Connexin isoform expression in smooth muscle cells and endothelial cells of hamster cheek pouch arterioles and retractor feed arteries

Abstract

Objective: Gap junction channels formed by connexin (Cx) protein subunits enable cell-to-cell conduction of vasoactive signals. Given the lack of quantitative measurements of Cx expression in microvascular endothelial cells (EC) and smooth muscle cells (SMC), the objective was to determine whether Cx expression differed between EC and SMC of resistance microvessels for which conduction is well-characterized.

Methods: Cheek pouch arterioles (CPA) and retractor feed arteries (RFA) were hand-dissected and dissociated to obtain SMC or endothelial tubes. In complementary experiments, small intestine was dissociated to obtain SMC. Following reverse transcription, quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) was performed by using specific primers and fluorescent probes for Cx37, Cx40, and Cx43. Smooth muscle alpha-actin (SMAA) and platelet endothelial cell adhesion molecule-1 (PECAM-1) served as respective reference genes.

Results: Transcript copy numbers were similar for each Cx isoform in EC from CPA and RFA (approximately 0.5 Cx/PECAM-1). For SMC, Cx43 transcript in CPA and RFA (< 0.1 Cx/SMAA) was less (p < 0.05) than that in small intestine (approximately 0.4 Cx/SMAA). Transcripts for Cx37 and Cx40 were also detected in SMC. Punctate immunolabeling for each Cx isoform was pronounced at EC borders and that for Cx43 was pronounced in SMC of small intestine. In contrast, Cx immunolabeling was not detected in SMC of CPA or RFA.

Conclusions: Connexin expression occurs primarily within the endothelium of arterioles and feed arteries, supporting a highly effective pathway for conducting vasoactive signals along resistance networks. The apparent paucity of Cx expression within SMC underscores discrete homocellular coupling and focal localization of myoendothelial gap junctions.

Figure 1. Co-Immunolabeling for SMAA and Cx isoforms in SMC from CPA and RFA

Immunolabeling for SMAA (red) and Cx isoforms (green) in single dissociated SMC from CPA (rows A-C) and RFA (rows D-F). Connexin isoforms were not detected in SMC from either vessel while SMAA was present in all SMC. DIC images of each cell are shown at left. Insets: negative controls in the absence of primary antibodies. Scale bar = 20 μm and applies to all panels.

Figure 2. Co-Imunolabeling for SMAA and Cx isoforms in SMC of small intestine

Immunolabeling for SMAA (red) and Cx isoforms (green) in whole-mount preparations of muscularis externa from the jejunum (rows A-C) and from single dissociated SMC (row D). Each row contains the same observed field. Arrowheads point to punctuate Cx43 staining between SMC in the intact tissue (A) and at the cell border of a single SMC (D). Neither Cx40 nor Cx37 were detected. DIC images for each preparation are shown at left. Insets: Negative controls in the absence of respective primary antibodies. Scale bar in last panel of row C = 20 μm and applies to all panels in rows A-C. Scale bar in last panel of row D = 50 μm and applies to all panels in row D.

Figure 3. Immunolabeling for Cx isoforms in endothelial tubes

Immunolabeling for Cx isoforms within regions of endothelial tubes isolated from CPA (panels A-D) and RFA (panels E-H). Arrowheads point to labeling around borders of individual EC. The DIC images taken at lower magnification serve to illustrate the shape of representative intact endothelial tubes from CPA (A) and RFA (E) but do not correspond to the same fields of view as immunofluorescence. Note prominence of EC nuclei. Inset: negative controls in the absence of primary antibodies. Scale bar in panel D = 20 μm and applies to panels B and C; Scale bar in panel H = 20 μm and applies to F and G. Scale bars in panels A and E = 50 μm. See Figure 4 for orientation of endothelial tube tube relative to SMC.

Figure 4. Co-Immunolabeling for SMAA and Cx43 following partial dissociation of RFA

(A) DIC image showing endothelial tube in which SMC have been dissociated from half of the vessel segment while the remaining SMC have been “loosened’’ but still surround the endothelial tube. Panels B–D are higher magnification of area enclosed by upper box in panel A. (B) DIC image showing SMC remaining in register along the vessel wall. (C) Immunolabeling for SMAA (red) is restricted to SMC. (D) Immunoreactivity for Cx43 occurs only at borders of EC along endothelial tube surrounded by SMC in panel C. Panels E–G are higher magnification of area enclosed by lower box in panel A. (E) All SMC have been dissociated from this region of the isolated vessel segment, which consists entirely of endothelial tube. (F) No labeling for SMAA is apparent in endothelial tube. (G) Cx43 immunoreactivity at EC borders recapitulates that shown in panel D. Scale bar in A = 150 μm. Scale bar in panel G = 20 μm and applies to panels B–G.

Figure 5. Quantification of Cx isoform expression in smooth muscle of CPA, RFA and small intestine

A. Transcript copy numbers for Cx isoforms in SMC from CPA and RFA normalized to SMAA. For SMC of both microvessels, levels of Cx43 were significantly greater (* p<0.05) than for Cx40 or Cx37 (n = 5-9 independent determinations for each gene). B. Transcript copy numbers for Cx isoforms in SMC from small intestine normalized to SMAA. The level of Cx43 transcript was significantly greater (* p<0.025) than that for Cx40 or Cx37. Summary data are means ± S.E. from 6 independent determinations.

Figure 6. Quantification of Cx isoform expression in endothelium of CPA and RFA

Normalized to PECAM-1, there were no significant differences in respective levels of Cx37, Cx40, or Cx43 transcript in either CPA or RFA, nor was there a difference between respective vessels. Summary data are means ± S.E. from 4-5 independent determinations.

Figure 7. Stability of total RNA

Total RNA increased in direct proportion to the number of cells sampled. Summary data are means ± S.E. from four independent experiments.