73 results on '"Stumpff, Friederike"'
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52. Isolated Cells of the Sheep Rumen and Omasum Express Maxi‐anion Channels Permeable to Acetate, Propionate and Butyrate
- Author
-
Stumpff, Friederike, primary, Georgi, Maria Ifigenia, additional, and Martens, Holger, additional
- Published
- 2010
- Full Text
- View/download PDF
53. Cultured ruminal epithelial cells express a large-conductance channel permeable to chloride, bicarbonate, and acetate
- Author
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Stumpff, Friederike, primary, Martens, Holger, additional, Bilk, Sabine, additional, Aschenbach, Jörg R., additional, and Gäbel, Gotthold, additional
- Published
- 2008
- Full Text
- View/download PDF
54. Ruminal epithelial cells express high conductance chloride channel
- Author
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Stumpff, Friederike, primary, Martens, Holger, additional, and Gaebel, Gotthold, additional
- Published
- 2008
- Full Text
- View/download PDF
55. Activation of Neuroendocrine L-Type Channels (α1D Subunits) in Retinal Pigment Epithelial Cells and Brain Neurons by pp60c-src
- Author
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Strauss, Olaf, primary, Buss, Foline, additional, Rosenthal, Rita, additional, Fischer, Dirk, additional, Mergler, Stefan, additional, Stumpff, Friederike, additional, and Thieme, Hagen, additional
- Published
- 2000
- Full Text
- View/download PDF
56. Regulation of Trabecular Meshwork Contractility
- Author
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Stumpff, Friederike, primary and Wiederholt, Michael, additional
- Published
- 2000
- Full Text
- View/download PDF
57. Modulation of sheep ruminal urea transport by ammonia and pH.
- Author
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Zhongyan Lu, Stumpff, Friederike, Deiner, Carolin, Rosendahl, Julia, Braun, Hannah, Aschenbach, Jörg R., Martens, Holger, and Abdoun, Khalid
- Subjects
- *
UREA , *AMMONIA , *RUMEN (Ruminants) , *MEMBRANE transport proteins , *MICROELECTRODES - Abstract
Ruminal fermentation products such as short-chain fatty acids (SCFA) and CO2 acutely stimulate urea transport across the ruminal epithelium in vivo, whereas ammonia has inhibitory effects. Uptake and signaling pathways remain obscure. The ruminal expression of SLC14a1 (UT-B) was studied using polymerase chain reaction (PCR). The functional short-term effects of ammonia on cytosolic pH (pHi) and ruminal urea transport across native epithelia were investigated using pH-sensitive microelectrodes and via flux measurements in Ussing chambers. Two variants (UT-B1 and UT-B2) could be fully sequenced from ovine ruminal cDNA. Functionally, transport was passive and modulated by luminal pH in the presence of SCFA and CO2, rising in response to luminal acidification to a peak value at pH 5.8 and dropping with further acidification, resulting in a bell-shaped curve. Presence of ammonia reduced the amplitude, but not the shape of the relationship between urea flux and pH, so that urea flux remained maximal at pH 5.8. Effects of ammonia were concentration dependent, with saturation at 5 mmol/l. Clamping the transepithelial potential altered the inhibitory potential of ammonia on urea flux. Ammonia depolarized the apical membrane and acidified pHi, suggesting that, at physiological pH (<7), uptake of NH4+into the cytosol may be a key signaling event regulating ruminal urea transport. We conclude that transport of urea across the ruminal epithelium involves proteins subject to rapid modulation by manipulations that alter pHi and the cytosolic concentration of NH4+. Implications for epithelial and ruminal homeostasis are discussed. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
58. Measuring Ca2+ binding to short chain fatty acids and gluconate with a Ca2+ electrode: Role of the reference electrode.
- Author
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Stumpff, Friederike and McGuigan, John A.S.
- Subjects
- *
CALCIUM ions , *FATTY acids , *GLUCONIC acid , *ION selective electrodes , *IONIC solutions , *OSMOLAR concentration - Abstract
Abstract: Many organic anions bind free Ca2+, the total concentration of which must be adjusted in experimental solutions. Because published values for the apparent dissociation constant (K app) describing the Ca2+ affinity of short chain fatty acids (SCFAs) and gluconate are highly variable, Ca2+ electrodes coupled to either a 3M KCl or a Na+ selective electrode were used to redetermine K app. All solutions contained 130mM Na+, whereas the concentration of the studied anion was varied from 15 to 120mM, replacing Cl− that was decreased concomitantly to maintain osmolarity. This induces changes in the liquid junction potential (LJP) at the 3M KCl reference electrode, leading to a systematic underestimation of K app if left uncorrected. Because the Na+ concentration in all solutions was constant, a Na+ electrode was used to directly measure the changes in the LJP at the 3 M KCl reference, which were under 5mV but twice those predicted by the Henderson equation. Determination of K app either after correction for these LJP changes or via direct reference to a Na+ electrode showed that SCFAs do not bind Ca2+ and that the K app for the binding of Ca2+ to gluconate at pH 7.4, ionic strength 0.15M, and 23°C was 52.7mM. [Copyright &y& Elsevier]
- Published
- 2014
- Full Text
- View/download PDF
59. Calculated and measured [Ca2+] in buffers used to calibrate Ca2+ macroelectrodes
- Author
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McGuigan, John A.S. and Stumpff, Friederike
- Subjects
- *
CALCIUM ions , *BUFFER solutions , *DISSOCIATION (Chemistry) , *LIGANDS (Biochemistry) , *ELECTRODES , *ESTIMATION theory , *GLYCINE - Abstract
Abstract: The ionized concentration of calcium in physiological buffers ([Ca2+]) is normally calculated using either tabulated constants or software programs. To investigate the accuracy of such calculations, the [Ca2+] in EGTA [ethylene glycol-bis(β-aminoethylether)-N,N,N|,N|-tetraacetic acid], BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N|,N|-tetraacetic acid], HEDTA [N-(2-hydroxyethyl)-ethylenediamine-N,N|,N|-triacetic acid], and NTA [N,N-bis(carboxymethyl)glycine] buffers was estimated using the ligand optimization method, and these measured values were compared with calculated values. All measurements overlapped in the pCa range of 3.51 (NTA) to 8.12 (EGTA). In all four buffer solutions, there was no correlation between measured and calculated values; the calculated values differed among themselves by factors varying from 1.3 (NTA) to 6.9 (EGTA). Independent measurements of EGTA purity and the apparent dissociation constants for HEDTA and NTA were not significantly different from the values estimated by the ligand optimization method, further substantiating the method. Using two calibration solutions of pCa 2.0 and 3.01 and seven buffers in the pCa range of 4.0–7.5, calibration of a Ca2+ electrode over the pCa range of 2.0–7.5 became a routine procedure. It is proposed that such Ca2+ calibration/buffer solutions be internationally defined and made commercially available to allow the precise measurement of [Ca2+] in biology. [Copyright &y& Elsevier]
- Published
- 2013
- Full Text
- View/download PDF
60. Modulation of urea transport across sheep rumen epithelium in vitro by SCFA and CO2.
- Author
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Abdoun, Khalid, Stumpff, Friederike, Rabbani, Imtiaz, and Martens, Holger
- Subjects
- *
UREA , *GASTROINTESTINAL system , *FATTY acids , *EPITHELIUM , *SHEEP as laboratory animals , *MICROELECTRODES , *PERMEABILITY , *RUMEN (Ruminants) - Abstract
Urea transport across the gastrointestinal tract involves transporters of the urea transporter-B group, the regulation of which is poorly understood. The classical stimulatory effect of CO2 and the effect of short-chain fatty acids (SCFA) on the ruminal recycling of urea were investigated by using Ussing chamber and microelectrode techniques with isolated ruminal epithelium of sheep. The flux of urea was found to be phloretin sensitive and passive. At a luminal pH of 6.4, but not at 7.4, the addition of SCFA (40 mmol/l) or CO2/HCO3- (10% and 25 mmol/l) led to a fourfold increase in urea flux. The stepwise reduction of luminal pH in the presence of SCFA from 7.4 to 5.4 led to a bell-shaped modification of urea transport, with a maximum at pH 6.2. Lowering the pH in the absence of SCFA or CO2 had no effect. Inhibition of Na+/H+ exchange increased urea flux at pH 7.4, with a decrease being seen at pH 6.4. In experiments with double-barreled, pH-sensitive microelectrodes, we confirmed the presence of an apical pH microclimate and demonstrated the acidifying effects of SCFA on the underlying epithelium. We confirm that the permeability of the ruminal epithelium to urea involves a phloretin-sensitive pathway. We present clear evidence for the regulation of urea transport by strategies that alter intracellular pH, with permeability being highest after a moderate decrease. The well-known postprandial stimulation of urea transport to the rumen in vivo may involve acute pH-dependent effects of intraruminal SCFA and CO2 on the function of existing urea transporters. [ABSTRACT FROM AUTHOR]
- Published
- 2010
- Full Text
- View/download PDF
61. Modulation of urea transport across sheep rumen epithelium in vitro by SCFA and CO2.
- Author
-
Abdoun, Khalid, Stumpff, Friederike, Rabbani, Imtiaz, and Martens, Holger
- Subjects
UREA ,GASTROINTESTINAL system ,FATTY acids ,EPITHELIUM ,SHEEP as laboratory animals ,MICROELECTRODES ,PERMEABILITY ,RUMEN (Ruminants) - Abstract
Urea transport across the gastrointestinal tract involves transporters of the urea transporter-B group, the regulation of which is poorly understood. The classical stimulatory effect of CO
2 and the effect of short-chain fatty acids (SCFA) on the ruminal recycling of urea were investigated by using Ussing chamber and microelectrode techniques with isolated ruminal epithelium of sheep. The flux of urea was found to be phloretin sensitive and passive. At a luminal pH of 6.4, but not at 7.4, the addition of SCFA (40 mmol/l) or CO2 /HCO3 - (10% and 25 mmol/l) led to a fourfold increase in urea flux. The stepwise reduction of luminal pH in the presence of SCFA from 7.4 to 5.4 led to a bell-shaped modification of urea transport, with a maximum at pH 6.2. Lowering the pH in the absence of SCFA or CO2 had no effect. Inhibition of Na+ /H+ exchange increased urea flux at pH 7.4, with a decrease being seen at pH 6.4. In experiments with double-barreled, pH-sensitive microelectrodes, we confirmed the presence of an apical pH microclimate and demonstrated the acidifying effects of SCFA on the underlying epithelium. We confirm that the permeability of the ruminal epithelium to urea involves a phloretin-sensitive pathway. We present clear evidence for the regulation of urea transport by strategies that alter intracellular pH, with permeability being highest after a moderate decrease. The well-known postprandial stimulation of urea transport to the rumen in vivo may involve acute pH-dependent effects of intraruminal SCFA and CO2 on the function of existing urea transporters. [ABSTRACT FROM AUTHOR]- Published
- 2010
- Full Text
- View/download PDF
62. Basolateral Mg²+;/Na+ exchange regulates apical nonselective cation channel in sheep rumen epithelium via cytosolic Mg²+;.
- Author
-
Leonhard-Marek, Sabine, Stumpff, Friederike, Brinkmann, Inge, Breves, Gerhard, and Martens, Holger
- Subjects
- *
MAGNESIUM , *CATIONS , *IONS , *ION channels , *MAGNESIUM metabolism , *HYPOMAGNESEMIA - Abstract
High potassium diets lead to an inverse regulation of sodium and magnesium absorption in ruminants, suggesting some form of cross talk. Previous Ussing chamber experiments have demonstrated a divalent sensitive Na+ conductance in the apical membrane of ruminal epithelium. Using patch-clamped ruminal epithelial cells, we could observe a divalent sensitive, non- selective cation conductance (NSCC) with K+ permeability > Cs+ permeability > Na+ permeability. Conductance increased and rectification decreased when either Mg2+ or both Ca2+ and Mg2+ were removed from the internal or external solution or both. The conductance could be blocked by Ba2+, but not by tetraethylammonium (TEA). Subsequently, we studied this conductance measured as short-circuit current (Isc) in Ussing chambers. Forskolin, IBMX, and theophylline are known to block both Isc and Na transport across ruminal epithelium in the presence of divalent cations. When the NSCC was stimulated by removing mucosal calcium, an initial decrease in Isc was followed by a subsequent increase. The cAMP-mediated increase in Ix was reduced by low serosal Na+ and serosal addition of imipramine or serosal amiloride and depended on the availability of mucosal magnesium. Luminal amiloride had no effect. Flux studies showed that low serosal Na+ reduced 28Mg fluxes from mucosal to serosal. The data suggest that cAMP stimulates basolateral Na+/Mg2+ exchange, reducing cytosolic Mg. This increases sodium uptake through a magnesium-sensitive NSCC in the apical membrane. Likewise, the reduction in magnesium uptake that follows ingestion of high potassium fodder may facilitate sodium absorption, as observed in studies of ruminal osmoregulation. Possibly, grass tetany (hypomagnesemia) is a side effect of this useful mechanism. [ABSTRACT FROM AUTHOR]
- Published
- 2005
- Full Text
- View/download PDF
63. Flufenamic acid enhances current through maxi-K channels in thetrabecular meshwork of the eye.
- Author
-
Stumpff, Friederike, Boxberger, Marianne, Thieme, Hagen, Strauß, Olaf, and Wiederholt, Michael
- Subjects
- *
NONSTEROIDAL anti-inflammatory agents , *POTASSIUM channels , *SMOOTH muscle , *BIOCHEMICAL mechanism of action , *CHEMICALS , *DRUGS - Abstract
PURPOSE. Flufenamic acid relaxes trabecular meshwork,a smooth muscle-like tissue involved in the regulation of ocular outflow inthe eye. In this study, we attempted to determine if ionic channels are involvedin this response. METHODS. Cultured human (HTM) and bovine (BTM) trabecular meshwork cellswere investigated using the patch-clamp technique. RESULTS. In trabecular meshwork, flufenamic acid (10[sup -5] M) reversibly stimulated outward current to 406 ± 71% of initial outwardcurrent level in BTM (n = 10) and 294 ± 75% of initial current levelin HTM (n = 12) in all cells investigated; no significant differences emerged.The response was dosage-dependent. Replacement of potassium in all solutionseliminated the response to flufenamic acid (n = 4, BTM). Blocking K[sub ATP] channels with glibenclamide (10[sup -5] M, n = 6) and small-conductancecalcium-activated potassium channels with apamin (10[sup -6] M,n = 5) had no effect. A direct effect on calcium channels could also not bedetected. Blockage of the large-conductance calcium-activated potassium channel(maxi-K) by iberiotoxin (10[sup -7] M) suppressed 87 ± 9%(n = 6; HTM) and 91 ± 10% (n = 6; BTM) of the response. Depleting thecells of calcium did not significantly alter the response to flufenamic acid. CONCLUSIONS. Flufenamic acid stimulates maxi-K channels in trabecular meshworkof both human and bovine origin. This should lead to hyperpolarization, closureof L-type channels and lowered cytosolic calcium levels, possibly explainingthe relaxation observed in response to this substance. [ABSTRACT FROM AUTHOR]
- Published
- 2001
- Full Text
- View/download PDF
64. The TRPA1 Agonist Cinnamaldehyde Induces the Secretion of HCO 3 − by the Porcine Colon.
- Author
-
Manneck, David, Manz, Gisela, Braun, Hannah-Sophie, Rosendahl, Julia, Stumpff, Friederike, and Vlachova, Viktorie
- Subjects
GASTROINTESTINAL system ,SECRETION ,SHORT circuits ,INFLAMMATORY bowel diseases ,COLON (Anatomy) ,QUINIDINE ,JEJUNUM - Abstract
A therapeutic potential of the TRPA1 channel agonist cinnamaldehyde for use in inflammatory bowel disease is emerging, but the mechanisms are unclear. Semi-quantitative qPCR of various parts of the porcine gastrointestinal tract showed that mRNA for TRPA1 was highest in the colonic mucosa. In Ussing chambers, 1 mmol·L
−1 cinnamaldehyde induced increases in short circuit current (ΔIsc ) and conductance (ΔGt ) across the colon that were higher than those across the jejunum or after 1 mmol·L−1 thymol. Lidocaine, amiloride or bumetanide did not change the response. The application of 1 mmol·L−1 quinidine or the bilateral replacement of 120 Na+ , 120 Cl− or 25 HCO3 − reduced ΔGt , while the removal of Ca2+ enhanced ΔGt with ΔIsc numerically higher. ΔIsc decreased after 0.5 NPPB, 0.01 indometacin and the bilateral replacement of 120 Na+ or 25 HCO3 − . The removal of 120 Cl− had no effect. Cinnamaldehyde also activates TRPV3, but comparative measurements involving patch clamp experiments on overexpressing cells demonstrated that much higher concentrations are required. We suggest that cinnamaldehyde stimulates the secretion of HCO3 − via apical CFTR and basolateral Na+ -HCO3 − cotransport, preventing acidosis and damage to the epithelium and the colonic microbiome. Signaling may involve the opening of TRPA1, depolarization of the epithelium and a rise in PGE2 following a lower uptake of prostaglandins via OATP2A1. [ABSTRACT FROM AUTHOR]- Published
- 2021
- Full Text
- View/download PDF
65. Role of HCO3- in regulation of cytoplasmic pH in ciliary epithelial cells.
- Author
-
HELBIG, HORST, KORBMACHER, CHRISTOPH, STUMPFF, FRIEDERIKE, COCA-PRADOS, MIGUEL, and WIEDERHOLT, MICHAEL
- Published
- 1989
- Full Text
- View/download PDF
66. Na+/H+ exchange regulates intracellular pH in a cell clone derived from bovine pigmented ciliary epithelium.
- Author
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Helbig, Horst, Korbmacher, Christoph, Stumpff, Friederike, Coca-Prados, Miguel, and Wiederholt, Michael
- Published
- 1988
- Full Text
- View/download PDF
67. Role of HC03 in regulation of cytoplasmic pH in ciliary epithelial cells
- Author
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Stumpff, Friederike, Helbig, Horst, Korbmacher, Christoph, Coca-Prados, Miguel, and Wiederholt, Michael
- Subjects
chloridebicarbonate exchange ,ciliary epithelium ,intracellular pH ,sodium-bicarbonate cotransport ,tissue culture - Published
- 1989
68. Na+/H+ exchange regulates intracellular pH in a cell clone derived from bovine pigmented ciliary epithelium
- Author
-
Helbig, Horst, primary, Korbmacher, Christoph, additional, Stumpff, Friederike, additional, Coca‐Prados, Miguel, additional, and Wiederholt, Michael, additional
- Published
- 1988
- Full Text
- View/download PDF
69. Beyond Ca2+ signalling: the role of TRPV3 in the transport of NH4+.
- Author
-
Liebe, Hendrik, Liebe, Franziska, Sponder, Gerhard, Hedtrich, Sarah, and Stumpff, Friederike
- Subjects
- *
TRPV cation channels , *OVUM , *INTESTINES , *GENETIC overexpression , *EPITHELIUM , *SKIN permeability - Abstract
Mutations of TRPV3 lead to severe dermal hyperkeratosis in Olmsted syndrome, but whether the mutants are trafficked to the cell membrane or not is controversial. Even less is known about TRPV3 function in intestinal epithelia, although research on ruminants and pigs suggests an involvement in the uptake of NH4+. It was the purpose of this study to measure the permeability of the human homologue (hTRPV3) to NH4+, to localize hTRPV3 in human skin equivalents, and to investigate trafficking of the Olmsted mutant G573S. Immunoblotting and immunostaining verified the successful expression of hTRPV3 in HEK-293 cells and Xenopus oocytes with trafficking to the cell membrane. Human skin equivalents showed distinct staining of the apical membrane of the top layer of keratinocytes with cytosolic staining in the middle layers. Experiments with pH-sensitive microelectrodes on Xenopus oocytes demonstrated that acidification by NH4+ was significantly greater when hTRPV3 was expressed. Single-channel measurements showed larger conductances in overexpressing Xenopus oocytes than in controls. In whole-cell experiments on HEK-293 cells, both enantiomers of menthol stimulated influx of NH4+ in hTRPV3 expressing cells, but not in controls. Expression of the mutant G573S greatly reduced cell viability with partial rescue via ruthenium red. Immunofluorescence confirmed cytosolic expression, with membrane staining observed in a very small number of cells. We suggest that expression of TRPV3 by epithelia may have implications not just for Ca2+ signalling, but also for nitrogen metabolism. Models suggesting how influx of NH4+ via TRPV3 might stimulate skin cornification or intestinal NH4+ transport are discussed. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
70. The bovine TRPV3 as a pathway for the uptake of Na+, Ca2+, and NH4+.
- Author
-
Schrapers, Katharina T., Sponder, Gerhard, Liebe, Franziska, Liebe, Hendrik, and Stumpff, Friederike
- Subjects
- *
TRP channels , *PATCH-clamp techniques (Electrophysiology) , *SODIUM channels , *INTRACELLULAR calcium , *AMMONIA - Abstract
Absorption of ammonia from the gastrointestinal tract results in problems that range from hepatic encephalopathy in humans to poor nitrogen efficiency of cattle with consequences for the global climate. Previous studies on epithelia and cells from the native ruminal epithelium suggest functional involvement of the bovine homologue of TRPV3 (bTRPV3) in ruminal NH4+ transport. Since the conductance of TRP channels to NH4+ has never been studied, bTRPV3 was overexpressed in HEK-293 cells and investigated using the patch-clamp technique and intracellular calcium imaging. Control cells contained the empty construct. Divalent cations blocked the conductance for monovalent cations in both cell types, with effects higher in cells expressing bTRPV3. In bTRPV3 cells, but not in controls, menthol, thymol, carvacrol, or 2-APB stimulated whole cell currents mediated by Na+, Cs+, NH4+, and K+, with a rise in intracellular Ca2+ observed in response to menthol. While only 25% of control patches showed single-channel events (with a conductance of 40.8 ± 11.9 pS for NH4+ and 25.0 ± 5.8 pS for Na+), 90% of bTRPV3 patches showed much larger conductances of 127.8 ± 4.2 pS for Na+, 240.1 ± 3.6 pS for NH4+, 34.0 ± 1.7 pS for Ca2+, and ~ 36 pS for NMDG+. Open probability, but not conductance, rose with time after patch excision. In conjunction with previous research, we suggest that bTRPV3 channels may play a role in the transport of Na+, K+, Ca2+ and NH4+ across the rumen with possible repercussions for understanding the function of TRPV3 in other epithelia. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
71. The TRPA1 Agonist Cinnamaldehyde Induces the Secretion of HCO 3 - by the Porcine Colon.
- Author
-
Manneck D, Manz G, Braun HS, Rosendahl J, and Stumpff F
- Subjects
- Acrolein pharmacology, Animals, Epithelial Cells drug effects, Gastrointestinal Tract drug effects, Swine, Acrolein analogs & derivatives, Antineoplastic Agents, Phytogenic pharmacology, Bicarbonates metabolism, Epithelial Cells metabolism, Gastrointestinal Tract metabolism, TRPA1 Cation Channel agonists
- Abstract
A therapeutic potential of the TRPA1 channel agonist cinnamaldehyde for use in inflammatory bowel disease is emerging, but the mechanisms are unclear. Semi-quantitative qPCR of various parts of the porcine gastrointestinal tract showed that mRNA for TRPA1 was highest in the colonic mucosa. In Ussing chambers, 1 mmol·L
-1 cinnamaldehyde induced increases in short circuit current (ΔIsc ) and conductance (ΔGt ) across the colon that were higher than those across the jejunum or after 1 mmol·L-1 thymol. Lidocaine, amiloride or bumetanide did not change the response. The application of 1 mmol·L-1 quinidine or the bilateral replacement of 120 Na+ , 120 Cl- or 25 HCO3 - reduced ΔGt , while the removal of Ca2+ enhanced ΔGt with ΔIsc numerically higher. ΔIsc decreased after 0.5 NPPB, 0.01 indometacin and the bilateral replacement of 120 Na+ or 25 HCO3 - . The removal of 120 Cl- had no effect. Cinnamaldehyde also activates TRPV3, but comparative measurements involving patch clamp experiments on overexpressing cells demonstrated that much higher concentrations are required. We suggest that cinnamaldehyde stimulates the secretion of HCO3 - via apical CFTR and basolateral Na+ -HCO3 - cotransport, preventing acidosis and damage to the epithelium and the colonic microbiome. Signaling may involve the opening of TRPA1, depolarization of the epithelium and a rise in PGE2 following a lower uptake of prostaglandins via OATP2A1.- Published
- 2021
- Full Text
- View/download PDF
72. The bovine TRPV3 as a pathway for the uptake of Na + , Ca 2+ , and NH 4 +
- Author
-
Schrapers KT, Sponder G, Liebe F, Liebe H, and Stumpff F
- Subjects
- Animals, Biological Transport, Active, Cattle, Cloning, Molecular, HEK293 Cells, Humans, Patch-Clamp Techniques, TRPV Cation Channels genetics, Ammonium Sulfate metabolism, Calcium metabolism, Sodium metabolism, TRPV Cation Channels metabolism
- Abstract
Absorption of ammonia from the gastrointestinal tract results in problems that range from hepatic encephalopathy in humans to poor nitrogen efficiency of cattle with consequences for the global climate. Previous studies on epithelia and cells from the native ruminal epithelium suggest functional involvement of the bovine homologue of TRPV3 (bTRPV3) in ruminal NH4+ transport. Since the conductance of TRP channels to NH4+ has never been studied, bTRPV3 was overexpressed in HEK-293 cells and investigated using the patch-clamp technique and intracellular calcium imaging. Control cells contained the empty construct. Divalent cations blocked the conductance for monovalent cations in both cell types, with effects higher in cells expressing bTRPV3. In bTRPV3 cells, but not in controls, menthol, thymol, carvacrol, or 2-APB stimulated whole cell currents mediated by Na+, Cs+, NH4+, and K+, with a rise in intracellular Ca2+ observed in response to menthol. While only 25% of control patches showed single-channel events (with a conductance of 40.8 ± 11.9 pS for NH4+ and 25.0 ± 5.8 pS for Na+), 90% of bTRPV3 patches showed much larger conductances of 127.8 ± 4.2 pS for Na+, 240.1 ± 3.6 pS for NH4+, 34.0 ± 1.7 pS for Ca2+, and ~ 36 pS for NMDG+. Open probability, but not conductance, rose with time after patch excision. In conjunction with previous research, we suggest that bTRPV3 channels may play a role in the transport of Na+, K+, Ca2+ and NH4+ across the rumen with possible repercussions for understanding the function of TRPV3 in other epithelia.
- Published
- 2018
- Full Text
- View/download PDF
73. Modulation of sheep ruminal urea transport by ammonia and pH.
- Author
-
Lu Z, Stumpff F, Deiner C, Rosendahl J, Braun H, Abdoun K, Aschenbach JR, and Martens H
- Subjects
- Animals, Biological Transport drug effects, Carbon Dioxide metabolism, Dose-Response Relationship, Drug, Fatty Acids, Volatile metabolism, Female, Hydrogen-Ion Concentration, Membrane Transport Proteins metabolism, Rumen drug effects, Signal Transduction drug effects, Urea Transporters, Ammonia pharmacology, Protons, Rumen metabolism, Sheep metabolism, Urea metabolism
- Abstract
Ruminal fermentation products such as short-chain fatty acids (SCFA) and CO2 acutely stimulate urea transport across the ruminal epithelium in vivo, whereas ammonia has inhibitory effects. Uptake and signaling pathways remain obscure. The ruminal expression of SLC14a1 (UT-B) was studied using polymerase chain reaction (PCR). The functional short-term effects of ammonia on cytosolic pH (pHi) and ruminal urea transport across native epithelia were investigated using pH-sensitive microelectrodes and via flux measurements in Ussing chambers. Two variants (UT-B1 and UT-B2) could be fully sequenced from ovine ruminal cDNA. Functionally, transport was passive and modulated by luminal pH in the presence of SCFA and CO2, rising in response to luminal acidification to a peak value at pH 5.8 and dropping with further acidification, resulting in a bell-shaped curve. Presence of ammonia reduced the amplitude, but not the shape of the relationship between urea flux and pH, so that urea flux remained maximal at pH 5.8. Effects of ammonia were concentration dependent, with saturation at 5 mmol/l. Clamping the transepithelial potential altered the inhibitory potential of ammonia on urea flux. Ammonia depolarized the apical membrane and acidified pHi, suggesting that, at physiological pH (< 7), uptake of NH4 (+) into the cytosol may be a key signaling event regulating ruminal urea transport. We conclude that transport of urea across the ruminal epithelium involves proteins subject to rapid modulation by manipulations that alter pHi and the cytosolic concentration of NH4 (+). Implications for epithelial and ruminal homeostasis are discussed., (Copyright © 2014 the American Physiological Society.)
- Published
- 2014
- Full Text
- View/download PDF
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