α1D-Adrenergic receptor insensitivity is associated with alterations in its expression and distribution in cultured vascular myocytes

Abstract

Aim: It is unclear why α 1D -adrenergic receptors (α 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 α 1 -adrenergic functional performance between the vascular smooth muscle and myocytes. Methods: The α 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 µmol), 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 α 1D -ARs, the major receptor subtype responsible for the α 1 -adrenergic regulation of aortic contraction, were distributed both intracellularly and at the cell membrane in freshly dispersed aortic myocytes, similar to the α 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 α 1D -ARs were found in the cytoplasm. Importantly, treating the culture medium with charcoal/dextran caused the reappearance of α 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 α 1D -AR total protein expression and disappearance from the cell surface contribute to the insensitivity of cultured vascular smooth muscle cells to α 1 -adrenergic receptor activation.

α-Dystrobrevin-1 recruits α-catulin to the α 1D -adrenergic receptor/dystrophin-associated protein complex signalosome

“…Indeed, recent studies support the idea that α 1D -ARs are expressed at the plasma membrane and intracellularly in native tissues (11,30), but it remains unclear how the DAPC is regulated to alter α 1D -AR localization. One study of particular interest revealed that α 1D -ARs are clearly expressed both at the plasma membrane and intracellularly in freshly dissociated aortic smooth muscle cells but become completely intracellular and nonfunctional with culturing (31). Thus, it is likely that unidentified signals are required to maintain sequestration of α 1D -ARs at their proper sites in vivo.…”

Bladder smooth muscle organ culture preparation maintains the contractile phenotype

“…These proteins include h-caldesmon (29,69), calponin (44), smooth muscle myosin isoforms (2), and ␣-actin (65). Moreover, culturing smooth muscle cells lowers the expression of calcium channels and receptors, thus decreasing depolarization-induced contractions (26,27,67) and agonist-induced contractions (22,68), respectively.…”

Mitochondrial Fission of Smooth Muscle Cells Is Involved in Artery Constriction

“…In fact, even in primary arterial smooth muscle cells, the response to phenylephrine was also lost after cell culturing. 39 Therefore, we only summarized the role of mitochondrial fission in high K + (membrane depolarization)-induced vasoconstriction in Figure S12.…”