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Attenuated, flow-induced ATP release contributes to absence of flow-sensitive, purinergic Cai2+ signaling in human ADPKD cyst epithelial cells.

ABSTRACT: Flow-induced cytosolic Ca2+ Ca(i)2+ signaling in renal tubular epithelial cells is mediated in part through P2 receptor (P2R) activation by locally released ATP. The ability of P2R to regulate salt and water reabsorption has suggested a possible contribution of ATP release and paracrine P2R activation to cystogenesis and/or enlargement in autosomal dominant polycystic kidney disease (ADPKD). We and others have demonstrated in human ADPKD cyst cells the absence of flow-induced Ca(i)2+ signaling exhibited by normal renal epithelial cells. We now extend these findings to primary and telomerase-immortalized normal and ADPKD epithelial cells of different genotype and of both proximal and distal origins. Flow-induced elevation of Ca(i)2+ concentration ([Ca2+](i)) was absent from ADPKD cyst cells, but in normal cells was mediated by flow-sensitive ATP release and paracrine P2R activation, modulated by ecto-nucleotidase activity, and abrogated by P2R inhibition or extracellular ATP hydrolysis. In contrast to the elevated ATP release from ADPKD cells in static isotonic conditions or in hypotonic conditions, flow-induced ATP release from cyst cells was lower than from normal cells. Extracellular ATP rapidly reduced thapsigargin-elevated [Ca2+](i) in both ADPKD cyst and normal cells, but cyst cells lacked the subsequent, slow, oxidized ATP-sensitive [Ca2+](i) recovery present in normal cells. Telomerase-immortalized cyst cells also exhibited altered CD39 and P2X7 mRNA levels. Thus the loss of flow-induced, P2R-mediated Ca(i)2+ signaling in human ADPKD cyst epithelial cells was accompanied by reduced flow-sensitive ATP release, altered purinergic regulation of store-operated Ca2+ entry, and altered expression of gene products controlling extracellular nucleotide signaling.

SUBMITTER: Xu C

PROVIDER: S-EPMC2692447 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Attenuated, flow-induced ATP release contributes to absence of flow-sensitive, purinergic Cai2+ signaling in human ADPKD cyst epithelial cells.

Xu Chang C Shmukler Boris E BE Nishimura Katherine K Kaczmarek Elzbieta E Rossetti Sandro S Harris Peter C PC Wandinger-Ness Angela A Bacallao Robert L RL Alper Seth L SL

American journal of physiology. Renal physiology 20090225 6

Flow-induced cytosolic Ca2+ Ca(i)2+ signaling in renal tubular epithelial cells is mediated in part through P2 receptor (P2R) activation by locally released ATP. The ability of P2R to regulate salt and water reabsorption has suggested a possible contribution of ATP release and paracrine P2R activation to cystogenesis and/or enlargement in autosomal dominant polycystic kidney disease (ADPKD). We and others have demonstrated in human ADPKD cyst cells the absence of flow-induced Ca(i)2+ signaling e ...[more]

PMID: 19244404

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Project description:BackgroundAugmentation of tissue blood flow by therapeutic ultrasound is thought to rely on convective shear. Microbubble contrast agents that undergo ultrasound-mediated cavitation markedly amplify these effects. We hypothesized that purinergic signaling is responsible for shear-dependent increases in muscle perfusion during therapeutic cavitation.MethodsUnilateral exposure of the proximal hindlimb of mice (with or without ischemia produced by iliac ligation) to therapeutic ultrasound (1.3 MHz, mechanical index 1.3) was performed for 10 minutes after intravenous injection of 2×108 lipid microbubbles. Microvascular perfusion was evaluated by low-power contrast ultrasound perfusion imaging. In vivo muscle ATP release and in vitro ATP release from endothelial cells or erythrocytes were assessed by a luciferin-luciferase assay. Purinergic signaling pathways were assessed by studying interventions that (1) accelerated ATP degradation; (2) inhibited P2Y receptors, adenosine receptors, or KATP channels; or (3) inhibited downstream signaling pathways involving endothelial nitric oxide synthase or prostanoid production (indomethacin). Augmentation in muscle perfusion by ultrasound cavitation was assessed in a proof-of-concept clinical trial in 12 subjects with stable sickle cell disease.ResultsTherapeutic ultrasound cavitation increased muscle perfusion by 7-fold in normal mice, reversed tissue ischemia for up to 24 hours in the murine model of peripheral artery disease, and doubled muscle perfusion in patients with sickle cell disease. Augmentation in flow extended well beyond the region of ultrasound exposure. Ultrasound cavitation produced an ≈40-fold focal and sustained increase in ATP, the source of which included both endothelial cells and erythrocytes. Inhibitory studies indicated that ATP was a critical mediator of flow augmentation that acts primarily through either P2Y receptors or adenosine produced by ectonucleotidase activity. Combined indomethacin and inhibition of endothelial nitric oxide synthase abolished the effects of therapeutic ultrasound, indicating downstream signaling through both nitric oxide and prostaglandins.ConclusionsTherapeutic ultrasound using microbubble cavitation to increase muscle perfusion relies on shear-dependent increases in ATP, which can act through a diverse portfolio of purinergic signaling pathways. These events can reverse hindlimb ischemia in mice for >24 hours and increase muscle blood flow in patients with sickle cell disease.Clinical trial registrationURL: http://clinicaltrials.gov. Unique identifier: NCT01566890.

Project description:Hyperpigmentation stimulated by ultraviolet (UV)-induced melanin overproduction causes various cosmetic problems. UV radiation's activation of the cyclic adenosine monophosphate (cAMP)-mediated cAMP-dependent protein kinase (PKA)/cAMP response element-binding protein (CREB)/microphthalmia-associated transcription factor (MITF) pathway is the main pathway for melanogenesis. However, the secretion of adenosine triphosphate (ATP) from keratinocytes due to UV radiation also leads to melanogenesis. Adenosine, converted from ATP by CD39 and CD73, can activate adenylate cyclase (AC) activity and increase intracellular cAMP expression. cAMP-mediated PKA activation results in dynamic mitochondrial changes that affect melanogenesis via ERK. We evaluated whether radiofrequency (RF) irradiation could decrease ATP release from keratinocytes and suppress the expression of CD39, CD73, and A2A/A2B adenosine receptors (ARs) and the activity of AC and downregulate the PKA/CREB/MITF pathway, which would eventually decrease melanogenesis in vitro in UV-irradiated cells and animal skin. Our results indicate that RF decreased ATP release from UVB-irradiated keratinocytes. When conditioned media (CM) from UVB-irradiated keratinocytes (CM-UVB) were administered to melanocytes, the expressions of CD39, CD73, A2A/A2BARs, cAMP, and PKA increased. However, the expression of these factors decreased when CM from UVB and RF-irradiated keratinocytes (CM-UVB/RF) was administered to melanocytes. The phosphorylation of DRP1 at Ser637, which inhibits mitochondrial fission, increased in UVB-irradiated animal skin and was decreased by RF irradiation. The expression of ERK1/2, which can degrade MITF, was increased using RF treatment in UVB-irradiated animal skin. Tyrosinase activity and melanin levels in melanocytes increased following CM-UVB administration, and these increases were reversed after CD39 silencing. Tyrosinase activity and melanin levels in melanocytes were decreased by CM-UVB/RF irradiation. In conclusion, RF irradiation decreased ATP release from keratinocytes and the expressions of CD39, CD73, and A2A/A2BARs, which decreased AC activity in melanocytes. RF irradiation downregulated the cAMP-mediated PKA/CREB/MITF pathway and tyrosinase activity, and these inhibitory effects can be mediated via CD39 inhibition.

Dataset Information

Attenuated, flow-induced ATP release contributes to absence of flow-sensitive, purinergic Cai2+ signaling in human ADPKD cyst epithelial cells.

Publications

Attenuated, flow-induced ATP release contributes to absence of flow-sensitive, purinergic Cai2+ signaling in human ADPKD cyst epithelial cells.

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