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(Circulation Research. 2007;100:931.)
© 2007 American Heart Association, Inc.

Editorials

Closing the Gap at Hot Spots

Cor de Wit

From the Institut für Physiologie, Universität Lübeck, Germany.

Correspondence to Dr Cor de Wit, Institut für Physiologie, Universität Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany. E-mail dewit{at}uni-luebeck.de

See related article, pages 1026–1035

Key Words: connexin • Ca²⁺ microdomains • Na⁺/K⁺-pump • Na⁺-Ca²⁺-exchange

Coordination of cellular behavior is indispensable for vascular function. Because of the structural design of the vascular tree only an orchestrated diameter change along the vessel length increases conductance significantly and ensures optimal tissue perfusion. Mechanisms that suffice these needs include locally generated signals at upstream sites which reflect downstream requirements (flow-induced dilation)¹ and longitudinal signal transmission through communication channels residing in the vessel wall itself which enable the synchronisation of cellular behavior. This latter is achieved by homocellular gap junctions which are composed of connexins that connect adjacent cells by intercellular low-resistance channels establishing a functional syncitium.² Electrical communication through these channels is experimentally studied by locally initiated vasomotor responses which conduct along the vessel wall and promote synchronized dilations or constrictions of arteriolar segments reflecting the coordination of cellular behavior.³ Longitudinal communication through gap junctions is also required for the synchronisation of intracellular Ca²⁺ ([Ca²⁺]_i) changes in smooth muscle and subsequent vasomotion, ie, rhythmic oscillations of vascular diameter. Although the physiological significance of vasomotion is still to be clearly defined it depends critically on Ca²⁺ release from intracellular stores which occurs in an oscillating fashion and the synchronization of [Ca²⁺]_i transients through gap junctional communication.^4,5 In addition, endothelial and smooth muscle cells are coupled heterocellularly (myoendothelial junctions) allowing the direct spread of current from the endothelium to the smooth muscle⁶ or other signaling molecules.⁷

The importance of gap junctional communication in vascular function contrasts with the poor knowledge of its regulation in vessels which is complicated by the existence of functional and structural different types of gap junctions. Moreover, longterm regulation because of degradation or upregulation of connexins has to be separated from shortterm effects which reflect an alteration of channel conductance or permeability in response to an altered phosphorylation state of connexins. Although the literature on the regulation of connexins is abundant, only a few observations on the shortterm regulation in vascular tissue have been published. For example, cAMP enhances myoendothelial, homocellular smooth muscle, and interendothelial gap junctional coupling,⁸ a pathway that is activated by 11,12-epoxyeicosatrienoic acid.⁹ An inhibition was reported for inflammatory mediators, which selectively attenuated the permeability of myoendothelial junctions in vitro.¹⁰ Sepsis also reduced electrical intercellular coupling and the propagation of conducted constrictions in vivo,¹¹ possibly in an NO-dependent fashion.¹² Such an inhibition of the propagation of constrictions can also be exerted by the physiologically released amounts of NO most likely reflecting inhibition of homocellular smooth muscle coupling.¹³ Gap junctions are associated and interact with a variety of proteins that target and deliver them to specific membrane microdomains eg, adherens-junction proteins.¹⁴ However, in these microdomains regulatory proteins may also be located creating a spatially confined microenvironment which allows a selective control of gap junctional communication.

In this issue of Circulation Research, Matchkov et al¹⁵ report that the activity of the Na⁺/K⁺-pump in conjunction with the Na⁺-Ca²⁺-exchanger keeps gap junction channels in an open state giving rise to a coordinated activity of the smooth muscle cells as reflected by synchronous [Ca²⁺]_i transients. Such a synchronous behavior of the vascular smooth muscle cells led to rhythmic contractions (vasomotion) in the intact vessel (small mesenteric artery). Interference with Na⁺/K⁺-pump or Na⁺-Ca²⁺-exchanger activity caused localized Ca²⁺ increases at the periphery of the cell. These spatially confined Ca²⁺ signals uncoupled the smooth muscle cells, desynchronised [Ca²⁺]_i transients, and prevented rhythmic contractions (Figure).

View larger version (17K): [in this window] [in a new window]   The Na+/K+-pump provides the driving force for Ca2+ extrusion by the Na+-Ca2+-exchanger (NCX), which prevents local Ca2+ accumulation and keeps gap junctions in an open state. This allows synchronisation of [Ca2+]i transients in smooth muscle attributed to a transfer of membrane potential (EM) oscillations ultimately giving rise to rhythmic contractions of the vessel (A). Interfering with the activity of the Na+/K+-pump by the interventions depicted alters the Na+ gradient and inhibits or reverses NCX, which increases Ca2+ in a local fashion. The local elevation of Ca2+ causes the closure of gap junctions by an unknown mechanism thus preventing synchronization of [Ca2+]i transients and vasomotion (B). The same effect was observed after blockade of NCX (SEA0400).

The authors used an isolated small artery from the rat mesentery and cultured smooth muscle cells (A7r5) to study the regulation of gap junctional communication which was assessed by functional parameters in the intact vessel (Ca²⁺ transients and vasomotion) or direct measurement of electrical coupling (membrane capacitance) in a cell system. The events initiating vasomotion in this model have been previously described by this group¹⁶ and include an oscillating Ca²⁺ release and the resultant activation of a depolarising current, which gives rise to oscillations of the membrane potential that can act as a synchronizing signal if sufficient in amplitude and transmitted through gap junctions.¹⁷ Thus, this model provides the opportunity to study synchronization of cellular behavior. Blockade of the Na⁺/K⁺-pump with ouabain desynchronised [Ca²⁺]_i transients in smooth muscle cells and abrogated vasomotion. This effect seems to depend on pump inhibition because the reduction of the extracellular Na⁺-concentration had similar effects. The observation of a concomitant increase of Ca²⁺ in the periphery of the cell led the authors to speculate that the Na⁺-Ca²⁺-exchange is spatially located with and functionally coupled to the activity of the Na⁺/K⁺-pump as has been demonstrated in the heart and in the vasculature.¹⁸

Cultured A7r5 cells form gap junctions, are electrically coupled, and allow easy control of ion concentrations using patch clamp. Matchkov et al showed that ATP-depletion, omission of extracellular K⁺ (in the presence of K_ATP-channel blockade or substitution of intracellular K⁺ by Cs⁺ to avoid supplying the Na⁺/K⁺-pump with K⁺ from the interior of the cell), and a reduction of the extracellular Na⁺-concentration inhibited coupling. These interventions have in common that the Na⁺/K⁺-pump is inhibited and thus verify the regulatory effect on gap junctional communication. In contrast, the omission of intracellular Na⁺ which also blocks the Na⁺/K⁺-pump leaves coupling intact and prevents the effect of ouabain suggesting that intracellular Na⁺ is involved in the signaling, possibly by inhibiting or reversing Na⁺-Ca²⁺-exchange (ie, Na⁺ extrusion and Ca²⁺ influx). Therefore, the contribution of the Na⁺-Ca²⁺-exchanger was verified pharmacologically (also shown to be effective in the intact vessel) or by altering driving forces (manipulating the membrane potential in conjunction with the Na⁺ gradient). In line with the hypothesis these interventions (inhibiting forward mode or switching to reverse mode) uncoupled the cells. Both approaches should increase Ca²⁺ in the microdomain. The authors indeed demonstrated such rises in Ca²⁺ in subdomains along the cell membrane after ouabain which were remarkbly stable in time and place. Buffering Ca²⁺ prevented the inhibitory effect of Na⁺/K⁺-pump and Na⁺-Ca²⁺-exchanger blockade on gap junctional communication. In contrast, global increases in Ca²⁺ induced by vasopressin accompanied by more variable peripheral Ca²⁺ rises did not affect gap junctional communication which accentuates the importance of microdomains to regulate gap junctional communication.

The Na⁺/K⁺-pump consists of catalytic - and regulatory ß-subunits and for both subunits different isoforms have been characterized. Matchkov et al used a concentration of ouabain that selectively inhibits the high affinity 2- and 3-isoforms thereby possibly revealing another physiological implication of different isoforms. Interestingly enough, genetically engineered mice which expressed lower amounts only of the 2-isoform exhibited an elevated blood pressure and their vessels displayed enhanced contractility to pressure, whereas animals with a reduced amount of the 1-isoform remained normotensive.¹⁹ If this is also related to an alteration of gap junctional communication is at present unclear. Because vascular cells express four connexin (Cx) isoforms named according to their molecular weight (Cx37, Cx40, Cx43, Cx45) a key question, having identified a modulation of gap junctional communication, is which connexin is affected. Previous work from the author’s laboratory localized Cx37 in smooth muscle (and Cx37 as well as Cx40 in the endothelium) of the mesenteric artery.¹⁷ In contrast, Cx40 and Cx43 have been found to be abundantly expressed in A7r5 cells.²⁰ Although this is at first confusing it could be speculated that all connexins are sensitive to regulation by local Ca²⁺, as indicated by the need for a combination of the so-called connexin-mimetic peptides to abrogate vasomotion despite their suggested ability to specifically block connexin subtypes.¹⁷ In conclusion, the work by Matchkov and colleagues shines important light on the regulation of gap junctional communication in the vasculature: In microdomains Ca²⁺ is able to functionally uncouple smooth muscle cells possibly because connexins and gap junctions colocalize with regulatory pumps creating a microenvironment. Alternatively, they might be controlled by the Ca²⁺ store loaded by the interaction of the Na⁺/K⁺-pump (2, 3), Na⁺-Ca²⁺-exchanger, and Ca²⁺ pumps in the restricted space between plasma membrane and sarcoplasmic reticulum.¹⁸ Perhaps other proteins are likewise spatially connected to connexins allowing easier interaction, eg, K⁺-channels.²¹ The importance of microdomains reminds one of an old adage: location, location, location.

	Acknowledgments

 
Source of Funding

Studies performed in the author’s own laboratory were supported by the Deutsche Forschungsgemeinschaft (WI 2071/1-1).

Disclosures

None.

	Footnotes

 
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.

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Related Article:

Interaction Between Na⁺/K⁺-Pump and Na⁺/Ca²⁺-Exchanger Modulates Intercellular Communication

Vladimir V. Matchkov, Helena Gustafsson, Awahan Rahman, Donna M. Briggs Boedtkjer, Sarah Gorintin, Anne Kirstine Hansen, Elena V. Bouzinova, Helle A. Praetorius, Christian Aalkjaer, and Holger Nilsson
Circ. Res. 2007 100: 1026-1035. [Abstract] [Full Text] [PDF]

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