The existence of endothelium-derived vascular relaxant factor (EDRF) was postulated by Furchgott and colleagues when they observed that acetylcholine paradoxically relaxed preconstricted aortic strip preparations by an endothelium-dependent mechanism. This phenomenon has since been demonstrated in different blood vessels and mammalian species and it can be elicited by several other agents. EDRF has been thought to be a humoral agent, a lipoxygenase derivative and possibly a free radical. In the study reported here, by using aortic preparations from the rabbit, alone and in cascade experiments with isolated perfused coronary preparations, we demonstrate definitively that EDRF is a humoral agent. It is released from unstimulated aortic preparations containing endothelium, its release can be stimulated for prolonged periods by acetylcholine, and it is not a lipoxygenase derivative or free radical but an unstable compound with a carbonyl group at or near its active site.

We have developed a mathematical model of arterial vasomotion in which irregular rhythmic activity is generated by the nonlinear interaction of intracellular and membrane oscillators that depend on cyclic release of Ca2+ from internal stores and cyclic influx of extracellular Ca2+, respectively. Four key control variables were selected on the basis of the pharmacological characteristics of histamine-induced vasomotion in rabbit ear arteries: Ca2+ concentration in the cytosol, Ca2+ concentration in ryanodine-sensitive stores, cell membrane potential, and the open state probability of Ca2+-activated K+ channels. Although not represented by independent dynamic variables, the model also incorporates Na+/Ca2+exchange, the Na+-K+-ATPase, Cl− fluxes, and Ca2+ efflux via the extrusion ATPase. Simulations reproduce a wide spectrum of experimental observations, including 1) the effects of interventions that modulate the functionality of Ca2+ stores and membrane ion channels, 2) paradoxes such as the apparently unpredictable dual action of Ca2+ antagonists and low extracellular Na+ concentration, which can abolish vasomotion or promote the appearance of large-amplitude oscillations, and 3) period-doubling, quasiperiodic, and intermittent routes to chaos. Nonlinearity is essential to explain these diverse patterns of experimental vascular response.

Synthetic peptides homologous to the Gap 26 and Gap 27 domains of the first and second extracellular loops of the major vascular connexins (Cx37, Cx40, and Cx43) have been used to investigate the role of gap junctions in endothelium-derived hyperpolarizing factor (EDHF)-type relaxations of the rat hepatic artery. These peptides were designated 37,40Gap 26, 43Gap 26, 37,43Gap 27, and 40Gap 27, according to connexin specificity. When administered at 600 microM, none of the peptides individually affected maximal EDHF-type relaxations to ACh. By contrast, at 300 microM each, paired peptide combinations targeting more than one connexin subtype attenuated relaxation by up to 50%, and responses were abolished by the triple peptide combination 43Gap 26 + 40Gap 27 + 37,43Gap 27. In parallel experiments with A7r5 cells expressing Cx40 and Cx43, neither 43Gap 26 nor 40Gap 27 affected intercellular diffusion of Lucifer yellow individually but, in combination, significantly attenuated dye transfer. The findings confirm that functional cell-cell coupling may depend on more than one connexin subtype and demonstrate that direct intercellular communication via gap junctions constructed from Cx37, Cx40, and Cx43 underpins EDHF-type responses in the rat hepatic artery.

Constriction of vascular smooth muscle in response to the stimulus of raised intravascular pressure--the myogenic response--represents a positive feedback mechanism which, if unopposed, could theoretically lead to instability in the intact circulation. Dilation in response to increased intraluminal flow would provide an opposing feedback mechanism which could confer overall stability. Flow-dependent dilation in conduit vessels is mediated by endothelium-derived relaxing factor (EDRF), but the relationship between flow and EDRF activity has not been studied in resistance vessels in situ. We here demonstrate that EDRF can coordinate the aggregate hydrodynamic properties of an intact network. Under control conditions, EDRF maintains a fourth-power relationship between diameter and flow so that the pressure gradient in each vessel asymptotically approaches a constant value at high flow rates. Basal EDRF release may also maintain a similar spatial distribution of flow at different flow rates, even under conditions of moderate pharmacological constriction.

We have investigated the role of cAMP in NO-and prostanoidindependent relaxations that are widely attributed to an endothelium-derived hyperpolarizing factor (EDHF). Under control conditions EDHF-type relaxations evoked by acetylcholine (ACh) in rabbit iliac arteries were transient, but in the presence of the cAMP phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) or the cell permeant cAMP analog 8-bromo-cAMP, relaxations became sustained with their maxima potentiated Ϸ2-fold. Relaxation was associated with transient Ϸ1.5-fold elevations in smooth muscle cAMP levels with both mechanical and nucleotide responses being abolished by interrupting gap junctional communication with the connexin-mimetic peptide Gap 27 and by endothelial denudation. However, IBMX induced a sustained endothelium-independent Ϸ2-fold rise in cAMP levels, which was not further amplified by ACh, suggesting that the contribution of cAMP to the EDHF phenomenon is permissive. After selective loading of the endothelium with calcein AM, direct transfer of dye from the endothelium to the media was enhanced by IBMX or 8-bromo-cAMP, but not by 8-bromo-cGMP, whereas Gap 27 promoted sequestration within the intima. ACh-induced hyperpolarizations of subintimal smooth muscle in arterial strips with intact endothelium were abolished by Gap 27 and the adenylyl cyclase inhibitor 2 ,5 -dideoxyadenosine but were unaffected by IBMX. By contrast, in strips partially denuded of endothelium, IBMX enhanced the transmission of hyperpolarization from the endothelium to remote smooth muscle cells. These findings support the hypothesis that endothelial hyperpolarization underpins the EDHF phenomenon, with cAMP governing subsequent electrotonic signaling via both myoendothelial and homocellular smooth muscle gap junctions.connexin ͉ acetylcholine ͉ IBMX ͉ 8-bromo-cAMP ͉ cGMP

-The extracellular calcium-sensing receptor CaSR is expressed in blood vessels where its role is not completely understood. In this study, we tested the hypothesis that the CaSR expressed in vascular smooth muscle cells (VSMC) is directly involved in regulation of blood pressure and blood vessel tone. Mice with targeted CaSR gene ablation from vascular smooth muscle cells (VSMC) were generated by breeding exon 7 LoxP-CaSR mice with animals in which Cre recombinase is driven by a SM22␣ promoter (SM22␣-Cre). Wire myography performed on Cre-negative [wild-type (WT)] and Crepositive SM22␣ CaSR ⌬flox/⌬flox [knockout (KO)] mice showed an endothelium-independent reduction in aorta and mesenteric artery contractility of KO compared with WT mice in response to KCl and to phenylephrine. Increasing extracellular calcium ion (Ca 2ϩ ) concentrations (1-5 mM) evoked contraction in WT but only relaxation in KO aortas. Accordingly, diastolic and mean arterial blood pressures of KO animals were significantly reduced compared with WT, as measured by both tail cuff and radiotelemetry. This hypotension was mostly pronounced during the animals' active phase and was not rescued by either nitric oxide-synthase inhibition with nitro-L-arginine methyl ester or by a high-salt-supplemented diet. KO animals also exhibited cardiac remodeling, bradycardia, and reduced spontaneous activity in isolated hearts and cardiomyocyte-like cells. Our findings demonstrate a role for CaSR in the cardiovascular system and suggest that physiologically relevant changes in extracellular Ca 2ϩ concentrations could contribute to setting blood vessel tone levels and heart rate by directly acting on the cardiovascular CaSR. calcium-sensing receptor; CaSR; vascular smooth muscle cells; blood pressure regulation; G protein-coupled receptor; blood vessel tone regulation THE EXTRACELLULAR CALCIUM -sensing receptor CaSR was the first G protein-coupled receptor identified that has an ion, Ca 2ϩ

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