Human Keratinocytes Express Multiple P2Y-Receptors: Evidence for Functional P2Y1, P2Y2, and P2Y4 Receptors Helen E. Burrell, Wayne B. Bowler, James A.

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Presentation transcript:

Human Keratinocytes Express Multiple P2Y-Receptors: Evidence for Functional P2Y1, P2Y2, and P2Y4 Receptors Helen E. Burrell, Wayne B. Bowler, James A. Gallagher, Graham R. Sharpe Journal of Investigative Dermatology Volume 120, Issue 3, Pages 440-447 (March 2003) DOI: 10.1046/j.1523-1747.2003.12050.x Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 NHK express mRNA for P2Y1, P2Y2, P2Y4, and P2Y6 receptors. PCR products for P2Y1 (237bp), P2Y2 (344bp), P2Y4 (411bp), and P2Y6 (463bp) after 35 cycles at 58°C were obtained using cDNA from NHK, and cDNA integrity was confirmed by amplification of GAPDH (520bp) after 35 cycles at 60°C. Journal of Investigative Dermatology 2003 120, 440-447DOI: (10.1046/j.1523-1747.2003.12050.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 HaCaT cells express mRNA for P2Y1, P2Y2, P2Y4, and P2Y6 receptors. PCR products for P2Y1 (237bp), P2Y2 (344bp), P2Y4 (411bp), and P2Y6 (463bp) after 35 cycles at 58°C were obtained using cDNA from HaCaT cells, and cDNA integrity was confirmed by amplification of GAPDH (520bp) after 35 cycles at 60°C. Journal of Investigative Dermatology 2003 120, 440-447DOI: (10.1046/j.1523-1747.2003.12050.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 P2Y2 and P2Y4 receptor expression is downregulated in differentiated NHK after being cultured in elevated Ca2+ for 24h. NHK, which are normally cultured in medium containing 70μM Ca2+, were compared to differentiated NHK, which had been cultured for 24h in the presence of 200μM and 1mM Ca2+. Control lanes are NHK in 70μM Ca2+ prior to Ca2+ treatment (0 h). PCR products for P2Y1 (237bp), P2Y2 (344bp), P2Y4 (411bp), and P2Y6 (463bp) after 35 cycles at 58°C and GAPDH (520bp) after 30 cycles at 60°C were obtained using cDNA from NHK. Journal of Investigative Dermatology 2003 120, 440-447DOI: (10.1046/j.1523-1747.2003.12050.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 ATP and UTP elevate Cai by equal amounts in NHK. NHK were loaded with calcium-free fura-2 by incubation with 5μM fura-2AM for 30min. Measurements were made from single cells within colonies of approximately four to eight cells. The ratio of emitted light at 510nm was recorded after excitation at 350nm and 380nm. Baseline Cai measurements were recorded for 60s prior to addition of ADP, ATP, or UTP (10μM). The graph shows a typical response observed in six different cells. Journal of Investigative Dermatology 2003 120, 440-447DOI: (10.1046/j.1523-1747.2003.12050.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 ATP and UTP increase cell number by approximately equal amounts in comparison with the control in NHK. To investigate the cellular consequences of an increase in Cai on NHK, proliferation assays involving cell counting were carried out. NHK were plated out at 1×105 cells per 6cm Petri dish and allowed to settle for 72h prior to stimulation. ATP (diagonal hatching) and UTP (solid shading) (0.1–10μM) were added for a further 24h before cell counting with a Coulter counter model ZM. Cell numbers are expressed as mean cell number (no shading) (n=4 for each treatment, and results are typical of responses observed in four separate experiments). Increased proliferation is inferred by the increase in cell numbers in comparison with the control. The asterisk denotes significance p<0.01 (Student's t test). Journal of Investigative Dermatology 2003 120, 440-447DOI: (10.1046/j.1523-1747.2003.12050.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 ATP and UTP dose-dependently increased Cai in HaCaT cells. ATP and UTP (1–10μM) were introduced to HaCaT cells loaded with calcium-free fura-2 by incubation with 5μM fura-2AM for 30min. Measurements were made from single cells within colonies of approximately four to eight cells, and the ratio of emitted light at 510nm was recorded after excitation at 350nm and 380nm. Baseline measurements were recorded for 60s prior to addition of extracellular nucleotides. Ratios were converted into Cai concentrations by comparison with a calibration curve obtained using standard concentrations of calcium, and the baseline measurements were subtracted from the peak values. Results are expressed as mean ±SEM [n=7 for each UTP concentration (solid shading) and n = 4 for each ATP concentration (diagonal hatching)]. Journal of Investigative Dermatology 2003 120, 440-447DOI: (10.1046/j.1523-1747.2003.12050.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 P2Y4 receptors but not P2Y6 are functional in HaCaT cells. ATP (5–10μM) followed by UDP (10μM) and UTP (10μM) were introduced to fura-2 loaded HaCaT cells. Measurements were made from single cells for approximately 100s before addition of the next extracellular nucleotide. The ratio of emitted light at 510nm was recorded after excitation at 350nm and 380nm. Similar results were obtained from n=13 cells. Journal of Investigative Dermatology 2003 120, 440-447DOI: (10.1046/j.1523-1747.2003.12050.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 ATP and UTP significantly increase cell numbers in HaCaT cells in comparison with the control. HaCaT cells were seeded at a density of 1×105 cells per 6cm Petri dish and allowed to settle for 24h prior to stimulation by ATP or UTP (0.1–10μM). Proliferation was determined using a Coulter counter (model ZM) to obtain mean cell numbers after a further 24h incubation with agonist. Figures are representative of four separate studies. An asterisk denotes significant differences from control at p<0.05, a double asterisk denotes significant differences at p<0.005, n=8 for controls and n=4 for treatments (Student's t test). Journal of Investigative Dermatology 2003 120, 440-447DOI: (10.1046/j.1523-1747.2003.12050.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 9 P2Y1 receptors are functional and coexpressed with either P2Y2 or P2Y4 receptors in HaCaT cells. 2MeSADP (1μM) was introduced to fura-2 loaded HaCaT cells, and the ratio of emitted light at 510nm was recorded from single cells after excitation at 350nm and 380nm. Following stimulation by 2MeSADP, the same concentration of UTP (1μM) was introduced. Results are typical of n=6 similar recordings. Journal of Investigative Dermatology 2003 120, 440-447DOI: (10.1046/j.1523-1747.2003.12050.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 10 P2Y2 receptors are functional in HaCaT cells. Equimolar concentrations of UTP and ATP (5μM) were introduced to fura-2 loaded HaCaT cells and measurements of the ratio of emitted light at 510nm was recorded in single cells after excitation at 350nm and 380nm. A higher concentration of ADP (10μM) was added to examine the coexpression of P2Y2 and P2Y1 receptors. Results are typical of n=4 similar recordings. Journal of Investigative Dermatology 2003 120, 440-447DOI: (10.1046/j.1523-1747.2003.12050.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 11 UTP induces calcium oscillations in HaCaT cells. HaCaT cells were loaded with calcium-free fura-2 by incubation with 5μM fura-2AM for 30min and stimulated with UTP. Measurements were made from single cells within colonies of approximately four to eight cells. The ratio of emitted light at 510nm was recorded after excitation at 350nm and 380nm. Cells were allowed to oscillate for at least four cycles before re-stimulation with an increasing concentration of agonist. Results are typical of n=15 similar recordings. Journal of Investigative Dermatology 2003 120, 440-447DOI: (10.1046/j.1523-1747.2003.12050.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 12 Thapsigargin causes cessation of calcium oscillations in HaCaT cells. Fura-2 loaded HaCaT cells were stimulated with ATP or UTP (1–10μM) in order to induce calcium oscillations, and thapsigargin (1μM) was added immediately after the Cai level had returned to the baseline. Measurements were made from single cells and the ratio of emitted light at 510nm was recorded after excitation at 350nm and 380nm. Results are typical of n=3 similar recordings. Journal of Investigative Dermatology 2003 120, 440-447DOI: (10.1046/j.1523-1747.2003.12050.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions