca2+ levels ([Ca2+]i) in monolayers. PA potentiated the effect of carbachol on [Ca2+]i and Cl- secretion, although it did not stimulate Cl- secretion by itself. PA had divergent effects on cyclic nucleotide-dependent Cl- secretion. It delayed Cl- secretion induced by vasoactive intestinal polypeptide [VIP, adenosine 3',5'-cyclic monophosphate (cAMP) dependent] but potentiated that induced by the heat-stable enterotoxin of Escherichia coli (STa; guanosine 3',5'-cyclic monophosphate dependent). PA did not alter AMP or GMP levels, suggesting that PA acts at a site distal to the generation of these second messengers. PA caused a slight increase in phosphorylation of protein kinase C substrates but not of cAMP-dependent protein kinase substrates. However, PA is probably not acting through a classical protein kinase C pathway, because we have previously shown that phorbol esters inhibit carbachol's actions, and the protein kinase C inhibitor staurosporine failed to block the effect of PA on VIP- or STa-stimulated Cl- secretion. Thus PA differentially regulates stimulated Cl- secretion in T84 cells, depending on the nature of the agonist.
Sulfate concentrations and transport in human bronchial epithelial cells
Authors: Mohapatra N. K. , Cheng P. W. , Parker J. C. , Paradiso A. M. , Yankaskas J. R. , Boucher R. C. , Boat T. F.
Published: December 2017

Inorganic sulfate concentrations in the cytoplasm of human bronchial epithelial cells exceeded levels in the bathing medium under all circumstances tested. Cell sulfate concentrations were directly related to medium sulfate concentrations and inversely related to medium chloride concentrations. In physiological media there was a sulfate compartment of approximately 0.3 mM that exchanged very slowly with extracellular sulfate. In media lacking chloride, sulfate was accumulated by the cells to a level as high as 2 mM. Sulfate uptake was markedly inhibited by external chloride and by stilbene sulfonic acid derivatives but was not affected by sodium in the medium. Efflux of 35SO4(2-) was stimulated by both chloride and sulfate in the bathing medium but inhibited by stilbenes. The following compounds had no effect on sulfate movements: phorbol esters, adenosine 3',5'-cyclic monophosphate derivatives, and okadaic acid. Changes in medium tonicity were likewise without effect. Our results suggest that human bronchial epithelial cells maintain a steady-state disequilibrium for inorganic sulfate. Furthermore, sulfate appears to exist in at least two compartments in the cells: one that is slowly exchangeable with sulfate in the medium and another exchangeable compartment that is of negligible size in physiological media but that becomes very large in media lacking chloride. Sulfate is transported by an anion exchanger of broad specificity that is not influenced by substances known to modulate chloride channels.
Distinct regulation of Na+ reabsorption and Cl- secretion by arginine vasopressin in the amphibian cell line A6
Authors: Chalfant M. L. , Coupaye-Gerard B. , Kleyman T. R.
Published: December 2017

The neurohypophysial peptide arginine vasopressin (AVP) increases Na+ absorption across A6 epithelia. In addition to the positive natriferic response, AVP increases net basolateral to apical Cl- flux. The time course of activation of electrogenic ion transport in A6 epithelia was examined by measuring transepithelial short-circuit current (ISC). Basolateral application of AVP (0.1 U/ml) or forskolin (10 microM) affects ISC in a biphasic manner. Shortly after addition of AVP, an early (transient) phase is observed in which ISC is rapidly stimulated, reaching a peak value at 1.4 +/- 0.1 min. A subsequent decrease in current is interrupted by a slower, late phase in which ISC reaches a peak 23 +/- 3 min after addition of AVP. The late increase in ISC is sustained over the remainder of the 40-min period of observation. The time course of ISC stimulation by forskolin is qualitatively similar. Replacement of external Cl- by aspartate lowers baseline transport nearly 40%, strongly blunts the early phase of ISC stimulation, and retains the late increase. Addition of amiloride (10 microM) to the apical bath before AVP or forskolin stimulation of ISC eliminates the late increase of ISC. Steady-state amiloride-insensitive ISC activated under these conditions was sensitive to apical application of the Cl- channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoate (20 microM) and niflumic acid (100 microM). 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (1 mM) was not an effective inhibitor of this current. Basolateral bumetanide (100 microM) inhibited baseline ISC and reduced both the peak transient and steady-state amiloride-insensitive ISC.(ABSTRACT TRUNCATED AT 250 WORDS)
Regulation of K secretion across the porcine gallbladder epithelium
Authors: DuVall M. D. , O'Grady S. M.
Published: December 2017

Porcine gallbladder epithelium from the neck and the fundus of the organ was stripped of serosal muscle and mounted in Ussing chambers to investigate the mechanisms of K secretion. The sensitivity to K channel blockers and regulation by norepinephrine (NE), adenosine 3',5'-cyclic monophosphate (cAMP), and increases in intracellular Ca concentration ([Ca]) were studied. The porcine gallbladder secretes K (approximately 0.8 mu eq/cm2.h) under basal conditions. Mucosal tetraethylammonium (TEA) produced a concentration-dependent increase in short-circuit current (Isc) and inhibited the unidirectional serosal-to-mucosal 86Rb flux JsmRb, resulting in a > 60% reduction in net Rb secretion. In contrast, serosal Ba produced a concentration-dependent decrease in Isc and stimulated JsmRb, resulting in a > 200% increase in net Rb secretion. NE inhibited JsmRb and net Rb secretion in both regions. In the fundic region the mucosal-to-serosal Rb flux (JmsRb) was also significantly increased, suggesting that active K absorption was activated. Exogenous cAMP increased JsmRb and net Rb secretion by > 85% in both regions. This increase in net Rb secretion was blocked by mucosal TEA but unaffected by NE. The Ca ionophore ionomycin also increased JsmRb and net Rb secretion and reduced the Isc by approximately 50%. Neither mucosal TEA nor Ba blocked changes in steady-state Rb secretion induced by ionomycin. Although both serosal Ba and ionomycin produced significant reductions in Isc, the effects of Ba were blocked by ionomycin pretreatment. These findings indicate that basal K secretion occurs through TEA-sensitive apical K channels and is regulated by intracellular cAMP. NE likely reduces K secretion by decreasing intracellular concentration of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)
Cl- channels of the gastric parietal cell that are active at low pH
Authors: Cuppoletti J. , Baker A. M. , Malinowska D. H.
Published: December 2017

HCl secretion across mammalian gastric parietal cell apical membrane may involve Cl- channels. H(+)-K(+)-ATPase-containing membranes isolated from gastric mucosa of histamine-stimulated rabbits were fused to planar lipid bilayers. Channels were recorded with symmetric 800 mM CsCl solutions, pH 7.4. A linear current-voltage (I-V) relationship was obtained, and conductance was 28 +/- 1 pS at 800 mM CsCl. Conductance was 6.9 +/- 2 pS at 150 mM CsCl. Reversal potential was +22 mV with a fivefold cis-trans CsCl concentration gradient, indicating that the channel was anion selective with a discrimination ratio of 6:1 for Cl- over Cs+. Anion selectivity of the channel was I- > Cl- > or = Br- > NO3-, and gluconate was impermeant. Channels obtained at pH 7.4 persisted when pH of medium bathing the trans side of the bilayer (pHtrans) was reduced to pH 3, without a change in conductance, linearity of I-V relationship, or ion selectivity. In contrast, asymmetric reduction of pH of medium bathing the cis side of the bilayer from 7.4 to 3 always resulted in loss of channel activity. At pH 7.4, open probability (Po) of the channel was voltage dependent, i.e., predominantly open at +80 mV but mainly closed at -80 mV. In contrast, with low pHtrans, channel Po at -80 mV was increased 3.5-fold. The Cl- channel was Ca2+ indifferent. In absence of ionophores, ion selectivity for support of H(+)-K(+)-ATPase activity and H+ transport was consistent with that exhibited by the channel and could be limited by substitution with NO3-, whereas maximal H(+)-K(+)-ATPase activity was indifferent to anion present, demonstrating that anion transport can be rate limiting. Cl- channels with similar characteristics (conductance, linear I-V relationship, and ion selectivity) were also present in H(+)-K(+)-ATPase-containing vesicles isolated from resting (cimetidine-treated) gastric mucosa, exhibiting at -80 mV a pH-independent approximately 3.5-fold lower Po than stimulated vesicle channels. At -80 mV, reduction of pHtrans increased Po of both resting and stimulated Cl- channels by five- to sixfold. Changing membrane potential from 0 to -80 mV across stimulated vesicles increased Cl- channel activity an additional 10-fold.(ABSTRACT TRUNCATED AT 400 WORDS)
Evidence for the existence of F2-isoprostane receptors on rat vascular smooth muscle cells
Authors: Fukunaga M. , Makita N. , Roberts L. J. , Morrow J. D. , Takahashi K. , Badr K. F.
Published: December 2017

The isoprostanes are nonenzymatically generated prostanoids synthesized in vivo in humans and rats through reactions catalyzed by free oxygen radicals. 8-Epi-prostaglandin F2 alpha (8-epi-PGF2 alpha), an F2-isoprostane, is a potent smooth muscle constrictor. A thromboxane A2 (TxA2) receptor antagonist, SQ 29548, blocks renal vasoconstriction during 8-epi-PGF2 alpha administration in rats. With the use of cultured rat aortic smooth muscle cells, we found specific binding sites for [3H]SQ 29548 and for [125I]BOP, a TxA2 agonist. Both ligands were displaced from these binding sites by 8-epi-PGF2 alpha, although with significantly lesser potency than nonlabeled SQ 29548, I-BOP, or U-46619, a TxA2 agonist. In contrast, 8-epi-PGF2 alpha stimulated inositol 1,4,5-trisphosphate production and DNA synthesis in these cells with significantly greater potency than any TxA2 agonist, effects only partially inhibited by SQ 29548. In human TxA2 receptor cDNA-transfected cells, competition by 8-epi-PGF2 alpha for specific [3H]SQ 29548 binding was negligible. Thus 8-epi-PGF2 alpha probably exerts its biological actions in vascular smooth muscle through activation of receptor sites related to but distinct from TxA2 receptors. The existence of such binding sites suggests novel avenues for investigation into the biology of TxA2 and of free radical-mediated tissue injury.
Downregulation of endothelin receptors by autocrine production of endothelin-1
Authors: Clozel M. , Loffler B. M. , Breu V. , Hilfiger L. , Maire J. P. , Butscha B.
Published: December 2017

The potent vasoconstrictor endothelin-1 (ET-1) is a paracrine, but also autocrine, factor for some types of cells. The goal of our study was to evaluate whether the receptor population in cells expressing endothelin receptor subtype A (rat mesangial cells) or endothelin receptor subtype B (human and rat endothelial cells) was affected by the autocrine production of ET-1. We therefore studied maximal binding capacity of 125I-labeled ET-1 in the presence or absence of the metalloprotease inhibitors phosphoramidon, which blocks the intracellular processing of Big ET-1 to ET-1, and thiorphan, which does not block this conversion. Phosphoramidon inhibited the release of ET-1 by human umbilical vein endothelial cells, rat aortic endothelial cells, and rat mesangial cells, and increased 1.4- to 17-fold the maximal binding capacity in the three types of cells. Thiorphan affected neither ET-1 release nor binding. The increase in receptor binding by phosphoramidon was associated with an increase in the functional effect of ET-1, as measured by arachidonic acid release in rat mesangial cells. We conclude that autocrine production of ET-1 decreases, either by binding or by downregulation, the number of binding sites available for ET-1 of paracrine or systemic sources. This aspect of modulation of the vasoconstrictor effect of endothelin should be considered in pathological situations or after endothelin-converting-enzyme inhibition.
Distinct pathways to refill ACh-sensitive internal Ca2+ stores in canine airway smooth muscle
Authors: Bourreau J. P. , Kwan C. Y. , Daniel E. E.
Published: December 2017

The ability of extracellular Ca2+ to refill internal Ca2+ stores of canine tracheal smooth muscle after a prior depletion by acetylcholine (ACh) was assessed using a novel sarcoplasmic reticulum (SR) Ca2+ pump inhibitor, cyclopiazonic acid (CPA). The transient contraction induced by ACh in a medium free of Ca2+ was used as an index for the content of agonist-sensitive intracellular Ca2+ stores. CPA inhibited in a concentration-dependent manner the refilling of the stores occurring during high KCl stimulation, and this inhibitory effect was independent of the external Ca2+ concentration. On the other hand, CPA was less effective in inhibiting the refilling occurring during prolonged ACh stimulation, especially when external Ca2+ concentration was raised. At 5.0 mM external Ca2+ or when 0.1 microM BAY 8644 was present in the medium, CPA was ineffective in inhibiting the refilling occurring during prolonged ACh stimulation. The maximum ACh-induced contraction in Ca(2+)-containing medium was independent of the extent of internal store Ca2+ load in the absence of L-type Ca2+ channel blocker but was highly dependent on the extent of internal Ca2+ load in the presence of the Ca2+ channel blocker. Hyperpolarization of the plasma membrane with the K+ channel opener cromakalim reduced the amplitude of ACh tonic contraction. Subsequent addition of nifedipine further reduced ACh tonic contraction. It is concluded that two different pathways for external Ca2+ are used to refill ACh-sensitive internal stores. One involves active Ca2+ uptake via a CPA-sensitive Ca2+ pump, and the other involves a CPA-insensitive pathway whose nature remains to be determined.
Na(+)-dependent sulfate transport in opossum kidney cells is DIDS sensitive
Authors: Tenenhouse H. S. , Martel J.
Published: December 2017

Sulfate transport was examined in OK/E cells, a clonal subline of opossum kidney cells that express several differentiated functions of the proximal tubule. Extracellular Na+ stimulated [35S]sulfate uptake five- to sixfold. Hill analysis demonstrating the dependence of sulfate uptake on Na+ concentration yielded a Hill coefficient of 1.5 and a Michaelis constant (KNa+) of 23 mM. Na(+)-dependent sulfate uptake was increased by lowering the pH from 7.4 to 6.4, decreased by raising the pH to 8.4 and inhibited by a 10-fold molar excess of SO3(2-), S2O3(2-) and CrO4(2-), but not by phosphate. The Na(+)-mediated component of sulfate uptake was saturable and kinetic parameters were estimated [Michaelis constant (Km) = 2.4 +/- 0.2 mM and maximum velocity (Vmax) = 125 +/- 15 pmol.mg protein-1.min-1]. Omitting extracellular Cl- resulted in a significant increase in the affinity of the carrier for sulfate (Km = 0.5 mm), without changing Vmax, consistent with competitive inhibition by Cl-. Na(+)-dependent sulfate uptake in OK/E cells was also inhibited by HCO3- [half-maximal inhibitory concentration (IC50) = 7 mM], 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, IC50 = 0.9 microM), 0.5 mM picrylsulfonic acid and 0.1 mM ethacrynic acid, but not by 1 mM amiloride. Na(+)-dependent, DIDS-sensitive sulfate uptake was also expressed in the parental OK cell line and was not influenced by serum or 3,3',5-triiodo-L-thyronine. We conclude that Na(+)-dependent sulfate uptake in OK/E cells observes many of the features of Na(+)-sulfate cotransport in the renal brush-border membrane and provides a useful model to investigate the regulation of renal sulfate transport.
WW-781, a potent reversible inhibitor of red cell Cl- flux, binds to band 3 by a two-step mechanism
Authors: Raha N. M. , Spinelli L. J. , Knauf P. A.
Published: December 2017

WW-781 ([3-methyl-1-p-sulfophenyl-5-pyrazolone-(4)]-[1,3-dibutylbarbit uric acid]-pentamethine oxonol), a fluorescent dye that has been used for measuring membrane potentials by optical methods, inhibits human red blood cell Cl- exchange, which is mediated by the membrane protein known as band 3 or capnophorin. The inhibition is slowly reversible upon removal of WW-781 from the medium, with a half time of approximately 4.7 min in 150 mM Cl- medium at 0 degrees C. The mechanism of inhibition by WW-781 involves a two-step binding reaction. WW-781 binds rapidly to band 3 to form an initial complex, which can also rapidly dissociate. Formation of this initial complex is followed by the much slower formation of a second complex (with a rate constant of approximately 1.1 min-1), probably involving a protein conformational change, through which WW-781 is more tightly bound to band 3. At low concentrations, WW-781 inhibits Cl- exchange with a stoichiometry of 1 WW-781 molecule per band 3 monomer, suggesting that under these conditions the binding of WW-781 is highly selective for the band 3 protein.
Culture-induced alterations in alveolar type II cell Na+ conductance
Authors: Yue G. , Hu P. , Oh Y. , Jilling T. , Shoemaker R. L. , Benos D. J. , Cragoe E. J. , Matalon S.
Published: December 2017

Changes in Na+ transport in rat alveolar type II (ATII) cells during culture were quantified and related to alterations in spatial distribution of proteins antigenically related to amiloride-sensitive Na+ channels. Adult rat ATII cells were cultured for periods ranging from 24 to 96 h. When patch clamped in the whole cell mode, both freshly isolated and cultured ATII cells exhibited outwardly rectified Na+ currents. At 0 and 24 h in culture, these currents were equally inhibited by amiloride, benzamil, and 5-(N-ethyl-N-isopropyl)-2',4'-amiloride (inhibitory constant approximately 1 microM). These conductive pathways were equally permeable to Na+ and K+. Immunocytochemical localization at 0 or 24 h in culture revealed the presence of plasma membrane antigenic sites; after 48 h, the appearance of intracellular antigenic sites increased significantly. A single band of molecular mass 135 kDa in membrane proteins of freshly isolated ATII cells was recognized in Western blots; at 48 h in culture, two lower bands with molecular masses of 75 and 65 kDa were detected in either membrane or cytoplasmic proteins. Photolabeling with 2'-methoxy-5'-nitrobenzamil showed that the 135-, 75-, and 65-kDa bands contained amiloride-binding sites. These results suggest the presence of low amiloride affinity conductive pathways in freshly isolated and cultured ATII cells. Culturing ATII cells resulted in internalization and possible breakdown of these pathways and decreased Na+ transport.
Pharmacological characterization of muscarinic receptors in neonatal rat cardiomyocytes
Authors: Yang C. M. , Chen F. F. , Sung T. C. , Hsu H. F. , Wu D.
Published: December 2017

[N-methyl-3H]scopolamine methylchloride ([3H]NMS) was used to characterize the muscarinic receptors (mAChRs) in the intact cardiomyocytes. The specific binding of [3H]NMS was proportional to cell concentration, saturable with respect to [3H]NMS concentration, and time dependent. Scatchard analysis of binding isotherms showed that [3H]NMS bound to the freshly isolated and cultured cardiomyocytes with dissociation constants of 275 +/- 64 and 207 +/- 20 pM as well as maximum receptor densities of 0.13 +/- 0.09 and 5.36 +/- 0.20 fmol/10(5) cells, respectively. Heterogeneity of mAChRs was demonstrated by competitive binding experiments against [3H]NMS with M2 and M3 antagonists. These receptors (80%) exhibited high affinities for 11-([2-[(diethylamino)methyl]-1-piperidinyl]-acetyl)-5,11-dihydro- 6H-pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX-116) and methoctramine similar to those of M2 subtype. The low-affinity M2 antagonist binding constants were close to those reported for M3 receptors and possessed high affinity for 4-diphenylacetoxyl-N-methylpiperidine (4-DAMP) and hexahydrosiladifenidol. On the basis of biochemical studies, AF-DX-116 blocked adenosine 3',5'-cyclic monophosphate (cAMP) inhibition with high affinity (pKB 7.4), while it antagonized inositol phosphate formation with low affinity (pKB 6.5). 4-DAMP possessed high affinity in blocking inositol phosphate formation (pKB 9.0) and low affinity for antagonism of cAMP inhibition (pKB 7.7). Although no other muscarinic receptor mRNA has been detected in these cells, these data suggest the presence of a second population of mAChRs, which may not be identical to the classical cardiac "M2" receptors.
Ca-dependent action of cocaine on K current in freshly dissociated dorsal root ganglia from rats
Authors: Grossie J.
Published: December 2017

The actions of cocaine on K currents were analyzed in enzymatically dissociated neurons from adult rat dorsal root ganglia. The whole cell voltage clamp configuration was used to determine that in the presence of Ca cocaine inhibited a K conductance that was activated from a holding potential of -55 mV. The cocaine concentration that decreased K currents half-maximally was 18 microM. At a concentration of 40 microM, cocaine had no significant effect on K current remaining in the absence of external Ca, whereas at 80 microM a significant 17% depression of this current was observed. Large transient K currents occurring with depolarizations from -90 mV were also resistant to 40 microM cocaine, suggesting that cocaine specifically inhibits a Ca-dependent K current. Ca currents elicited from a holding potential of -55 mV were largely unaltered by 40 microM cocaine. The data suggest that cocaine may be a useful tool for experimental blockage of Ca-dependent K currents and also provide additional insight into the overall actions and toxicity of the drug.
Disulfide-linked aggregation of thyroglobulin normally occurs during nascent protein folding
Authors: Kim P. S. , Kim K. R. , Arvan P.
Published: December 2017

In the endoplasmic reticulum (ER) of cultured porcine thyrocytes, newly synthesized thyroglobulin (Tg, the precursor in thyroid hormone synthesis) initially forms protein aggregates, which are dissolved into monomers and then assembled to dimers, before intracellular transport and secretion. However, studies suggest that in different physiological states and in different cells, folding efficiency in the ER may vary; with this in mind we have set out to further characterize the phenomenon of nascent Tg aggregation. In primary cultured thyrocytes, fresh thyroid follicular tissue (of porcine and rat origin), and the FRTL-5 cell line, nascent Tg appears transiently aggregated with mispaired, interchain disulfide linkages. Using a cell lysis procedure that maximally inhibits proteolysis as well as artifactual disulfide formation, Tg aggregates of M(r) > or = 2,000,000 can be stably isolated by gel filtration. Furthermore, stimulation with thyrotropin and other hormones that enhance Tg production may alter but does not eliminate formation of these aggregates. We conclude that transient disulfide-linked aggregation occurs normally during Tg folding in the ER of thyroid epithelial cells.
Alterations in mitochondrial RNA expression after renal ischemia
Authors: Van Itallie C. M. , Van Why S. , Thulin G. , Kashgarian M. , Siegel N. J.
Published: December 2017

Ischemia and reperfusion damage mitochondrial structure and impair respiratory function. In this study, 45 min of renal ischemia followed by varying periods of reflow profoundly depressed the activity of several respiratory complexes in mitochondria isolated from rat kidneys. The respiratory complexes are composed of subunits encoded by both the nuclear and mitochondrial genomes. To determine the role of mitochondrial gene expression in recovery of respiratory function, expression of mitochondrial RNA was examined during reperfusion. Both mature and incompletely processed cytochrome b mRNA levels were depressed after 45 min of ischemia and 15 min of reflow; levels rebounded to above normal after 2 h of reflow and then declined over the next 22 h. Another mitochondrial RNA showed a similar pattern; in contrast, the levels of a nuclear-encoded subunit mRNA for a respiratory enzyme and of 28S rRNA were unchanged. These data demonstrate that renal ischemia followed by reperfusion alters mitochondrial RNA expression. We speculate that mitochondrial RNA turnover is increased in response to continuing injury and that recovery is accompanied by enhanced RNA synthesis.
Swelling-activated K fluxes in vascular endothelial cells: volume regulation via K-Cl cotransport and K channels
Authors: Perry P. B. , O'Neill W. C.
Published: December 2017

K efflux pathways responsible for regulatory volume decrease (RVD) were examined in bovine aortic endothelial cells. Hypotonic swelling produced a rapid and reversible threefold increase in bumetanide-insensitive 86Rb efflux. Swelling-activated 86Rb efflux was inhibited 43% when Cl was replaced with NO3, and this Cl-dependent efflux was inhibited by 1 mM furosemide. Neither Cl replacement nor furosemide inhibited the efflux stimulated by a Ca ionophore (A23187) in isotonic medium. Swelling-activated 86Rb efflux was also inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonate but not by dinitrostilbenedisulfonate. Cell swelling induced a volume-regulatory K loss that was incomplete in hypotonic medium but complete and more rapid when bumetanide was added or when cells were swollen isosmotically. K loss in the presence of bumetanide was partially blocked by furosemide. We conclude that two separate swelling-activated K fluxes mediate RVD in aortic endothelial cells: a Cl-dependent, furosemide-sensitive, but bumetanide-insensitive flux that is consistent with K-Cl cotransport, and a Cl-independent efflux that presumably is mediated by K channels.
Vacuolar-type H(+)-ATPases are functionally expressed in plasma membranes of human tumor cells
Authors: Martinez-Zaguilan R. , Lynch R. M. , Martinez G. M. , Gillies R. J.
Published: December 2017

Mammalian cells generally regulate their intracellular pH (pHi) via collaboration between Na(+)-H+ exchanger and HCO3- transport. In addition, a number of normal mammalian cells have been identified that express H(+)-adenosinetriphosphatases (ATPases) in their plasma membranes. Because tumor cells often maintain a high pHi, we hypothesized that they might functionally express H(+)-ATPases in their plasma membranes. In the first phase of the present study, we screened 19 normal and tumorigenic human cell lines for the presence of plasmalemmal H(+)-ATPase activity using bafilomycin A1 to inhibit V-type H(+)-ATPase and Sch-28080 to inhibit P-type H(+)-K(+)-ATPase. Bafilomycin A1 decreased pHi in the six tumor cell lines with the highest resting pHi in the absence of HCO3-. Sch-28080 did not affect pHi in any of the human cells. Simultaneous measurement of pH in the cytoplasm and in the endosomes/lysosomes localized the activity of bafilomycin to the plasma membrane in three cell lines. In the second phase of this study, these three cell lines were shown to recover from NH4(+)-induced acid loads in the absence of Na+. This recovery was inhibited by N-ethylmaleimide, bafilomycin A1, and ATP depletion and was not significantly affected by vanadate, Sch-28080, or hexamethyl amiloride. These results indicate that a vacuolar type H(+)-ATPase is expressed in the plasma membrane of some tumor cells.