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Alpha(1D)-adrenergic receptor insensitivity is associated with alterations in its expression and distribution in cultured vascular myocytes.

Fan LL, Ren S, Zhou H, Wang Y, Xu PX, He JQ, Luo DL - Acta Pharmacol. Sin. (2009)

Bottom Line: In the cultured aortic myocytes, however, in addition to a marked decrease in their protein expression relative to the aorta, most labeling signals for alpha(1D)-ARs were found in the cytoplasm.Importantly, treating the culture medium with charcoal/dextran caused the reappearance of alpha(1D)-ARs at the cell surface and a partial restoration of the Ca(2+) signal response to PE in approximately 30% of the cultured cells.Reduction in alpha(1D)-AR total protein expression and disappearance from the cell surface contribute to the insensitivity of cultured vascular smooth muscle cells to alpha(1)-adrenergic receptor activation.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pharmacology, Capital Medical University, Beijing, China.

ABSTRACT

Aim: It is unclear why alpha(1D)-adrenergic receptors (alpha(1D)-ARs) play a critical role in the mediation of peripheral vascular resistance and blood pressure in situ but function inefficiently when studied in vitro. The present study examined the causes for these inconsistencies in native alpha(1)-adrenergic functional performance between the vascular smooth muscle and myocytes.

Methods: The alpha(1)-adrenergic mediated contraction, Ca(2+) signaling and the subcellular receptor distribution were evaluated using the Fluo-4, BODIPY-FL prazosin and subtype-specific antibodies.

Results: Rat aortic rings and freshly dissociated myocytes displayed contractile and increased intracellular Ca(2+) responses to stimulation with phenylephrine (PE, 10 micromol), respectively. However, the PE-induced responses disappeared completely in cultured aortic myocytes, whereas PE-enhanced Ca(2+) transients were seen in cultured rat cardiac myocytes. Further studies indicated that alpha(1D)-ARs, the major receptor subtype responsible for the alpha(1)-adrenergic regulation of aortic contraction, were distributed both intracellularly and at the cell membrane in freshly dispersed aortic myocytes, similar to the alpha(1A)-AR subcellular localization in the cultured cardiomyocytes. In the cultured aortic myocytes, however, in addition to a marked decrease in their protein expression relative to the aorta, most labeling signals for alpha(1D)-ARs were found in the cytoplasm. Importantly, treating the culture medium with charcoal/dextran caused the reappearance of alpha(1D)-ARs at the cell surface and a partial restoration of the Ca(2+) signal response to PE in approximately 30% of the cultured cells.

Conclusion: Reduction in alpha(1D)-AR total protein expression and disappearance from the cell surface contribute to the insensitivity of cultured vascular smooth muscle cells to alpha(1)-adrenergic receptor activation.

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Differences in contractile and intracellular Ca2+ signal responses to α1-adrenergic receptor activation between the rat thoracic aorta and cultured aortic smooth muscle cells. (A) Stimulating rat aortic rings with PE or high KCl concentration (80 mmol) (indicated with an arrow) induced a tonic force in Ca2+ containing HBSS. Pretreatment of the rings with 1 μmol prazosin for 10 min abolished the effect of PE, but not that of high KCl. (B) PE or Ang II caused a transient aortic contraction in Ca2+-free medium, followed by a tonic constriction upon Ca2+ addition to the medium. (C and D) A [Ca2+]i increase upon stimulation with high KCl (C) or Ang II (D), but not with PE, in cultured aortic VSMCs loaded with fluo4. Numbers for each curve in all panels ranged from 6 to 12 separate experiments.
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fig1: Differences in contractile and intracellular Ca2+ signal responses to α1-adrenergic receptor activation between the rat thoracic aorta and cultured aortic smooth muscle cells. (A) Stimulating rat aortic rings with PE or high KCl concentration (80 mmol) (indicated with an arrow) induced a tonic force in Ca2+ containing HBSS. Pretreatment of the rings with 1 μmol prazosin for 10 min abolished the effect of PE, but not that of high KCl. (B) PE or Ang II caused a transient aortic contraction in Ca2+-free medium, followed by a tonic constriction upon Ca2+ addition to the medium. (C and D) A [Ca2+]i increase upon stimulation with high KCl (C) or Ang II (D), but not with PE, in cultured aortic VSMCs loaded with fluo4. Numbers for each curve in all panels ranged from 6 to 12 separate experiments.

Mentions: To investigate the native α1AR function at the tissue level, the non-subtype selective α1AR agonist phenylephrine (PE)-induced vasoconstriction was evaluated in rat aortic rings. Similar to the response induced by 80 mmol KCl depolarization, PE (10 μmol) evoked a strong tonic constriction in the vessels, which was completely abolished by prazosin, a specific antagonist for α1ARs (Figure 1A). In a Ca2+-free medium, a transient contractile response to PE was followed by a relaxation to the baseline level, suggesting an internal Ca2+ release-related constriction. Upon the re-addition of extracellular CaCl2 (2.0 mmol) in the presence of PE, further sustained force was induced (Figure 1B). This force could be decreased significantly by nifedipine (1 μmol, data not shown), demonstrating an extracellular Ca2+-dependent constriction of this part. Additionally, as a reference for vasoconstriction regulation among GPCR members, angiotensin II (Ang II), an agonist of the angiotensin receptor, was also examined in this study; Ang II produced similar responses to PE in the rings (Figure 1B). Therefore, these data demonstrate a common characteristic of Ca2+-dependent vasoconstriction by the activation of two different GPCR members, consistent with previous studies on arteries25, 27, 28.


Alpha(1D)-adrenergic receptor insensitivity is associated with alterations in its expression and distribution in cultured vascular myocytes.

Fan LL, Ren S, Zhou H, Wang Y, Xu PX, He JQ, Luo DL - Acta Pharmacol. Sin. (2009)

Differences in contractile and intracellular Ca2+ signal responses to α1-adrenergic receptor activation between the rat thoracic aorta and cultured aortic smooth muscle cells. (A) Stimulating rat aortic rings with PE or high KCl concentration (80 mmol) (indicated with an arrow) induced a tonic force in Ca2+ containing HBSS. Pretreatment of the rings with 1 μmol prazosin for 10 min abolished the effect of PE, but not that of high KCl. (B) PE or Ang II caused a transient aortic contraction in Ca2+-free medium, followed by a tonic constriction upon Ca2+ addition to the medium. (C and D) A [Ca2+]i increase upon stimulation with high KCl (C) or Ang II (D), but not with PE, in cultured aortic VSMCs loaded with fluo4. Numbers for each curve in all panels ranged from 6 to 12 separate experiments.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4007500&req=5

fig1: Differences in contractile and intracellular Ca2+ signal responses to α1-adrenergic receptor activation between the rat thoracic aorta and cultured aortic smooth muscle cells. (A) Stimulating rat aortic rings with PE or high KCl concentration (80 mmol) (indicated with an arrow) induced a tonic force in Ca2+ containing HBSS. Pretreatment of the rings with 1 μmol prazosin for 10 min abolished the effect of PE, but not that of high KCl. (B) PE or Ang II caused a transient aortic contraction in Ca2+-free medium, followed by a tonic constriction upon Ca2+ addition to the medium. (C and D) A [Ca2+]i increase upon stimulation with high KCl (C) or Ang II (D), but not with PE, in cultured aortic VSMCs loaded with fluo4. Numbers for each curve in all panels ranged from 6 to 12 separate experiments.
Mentions: To investigate the native α1AR function at the tissue level, the non-subtype selective α1AR agonist phenylephrine (PE)-induced vasoconstriction was evaluated in rat aortic rings. Similar to the response induced by 80 mmol KCl depolarization, PE (10 μmol) evoked a strong tonic constriction in the vessels, which was completely abolished by prazosin, a specific antagonist for α1ARs (Figure 1A). In a Ca2+-free medium, a transient contractile response to PE was followed by a relaxation to the baseline level, suggesting an internal Ca2+ release-related constriction. Upon the re-addition of extracellular CaCl2 (2.0 mmol) in the presence of PE, further sustained force was induced (Figure 1B). This force could be decreased significantly by nifedipine (1 μmol, data not shown), demonstrating an extracellular Ca2+-dependent constriction of this part. Additionally, as a reference for vasoconstriction regulation among GPCR members, angiotensin II (Ang II), an agonist of the angiotensin receptor, was also examined in this study; Ang II produced similar responses to PE in the rings (Figure 1B). Therefore, these data demonstrate a common characteristic of Ca2+-dependent vasoconstriction by the activation of two different GPCR members, consistent with previous studies on arteries25, 27, 28.

Bottom Line: In the cultured aortic myocytes, however, in addition to a marked decrease in their protein expression relative to the aorta, most labeling signals for alpha(1D)-ARs were found in the cytoplasm.Importantly, treating the culture medium with charcoal/dextran caused the reappearance of alpha(1D)-ARs at the cell surface and a partial restoration of the Ca(2+) signal response to PE in approximately 30% of the cultured cells.Reduction in alpha(1D)-AR total protein expression and disappearance from the cell surface contribute to the insensitivity of cultured vascular smooth muscle cells to alpha(1)-adrenergic receptor activation.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pharmacology, Capital Medical University, Beijing, China.

ABSTRACT

Aim: It is unclear why alpha(1D)-adrenergic receptors (alpha(1D)-ARs) play a critical role in the mediation of peripheral vascular resistance and blood pressure in situ but function inefficiently when studied in vitro. The present study examined the causes for these inconsistencies in native alpha(1)-adrenergic functional performance between the vascular smooth muscle and myocytes.

Methods: The alpha(1)-adrenergic mediated contraction, Ca(2+) signaling and the subcellular receptor distribution were evaluated using the Fluo-4, BODIPY-FL prazosin and subtype-specific antibodies.

Results: Rat aortic rings and freshly dissociated myocytes displayed contractile and increased intracellular Ca(2+) responses to stimulation with phenylephrine (PE, 10 micromol), respectively. However, the PE-induced responses disappeared completely in cultured aortic myocytes, whereas PE-enhanced Ca(2+) transients were seen in cultured rat cardiac myocytes. Further studies indicated that alpha(1D)-ARs, the major receptor subtype responsible for the alpha(1)-adrenergic regulation of aortic contraction, were distributed both intracellularly and at the cell membrane in freshly dispersed aortic myocytes, similar to the alpha(1A)-AR subcellular localization in the cultured cardiomyocytes. In the cultured aortic myocytes, however, in addition to a marked decrease in their protein expression relative to the aorta, most labeling signals for alpha(1D)-ARs were found in the cytoplasm. Importantly, treating the culture medium with charcoal/dextran caused the reappearance of alpha(1D)-ARs at the cell surface and a partial restoration of the Ca(2+) signal response to PE in approximately 30% of the cultured cells.

Conclusion: Reduction in alpha(1D)-AR total protein expression and disappearance from the cell surface contribute to the insensitivity of cultured vascular smooth muscle cells to alpha(1)-adrenergic receptor activation.

Show MeSH
Related in: MedlinePlus