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First published online October 22, 2008
doi: 10.1242/10.1242/jcs.037481

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Localized expression of an Ins(1,4,5)P₃ receptor at the myoendothelial junction selectively regulates heterocellular Ca²⁺ communication

Robert M. Berne Cardiovascular Research Center, and Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, P.O. Box 801394, Charlottesville, VA 22908, USA

View larger version (78K): [in this window] [in a new window]   Fig. 1. Phenotypic verification and connexin expression in cremasteric vascular-cell co-culture. En face staining for acetylated low-density lipoprotein (LDL) demonstrated ECs on the top of the Transwell insert, with no observed staining for -actin (A). (A) Transverse sections of the VCCC (top is ECs, bottom is VSMCs) double stained with phalloidin (F-actin, green) and VE-cadherin (red) demonstrated distinct EC localization in the pores of the Transwell. In the adjacent immunoblot, CD31 stained only the EC isolates. (B) When examining VSMCs en face, acetylated LDL was not detected; however, -actin was found throughout the monolayers. Transverse sections of VCCC revealed VSMC extensions, which stained with desmin (red), following the F-actin (green) within the pores of the Transwell. The immunoblot, which was probed for desmin, demonstrates that only VSMC isolates express this phenotypic marker. (C-F) In transverse sections of the cremasteric VCCCs, Cx37 (C), Cx40 (D), Cx43 (E) and Cx45 (F) were observed. Arrows demonstrate connexin expression within the pores of the Transwell. (G) Single pores of the Transwell were double stained for both Cx40 (red) and Cx43 (green) to demonstrate a lack of re-arrangement of connexins at the in vitro MEJ when Cx43 or Cx40 was eliminated. Scale bars: 75 µm (en face view in A,B); 10 µm (transverse view from A,B; C-F); 2.5 µm (G).

View larger version (25K): [in this window] [in a new window]   Fig. 2. Effects of different connexins on Ca2+ communication between ECs and VSMCs. In the cartoon at the top of the figure, the pipette indicates the stimulated cell type (ECs top, VSMCs bottom). In control conditions (A,B), stimulation of ECs (A) resulted in an increase in EC [Ca2+]i and, after a short delay, an increase in VSMC [Ca2+]i. (B) Stimulation of VSMCs resulted in an increase in VSMC [Ca2+]i and, after a short delay, an increase in EC [Ca2+]i. (C,D) When ECs from Cx40–/– animals were used, stimulation of ECs (C) resulted in an increase in VSMC [Ca2+]i and stimulation of VSMCs (D) caused an increase in EC [Ca2+]i. (E,F) ECs with Cx43 siRNA were also tested and EC stimulation of these cells resulted in an increase in VSMC [Ca2+]i (E). Stimulation of VSMCs increased EC [Ca2+]i (F). (G,H) When both Cx40 and Cx43 were deleted from the ECs, stimulation of ECs resulted in an increase in EC [Ca2+]i, but not VSMC [Ca2+]i (G), and stimulation of VSMCs caused only an increase in VSMC [Ca2+]i, and not EC [Ca2+]i (H). (I,J) This observation was also evident when Cx43 was deleted from VSMCs: after stimulation of ECs, there was no increase in [Ca2+]i in VSMCs (I) and, after stimulation of VSMCs, no increase in EC [Ca2+]i was observed (J). (K,L) Lastly, when the gap-junction inhibitor 18 -GA was used, stimulation of ECs was unable to produce an increase in VSMC [Ca2+]i (K) and stimulation of VSMCs was unable to produce an increase in EC [Ca2+]i (L). *P<0.05.

View larger version (36K): [in this window] [in a new window]   Fig. 3. Directional Ins(1,4,5)P3 persists regardless of connexin deletion. In the cartoons on the left, the pipette indicates the stimulated cell type (EC top, VSMC bottom), blue represents BAPTA and green represents XPC. In control conditions (A), stimulation of BAPTA-loaded ECs resulted in no increase in [Ca2+]i in either cell type. There was also no increase in [Ca2+]i in either cell type when ECs with Cx43 siRNA that were loaded with BAPTA were stimulated (B), or when Cx40–/– ECs were loaded with BAPTA and stimulated (C). However, after stimulation of VSMCs loaded with BAPTA in control conditions (D), ECs responded with an increase in [Ca2+]i. When Cx43 siRNA was added to the ECs (E), an increase in EC [Ca2+]i was still observed after VSMC stimulation. When ECs from Cx40–/– mice were used (F), an increase in EC [Ca2+]i was observed after VSMC stimulation. The addition of XPC to the ECs (G-I) eliminated the increase in EC [Ca2+]i after VSMC stimulation in control conditions (G), when Cx43 siRNA was applied to ECs (H) or when Cx40–/– ECs were used (I). *P<0.05.

View larger version (25K): [in this window] [in a new window]   Fig. 4. Ins(1,4,5)P3-R1 is selectively localized to the EC side of the myoendothelial junction in vivo. Immunocytochemistry on cremasteric muscles (A,C,E) and immunoblots of freshly isolated cremasteric ECs and VSMCs (cre-EC, cre-VSMC; B,D,F) were stained with Ins(1,4,5)P3-R1 (A,B), Ins(1,4,5)P3-R2 (C,D) or Ins(1,4,5)P3-R3 (E,F). (A,C,E) Red is the Ins(1,4,5)P3-R isoform and green is the autofluorescence of the internal elastic lamina. (G) Using protein quantification of antibody detection on actin bridges from in situ cremasteric arterioles (e.g. Isakson et al., 2008), only the Ins(1,4,5)P3-R1 isoform was present in significant quantities. *P<0.05. (H) At the TEM level, Ins(1,4,5)P3-R1 (10-nm gold particles) was localized to the EC side of the MEJ, but not the VSMC (insert of enlarged MEJ; H). Scale bars: 20 µm (A,C,E); 2 µm (H), 1 µm (insert in H).

View larger version (68K): [in this window] [in a new window]   Fig. 5. Selective knockdown of the Ins(1,4,5)P3-R1 isoform from the ECs by using the cremasteric VCCC. (A) Immunoblots of ECs or VSMCs from the VCCC that were stained for Ins(1,4,5)P3-R1, Ins(1,4,5)P3-R2 or Ins(1,4,5)P3-R3. Representative β-actin staining from stripped immunoblots is also shown. Experiments included lanes loaded with samples from control conditions, the addition of Ins(1,4,5)P3-R1 siRNA to ECs only, or the addition of control siRNA (c-siRNA) to ECs only. (B) Quantification of ratioed immunoblots. *P<0.05. (C,D) Immunostain for Ins(1,4,5)P3-R1 on transverse sections of cremasteric VCCC before (C) and after (D) application of Ins(1,4,5)P3-R1 siRNA to the ECs; note the sparse staining for Ins(1,4,5)P3-R1 in the ECs, but the isoform is still observed in the VSMCs. Arrows demonstrate Ins(1,4,5)P3-R1 expression within the pores of the Transwell. (E,F) On the cremasteric VCCC, Ins(1,4,5)P3-R2 and Ins(1,4,5)P3-R3 show expression within the monolayers of the cells only. Scale bar: 10 µm.

View larger version (36K): [in this window] [in a new window]   Fig. 6. Selective knockdown of the Ins(1,4,5)P3-R1 isoform eliminates the Ins(1,4,5)P3 response in ECs. In the cartoons on the left, the pipette indicates the stimulated cell type (EC top, VSMC bottom), blue is BAPTA and `+' indicates the cell type that is loaded with siRNA. (A) When Ins(1,4,5)P3-R1 was selectively deleted from the ECs, stimulation of BAPTA-loaded VSMCs no longer elicited an increase in EC [Ca2+]i. (B) Knockdown of Ins(1,4,5)P3-R2 and Ins(1,4,5)P3-R3 using siRNA is demonstrated by the immunoblot, with β-actin loading controls shown directly underneath. (C,D) When Ins(1,4,5)P3-R2 was knocked down, a polarized increase in EC [Ca2+]i was still present (C), and the deletion of Ins(1,4,5)P3-R3 also failed to inhibit increases in EC [Ca2+]i (D). *P<0.05.

View larger version (33K): [in this window] [in a new window]   Fig. 7. Metabolic degradation of Ins(1,4,5)P3 determines directional responses. In the cartoons, the pipette indicates the stimulated cell type (EC top, VSMC bottom), blue is BAPTA, yellow is 5-PI, orange is XPC with 5-PI and `+' indicates the cell type that is loaded with siRNA. (A,B) BAPTA-loaded VSMCs were stimulated. When 5-PI was loaded into ECs treated with Ins(1,4,5)P3-R1 siRNA, an increase in EC [Ca2+]i was observed (A) and, when XPC was added to these cells, an increase in EC [Ca2+]i was no longer present (B). (C,D) BAPTA-loaded ECs were stimulated. With the addition of 5-PI to VSMCs, an increase in VSMC [Ca2+]i was observed after EC stimulation (C) and, when XPC was added to the VSMCs with 5-PI, the VSMC Ca2+ response to EC stimulation was abolished (D). *P<0.05. (E) In transverse sections of the cremasteric VCCC, pores of the Transwell demonstrate the presence of Ins(1,4,5)P3-R1 (green) and 5-phosphatase (red). The vertical white line indicates the location of the line scan. (F) The line scans correspond to the fluorescent intensity from each of the proteins tested in E. Scale bar: 10 µm.

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