Potentiation of Acetylcholine-lnduced Responses in Freshly Isolated Rabbit Aortic Endothelial Cells

Abstract

Acetylcholine (ACh)-induced membrane hyperpolarization was studied in freshly isolated endothelial cells from rabbit aorta. Ten µM ACh induced transient hyperpolarization due to the opening of Ca²⁺-sensitive K⁺ channels, sensitive to TEA and charybdotoxin (CTX). The membrane potential response was accompanied by an increase in intracellular Ca²⁺ [Ca²⁺]_i. Pretreatment of endothelial cells with 20 µM ATP, 0.2 µM bradykinin or 0.1 µM platelet-aggregating factor, which induced either a transient hyperpolarization or no response, changed the subsequent ACh-induced response to a large maintained hyperpolarization. This sustained membrane hyperpolarization was also due to the opening of Ca²⁺-activated K⁺ channels as confirmed by CTX and TEA blockade, and was related to elevated [Ca²⁺]_i measured by fura-2 fluorescence. Pertussis toxin blocked potentiation, indicating involvement of a G protein. The linkage to receptor-operated Ca²⁺ (ROC)-entry was suggested by observations that the maintained hyperpolarization during potentiation was dependent on extracellular Ca²⁺ and was abolished by the ROC blockers SKF-96365 and Ni²⁺. Inhibition of the Ca²⁺ pump of the endoplasmic reticulum mimicked the potentiating effect of the agonists. The results suggest that crosstalk between the agonists in endothelial cells involves Ca²⁺ movements and that this crosstalk is important for the generation of endothelial secretions.

Rat Coronary Endothelial Cell Membrane Potential Responses During Hypertension

“…[23][24][25] These large E m changes to SP and BK clearly contrast with the very slight hyperpolarizing responses to these peptides that we observed in current-clamped intact endothelial cells. They also contrast with the findings of Wang et al 19 in freshly isolated current-clamped endothelial cells of rat aorta studied by the perforated-patch method. In this endothelial preparation, BK (400 nmol/L) also elicited only a slight hyperpolarizing response, whereas ACH (10 mol/L) induced pronounced hyperpolarizations averaging 51 mV in the intact endothelial cells.…”

“…10,16 -19 This hyperpolarizing response has been linked to the elevation of cytosolic Ca 2ϩ levels in the endothelial cell and appears to be obligatory for the release of dilator factors. 12,18,19 For example, in bioassay experiments of rabbit aorta, Demirel et al 10 have shown that perfusion of ACH through endothelium-intact donor rings results in the relaxation of recipient rings denuded of endothelium. In this preparation, pharmacological block of K ϩ channels in the ACH-stimulated donor segment inhibited the downstream relaxation of the recipient ring, inferring that ACH activates endothelial K ϩ channels to mediate the release of dilator factors.…”

“…In this endothelial preparation, BK (400 nmol/L) also elicited only a slight hyperpolarizing response, whereas ACH (10 mol/L) induced pronounced hyperpolarizations averaging 51 mV in the intact endothelial cells. 19 Notably, the perforated-patch method as used in this study provides several distinct advantages over the use of microelectrode or standard whole-cell patch-clamp recording for monitoring E m levels in endothelial cells. 16 First, in contrast to standard whole-cell approaches, the perforated patch method permits electrical access to the cell interior for the dynamic recording of E m responses without interrupting the structural integrity and cytosolic signaling of the endothelial cell.…”

Store-operated Ca2+ entry (SOCE) pathways

“…Rearrangement of the microfilamentous cytoskeleton represents a potential mechanism underlying SOCE-induced shape change in endothelial cells (Moore et al 1998). Other studies have shown the involvement of calcium/calmodulin-dependent protein kinase II (d) in endothelial barrier dysfunction ) and indicated its influence on cytoskeletal organisation by affecting translocation of filamin (Wang et al 1996) and stress fiber formation (Borbiev et al 2003). In pulmonary arteries, involvement of the potential SOCE channel proteins TRPC1 and TRPC4 in inflammation-associated loss of endothelial barrier function has been suggested.…”

Delivery and Expression of Fluid Shear Stress-Inducible Promoters to the Vessel Wall: Applications for Cardiovascular Gene Therapy

“…The cells were passaged with trypsin-EDTA (Life Sciences, St. Petersburg, FL) and maintained in a humidified atmosphere of 5% CO 2 at 37°C. Rabbit aortic endothelial cells (RAECs) were prepared from freshly sacrificed rabbits as described (Wang et al, 1996).…”