Endothelium-dependent dilation

Oxygen and substrate requirements of the organs depend on work load and thus organ perfusion is continuously matched to the changing needs. This adaptation is achieved by an adoption of vascular diameters. The most pronounced changes in organ perfusion can be observed in skeletal muscle. Perfusion can increase up to 20-fold in this organ with heavy work load. For such large perfusion changes to occur the behaviour of the smallest arteries (arterioles) is of special importance, because the highest resistance resides in these vessels. The endothelial cells (EC, depicted in blue in the scheme at the bottom) are crucial for dilation in arterioles, because they release mediator substances that are strongly affecting the contractile status of the adjacent vascular smooth muscle cells (depicted in red, VSM). The dilating mediators include nitric oxide (NO), prostaglandins, and an additional principle, whose chemical identity is still elusive. Since this latter mechanism induces a hyperpolarization of the membrane potential in smooth muscle it is also termed 'endothelium-derived hyperpolarizing factor' (EDHF). The activity or release of EDHF itself relies on the hyperpolarization of endothelial cells achieved by activation of endothelial potassium channels (SKCa, IKCa), which allows potassium (K+) efflux, loss of positive ions, and thus a decrease of the membrane potential towards lower values (hyperpolarisation). This endothelial hyperpolarisation is transmited to the adjacent smooth muscle either chemically by a mediator or directly through channels connecting the two cell types (myoendothelial junctions). Thus, a hyperpolarisation of the smooth muscle is achieved that leads to relaxation. The release (or activation) of these endothelial dilator principles is modulated by mechanical (intravascular pressure, wall shear stress) and chemical substances (e.g. acetylcholine, Ach or bradykinin, Bk). These substances are therefore called endothelium-dependent dilators since their efficacy requires an intact endothelium.

We study the release and the relative importance of these factors as well as their interactions and intracellular signal transduction mechanisms in the vascular smooth muscle (e.g. NO and the activation of the soluble guanylyl cyclase (sGC) that produces the second messenger cGMP, see figure). Other vasoactive substanses act directly on smooth muscle cells, e.g. adenosine (ADO) through activation of ATP-depending potassium channels (KATP) and a smooth muscle hyperpolarisation. The figure further depicts intercellular channels (gap junctions), that are formed by connexins (Cx) and exhibit distinct resistances (given in MegaOhm). They allow communication between endothelial cells, between smooth muscle cells, but also allow communication between these two cell types.


Communication and coordination in vessels

A swarm of fish displays a collective behavior ('swarm behavior') and moves 'en masse' despite the huge number of individual animals. In analogy, organ function is supported by a huge number of cells that act in an orchestrated fashion. It is obvious that communication is required to achieve this vital goal. The communication channel is indeed a true channel that interlinks the cytosol of adjacent cells by a pore sealed against the extracellular space (see figure above). Many channels that cluster in a specific region of intimate cell contact bridge the remaining gap to form a low-resistance connection (gap junction) allowing exchange of ions and other small molecules. The modular bricks of these channels are connexins. Because charge (and membrane potential) is transferred, gap junctions convert many individual endothelial cells or many individual smooth muscle cells into a synchronously acting unit. Evidence for a communication along the vessel wall is presented below.

Endothelial mediators do not only induce dilation in the local vicinity of the site of release, but they have also a remote effect. This is especially the case for EDHF, which induces a local and a concomitant remote, distant dilation at up- and downstream sites. This is termed an ascending or conducted dilation which is shown in the scheme (right). Obviously the membrane potential is of special importance and it can be spread through intercellular contacts along the vessel wall. The cellular contacts enable the coordination of vascular behaviour over long distances, which leads to a steady uniform dilation or constriction over a comparably long distance. This coordination of diameter changes along the vessel is an indispensable requirement for large changes of blood flow. The cellular contacts are at the molecular level intercellular channels that are composed of connexin proteins. Cluster of these channels are termed gap junctions, which allow the communication along the endothelial or the smooth muscle cell layer and thus along the vascular tree. We are studying the role of different connexins as well as other mechanism that contribute to the conduction of vasomotor responses (dilation or constriction) along the arteriolar tree.

Localised stimulation of the arteriole with acetylcholin (ACh, pipette, right) induces a dilation at the stimulation site (top panel). This dilation spreads along the arteriole and can be also observed at the remote, upstream location (bottom panel). The spread along this distance occurs nearly instantaneous (< 1 s). (Diameter of the arterioles: about 30 µm, distance 1100 µm).


Gap junctions and connexins

Gap junctions are channels connecting adjacent cells and form a bridge to join the cytoplasma of neighboring cells. They consist of connexin proteins. Of these connexins (Cx) different subtypes exist and in vascular cells Cx40, Cx37, Cx43, and Cx45 are expressed. After labeling the expression of a connexin in the microcirculation the vascular tree with its branches is clearly visible (figure to the right, Cx in red, nuclei stained in blue) demonstrating the expression of Cx in vessels. Six of these proteins (similar or different subtypes) oligomerize to a hemichannel with a central pore. It is introduced into the plasma membrane. Docking with a hemichannel from the adjacent cell gives rise to a functional pore or channel. Through this channel ions can move along the gradients of their electrochemical forces (membrane potential, concentration). In addition, small molecules up to a molecular mass to 1 kDa can diffuse through these pores. By this means individual cells are connected to a functional syncytium, which acts in a coordinated fashion together. Connexin channels are important for the above mentioned conducted vasomotor responses because the deficiency of certain connexins results in a strong reduction of remote responses and thus to a deficit in the coordination of vascular behaviour.

Laboratory methods

1.Study of the microcirculation by means of intravitalmicroscopy and the observation of arterioles in the skeletal muscle enables the direct measurement of microcirculatory pressure, blood flow, arteriolar diameter and the membrane potential of vascular cells
Measurement of the membrane potential in arterioles in the microcirculation
(left: Endothelial cell; right: vascular smooth muscle cell)
2.Study of isolated vessels
(isometric force or isobaric diameter measurements, picture below)
Isobaric preparation:
After cannulation pressure is applied and the diameter of the vessel measured
Isometric preparation:
The developed force is measured
after initial optimal stretching
3. Telemetric pressure measurements in the intact animal

Selected publications


Wölfle SE, Schmidt VJ, Hoyer J, Köhler R, de Wit C
Prominent role of KCa3.1 in EDHF-type dilations and conducted responses in the microcirculation in vivo
Cardiovasc Res 82, 476-483, 2009


Wölfle SE, Schmidt VJ, Hoepfl B, Gebert A, Alcoléa S, Gros D, de Wit C
Connexin45 (Cx45) cannot replace the function of Cx40 in conducting endothelium-dependent dilations along arterioles
Circ Res 101, 1292-1299, 2007


Siegl D, Koeppen M, Wölfle SE, Pohl U, de Wit C
Myoendothelial coupling is not prominent in arterioles within the mouse cremaster microcirculation in vivo
Circ Res 97, 781-788, 2005


Wölfle SE, de Wit C
Intact endothelium-dependent dilation and conducted responses in resistance vessels of hypercholesterolemic mice in vivo
J Vasc Res 42: 475-482, 2005


Koeppen M, Feil R, Siegl D, Feil S, Hofmann F, Pohl U, de Wit C
cGMP-dependent protein kinase mediates NO- but not acetylcholine-induced dilations in resistance vessels in vivo
Hypertension 44: 952-955, 2004


de Wit C
Connexins pave the way for vascular communication
News Physiol Sci 19: 148-153, 2004


de Wit C, Roos F, Bolz SS, Pohl U
Lack of vascular connexin40 is associated with hypertension and irregular arteriolar vasomotion
Physiol Gen 13:169-177, 2003


Hoepfl B, Rodenwaldt B, Pohl U, de Wit C
EDHF, but not NO or prostaglandins, is critical to evoke a conducted dilation upon ACh in hamster arterioles
Am J Physiol 283: H996-H1004, 2002


de Wit, C, Roos F, Bolz St-S, Kirchhoff S, Krüger O, Willecke K, Pohl U
Impaired conduction of vasodilation along arterioles in connexin40 deficient mice
Circ Res 86: 649-655, 2000