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Culturing Primary Rat Inner Medullary Collecting Duct Cells
Published on: June 21, 2013
Phenotypic changes of adrenomedullin receptor components, RAMP2, and CRLR mRNA expression in cultured rat vascular
1Department of Obstetrics and Gynecology, School of Medicine, Fukuoka University, Fukuoka, 814-0180, Japan.
Abstract:
Adrenomedullin is known to inhibit cell proliferation in cultured rat vascular smooth muscle cells, through a cAMP-dependent process. The calcitonin receptor-like receptor could function as an adrenomedullin receptor when co-expressed with receptor activity-modifying protein 2. To determine whether vascular adrenomedullin receptor components, the calcitonin receptor-like receptor and the receptor activity-modifying protein 2, phenotypically change during in vitro culture conditions, we examined the expression of adrenomedullin receptor components, adrenomedullin-induced cAMP production, and the inhibition of cell proliferation in culture rat vascular smooth muscle cells during serial passages. The results demonstrated that the receptor activity-modifying protein 2 and calcitonin receptor-like receptor mRNAs increased in a passage-dependent manner in rat vascular smooth muscle cells. Furthermore, the responses of both the elevation of cAMP and the inhibition of cell proliferation became larger in vascular smooth muscle cells with an increasing number of passages. The results suggest that the increase in functional AM receptor during phenotypic change may in part contribute to the development of vascular lesions, such as in atherosclerosis.
Insights
Adrenomedullin receptor components increase with cell culture passages in rat vascular smooth muscle cells. This enhanced receptor function may contribute to vascular lesions like atherosclerosis.
Area of Science:
- Vascular Biology
- Molecular Endocrinology
- Cellular Physiology
Background:
- Adrenomedullin (AM) inhibits vascular smooth muscle cell proliferation via a cAMP-dependent pathway.
- The calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 2 (RAMP2) form the functional AM receptor.
- Phenotypic changes in vascular cells during culture may alter AM receptor expression and function.
Purpose of the Study:
- To investigate phenotypic changes in vascular adrenomedullin receptor components (CLR and RAMP2) under in vitro culture conditions.
- To assess the impact of serial passages on AM-induced cAMP production and cell proliferation inhibition.
- To determine the relationship between AM receptor expression and vascular cell behavior during prolonged culture.
Main Methods:
- Cultured rat vascular smooth muscle cells were subjected to serial passages.
- Expression levels of CLR and RAMP2 mRNAs were quantified using RT-PCR.
- Adrenomedullin-induced cAMP production was measured.
- Inhibition of cell proliferation by adrenomedullin was assessed.
Main Results:
- CLR and RAMP2 mRNA expression increased in a passage-dependent manner.
- Adrenomedullin-induced cAMP elevation and inhibition of cell proliferation were enhanced with increasing passages.
- Functional AM receptor activity increased as vascular smooth muscle cells underwent phenotypic changes in vitro.
Conclusions:
- Vascular smooth muscle cells exhibit increased adrenomedullin receptor expression and responsiveness during serial passaging in vitro.
- Upregulation of functional AM receptors during phenotypic drift may contribute to the pathogenesis of vascular diseases.
- These findings highlight the dynamic nature of vascular cell receptors and their potential role in disease development, such as atherosclerosis.
