cAMP facilitates EDHF-type relaxations in conduit arteries by enhancing electrotonic conduction via gap junctions Tudor M. Griffith*, Andrew T. Chaytor, Hannah J. Taylor, Beverley D. Giddings, and David H. Edwards Department of Diagnostic Radiology, Wales Heart Research Institute, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, United Kingdom Edited by Louis J. Ignarro, University of California, School of Medicine, Los Angeles, CA, and approved March 13, 2002 (received for review February 13, 2002) We have investigated the role of cAMP in NO- and prostanoid- endothelium-derived hyperpolarizing factor (EDHF) is released independent relaxations that are widely attributed to an endo- into the extracellular space by the endothelium on stimulation thelium-derived hyperpolarizing factor (EDHF). Under control con- with ACh to activate smooth muscle Kϩ channels and thereby ditions EDHF-type relaxations evoked by acetylcholine (ACh) in mediate relaxation (7, 8). However, bioassay experiments with rabbit iliac arteries were transient, but in the presence of the cAMP sandwich preparations of rabbit conduit arteries, constructed phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) or the from closely apposed strips of endothelium-intact and -denuded cell permeant cAMP analog 8-bromo-cAMP, relaxations became vessel, fail to demonstrate the release of a freely diffusible sustained with their maxima potentiated Ϸ2-fold. Relaxation was EDHF after administration of ACh (9–11). Because there is no associated with transient Ϸ1.5-fold elevations in smooth muscle gap junctional communication between the ‘‘donor’’ endothe- cAMP levels with both mechanical and nucleotide responses being lium and ‘‘detector’’ smooth muscle in such vessel composites, abolished by interrupting gap junctional communication with the direct endothelial-smooth muscle coupling thus appears central connexin-mimetic peptide Gap 27 and by endothelial denudation. to the EDHF-type response evoked by endothelium-dependent However, IBMX induced a sustained endothelium-independent agonists. Ϸ2-fold rise in cAMP levels, which was not further amplified by Previous studies have nevertheless provided evidence that ACh, suggesting that the contribution of cAMP to the EDHF EDHF-type responses evoked by ACh in rabbit conduit arteries phenomenon is permissive. After selective loading of the endo- may not be exclusively passive because relaxation is associated thelium with calcein AM, direct transfer of dye from the endothe- with endothelium-dependent elevations in smooth muscle lium to the media was enhanced by IBMX or 8-bromo-cAMP, but cAMP levels, and therefore attenuated by inhibition of adenylyl not by 8-bromo-cGMP, whereas Gap 27 promoted sequestration cyclase with P-site agonists such as 2Ј,5Ј-dideoxyadenosine within the intima. ACh-induced hyperpolarizations of subintimal (2Ј,5Ј-DDA) and inhibition of protein kinase A (12). Further- smooth muscle in arterial strips with intact endothelium were more, EDHF-type relaxations are potentiated by inhibition of -abolished by Gap 27 and the adenylyl cyclase inhibitor 2؅,5؅- cAMP hydrolysis with the phosphodiesterase inhibitor isobutyl dideoxyadenosine but were unaffected by IBMX. By contrast, in methylxanthine (IBMX), while remaining susceptible to the strips partially denuded of endothelium, IBMX enhanced the trans- combination of apamin plus charybdotoxin (12). Sensitivity to mission of hyperpolarization from the endothelium to remote these peptide toxins is a hallmark of the EDHF phenomenon and smooth muscle cells. These findings support the hypothesis that thought to reflect the opening of apamin-sensitive small con- ϩ endothelial hyperpolarization underpins the EDHF phenomenon, ductance K channels (SKCa) and charybdotoxin-sensitive large ϩ with cAMP governing subsequent electrotonic signaling via both and intermediate conductance K channels (BKCa and IKCa) myoendothelial and homocellular smooth muscle gap junctions. located on endothelial cells (1). Consistent with a role for gap junctions, EDHF-type relaxations and cAMP accumulation connexin ͉ acetylcholine ͉ IBMX ͉ 8-bromo-cAMP ͉ cGMP evoked by ACh are inhibited by synthetic connexin-mimetic peptides that interrupt intercellular communication in a ␣ t is widely recognized that agonists such as acetylcholine connexin-specific fashion and by 18 -glycyrrhetinic acid, an I(ACh) evoke endothelial hyperpolarization and smooth mus- aglycone that disrupts gap junction plaques at points of cell–cell cle relaxations that are independent of the synthesis of nitric contact (11–13). oxide (NO) and vasodilator prostanoids (1). In guinea pig and Because there is evidence that elevations in cAMP levels may hamster arterioles, hyperpolarizations induced either by ACh or rapidly enhance gap junctional communication (14–18), in the the injection of electrical current into the endothelium may be present study we have tested the hypothesis that this nucleotide transmitted to smooth muscle cells through a passive electro- underpins EDHF-type relaxations in conduit arteries by facili- tonic mechanism, and in the converse sense, action potentials tating the electrotonic spread of hyperpolarizing current from originating in smooth muscle cells can be detected in the the endothelium into and through the media. Mechanical re- endothelium (2–4). Such direct intercellular communication sponses were correlated with measurements of smooth muscle depends on the coupling of apposing cells by gap junctions, which cAMP levels and membrane potential, and modulation of gap are constructed from connexin (Cx) proteins and possess an junctional permeability by cAMP was assessed by a dye transfer aqueous central pore (5). In arterioles, myoendothelial gap junctions behave as simple ohmic resistors without rectification, so that complex electrical events are reduced in amplitude by This paper was submitted directly (Track II) to the PNAS office. Ј Ј Ј Ј 10–20% when conducted between the endothelium and media in Abbreviations: ACh, acetylcholine; 2 ,5 -DDA, 2 ,5 -dideoxyadenosine; IBMX, isobutyl- methylxanthine; EDHF, endothelium-derived hyperpolarizing factor; L-NAME, NG-nitro-L- either direction, but are preserved in temporal form (4). By arginine methyl ester; PE, phenylephrine. contrast, in thick-walled arteries, it has been argued that the *To whom reprint requests should be addressed. E-mail: griffi[email protected]. endothelium cannot act as a significant source of hyperpolarizing The publication costs of this article were defrayed in part by page charge payment. This current because the large bulk of the media will result in current article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. dissipation (6). An alternative hypothesis, therefore, is that an §1734 solely to indicate this fact. 6392–6397 ͉ PNAS ͉ April 30, 2002 ͉ vol. 99 ͉ no. 9 www.pnas.org͞cgi͞doi͞10.1073͞pnas.092089799 Downloaded by guest on September 27, 2021 technique whereby the entire endothelial monolayer was loaded with 300 ␮M L-NAME and 10 ␮M indomethacin for 60 min with the cell permeant tracer calcein AM. In addition to followed, additionally, by either 600 ␮M Gap 27, 20 ␮M IBMX, providing electrical continuity, gap junctions allow the transfer 500 ␮M 8-bromo-cAMP, or 500 ␮M 8-bromo-cGMP for 30 min. of molecules Ͻ1,000 Da in size between coupled cells (5), so that The preparations were allowed to return to room temperature, after intracellular cleavage of the acetoxymethyl moiety respon- and 10 ␮M calcein AM (Molecular Probes) was perfused sible for endothelial uptake, a reduction in molecular mass (from through the lumen for 30 min before washout with dye-free 1,000 to 623 Da) allows diffusion of calcein into the media. buffer at 35°C for 30 min. In control experiments arteries were Connexins may be classified on the basis of molecular mass (in perfused with 10 ␮M calcein, which is membrane impermeable. kDa) with three main subtypes (Cxs 37, 40, and 43) being found The vessels were subsequently removed from the myograph and in endothelial and vascular smooth muscle cells in rabbit arteries fixed in 0.1 M PBS containing 0.2% glutaraldehyde and 2% (19). In the present study, a peptide homologous to the Gap 27 formalin for 90 min before cryopreservation in OCT compound domain of the second extracellular loop of Cxs 37 and 43 was (Agar Scientific, Stanstead, UK), cooled by liquid N2. Cryosec- used to interrupt gap junctional communication, and has pre- ␮ viously been shown to inhibit EDHF-type responses through a tions of transverse rings (10 m thick) were prepared and receptor-independent mechanism that does not involve nonspe- mounted onto poly-L-lysine-coated slides under Fluorsave (Cal- cific effects on contraction, relaxation, or hyperpolarization biochem) and imaged with a TCS four-dimensional confocal (20–22). laser scanning system (Leica) with the filters set for 490 nm excitation and 525 nm emission. A gallery of several images was Materials and Methods collected at 0.5-␮m steps for each sample followed by image Iliac and femoral arteries were removed from male New Zealand processing by using Leica SCANWARE software to obtain a White rabbits (2–2.5 kg) killed with sodium pentobarbitone (120 maximum projection image. A pixel intensity profile across the ͞ ͞ mg kg; i.v.), transferred to oxygenated (95% O2 5% CO2,pH vessel wall was then obtained with MATLAB software and fitted 7.4) Holmans buffer (composition in mM: 120 NaCl, 5 KCl, 2.5 to a monoexponential to derive a space constant