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Defining the roles of arrestin2 and arrestin3 in vasoconstrictor receptor desensitization in hypertension.

Willets JM, Nash CA, Rainbow RD, Nelson CP, Challiss RA - Am. J. Physiol., Cell Physiol. (2015)

Bottom Line: Desensitization of UTP-stimulated vessel contractions was increased in 12-wk-old (but not 6-wk-old) SHR animals.In conclusion, arrestin2 and 3 expression is elevated in resistance arteries during the emergence of the early hypertensive phenotype, which underlies an enhanced ability to desensitize vasoconstrictor signaling and vessel contraction.Such regulatory changes may act to compensate for increased vasoconstrictor-induced vessel contraction.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, United Kingdom; and jmw23@leicester.ac.uk.

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Assessment of ET1 and UTP-stimulated phospholipase C (PLC) signaling desensitization in SHR and WKY MSMC from 6-wk-old animals. MSMC were transfected with pleckstrin homology domain of PLCδ1 tagged with enhanced green fluorescent protein (eGFP-PH; 0.5 μg) before being subjected to the following desensitization protocols: For ET1, MSMC were stimulated with ET1 (50 nM, 30 s; R1); with 5 min washout before a second challenge (50 nM, 30 s; R2), while for UTP, MSMC were challenged with an ∼EC50 UTP concentration (10 μM) for 30 s before (R1) and after (R2) addition of a maximal UTP concentration (Rmax: 100 μM, for 1 min). Representative traces are shown from single cells isolated from WKY (A and C) and SHR (B and D) treated with either ET1 (A and B) or UTP (C and D). Receptor desensitization was determined as the relative change in R2 response compared with R1. Cumulative data (E) are expressed as means ± SE for the % change in R2 relative to R1; n = 7–20 cells from ≥6 separate animals. IP3, inositol 1,4,5-trisphosphate.
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Figure 5: Assessment of ET1 and UTP-stimulated phospholipase C (PLC) signaling desensitization in SHR and WKY MSMC from 6-wk-old animals. MSMC were transfected with pleckstrin homology domain of PLCδ1 tagged with enhanced green fluorescent protein (eGFP-PH; 0.5 μg) before being subjected to the following desensitization protocols: For ET1, MSMC were stimulated with ET1 (50 nM, 30 s; R1); with 5 min washout before a second challenge (50 nM, 30 s; R2), while for UTP, MSMC were challenged with an ∼EC50 UTP concentration (10 μM) for 30 s before (R1) and after (R2) addition of a maximal UTP concentration (Rmax: 100 μM, for 1 min). Representative traces are shown from single cells isolated from WKY (A and C) and SHR (B and D) treated with either ET1 (A and B) or UTP (C and D). Receptor desensitization was determined as the relative change in R2 response compared with R1. Cumulative data (E) are expressed as means ± SE for the % change in R2 relative to R1; n = 7–20 cells from ≥6 separate animals. IP3, inositol 1,4,5-trisphosphate.

Mentions: The extent of ET1- and UTP-induced PLC signaling desensitization was examined in MSMC transfected with eGFP-PH (0.5 μg) and loaded with the Ca2+-sensitive dye Fura-Red (4 μM, 60 min) to allow simultaneous measurement of changes in cellular IP3 and [Ca2+]i, using standard desensitization protocols (21, 22). For ET1, MSMC were challenged with a short desensitizing pulse of ET1 (50 nM, for 30 s, R1) followed by a wash period (5 min) and then a second ET1 challenge (50 nM, 30 s, R2). For cells prepared from 6-wk-old animals, initial ET1 challenge induced rapid, but transient translocations of eGFP-PH from the plasma membrane to the cytoplasm. Changes in eGFP-PH fluorescence were paralleled by changes in Fura-Red fluorescence, and responses returned to baseline within 5 min of agonist washout (Fig. 5, A and B). Changes in eGFP-PH and Fura-Red signals were reduced during a second (R2) ET1 challenge, indicative of diminished IP3 and Ca2+ increases in response to agonist re-challenge. For UTP, MSMC were challenged with a submaximal (approximate EC50) concentration of UTP (10 μM), for 30 s before (R1) and after (R2) addition of a maximal UTP concentration (100 μM, for 1 min, Rmax; to induce receptor desensitization). R2 values were attenuated compared with R1, indicating a desensitization of UTP-stimulated PLC signaling (Fig. 5, C and D).


Defining the roles of arrestin2 and arrestin3 in vasoconstrictor receptor desensitization in hypertension.

Willets JM, Nash CA, Rainbow RD, Nelson CP, Challiss RA - Am. J. Physiol., Cell Physiol. (2015)

Assessment of ET1 and UTP-stimulated phospholipase C (PLC) signaling desensitization in SHR and WKY MSMC from 6-wk-old animals. MSMC were transfected with pleckstrin homology domain of PLCδ1 tagged with enhanced green fluorescent protein (eGFP-PH; 0.5 μg) before being subjected to the following desensitization protocols: For ET1, MSMC were stimulated with ET1 (50 nM, 30 s; R1); with 5 min washout before a second challenge (50 nM, 30 s; R2), while for UTP, MSMC were challenged with an ∼EC50 UTP concentration (10 μM) for 30 s before (R1) and after (R2) addition of a maximal UTP concentration (Rmax: 100 μM, for 1 min). Representative traces are shown from single cells isolated from WKY (A and C) and SHR (B and D) treated with either ET1 (A and B) or UTP (C and D). Receptor desensitization was determined as the relative change in R2 response compared with R1. Cumulative data (E) are expressed as means ± SE for the % change in R2 relative to R1; n = 7–20 cells from ≥6 separate animals. IP3, inositol 1,4,5-trisphosphate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525080&req=5

Figure 5: Assessment of ET1 and UTP-stimulated phospholipase C (PLC) signaling desensitization in SHR and WKY MSMC from 6-wk-old animals. MSMC were transfected with pleckstrin homology domain of PLCδ1 tagged with enhanced green fluorescent protein (eGFP-PH; 0.5 μg) before being subjected to the following desensitization protocols: For ET1, MSMC were stimulated with ET1 (50 nM, 30 s; R1); with 5 min washout before a second challenge (50 nM, 30 s; R2), while for UTP, MSMC were challenged with an ∼EC50 UTP concentration (10 μM) for 30 s before (R1) and after (R2) addition of a maximal UTP concentration (Rmax: 100 μM, for 1 min). Representative traces are shown from single cells isolated from WKY (A and C) and SHR (B and D) treated with either ET1 (A and B) or UTP (C and D). Receptor desensitization was determined as the relative change in R2 response compared with R1. Cumulative data (E) are expressed as means ± SE for the % change in R2 relative to R1; n = 7–20 cells from ≥6 separate animals. IP3, inositol 1,4,5-trisphosphate.
Mentions: The extent of ET1- and UTP-induced PLC signaling desensitization was examined in MSMC transfected with eGFP-PH (0.5 μg) and loaded with the Ca2+-sensitive dye Fura-Red (4 μM, 60 min) to allow simultaneous measurement of changes in cellular IP3 and [Ca2+]i, using standard desensitization protocols (21, 22). For ET1, MSMC were challenged with a short desensitizing pulse of ET1 (50 nM, for 30 s, R1) followed by a wash period (5 min) and then a second ET1 challenge (50 nM, 30 s, R2). For cells prepared from 6-wk-old animals, initial ET1 challenge induced rapid, but transient translocations of eGFP-PH from the plasma membrane to the cytoplasm. Changes in eGFP-PH fluorescence were paralleled by changes in Fura-Red fluorescence, and responses returned to baseline within 5 min of agonist washout (Fig. 5, A and B). Changes in eGFP-PH and Fura-Red signals were reduced during a second (R2) ET1 challenge, indicative of diminished IP3 and Ca2+ increases in response to agonist re-challenge. For UTP, MSMC were challenged with a submaximal (approximate EC50) concentration of UTP (10 μM), for 30 s before (R1) and after (R2) addition of a maximal UTP concentration (100 μM, for 1 min, Rmax; to induce receptor desensitization). R2 values were attenuated compared with R1, indicating a desensitization of UTP-stimulated PLC signaling (Fig. 5, C and D).

Bottom Line: Desensitization of UTP-stimulated vessel contractions was increased in 12-wk-old (but not 6-wk-old) SHR animals.In conclusion, arrestin2 and 3 expression is elevated in resistance arteries during the emergence of the early hypertensive phenotype, which underlies an enhanced ability to desensitize vasoconstrictor signaling and vessel contraction.Such regulatory changes may act to compensate for increased vasoconstrictor-induced vessel contraction.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, United Kingdom; and jmw23@leicester.ac.uk.

Show MeSH
Related in: MedlinePlus