Detection of Endothelin Receptors in Human Coronary Artery Vascular Smooth Muscle Cells But Not Endothelial Cells by Using Electron Microscope Autoradiography

Extracellular adenosine modulates cardiovascular and renal function. While measuring extracellular purines in biological samples, we observed a correlation between levels of adenosine and guanosine. This observation led us to test the hypothesis that extracellular guanosine regulates extracellular adenosine levels in the cardiovascular and renal systems. Rat preglomerular vascular smooth muscle cells in culture were incubated with adenosine and/or guanosine. In the absence of added adenosine, exogenous guanosine (30 μmol/L) had little effect on extracellular adenosine levels, indicating that extracellular guanosine does not trigger the release or production of adenosine. Without added guanosine and 1 hour after adding 3 μmol/L of exogenous adenosine, extracellular adenosine levels were only 0.125 ± 0.020 μmol/L, indicating rapid disposition of extracellular adenosine by a monolayer of cells. In contrast, extracellular adenosine levels 1 hour after adding 3 μmol/L of adenosine plus guanosine (30 μmol/L) were 1.173 ± 0.061 μmol/L (9-fold higher; p<0.0001), indicating slow disposition of extracellular adenosine in the presence of extracellular guanosine. Extracellular guanosine impeded the disposition of extracellular adenosine not only in preglomerular vascular smooth muscle cells, but also in rat preglomerular vascular endothelial cells, mesangial cells, cardiac fibroblasts and kidney epithelial cells, as well as in human aortic vascular smooth muscle cells, coronary artery vascular smooth muscle cells and coronary artery endothelial cells. In rats, infusions of guanosine per se had little effect on cardiovascular/renal variables, yet markedly enhanced the effects of co-infusions of adenosine. For example, in control rats, adenosine (0.3 μmol/kg/min) only modestly decreased mean arterial blood pressure (from 114 ± 4 to 100 ± 4 mm Hg). In contrast, in guanosine-treated rats (10 μmol/kg/min), adenosine profoundly decreased blood pressure (from 109 ± 4 to 79 ± 3 mm Hg; p<0.0001 vs non-guanosine treated group). Conclusion: Extracellular guanosine powerfully regulates extracellular adenosine levels by altering adenosine disposition and this occurs in many, perhaps most, cell types in the cardiovascular system and kidneys.

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Abstract 180: The Myristolated Alanine-Rich C Kinase Substrate (MARCKS) Differentially Regulates Proliferation of Vascular Smooth Muscle Cells and Endothelial Cells through Affecting Protein Stability and Proteolytic Processing of the Kinase Interacting with Stathmin (KIS)

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.180 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Dan Yu ◽

Charles Drucker ◽

Rajabrata Sarkar ◽

Dudley K Strickland ◽

Thomas S Monahan

Keyword(s):

Endothelial Cells ◽

Smooth Muscle ◽

Coronary Artery ◽

Smooth Muscle Cells ◽

Protein Stability ◽

Muscle Cells ◽

Cell Types ◽

Proteolytic Processing ◽

Functional Domain ◽

Human Coronary Artery

Objective: Presently, the antiproliferative agents used in drug eluting stents and drug coated balloons inhibit both VSMC and endothelial cell (EC) proliferation, and thus these patients require dual antiplatelet therapy indefinitely. Identification of a VSMC-specific target to prevent proliferation represents a significant unmet clinical need. Previously we found that knockdown of MARCKS arrests VSMC proliferation through a p27 kip1 -dependent mechanism. Interestingly MARCKS knockdown increases EC proliferation. p27 kip1 is phosphorylated by KIS allowing it to exit the nucleus and be degraded. Here we seek to understand how MARCKS influences KIS protein expression in these two cell types. Approach and Results: We performed siRNA-mediated knock down of MARCKS in human coronary artery endothelial cells (CAECs) and human coronary artery smooth muscle cells (CASMCs). MARCKS knockdown did not affect KIS mRNA expression as determined with RT-PCR in either cell type. KIS protein stability was evaluated in the presence of cyclohexamide with Western blot. In CAECs, MARCKS knockdown increased KIS stability, however, in CASMCs, MARCKS knockdown significantly decreased KIS protein stability. In CASMCs, MARCKS knockdown significantly increased KIS ubiquitinization where as in CAECs, MARCKS knockdown decreased KIS ubiquitinization. Interestingly, the well-studied functional domain of MARCKS(ED domain) is not directly involved in KIS regulation. MARCKS mutants (S4G and S4D) rescued proliferation in VSMCs. MARCKS knockdown in vivo in the murine femoral wire injury model resulted in decreased medial bromodeoxyuridine (BrdU) integration and neointima formation. MARCKS knockdown enhanced endothelial barrier function recovery four days after injury as assessed by Evans Blue integration. Conclusions: MARCKS differentially regulates the protein stability and proteolytic processing of KIS in VSMCs and ECs. The differential interaction of MARCKS and KIS likely explains the observed difference in proliferation observed with MARCKS knockdown in these two cell types.

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