Pharmacological dissection of the cellular mechanisms associated to the spontaneous and the mechanically stimulated ATP release by mesentery endothelial cells: roles of thrombin and TRPV

Abstract

Endothelial cells participate in extracellular ATP release elicited by mechanosensors. To characterize the dynamic interactions between mechanical and chemical factors that modulate ATP secretion by the endothelium, we assessed and compared the mechanisms participating in the spontaneous (basal) and mechanically stimulated secretion using primary cultures of rat mesentery endothelial cells. ATP/metabolites were determined in the cell media prior to (basal) and after cell media displacement or a picospritzer buffer puff used as mechanical stimuli. Mechanical stimulation increased extracellular ATP that peaked within 1 min, and decayed to basal values in 10 min. Interruption of the vesicular transport route consistently blocked the spontaneous ATP secretion. Cells maintained in media lacking external Ca elicited a spontaneous rise of extracellular ATP and adenosine, but failed to elicit a further extracellular ATP secretion following mechanical stimulation. 2-APB, a TRPV agonist, increased the spontaneous ATP secretion, but reduced the mechanical stimulation-induced nucleotide release. Pannexin1 or connexin blockers and gadolinium, a Piezo1 blocker, reduced the mechanically induced ATP release without altering spontaneous nucleotide levels. Moreover, thrombin or related agonists increased extracellular ATP secretion elicited by mechanical stimulation, without modifying spontaneous release. In sum, present results allow inferring that the spontaneous, extracellular nucleotide secretion is essentially mediated by ATP containing vesicles, while the mechanically induced secretion occurs essentially by connexin or pannexin1 hemichannel ATP transport, a finding fully supported by results from Panx1 rodents. Only the latter component is modulated by thrombin and related receptor agonists, highlighting a novel endothelium-smooth muscle signaling role of this anticoagulant.

Increased ATP and ADO Overflow From Sympathetic Nerve Endings and Mesentery Endothelial Cells Plus Reduced Nitric Oxide Are Involved in Diabetic Neurovascular Dysfunction

“…To further extend the notion that the vasodilator effect of ATP is associated with NO production, we next examined, using cultured ECs from the rat mesentery, whether ATP secretion elicited by mechanical stimuli such as CMD ( Donoso et al, 2018 ) is associated with NO production. This procedure somehow mimics shear stress forces detected by ECs which respond eliciting extracellular ATP secretion and thereafter NO production.…”

“…Thereafter, the cell suspension was centrifuged at 126 g at 4°C for 10 min to eliminate the mesentery adipose tissue and gross tissue debris. The cellular pellet was dissolved in M-199 medium plus antibiotics and next centrifuged at 126 g and 4°C for 5 min, re-suspended in 36 mL of medium 199 containing antibiotics and supplemented with: 20% fetal bovine serum (FBS) plus 20 μg/mL endothelial cell growth supplement (ECGS, Donoso et al, 2018 ). In addition, ECs isolated from the STZ-treated rats, were cultured in medium added with 25 mM glucose to maintain similar in vivo conditions.…”

“…To quantify the release of extracellular ATP/metabolites to the cell media, primary EC cultures were mechanically stimulated using the cell media displacement (CMD) procedure reported by Donoso et al (2018) . This protocol was adapted from the original report by Lazarowski et al (1997) .…”

“…This physiological preparation allows, on the one hand, assessing the functionality of this vascular bed by determining the changes in perfusion pressure, which indicates, the vascular tone of the arterial mesenteric territory ( Buvinic and Huidobro-Toro, 2001 ). In addition, this preparation allows generating primary cultures of ECs as a relevant model to study ATP release ( Donoso et al, 2018 ) and NO production in greater detail.…”

Potent Vasodilator and Cellular Antioxidant Activity of Endemic Patagonian Calafate Berries (Berberis microphylla) with Nutraceutical Potential

“…CAA determinations used endothelial cells from the rat mesentery. Primary cultures from the mesentery were isolated as reported Donoso et al [39]; the cell pellet obtained was resuspended in supplemented growth medium 199 (ECGS 2% v / v , fetal bovine serum 20% v / v ). These cells were maintained for 5 h at 37 °C and 5% CO 2 , then the medium was removed, and the cells were washed with phosphate buffer saline.…”