Hyperpolarization and increased free calcium in acetylcholine-stimulated endothelial cells

Busse, Rudi; Fichtner, H.; Lückhoff, Andreas; Kohlhardt, M.

doi:10.1152/ajpheart.1988.255.4.h965

Cited by 122 publications

(93 citation statements)

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“…Thus, it is reasonable to speculate that TRPV4 activity might be the underlying mechanism for the reported endotheliumdependent smooth muscle cell depolarization when K Ca 2.3 and K Ca 3.1 were inhibited. 19,20 However, note that acetylcholine was reported to induce membrane hyperpolarization without initial depolarization in some endothelial cells, 21 which is different from our results. This discrepancy could be related to variations in vascular beds or TRPV4 expression level.…”

Section: Discussioncontrasting

confidence: 99%

Functional Role of TRPV4-K _Ca 2.3 Signaling in Vascular Endothelial Cells in Normal and Streptozotocin-Induced Diabetic Rats

Du²,

Zhang³

et al. 2013

Hypertension

109

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15However, in some other pathological conditions, including congestive heart failure, hypercholesterolemia, ischemiareperfusion, and restenosis after coronary angioplasty, the NO-mediated relaxant response is impaired, whereas the EDHFmediated relaxant response is increased as a compensation. 15Abstract-The small conductance and intermediate conductance Ca 2+-activated K + channels are known to be involved in the endothelium-dependent hyperpolarization. Ca 2+ entry into endothelial cells stimulates these channels, causing membrane hyperpolarization in endothelial cells and underlying smooth muscle cells. In the present study, with the use of coimmunoprecipitation and double immunolabeling methods, we demonstrated a physical interaction of transient receptor potential vanilloid 4 (TRPV4) with K Ca 2.3 in rat mesenteric artery endothelial cells. Acetylcholine and 4α-PDD mainly acted through TRPV4-K Ca 2.3 pathway to induce smooth muscle hyperpolarization and vascular relaxation. K Ca 3.1 was also involved in the process but at a much lesser degree than that of K Ca 2.3. Stimulating TRPV4-K Ca 2.3 signaling pathway also increased local blood flow in mesenteric beds and reduced systemic blood pressure in anesthetized rats. In streptozotocin-induced diabetic rats, the expression levels of TRPV4 and K Ca 2.3 were reduced, which could be an underlying reason for the dysfunction of endothelium-dependent hyperpolarization in these animals. These results demonstrated an important physiological and pathological role of TRPV4-K Ca 2. However, the molecular mechanism of altered EDHF responses in these disease states is poorly understood.In the present study, we identified a previously unknown physical association between TRPV4 and K Ca 2.3 and uncovered the functional role of this TRPV4-K Ca 2.3 signaling pathway in smooth muscle hyperpolarization and relaxation. We also found that reduced expression levels of TRPV4 and K Ca 2.3 could be an underlying mechanism for EDHF dysfunction in diabetic rats. Materials and MethodsSee the Methods section in the online-only Data Supplement for details. Cell Preparation and CultureAll animal experiments were conducted in accordance with the regulation of the US National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals. Primary mesenteric artery endothelial cells (MAECs) were isolated from male Sprague-Dawley rats, cultured for 3 to 5 days, and used for experiments without passage. Immunostaining, Immunoprecipitation, and ImmunoblotsDouble immunolabeling was performed in rat MAECs using anti-TRPV4 antibody together with either anti-K Ca 2.3 or anti-K Ca 3.1 antibody. Artery sections were stained with either anti-TRPV4 or anti-K Ca 2.3 antibody. Immunoprecipitation and immunoblots were performed following the protocol described elsewhere with slight modifications. . The membrane potentials of MAECs were also measured using perforated wholecell patch clamp with an EPC-9 amplifier. High-impedance sharp microelectrodes were used to measure smooth muscle cell membran...

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Section: Discussioncontrasting

confidence: 99%

Functional Role of TRPV4-K _Ca 2.3 Signaling in Vascular Endothelial Cells in Normal and Streptozotocin-Induced Diabetic Rats

Du²,

Zhang³

et al. 2013

Hypertension

109

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“…However, in cultured porcine endothelium and isolated rabbit aorta K'a channels contribute to the EDRF response to flow but not that to agonists (Cooke et al 1991 b;. The explanation for this species difference may be that hyperpolarization of rabbit aortic endothelium by acetylcholine is very transient (3-5 s), while that of the guinea-pig coronary artery is sustained (> 25 min) (Busse, Fichtner, Luckhoff & Kohlhardt, 1988).…”

Section: Flow-induced Release Of Endothelium-dependent Agonistsmentioning

confidence: 99%

Modulation of blood flow and tissue perfusion by endothelium‐derived relaxing factor

Tm¹

1994

Experimental Physiology

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“…Evidence for the presence of muscarinic receptors on endothelial cells has been accumulating recently. Thus, acetylcholine has been shown to induce hyperpolarization of cultured endothelial cells (Busse et al, 1988;Olesen et al, 1988) and to increase their intracellular calcium concentration six to seven fold (Danthuluri et al, 1989). Muscarinic receptors were found to be dispersed on true capillary endothelium of the gastric mucosa by electron microscopic autoradiography (Nakamura et al, 1984).…”

Section: Introductionmentioning

confidence: 97%

Radioligand binding to muscarinic receptors of bovine aortic endothelial cells

Brünner

Kukovetz

1991

British J Pharmacology

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Muscarinic receptors on endothelial cells of bovine thoracic aorta were characterized by binding assays in which (−)‐[3H]‐N‐methyl quinuclidinyl benzilate ([3H]‐NMeQNB) was used as radioligand. Binding of [3H]‐NMeQNB to crude membranes of freshly isolated endothelial cells was atropine‐displaceable and of high affinity (KD = 0.48 nm) to a single class of sites (maximum binding capacity: 14 ± 3 fmol mg−1 protein). Stereospecificity of the binding sites was demonstrated in experiments in which [3H]‐NMeQNB binding was inhibited by dexetimide in the nanomolar range (KI = 0.63 nm) and by levetimide, its stereoisomer in the micromolar range (KI = 3.2 μm) (selectivity factor: ∼5000). Drug competition curves indicated a single class of binding sites for antagonists and the following apparant affinities (KI, nm): methyl atropine: 1.1; 4‐diphenylacetoxy N‐methyl piperidine methyl bromide (4‐DAMP): 3.4; pirenzepine: 16; 11‐[2‐(diethylamino‐methyl)‐1‐piperidinyl‐acetyl]‐5,11‐dihydro‐6H‐pyrido(2,3‐b)1,4‐benzodiazepine‐6‐one (AF‐DX 116): 2.500. Competition of acetylcholine with [3H]‐NMeQNB was best described by two affinity sites (or states) (KH = 0.82 μm, KL = 1.6 μm). In the presence of guanylimido diphosphate [Gpp(NH)p] (100 μm), acetylcholine affinity (IC50) was slightly, but significantly reduced (factor ∼4). Binding of [3H]‐NMeQNB to freshly harvested intact cells was also atropine‐displaceable, stereospecific (selectivity factor: ∼3500) and of high affinity (KD = 0.35 nm). The maximum binding capacity (9 ± 2 fmol mg−1 total cell protein) was comparable to that of membranes and corresponded to ∼900 binding sites per endothelial cell. Binding to enzymatically harvested and cultured endothelial cells, or membranes derived therefrom, showed no atropine‐displaceable binding. The results suggest that (1) bovine aortic endothelial cells contain muscarinic binding sites with all necessary criteria of functional muscarinic receptors; (2) the receptor most closely corresponds to the M1 subtype and is of comparatively very low density, and (3) cultured endothelial cells lose their receptors during isolation or culture procedures.

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Hyperpolarization and increased free calcium in acetylcholine-stimulated endothelial cells

Cited by 122 publications

References 0 publications

Functional Role of TRPV4-K _Ca 2.3 Signaling in Vascular Endothelial Cells in Normal and Streptozotocin-Induced Diabetic Rats

Functional Role of TRPV4-K _Ca 2.3 Signaling in Vascular Endothelial Cells in Normal and Streptozotocin-Induced Diabetic Rats

Modulation of blood flow and tissue perfusion by endothelium‐derived relaxing factor

Radioligand binding to muscarinic receptors of bovine aortic endothelial cells

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Hyperpolarization and increased free calcium in acetylcholine-stimulated endothelial cells

Cited by 122 publications

References 0 publications

Functional Role of TRPV4-K Ca 2.3 Signaling in Vascular Endothelial Cells in Normal and Streptozotocin-Induced Diabetic Rats

Functional Role of TRPV4-K Ca 2.3 Signaling in Vascular Endothelial Cells in Normal and Streptozotocin-Induced Diabetic Rats

Modulation of blood flow and tissue perfusion by endothelium‐derived relaxing factor

Radioligand binding to muscarinic receptors of bovine aortic endothelial cells

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Product

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About

Functional Role of TRPV4-K _Ca 2.3 Signaling in Vascular Endothelial Cells in Normal and Streptozotocin-Induced Diabetic Rats

Functional Role of TRPV4-K _Ca 2.3 Signaling in Vascular Endothelial Cells in Normal and Streptozotocin-Induced Diabetic Rats