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


CONCLUSIONS
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.
Fan
Lin-lin
LL
Departments of Pharmacology, Capital Medical University, Beijing, China.
Ren
Shuang
S
Zhou
Hong
H
Wang
Ying
Y
Xu
Ping-xiang
PX
He
Jun-qi
JQ
Luo
Da-li
DL
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000818:Animals; D001011:Aorta / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism; D002606:Charcoal / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D003911:Dextrans / Q000494:pharmacology; D051379:Mice; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology*; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D009924:Organ Culture Techniques; D010656:Phenylephrine / Q000494:pharmacology; D051381:Rats; D018340:Receptors, Adrenergic, alpha-1 / Q000378:metabolism*
C547720:Adra1d protein, mouse; C547721:Adra1d protein, rat; D000316:Adrenergic alpha-Agonists; D003911:Dextrans; D018340:Receptors, Adrenergic, alpha-1; D002606:Charcoal; D010656:Phenylephrine