The Experts below are selected from a list of 102 Experts worldwide ranked by ideXlab platform
P D Bell - One of the best experts on this subject based on the ideXlab platform.
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ATP as a mediator of Macula Densa Cell signalling
Purinergic Signalling, 2009Co-Authors: P D Bell, Peter Komlosi, Zhi-ren ZhangAbstract:Within each nephro-vascular unit, the tubule returns to the vicinity of its own glomerulus. At this site, there are specialised tubular Cells, the Macula Densa Cells, which sense changes in tubular fluid composition and transmit information to the glomerular arterioles resulting in alterations in glomerular filtration rate and blood flow. Work over the last few years has characterised the mechanisms that lead to the detection of changes in luminal sodium chloride and osmolality by the Macula Densa Cells. These Cells are true “sensor Cells” since intraCellular ion concentrations and membrane potential reflect the level of luminal sodium chloride concentration. An unresolved question has been the nature of the signalling molecule(s) released by the Macula Densa Cells. Currently, there is evidence that Macula Densa Cells produce nitric oxide via neuronal nitric oxide synthase (nNOS) and prostaglandin E_2 (PGE_2) through cyclooxygenase 2 (COX 2)-microsomal prostaglandin E synthase (mPGES). However, both of these signalling molecules play a role in modulating or regulating the Macula-tubuloglomerular feedback system. Direct Macula Densa signalling appears to involve the release of ATP across the basolateral membrane through a maxi-anion channel in response to an increase in luminal sodium chloride concentration. ATP that is released by Macula Densa Cells may directly activate P2 receptors on adjacent mesangial Cells and afferent arteriolar smooth muscle Cells, or the ATP may be converted to adenosine. However, the critical step in signalling would appear to be the regulated release of ATP across the basolateral membrane of Macula Densa Cells.
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unraveling the relationship between Macula Densa Cell volume and luminal solute concentration osmolality
Kidney International, 2006Co-Authors: Peter Komlosi, Attila Fintha, P D BellAbstract:At the Macula Densa, flow-dependent changes in luminal composition lead to tubuloglomerular feedback and renin release. Apical entry of sodium chloride in both Macula Densa and cortical thick ascending limb (cTAL) Cells occurs via furosemide-sensitive sodium–chloride–potassium cotransport. In Macula Densa, apical entry of sodium chloride leads to changes in Cell volume, although there are conflicting data regarding the directional change in Macula Densa Cell volume with increases in luminal sodium chloride concentration. To further assess volume changes in Macula Densa Cells, cTAL-glomerular preparations were isolated and perfused from rabbits, and Macula Densa Cells were loaded with fluorescent dyes calcein and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p -toluenesulfonate. Cell volume was determined with wide-field and multiphoton fluorescence microscopy. Increases in luminal sodium chloride concentration from 0 to 80mmol/l at constant osmolality led to Cell swelling in Macula Densa and cTAL Cells, an effect that was blocked by luminal application of furosemide. However, increases in luminal sodium chloride concentration from 0 to 80mmol/l with concomitant increases in osmolality caused sustained decreases in Macula Densa Cell volume but transient increases in cTAL Cell volume. Increases in luminal osmolality with urea also resulted in Macula Densa Cell shrinkage. These studies suggest that, under physiologically relevant conditions of concurrent increases in luminal sodium chloride concentration and osmolality, there is Macula Densa Cell shrinkage, which may play a role in the Macula Densa Cell signaling process.
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Unraveling the relationship between Macula Densa Cell volume and luminal solute concentration/osmolality.
Kidney international, 2006Co-Authors: Peter Komlosi, Attila Fintha, P D BellAbstract:At the Macula Densa, flow-dependent changes in luminal composition lead to tubuloglomerular feedback and renin release. Apical entry of sodium chloride in both Macula Densa and cortical thick ascending limb (cTAL) Cells occurs via furosemide-sensitive sodium–chloride–potassium cotransport. In Macula Densa, apical entry of sodium chloride leads to changes in Cell volume, although there are conflicting data regarding the directional change in Macula Densa Cell volume with increases in luminal sodium chloride concentration. To further assess volume changes in Macula Densa Cells, cTAL-glomerular preparations were isolated and perfused from rabbits, and Macula Densa Cells were loaded with fluorescent dyes calcein and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p -toluenesulfonate. Cell volume was determined with wide-field and multiphoton fluorescence microscopy. Increases in luminal sodium chloride concentration from 0 to 80mmol/l at constant osmolality led to Cell swelling in Macula Densa and cTAL Cells, an effect that was blocked by luminal application of furosemide. However, increases in luminal sodium chloride concentration from 0 to 80mmol/l with concomitant increases in osmolality caused sustained decreases in Macula Densa Cell volume but transient increases in cTAL Cell volume. Increases in luminal osmolality with urea also resulted in Macula Densa Cell shrinkage. These studies suggest that, under physiologically relevant conditions of concurrent increases in luminal sodium chloride concentration and osmolality, there is Macula Densa Cell shrinkage, which may play a role in the Macula Densa Cell signaling process.
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Macula Densa Cell signaling.
Annual review of physiology, 2003Co-Authors: P D Bell, Jean-yves Lapointe, János Peti-peterdiAbstract:Macula Densa Cells are renal sensor elements that detect changes in distal tubular fluid composition and transmit signals to the glomerular vascular elements. This tubuloglomerular feedback mechanism plays an important role in regulating glomerular filtration rate and blood flow. Macula Densa Cells detect changes in luminal sodium chloride concentration through a complex series of ion transport-related intraCellular events. NaCl entry via a Na:K:2Cl cotransporter and Cl exit through a basolateral channel lead to Cell depolarization and increases in cytosolic calcium. Na/H exchange (NHE2) results in Cell alkalization, whereas intraCellular [Na] is regulated by an apically located H(Na)-K ATPase and not by the traditional basolateral Na:K ATPase. Communication from Macula Densa Cells to the glomerular vascular elements involves ATP release across the Macula Densa basolateral membrane through a maxi-anion channel. The adaptation of multi-photon microscopy is providing new insights into Macula Densa-glomerular signaling.
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Purinergic Receptor Signaling at the Basolateral Membrane of Macula Densa Cells
Journal of the American Society of Nephrology : JASN, 2002Co-Authors: Ruisheng Liu, P D Bell, János Peti-peterdi, Gergly Kovacs, Alf Johansson, A. Erik G. PerssonAbstract:ABSTRACT. Purinergic receptors are important in the regulation of renal hemodynamics; therefore, this study sought to determine if such receptors influence Macula Densa Cell function. Isolated glomeruli containing Macula Densa Cells, with and without the cortical thick ascending limb, were loaded with the Ca 2+ sensitive indicators, Fura Red (confocal microscopy) or fura 2 (conventional video image analysis). Studies were performed on an inverted microscope in a chamber with a flow-through perfusion system. Changes in cytosolic calcium concentration ([Ca 2+ ] i ) from exposed Macula Densa plaques were assessed upon addition of adenosine, ATP, UTP, ADP, or 2-methylthio-ATP (2- MeS-ATP) for 2 min added to the bathing solution. There was no change in [Ca 2+ ] i with addition of adenosine (10 −7 to 10 −3 M). UTP and ATP (10 −4 M) caused [Ca 2+ ] i to increase by 268 ± 40 nM ( n = 21) and 295 ± 53 nM ( n = 21), respectively, whereas in response to 2MesATP and ADP, [Ca 2+ ] i increased by only 67 ± 13 nM ( n = 8) and 93 ± 36 nM ( n = 14), respectively. Dose response curve for ATP (10 −7 to 10 −3 M) added in bath showed an EC 50 of 15 μM. No effect on Macula Densa [Ca 2+ ] i was seen when ATP was added from the lumen. ATP caused similar increases in Macula Densa [Ca 2+ ] i in the presence or absence of bath Ca 2+ and addition of 5 mM ethyleneglycotetraacetic acid (EGTA). Suramin (an antagonist of P2X and P2Y receptors) completely inhibited ATP-induced [Ca 2+ ] i dynamics. Also, ATP-Ca 2+ responsiveness was prevented by the phospholipase C inhibitor, U-73122, but not by its inactive analog, U-73343. These results suggest that Macula Densa Cells possess P2Y 2 purinergic receptors on basolateral but not apical membranes and that activation of these receptors results in the mobilization of Ca 2+ .
Katsumasa Kawahara - One of the best experts on this subject based on the ideXlab platform.
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Low [NaCl]-induced neuronal nitric oxide synthase (nNOS) expression and NO generation are regulated by intraCellular pH in a mouse Macula Densa Cell line (NE-MD)
The Journal of Physiological Sciences, 2009Co-Authors: Hideaki Kawada, Yukiko Yasuoka, Hidekazu Fukuda, Katsumasa KawaharaAbstract:Changes in the luminal NaCl concentration ([NaCl]) at the Macula Densa (MD) modulate the tubuloglomerular feedback (TGF) responses via an affect on the release of nitric oxide (NO). This study was performed in a newly established mouse Macula Densa Cell line (NE-MD) to investigate the effects of lowering [NaCl] on the neuronal NO synthase (nNOS) protein expression and l -arginine (Arg)-induced NO release. Expression of nNOS protein and release of NO were evaluated by Western blot analysis and an NO-sensitive electrode, respectively. IntraCellular pH (pH_i) was monitored by the BCECF assay. Although there was weak staining of the nNOS protein expression, l -Arg-induced NO generation was negligible in normal (140 mM NaCl) solution. Both were significantly ( P
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Low [NaCl]-induced neuronal nitric oxide synthase (nNOS) expression and NO generation are regulated by intraCellular pH in a mouse Macula Densa Cell line (NE-MD).
The journal of physiological sciences : JPS, 2009Co-Authors: Hideaki Kawada, Yukiko Yasuoka, Hidekazu Fukuda, Katsumasa KawaharaAbstract:Changes in the luminal NaCl concentration ([NaCl]) at the Macula Densa (MD) modulate the tubuloglomerular feedback (TGF) responses via an affect on the release of nitric oxide (NO). This study was performed in a newly established mouse Macula Densa Cell line (NE-MD) to investigate the effects of lowering [NaCl] on the neuronal NO synthase (nNOS) protein expression and l-arginine (Arg)-induced NO release. Expression of nNOS protein and release of NO were evaluated by Western blot analysis and an NO-sensitive electrode, respectively. IntraCellular pH (pHi) was monitored by the BCECF assay. Although there was weak staining of the nNOS protein expression, l-Arg-induced NO generation was negligible in normal (140 mM NaCl) solution. Both were significantly (P < 0.05) increased either in the presence of furosemide (12 μM), an inhibitor of the Na+–K+–2Cl− cotransporter, or in a low (23 mM) Cl− solution. Furosemide- and low Cl−-induced NO generation was completely inhibited by 50 μM 7-nitroindasole (7-NI), a nNOS inhibitor. Moreover, these increases were significantly (P < 0.05) inhibited by the addition of 100 μM amiloride, an inhibitor of the Na+/H+ exchanger, or by its analogue 5-(N)-ethyl-N-isopropyl amiloride (EIPA), and also at a lower pH of 7.1. Furthermore, nNOS expression and NO release were not stimulated in as low as 19 mM Na+ solution. In conclusion, low [Cl−], but not low [Na+] in the lumen at the MD, increased nNOS protein expression and NO generation. Changes in the luminal [NaCl] may modulate the TGF system via an effect on the NO generation from the MD.
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Establishment of a mouse Macula Densa Cell line with an nNOS promoter driving EGFP expression.
The Japanese journal of physiology, 2005Co-Authors: Yukiko Yasuoka, Hideaki Kawada, Yoshiro Suzuki, Masahiro Sato, Hitoshi Endou, Masuo Obinata, Katsumasa KawaharaAbstract:We describe a unique method for establishing a functionally intact Macula Densa Cell line from immortalized renal Cells in culture. The Macula Densa is involved in the tubuloglomerular feedback (TGF) system in the kidney and specifically expresses neuronal nitric oxide synthase (nNOS). A 347 bp portion of the nNOS promoter was used to drive the expression of enhanced green fluorescence protein (EGFP). An immortalized distal tubule (DT) Cell line was derived from distal tubules microdissected from the kidneys of SV40 large T antigen transgenic mice. Immunofluorescence labeling using an antibody against nNOS revealed no specific EGFP expression in immunofluorescence-negative DT Cells. The established Cell line (NE-MD) showed a time-dependent increase in signals of the nNOS protein when they were incubated with 12 microM furosemide (an inhibitor of Na(+)-K(+)-2Cl(-) symporter) for 5 h. In conclusion, this newly developed Macula Densa Cell line will be useful in studies of the TGF stem.
Peter Komlosi - One of the best experts on this subject based on the ideXlab platform.
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ATP as a mediator of Macula Densa Cell signalling
Purinergic Signalling, 2009Co-Authors: P D Bell, Peter Komlosi, Zhi-ren ZhangAbstract:Within each nephro-vascular unit, the tubule returns to the vicinity of its own glomerulus. At this site, there are specialised tubular Cells, the Macula Densa Cells, which sense changes in tubular fluid composition and transmit information to the glomerular arterioles resulting in alterations in glomerular filtration rate and blood flow. Work over the last few years has characterised the mechanisms that lead to the detection of changes in luminal sodium chloride and osmolality by the Macula Densa Cells. These Cells are true “sensor Cells” since intraCellular ion concentrations and membrane potential reflect the level of luminal sodium chloride concentration. An unresolved question has been the nature of the signalling molecule(s) released by the Macula Densa Cells. Currently, there is evidence that Macula Densa Cells produce nitric oxide via neuronal nitric oxide synthase (nNOS) and prostaglandin E_2 (PGE_2) through cyclooxygenase 2 (COX 2)-microsomal prostaglandin E synthase (mPGES). However, both of these signalling molecules play a role in modulating or regulating the Macula-tubuloglomerular feedback system. Direct Macula Densa signalling appears to involve the release of ATP across the basolateral membrane through a maxi-anion channel in response to an increase in luminal sodium chloride concentration. ATP that is released by Macula Densa Cells may directly activate P2 receptors on adjacent mesangial Cells and afferent arteriolar smooth muscle Cells, or the ATP may be converted to adenosine. However, the critical step in signalling would appear to be the regulated release of ATP across the basolateral membrane of Macula Densa Cells.
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unraveling the relationship between Macula Densa Cell volume and luminal solute concentration osmolality
Kidney International, 2006Co-Authors: Peter Komlosi, Attila Fintha, P D BellAbstract:At the Macula Densa, flow-dependent changes in luminal composition lead to tubuloglomerular feedback and renin release. Apical entry of sodium chloride in both Macula Densa and cortical thick ascending limb (cTAL) Cells occurs via furosemide-sensitive sodium–chloride–potassium cotransport. In Macula Densa, apical entry of sodium chloride leads to changes in Cell volume, although there are conflicting data regarding the directional change in Macula Densa Cell volume with increases in luminal sodium chloride concentration. To further assess volume changes in Macula Densa Cells, cTAL-glomerular preparations were isolated and perfused from rabbits, and Macula Densa Cells were loaded with fluorescent dyes calcein and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p -toluenesulfonate. Cell volume was determined with wide-field and multiphoton fluorescence microscopy. Increases in luminal sodium chloride concentration from 0 to 80mmol/l at constant osmolality led to Cell swelling in Macula Densa and cTAL Cells, an effect that was blocked by luminal application of furosemide. However, increases in luminal sodium chloride concentration from 0 to 80mmol/l with concomitant increases in osmolality caused sustained decreases in Macula Densa Cell volume but transient increases in cTAL Cell volume. Increases in luminal osmolality with urea also resulted in Macula Densa Cell shrinkage. These studies suggest that, under physiologically relevant conditions of concurrent increases in luminal sodium chloride concentration and osmolality, there is Macula Densa Cell shrinkage, which may play a role in the Macula Densa Cell signaling process.
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Unraveling the relationship between Macula Densa Cell volume and luminal solute concentration/osmolality.
Kidney international, 2006Co-Authors: Peter Komlosi, Attila Fintha, P D BellAbstract:At the Macula Densa, flow-dependent changes in luminal composition lead to tubuloglomerular feedback and renin release. Apical entry of sodium chloride in both Macula Densa and cortical thick ascending limb (cTAL) Cells occurs via furosemide-sensitive sodium–chloride–potassium cotransport. In Macula Densa, apical entry of sodium chloride leads to changes in Cell volume, although there are conflicting data regarding the directional change in Macula Densa Cell volume with increases in luminal sodium chloride concentration. To further assess volume changes in Macula Densa Cells, cTAL-glomerular preparations were isolated and perfused from rabbits, and Macula Densa Cells were loaded with fluorescent dyes calcein and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p -toluenesulfonate. Cell volume was determined with wide-field and multiphoton fluorescence microscopy. Increases in luminal sodium chloride concentration from 0 to 80mmol/l at constant osmolality led to Cell swelling in Macula Densa and cTAL Cells, an effect that was blocked by luminal application of furosemide. However, increases in luminal sodium chloride concentration from 0 to 80mmol/l with concomitant increases in osmolality caused sustained decreases in Macula Densa Cell volume but transient increases in cTAL Cell volume. Increases in luminal osmolality with urea also resulted in Macula Densa Cell shrinkage. These studies suggest that, under physiologically relevant conditions of concurrent increases in luminal sodium chloride concentration and osmolality, there is Macula Densa Cell shrinkage, which may play a role in the Macula Densa Cell signaling process.
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Current mechanisms of Macula Densa Cell signalling.
Acta physiologica Scandinavica, 2004Co-Authors: Peter Komlosi, Attila Fintha, P. D. BellAbstract:Macula Densa Cells couple renal haemodynamics, glomerular filtration and renin release with tubular fluid salt and water reabsorption. These Cells detect changes in tubular fluid composition through a complex of intraCellular signalling events that are mediated by membrane transport pathways. Increases in luminal fluid sodium chloride concentration result in alterations in Cell sodium chloride concentration, cytosolic calcium, Cell pH, basolateral membrane depolarization and Cell volume. Macula Densa signalling then involves the production and release of specific paracrine signalling molecules at their basolateral membrane. Upon moderate increases in luminal sodium chloride concentration Macula Densa Cells release increasing amounts of ATP and decreasing amounts of prostaglandin E 2 , thereby increasing afferent arteriolar tone and decreasing the release of renin from granular Cells. On the other hand, further increases in luminal concentration stimulate the release of nitric oxide, which serve to prevent excessive tubuloglomerular feedback vasoconstriction. Paracrine signalling by the Macula Densa Cells therefore controls juxtaglomerular function, renal vascular resistance and participates in the regulation of renin release.
Hideaki Kawada - One of the best experts on this subject based on the ideXlab platform.
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Low [NaCl]-induced neuronal nitric oxide synthase (nNOS) expression and NO generation are regulated by intraCellular pH in a mouse Macula Densa Cell line (NE-MD)
The Journal of Physiological Sciences, 2009Co-Authors: Hideaki Kawada, Yukiko Yasuoka, Hidekazu Fukuda, Katsumasa KawaharaAbstract:Changes in the luminal NaCl concentration ([NaCl]) at the Macula Densa (MD) modulate the tubuloglomerular feedback (TGF) responses via an affect on the release of nitric oxide (NO). This study was performed in a newly established mouse Macula Densa Cell line (NE-MD) to investigate the effects of lowering [NaCl] on the neuronal NO synthase (nNOS) protein expression and l -arginine (Arg)-induced NO release. Expression of nNOS protein and release of NO were evaluated by Western blot analysis and an NO-sensitive electrode, respectively. IntraCellular pH (pH_i) was monitored by the BCECF assay. Although there was weak staining of the nNOS protein expression, l -Arg-induced NO generation was negligible in normal (140 mM NaCl) solution. Both were significantly ( P
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Low [NaCl]-induced neuronal nitric oxide synthase (nNOS) expression and NO generation are regulated by intraCellular pH in a mouse Macula Densa Cell line (NE-MD).
The journal of physiological sciences : JPS, 2009Co-Authors: Hideaki Kawada, Yukiko Yasuoka, Hidekazu Fukuda, Katsumasa KawaharaAbstract:Changes in the luminal NaCl concentration ([NaCl]) at the Macula Densa (MD) modulate the tubuloglomerular feedback (TGF) responses via an affect on the release of nitric oxide (NO). This study was performed in a newly established mouse Macula Densa Cell line (NE-MD) to investigate the effects of lowering [NaCl] on the neuronal NO synthase (nNOS) protein expression and l-arginine (Arg)-induced NO release. Expression of nNOS protein and release of NO were evaluated by Western blot analysis and an NO-sensitive electrode, respectively. IntraCellular pH (pHi) was monitored by the BCECF assay. Although there was weak staining of the nNOS protein expression, l-Arg-induced NO generation was negligible in normal (140 mM NaCl) solution. Both were significantly (P < 0.05) increased either in the presence of furosemide (12 μM), an inhibitor of the Na+–K+–2Cl− cotransporter, or in a low (23 mM) Cl− solution. Furosemide- and low Cl−-induced NO generation was completely inhibited by 50 μM 7-nitroindasole (7-NI), a nNOS inhibitor. Moreover, these increases were significantly (P < 0.05) inhibited by the addition of 100 μM amiloride, an inhibitor of the Na+/H+ exchanger, or by its analogue 5-(N)-ethyl-N-isopropyl amiloride (EIPA), and also at a lower pH of 7.1. Furthermore, nNOS expression and NO release were not stimulated in as low as 19 mM Na+ solution. In conclusion, low [Cl−], but not low [Na+] in the lumen at the MD, increased nNOS protein expression and NO generation. Changes in the luminal [NaCl] may modulate the TGF system via an effect on the NO generation from the MD.
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Establishment of a mouse Macula Densa Cell line with an nNOS promoter driving EGFP expression.
The Japanese journal of physiology, 2005Co-Authors: Yukiko Yasuoka, Hideaki Kawada, Yoshiro Suzuki, Masahiro Sato, Hitoshi Endou, Masuo Obinata, Katsumasa KawaharaAbstract:We describe a unique method for establishing a functionally intact Macula Densa Cell line from immortalized renal Cells in culture. The Macula Densa is involved in the tubuloglomerular feedback (TGF) system in the kidney and specifically expresses neuronal nitric oxide synthase (nNOS). A 347 bp portion of the nNOS promoter was used to drive the expression of enhanced green fluorescence protein (EGFP). An immortalized distal tubule (DT) Cell line was derived from distal tubules microdissected from the kidneys of SV40 large T antigen transgenic mice. Immunofluorescence labeling using an antibody against nNOS revealed no specific EGFP expression in immunofluorescence-negative DT Cells. The established Cell line (NE-MD) showed a time-dependent increase in signals of the nNOS protein when they were incubated with 12 microM furosemide (an inhibitor of Na(+)-K(+)-2Cl(-) symporter) for 5 h. In conclusion, this newly developed Macula Densa Cell line will be useful in studies of the TGF stem.
Yukiko Yasuoka - One of the best experts on this subject based on the ideXlab platform.
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Low [NaCl]-induced neuronal nitric oxide synthase (nNOS) expression and NO generation are regulated by intraCellular pH in a mouse Macula Densa Cell line (NE-MD)
The Journal of Physiological Sciences, 2009Co-Authors: Hideaki Kawada, Yukiko Yasuoka, Hidekazu Fukuda, Katsumasa KawaharaAbstract:Changes in the luminal NaCl concentration ([NaCl]) at the Macula Densa (MD) modulate the tubuloglomerular feedback (TGF) responses via an affect on the release of nitric oxide (NO). This study was performed in a newly established mouse Macula Densa Cell line (NE-MD) to investigate the effects of lowering [NaCl] on the neuronal NO synthase (nNOS) protein expression and l -arginine (Arg)-induced NO release. Expression of nNOS protein and release of NO were evaluated by Western blot analysis and an NO-sensitive electrode, respectively. IntraCellular pH (pH_i) was monitored by the BCECF assay. Although there was weak staining of the nNOS protein expression, l -Arg-induced NO generation was negligible in normal (140 mM NaCl) solution. Both were significantly ( P
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Low [NaCl]-induced neuronal nitric oxide synthase (nNOS) expression and NO generation are regulated by intraCellular pH in a mouse Macula Densa Cell line (NE-MD).
The journal of physiological sciences : JPS, 2009Co-Authors: Hideaki Kawada, Yukiko Yasuoka, Hidekazu Fukuda, Katsumasa KawaharaAbstract:Changes in the luminal NaCl concentration ([NaCl]) at the Macula Densa (MD) modulate the tubuloglomerular feedback (TGF) responses via an affect on the release of nitric oxide (NO). This study was performed in a newly established mouse Macula Densa Cell line (NE-MD) to investigate the effects of lowering [NaCl] on the neuronal NO synthase (nNOS) protein expression and l-arginine (Arg)-induced NO release. Expression of nNOS protein and release of NO were evaluated by Western blot analysis and an NO-sensitive electrode, respectively. IntraCellular pH (pHi) was monitored by the BCECF assay. Although there was weak staining of the nNOS protein expression, l-Arg-induced NO generation was negligible in normal (140 mM NaCl) solution. Both were significantly (P < 0.05) increased either in the presence of furosemide (12 μM), an inhibitor of the Na+–K+–2Cl− cotransporter, or in a low (23 mM) Cl− solution. Furosemide- and low Cl−-induced NO generation was completely inhibited by 50 μM 7-nitroindasole (7-NI), a nNOS inhibitor. Moreover, these increases were significantly (P < 0.05) inhibited by the addition of 100 μM amiloride, an inhibitor of the Na+/H+ exchanger, or by its analogue 5-(N)-ethyl-N-isopropyl amiloride (EIPA), and also at a lower pH of 7.1. Furthermore, nNOS expression and NO release were not stimulated in as low as 19 mM Na+ solution. In conclusion, low [Cl−], but not low [Na+] in the lumen at the MD, increased nNOS protein expression and NO generation. Changes in the luminal [NaCl] may modulate the TGF system via an effect on the NO generation from the MD.
-
Establishment of a mouse Macula Densa Cell line with an nNOS promoter driving EGFP expression.
The Japanese journal of physiology, 2005Co-Authors: Yukiko Yasuoka, Hideaki Kawada, Yoshiro Suzuki, Masahiro Sato, Hitoshi Endou, Masuo Obinata, Katsumasa KawaharaAbstract:We describe a unique method for establishing a functionally intact Macula Densa Cell line from immortalized renal Cells in culture. The Macula Densa is involved in the tubuloglomerular feedback (TGF) system in the kidney and specifically expresses neuronal nitric oxide synthase (nNOS). A 347 bp portion of the nNOS promoter was used to drive the expression of enhanced green fluorescence protein (EGFP). An immortalized distal tubule (DT) Cell line was derived from distal tubules microdissected from the kidneys of SV40 large T antigen transgenic mice. Immunofluorescence labeling using an antibody against nNOS revealed no specific EGFP expression in immunofluorescence-negative DT Cells. The established Cell line (NE-MD) showed a time-dependent increase in signals of the nNOS protein when they were incubated with 12 microM furosemide (an inhibitor of Na(+)-K(+)-2Cl(-) symporter) for 5 h. In conclusion, this newly developed Macula Densa Cell line will be useful in studies of the TGF stem.
