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Clusters of specialized detector cells provide sensitive and high fidelity receptor signaling in the intact endothelium.

Wilson C, Saunter CD, Girkin JM, McCarron JG - FASEB J. (2016)

Bottom Line: D., Girkin, J.M., McCarron, J.Clusters of specialized detector cells provide sensitive and high fidelity receptor signaling in the intact endothelium.

View Article: PubMed Central - PubMed

Affiliation: Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, United Kingdom; and.

No MeSH data available.


Related in: MedlinePlus

ACh-evoked Ca2+ responses and distribution of muscarinic receptors. (A) ACh-evoked Ca2+ signaling in the en face endothelial preparation. A) i) Images illustrating a field of endothelial cells and Ca2+ rises (green) in response to ACh [3 nM (left), 30 nM (middle), and 300 nM (right)]. Ca2+ rises occurred in discrete clusters of endothelial cells at lower ACh concentrations and in the majority of the field at the higher concentration. The cells shown are average images (gray) with Ca2+ activity overlaid (green). ii) Line trace of the Ca2+ changes from the same fields and ACh concentrations shown in i. B) Summarized data showing the total number of ACh responsive cells at each ACh concentration (EC50 = 27.3 nM; 95% confidence interval, 18.0–41.2 nM; n = 4). C) Total endothelial responses (activity) derived from the product of the number of active cells and mean response at each concentration) (EC50 = 52.5 nM; 95% confidence interval, 41.0–97.1 nM; n = 4). D) Immunohistochemical localization of endothelial M3 muscarinic AChR M3 in the endothelium of en face arterial preparations. Representative image (top left) illustrating that AChR M3 distribution was not uniform across the endothelium but more densely clustered to discrete regions (D, top right; yellow lines). In the same preparation, nuclei of endothelial cells were labeled with DAPI (D, bottom left). An overlay (D, bottom right) of endothelial nuclei (blue) with AChRM3 (red) staining shows the clustered localization of AChRM3 to particular regions of endothelium (bottom right; yellow lines). E) Negative control obtained by omitting the anti-AChRM3 (E, top panel). DAPI loading (E, bottom panel) shows cell nuclei positions. F) AChRM3s have increased occurrence in sensitive cells activated by ACh. Left panel, total endothelial Ca2+ activity (green; evoked by 30 nM ACh, left panel) overlaid on cells (gray) in an en face preparation. Right panel, immunohistochemical localization of endothelial AChRM3 (red) in the same field of endothelium (right panel) previously activated with ACh. Nuclei are shown in blue (DAPI staining). G) Summary data showing that AChRM3 are more densely localized in sensitive regions of endothelium (i.e., green in F) compared with those regions that are less sensitive to ACh (n = 3). *P < 0.05. Scale bars, 50 μm.
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Figure 5: ACh-evoked Ca2+ responses and distribution of muscarinic receptors. (A) ACh-evoked Ca2+ signaling in the en face endothelial preparation. A) i) Images illustrating a field of endothelial cells and Ca2+ rises (green) in response to ACh [3 nM (left), 30 nM (middle), and 300 nM (right)]. Ca2+ rises occurred in discrete clusters of endothelial cells at lower ACh concentrations and in the majority of the field at the higher concentration. The cells shown are average images (gray) with Ca2+ activity overlaid (green). ii) Line trace of the Ca2+ changes from the same fields and ACh concentrations shown in i. B) Summarized data showing the total number of ACh responsive cells at each ACh concentration (EC50 = 27.3 nM; 95% confidence interval, 18.0–41.2 nM; n = 4). C) Total endothelial responses (activity) derived from the product of the number of active cells and mean response at each concentration) (EC50 = 52.5 nM; 95% confidence interval, 41.0–97.1 nM; n = 4). D) Immunohistochemical localization of endothelial M3 muscarinic AChR M3 in the endothelium of en face arterial preparations. Representative image (top left) illustrating that AChR M3 distribution was not uniform across the endothelium but more densely clustered to discrete regions (D, top right; yellow lines). In the same preparation, nuclei of endothelial cells were labeled with DAPI (D, bottom left). An overlay (D, bottom right) of endothelial nuclei (blue) with AChRM3 (red) staining shows the clustered localization of AChRM3 to particular regions of endothelium (bottom right; yellow lines). E) Negative control obtained by omitting the anti-AChRM3 (E, top panel). DAPI loading (E, bottom panel) shows cell nuclei positions. F) AChRM3s have increased occurrence in sensitive cells activated by ACh. Left panel, total endothelial Ca2+ activity (green; evoked by 30 nM ACh, left panel) overlaid on cells (gray) in an en face preparation. Right panel, immunohistochemical localization of endothelial AChRM3 (red) in the same field of endothelium (right panel) previously activated with ACh. Nuclei are shown in blue (DAPI staining). G) Summary data showing that AChRM3 are more densely localized in sensitive regions of endothelium (i.e., green in F) compared with those regions that are less sensitive to ACh (n = 3). *P < 0.05. Scale bars, 50 μm.

Mentions: The variation in sensitivity to ACh in different regions of the endothelium may be explained by variations in the density of the ACh receptor (AChR) population. Regions most sensitive to ACh had a higher density of the muscarinic AChR M3 (Fig. 5). In initial experiments, similarities in the pattern of sensitivity to ACh and the distribution of AChRM3 was noted (Fig. 5). Subsequently, the pattern of endothelial Ca2+ signaling evoked by ACh and the distribution of AChRM3 receptors was examined in the same artery. Those regions most sensitive to a half-maximal effective concentration of ACh (30 nM) had a significantly (P < 0.05) increased number of AChM3 receptors (Fig. 5).


Clusters of specialized detector cells provide sensitive and high fidelity receptor signaling in the intact endothelium.

Wilson C, Saunter CD, Girkin JM, McCarron JG - FASEB J. (2016)

ACh-evoked Ca2+ responses and distribution of muscarinic receptors. (A) ACh-evoked Ca2+ signaling in the en face endothelial preparation. A) i) Images illustrating a field of endothelial cells and Ca2+ rises (green) in response to ACh [3 nM (left), 30 nM (middle), and 300 nM (right)]. Ca2+ rises occurred in discrete clusters of endothelial cells at lower ACh concentrations and in the majority of the field at the higher concentration. The cells shown are average images (gray) with Ca2+ activity overlaid (green). ii) Line trace of the Ca2+ changes from the same fields and ACh concentrations shown in i. B) Summarized data showing the total number of ACh responsive cells at each ACh concentration (EC50 = 27.3 nM; 95% confidence interval, 18.0–41.2 nM; n = 4). C) Total endothelial responses (activity) derived from the product of the number of active cells and mean response at each concentration) (EC50 = 52.5 nM; 95% confidence interval, 41.0–97.1 nM; n = 4). D) Immunohistochemical localization of endothelial M3 muscarinic AChR M3 in the endothelium of en face arterial preparations. Representative image (top left) illustrating that AChR M3 distribution was not uniform across the endothelium but more densely clustered to discrete regions (D, top right; yellow lines). In the same preparation, nuclei of endothelial cells were labeled with DAPI (D, bottom left). An overlay (D, bottom right) of endothelial nuclei (blue) with AChRM3 (red) staining shows the clustered localization of AChRM3 to particular regions of endothelium (bottom right; yellow lines). E) Negative control obtained by omitting the anti-AChRM3 (E, top panel). DAPI loading (E, bottom panel) shows cell nuclei positions. F) AChRM3s have increased occurrence in sensitive cells activated by ACh. Left panel, total endothelial Ca2+ activity (green; evoked by 30 nM ACh, left panel) overlaid on cells (gray) in an en face preparation. Right panel, immunohistochemical localization of endothelial AChRM3 (red) in the same field of endothelium (right panel) previously activated with ACh. Nuclei are shown in blue (DAPI staining). G) Summary data showing that AChRM3 are more densely localized in sensitive regions of endothelium (i.e., green in F) compared with those regions that are less sensitive to ACh (n = 3). *P < 0.05. Scale bars, 50 μm.
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Figure 5: ACh-evoked Ca2+ responses and distribution of muscarinic receptors. (A) ACh-evoked Ca2+ signaling in the en face endothelial preparation. A) i) Images illustrating a field of endothelial cells and Ca2+ rises (green) in response to ACh [3 nM (left), 30 nM (middle), and 300 nM (right)]. Ca2+ rises occurred in discrete clusters of endothelial cells at lower ACh concentrations and in the majority of the field at the higher concentration. The cells shown are average images (gray) with Ca2+ activity overlaid (green). ii) Line trace of the Ca2+ changes from the same fields and ACh concentrations shown in i. B) Summarized data showing the total number of ACh responsive cells at each ACh concentration (EC50 = 27.3 nM; 95% confidence interval, 18.0–41.2 nM; n = 4). C) Total endothelial responses (activity) derived from the product of the number of active cells and mean response at each concentration) (EC50 = 52.5 nM; 95% confidence interval, 41.0–97.1 nM; n = 4). D) Immunohistochemical localization of endothelial M3 muscarinic AChR M3 in the endothelium of en face arterial preparations. Representative image (top left) illustrating that AChR M3 distribution was not uniform across the endothelium but more densely clustered to discrete regions (D, top right; yellow lines). In the same preparation, nuclei of endothelial cells were labeled with DAPI (D, bottom left). An overlay (D, bottom right) of endothelial nuclei (blue) with AChRM3 (red) staining shows the clustered localization of AChRM3 to particular regions of endothelium (bottom right; yellow lines). E) Negative control obtained by omitting the anti-AChRM3 (E, top panel). DAPI loading (E, bottom panel) shows cell nuclei positions. F) AChRM3s have increased occurrence in sensitive cells activated by ACh. Left panel, total endothelial Ca2+ activity (green; evoked by 30 nM ACh, left panel) overlaid on cells (gray) in an en face preparation. Right panel, immunohistochemical localization of endothelial AChRM3 (red) in the same field of endothelium (right panel) previously activated with ACh. Nuclei are shown in blue (DAPI staining). G) Summary data showing that AChRM3 are more densely localized in sensitive regions of endothelium (i.e., green in F) compared with those regions that are less sensitive to ACh (n = 3). *P < 0.05. Scale bars, 50 μm.
Mentions: The variation in sensitivity to ACh in different regions of the endothelium may be explained by variations in the density of the ACh receptor (AChR) population. Regions most sensitive to ACh had a higher density of the muscarinic AChR M3 (Fig. 5). In initial experiments, similarities in the pattern of sensitivity to ACh and the distribution of AChRM3 was noted (Fig. 5). Subsequently, the pattern of endothelial Ca2+ signaling evoked by ACh and the distribution of AChRM3 receptors was examined in the same artery. Those regions most sensitive to a half-maximal effective concentration of ACh (30 nM) had a significantly (P < 0.05) increased number of AChM3 receptors (Fig. 5).

Bottom Line: D., Girkin, J.M., McCarron, J.Clusters of specialized detector cells provide sensitive and high fidelity receptor signaling in the intact endothelium.

View Article: PubMed Central - PubMed

Affiliation: Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, United Kingdom; and.

No MeSH data available.


Related in: MedlinePlus