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Loss of apical monocilia on collecting duct principal cells impairs ATP secretion across the apical cell surface and ATP-dependent and flow-induced calcium signals.

Hovater MB, Olteanu D, Hanson EL, Cheng NL, Siroky B, Fintha A, Komlosi P, Liu W, Satlin LM, Bell PD, Yoder BK, Schwiebert EM - Purinergic Signal. (2007)

Bottom Line: We compared these cells grown as polarized cell monolayers on permeable supports to the same cells where the apical monocilium was genetically rescued with the wild-type Tg737 gene that encodes Polaris, a protein essential to cilia formation.Principal cell monolayers with fully formed apical monocilia responded three- to fivefold greater to hypotonicity than mutant monolayers lacking monocilia.Mechanical stimulation was much less effective, however, on mutant orpk collecting duct principal cell monolayers that lacked apical central monocilia.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of Alabama at Birmingham, 1918 University Blvd., Birmingham, AL, 35294-0005, USA.

ABSTRACT
Renal epithelial cells release ATP constitutively under basal conditions and release higher quantities of purine nucleotide in response to stimuli. ATP filtered at the glomerulus, secreted by epithelial cells along the nephron, and released serosally by macula densa cells for feedback signaling to afferent arterioles within the glomerulus has important physiological signaling roles within kidneys. In autosomal recessive polycystic kidney disease (ARPKD) mice and humans, collecting duct epithelial cells lack an apical central cilium or express dysfunctional proteins within that monocilium. Collecting duct principal cells derived from an Oak Ridge polycystic kidney (orpk ( Tg737 ) ) mouse model of ARPKD lack a well-formed apical central cilium, thought to be a sensory organelle. We compared these cells grown as polarized cell monolayers on permeable supports to the same cells where the apical monocilium was genetically rescued with the wild-type Tg737 gene that encodes Polaris, a protein essential to cilia formation. Constitutive ATP release under basal conditions was low and not different in mutant versus rescued monolayers. However, genetically rescued principal cell monolayers released ATP three- to fivefold more robustly in response to ionomycin. Principal cell monolayers with fully formed apical monocilia responded three- to fivefold greater to hypotonicity than mutant monolayers lacking monocilia. In support of the idea that monocilia are sensory organelles, intentionally harsh pipetting of medium directly onto the center of the monolayer induced ATP release in genetically rescued monolayers that possessed apical monocilia. Mechanical stimulation was much less effective, however, on mutant orpk collecting duct principal cell monolayers that lacked apical central monocilia. Our data also show that an increase in cytosolic free Ca(2+) primes the ATP pool that is released in response to mechanical stimuli. It also appears that hypotonic cell swelling and mechanical pipetting stimuli trigger release of a common ATP pool. Cilium-competent monolayers responded to flow with an increase in cell Ca(2+) derived from both extracellular and intracellular stores. This flow-induced Ca(2+) signal was less robust in cilium-deficient monolayers. Flow-induced Ca(2+) signals in both preparations were attenuated by extracellular gadolinium and by extracellular apyrase, an ATPase/ADPase. Taken together, these data suggest that apical monocilia are sensory organelles and that their presence in the apical membrane facilitates the formation of a mature ATP secretion apparatus responsive to chemical, osmotic, and mechanical stimuli. The cilium and autocrine ATP signaling appear to work in concert to control cell Ca(2+). Loss of a cilium-dedicated autocrine purinergic signaling system may be a critical underlying etiology for ARPKD and may lead to disinhibition and/or upregulation of multiple sodium (Na(+)) absorptive mechanisms and a resultant severe hypertensive phenotype in ARPKD and, possibly, other diseases.

No MeSH data available.


Related in: MedlinePlus

Ionomycin-induced ATP release across the apical cell surface is markedly attenuated in mutant cilium-deficient versus genetically rescued cilium-competent orpk kidney cell monolayers. Six to nine cell monolayers were assessed at left for the summary data derived from the 6.5-mm diameter filter support preparation. The fold difference in ionomycin (2 μM, added in a 2-μl bolus along the side of the plastic wall into the apical medium)-induced ATP release in rescued cell monolayers over mutant monolayers is graphed for the dataset at left along with three additional datasets. Although the filter supports, amount of detection reagent, and magnitude of luminescence measured differed between preparation to preparation (n = 6 each), the fold difference in the ionomycin effect was constant. The asterisk reflects P < 0.05 by paired Student’s t-test; the cross reflects P < 0.05 significance by analysis of variance (ANOVA) and Tukey’s ad hoc test. The statistical analysis and results are given similarly in all other figures
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Fig2: Ionomycin-induced ATP release across the apical cell surface is markedly attenuated in mutant cilium-deficient versus genetically rescued cilium-competent orpk kidney cell monolayers. Six to nine cell monolayers were assessed at left for the summary data derived from the 6.5-mm diameter filter support preparation. The fold difference in ionomycin (2 μM, added in a 2-μl bolus along the side of the plastic wall into the apical medium)-induced ATP release in rescued cell monolayers over mutant monolayers is graphed for the dataset at left along with three additional datasets. Although the filter supports, amount of detection reagent, and magnitude of luminescence measured differed between preparation to preparation (n = 6 each), the fold difference in the ionomycin effect was constant. The asterisk reflects P < 0.05 by paired Student’s t-test; the cross reflects P < 0.05 significance by analysis of variance (ANOVA) and Tukey’s ad hoc test. The statistical analysis and results are given similarly in all other figures

Mentions: Ionomycin-stimulated ATP release is more robust in rescued versus mutant cell monolayers We first assessed the effect of an increase in intracellular free calcium (Ca2+) on ATP release. This first study was done in part because of the emerging role of the primary central monocilium in mediating Ca2+ transients, sparks, and waves in cell monolayers derived from extracellular and intracellular stores [17–30]. This cilium-derived Ca2+ signal is triggered by touch or flow across this organelle and is mediated, at least in part, by the polycystin proteins, PC-1 and PC-2 [17–30]. Polycystin-2 is a part of the TRPP subfamily of TRP genes and is a distant relative of the transient receptor potential or TRPC gene family of Ca2+ entry channels [17–30]. Ionomycin (2 μM) applied to the apical surface of well-polarized cell monolayers triggered a slow, monophasic rise in ATP release over 3–5 min (see typical time courses below in Figs. 6 and 8). This response was a similar phenotype to that observed in previous studies by our laboratory in human vascular endothelial cell monolayers [37]. In mutant monolayers deficient in monocilia, an increase in cell Ca2+ increased extracellular ATP from below 50 nM to 0.4 μM. Figure 2 shows data as bioluminescence in arbitrary light units (ALU) with corresponding estimated ATP for each step of the protocol based upon parallel standard curves with known amounts of ATP. In sharp contrast, rescued monolayers responded to ionomycin stimulation with a rise in secreted ATP from approximately 50 nM to 2 μM, three- to fivefold higher sustained amounts on average than in mutant monolayers. The data in Fig. 2 also group the data from the panels of mutant and rescued clones which were in agreement. Taken together, these data show that cell Ca2+-stimulated ATP release is more robust when a well-formed central monocilium is present. In fact, the “releasable” pool of ATP that is sensitive to Ca2+ appears intact in cilium-competent monolayers and deficient in cilium-deficient monolayers.Fig. 2


Loss of apical monocilia on collecting duct principal cells impairs ATP secretion across the apical cell surface and ATP-dependent and flow-induced calcium signals.

Hovater MB, Olteanu D, Hanson EL, Cheng NL, Siroky B, Fintha A, Komlosi P, Liu W, Satlin LM, Bell PD, Yoder BK, Schwiebert EM - Purinergic Signal. (2007)

Ionomycin-induced ATP release across the apical cell surface is markedly attenuated in mutant cilium-deficient versus genetically rescued cilium-competent orpk kidney cell monolayers. Six to nine cell monolayers were assessed at left for the summary data derived from the 6.5-mm diameter filter support preparation. The fold difference in ionomycin (2 μM, added in a 2-μl bolus along the side of the plastic wall into the apical medium)-induced ATP release in rescued cell monolayers over mutant monolayers is graphed for the dataset at left along with three additional datasets. Although the filter supports, amount of detection reagent, and magnitude of luminescence measured differed between preparation to preparation (n = 6 each), the fold difference in the ionomycin effect was constant. The asterisk reflects P < 0.05 by paired Student’s t-test; the cross reflects P < 0.05 significance by analysis of variance (ANOVA) and Tukey’s ad hoc test. The statistical analysis and results are given similarly in all other figures
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2377318&req=5

Fig2: Ionomycin-induced ATP release across the apical cell surface is markedly attenuated in mutant cilium-deficient versus genetically rescued cilium-competent orpk kidney cell monolayers. Six to nine cell monolayers were assessed at left for the summary data derived from the 6.5-mm diameter filter support preparation. The fold difference in ionomycin (2 μM, added in a 2-μl bolus along the side of the plastic wall into the apical medium)-induced ATP release in rescued cell monolayers over mutant monolayers is graphed for the dataset at left along with three additional datasets. Although the filter supports, amount of detection reagent, and magnitude of luminescence measured differed between preparation to preparation (n = 6 each), the fold difference in the ionomycin effect was constant. The asterisk reflects P < 0.05 by paired Student’s t-test; the cross reflects P < 0.05 significance by analysis of variance (ANOVA) and Tukey’s ad hoc test. The statistical analysis and results are given similarly in all other figures
Mentions: Ionomycin-stimulated ATP release is more robust in rescued versus mutant cell monolayers We first assessed the effect of an increase in intracellular free calcium (Ca2+) on ATP release. This first study was done in part because of the emerging role of the primary central monocilium in mediating Ca2+ transients, sparks, and waves in cell monolayers derived from extracellular and intracellular stores [17–30]. This cilium-derived Ca2+ signal is triggered by touch or flow across this organelle and is mediated, at least in part, by the polycystin proteins, PC-1 and PC-2 [17–30]. Polycystin-2 is a part of the TRPP subfamily of TRP genes and is a distant relative of the transient receptor potential or TRPC gene family of Ca2+ entry channels [17–30]. Ionomycin (2 μM) applied to the apical surface of well-polarized cell monolayers triggered a slow, monophasic rise in ATP release over 3–5 min (see typical time courses below in Figs. 6 and 8). This response was a similar phenotype to that observed in previous studies by our laboratory in human vascular endothelial cell monolayers [37]. In mutant monolayers deficient in monocilia, an increase in cell Ca2+ increased extracellular ATP from below 50 nM to 0.4 μM. Figure 2 shows data as bioluminescence in arbitrary light units (ALU) with corresponding estimated ATP for each step of the protocol based upon parallel standard curves with known amounts of ATP. In sharp contrast, rescued monolayers responded to ionomycin stimulation with a rise in secreted ATP from approximately 50 nM to 2 μM, three- to fivefold higher sustained amounts on average than in mutant monolayers. The data in Fig. 2 also group the data from the panels of mutant and rescued clones which were in agreement. Taken together, these data show that cell Ca2+-stimulated ATP release is more robust when a well-formed central monocilium is present. In fact, the “releasable” pool of ATP that is sensitive to Ca2+ appears intact in cilium-competent monolayers and deficient in cilium-deficient monolayers.Fig. 2

Bottom Line: We compared these cells grown as polarized cell monolayers on permeable supports to the same cells where the apical monocilium was genetically rescued with the wild-type Tg737 gene that encodes Polaris, a protein essential to cilia formation.Principal cell monolayers with fully formed apical monocilia responded three- to fivefold greater to hypotonicity than mutant monolayers lacking monocilia.Mechanical stimulation was much less effective, however, on mutant orpk collecting duct principal cell monolayers that lacked apical central monocilia.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of Alabama at Birmingham, 1918 University Blvd., Birmingham, AL, 35294-0005, USA.

ABSTRACT
Renal epithelial cells release ATP constitutively under basal conditions and release higher quantities of purine nucleotide in response to stimuli. ATP filtered at the glomerulus, secreted by epithelial cells along the nephron, and released serosally by macula densa cells for feedback signaling to afferent arterioles within the glomerulus has important physiological signaling roles within kidneys. In autosomal recessive polycystic kidney disease (ARPKD) mice and humans, collecting duct epithelial cells lack an apical central cilium or express dysfunctional proteins within that monocilium. Collecting duct principal cells derived from an Oak Ridge polycystic kidney (orpk ( Tg737 ) ) mouse model of ARPKD lack a well-formed apical central cilium, thought to be a sensory organelle. We compared these cells grown as polarized cell monolayers on permeable supports to the same cells where the apical monocilium was genetically rescued with the wild-type Tg737 gene that encodes Polaris, a protein essential to cilia formation. Constitutive ATP release under basal conditions was low and not different in mutant versus rescued monolayers. However, genetically rescued principal cell monolayers released ATP three- to fivefold more robustly in response to ionomycin. Principal cell monolayers with fully formed apical monocilia responded three- to fivefold greater to hypotonicity than mutant monolayers lacking monocilia. In support of the idea that monocilia are sensory organelles, intentionally harsh pipetting of medium directly onto the center of the monolayer induced ATP release in genetically rescued monolayers that possessed apical monocilia. Mechanical stimulation was much less effective, however, on mutant orpk collecting duct principal cell monolayers that lacked apical central monocilia. Our data also show that an increase in cytosolic free Ca(2+) primes the ATP pool that is released in response to mechanical stimuli. It also appears that hypotonic cell swelling and mechanical pipetting stimuli trigger release of a common ATP pool. Cilium-competent monolayers responded to flow with an increase in cell Ca(2+) derived from both extracellular and intracellular stores. This flow-induced Ca(2+) signal was less robust in cilium-deficient monolayers. Flow-induced Ca(2+) signals in both preparations were attenuated by extracellular gadolinium and by extracellular apyrase, an ATPase/ADPase. Taken together, these data suggest that apical monocilia are sensory organelles and that their presence in the apical membrane facilitates the formation of a mature ATP secretion apparatus responsive to chemical, osmotic, and mechanical stimuli. The cilium and autocrine ATP signaling appear to work in concert to control cell Ca(2+). Loss of a cilium-dedicated autocrine purinergic signaling system may be a critical underlying etiology for ARPKD and may lead to disinhibition and/or upregulation of multiple sodium (Na(+)) absorptive mechanisms and a resultant severe hypertensive phenotype in ARPKD and, possibly, other diseases.

No MeSH data available.


Related in: MedlinePlus