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Connexin channels provide a target to manipulate calcium dynamics and blood brain barrier permeability

Marijke De Bock UGent (2012)
abstract
A typical feature of multicellular organisms is their capacity to communicate with surrounding cells, coordinating organ function. In vertebrate tissues, the integration of cellular functions is mediated by gap junctions, i.e. cell-cell channels that connect the cytoplasm of two neigboring cells. These channels are composed of two half gap junction channels or ‘hemi’channels that, on their turn, enclose 6 connexin proteins. Over the years it has become evident that hemichannels are not mere building blocks of gap junction channels, but function by themselves as a tightly regulated conduit between the cell’s interior and the extracellular space. Connexin channels, both gap junctions and hemichannels, are implicated in many tissue functions and their contribution to, for instance, Ca2+ signaling (changes in the intracellular Ca2+ concentration [Ca2+]i) has been very well characterized. Ca2+ signals are highly organized in time and space, presenting as intracellular Ca2+ oscillations and intercellular Ca2+ waves respectively. Two mechanisms have been identified supporting the latter. The first mechanism relies on the diffusion of Ca2+ or Ca2+ mobilizing messengers through gap junction channels. Alternatively, paracrine signaling involves the release of a Ca2+ mobilizing messenger into the extracellular space where it binds on its corresponding receptor and activates downstream signaling pathways in neighboring cells. Connexin hemichannels are likely candidates for the release of these messengers. In addition to their proposed role in Ca2+ wave propagation, evidence is accruing that hemichannels may be involved in Ca2+ oscillations. The aim of this work was to define the mechanism by which hemichannels are involved in Ca2+ oscillations. Oscillations are based on positive and negative [Ca2+]i feedback on InsP3 receptor (InsP3R) opening and Ca2+ release from the endoplasmic reticulum. Connexin hemichannels too are Ca2+-permeable plasma membrane channels that are controlled by [Ca2+]i; therefore they may have an active contribution to the Ca2+ oscillation machinery as well. We applied different agonists that trigger Ca2+ oscillations and determined the involvement of connexin hemichannels in immortalized and long term cultured, primary brain endothelial cells that express Cx37 and Cx43. Bradykinin-triggered Ca2+ oscillations were inhibited by interfering with connexin channels making use of the pan-connexin channel inhibitor carbenoxolone, Cx37/Cx43 knockdown or Gap27, a peptide blocker of Cx37/Cx43 channels. Gap27 inhibition of the oscillations was rapid (within minutes), indicating the involvement of hemichannels (not gap junctions). Work with connexin hemichannel-permeable dyes provided evidence for bradykinin-triggered hemichannel opening and we furthermore found that a bradykinin-activated purinergic signaling loop contributes to the oscillations. In contrast, Ca2+ oscillations provoked by exposure to ATP were not affected by carbenoxolone or Gap27. In Madine Darby Canine Kidney (MDCK) cells expressing Cx32 and Cx43, bradykinin-induced oscillations were rapidly and reversibly inhibited by the connexin mimetic peptides 32Gap27/43Gap26 and by Cx43 gene silencing, while ATP-induced oscillations were again unaffected. These peptides also inhibited the bradykinin-triggered release of hemichannel-permeable dyes. Furthermore, bradykinin-induced oscillations, but not those induced by ATP, were sensitive to lowering extracellular Ca2+ to 0.5 mM. Alleviating the negative feedback of [Ca2+]i on InsP3Rs using CytC inhibited both bradykinin- and ATP-induced oscillations. Similar to the InsP3R, Cx32 and Cx43 hemichannels are activated by [Ca2+]i < 500 nM but are inhibited by higher concentrations. CT9 peptide (last 9 amino acids of the Cx43 C-terminal tail) removed the inhibition by [Ca2+]i > 500 nM. Unlike interfering with the bell-shaped dependence of InsP3Rs to [Ca2+]i with CytC, CT9 peptide only prevented bradykinin-induced oscillations. Furthermore, hemichannel opening was not sufficient to set off oscillations by itself but a contribution of hemichannels was crucial as their inhibition stopped the oscillations. Repetitive changes in [Ca2+]i are documented to initiate a myriad of cellular processes, but little is known on the effect of [Ca2+]i dynamics on blood-brain barrier (BBB) function. This barrier is present between the systemic circulation and the brain, and protects the nervous tissue from potentially toxic, circulating substances while securing a specialized environment for proper neuronal signaling. The BBB is formed by capillary endothelial cells that are characterized by an extremely low pinocytotic activity thus limiting non-specific transcellular access to the brain tissue. BBB endothelial cells are furthermore equipped with a tight and complex junctional network which, aided by the actin cytoskeleton, results in a restriction of the paracellular permeability. The latter route has been the subject of extensive research as an increase in paracellular permeability is often associated with a wide range of central nervous system pathologies and underlies brain edema and inflammation. In second instance we aimed to define a functional link for connexin-based Ca2+ dynamics (oscillations and waves) on BBB function. In particular, we explored the effects of hemichannel-supported Ca2+ oscillations on BBB endothelial permeability. Bradykinin triggered Ca2+ oscillations and increased endothelial permeability in immortalized and long term cultured, primary BBB endothelial cells. This was prevented by buffering intracellular Ca2+ changes with BAPTA indicating that Ca2+ oscillations are crucial in the permeability changes. Moreover, Gap27 inhibited the bradykinin-triggered endothelial permeability increase in in vitro and in vivo experiments. ATP, which induced oscillations that did not require hemichannels, did not disturb endothelial permeability. At the protein level we found bradykinin-induced alterations in the intermediate filament vimentin, but not in the tight junction proteins occludin and ZO-1. Again, these changes could be counteracted by Gap27 and were not found in cells treated with ATP. Exposing brain endothelial cells to low extracellular Ca2+ conditions triggered intercellular Ca2+ waves in the endothelial cultures. These waves elicited an increase in endothelial permeability that was inhibited by buffering [Ca2+]i changes, indicating a crucial role for [Ca2+]i changes, and by the connexin channel blocker Gap27. Although, the cell mass participating in either Ca2+ oscillations or Ca2+ waves was comparable, the permeability-increase triggered by low extracellular Ca2+ conditions largely exceeded that brought about by bradykinin, suggesting that intercellular Ca2+ waves are more efficient in modulating barrier function. Inhibiting protein kinase C, Ca2+/calmodulin-dependent kinase II and actomyosin contraction interfered with the permeability-increase brought about by Ca2+-free solution but did not influence the permeability increase triggered by bradykinin. Collectively, our data indicate that connexin hemichannels contribute to bradykinin-induced oscillations by allowing Ca2+-entry and/or release of ATP that acts in an autocrine manner, and that such hemichannel-supported oscillations increase BBB permeability. Additionally, intercellular Ca2+ waves that propagate by means of the different connexin channels result in more pronounced changes in BBB permeability. Currently, there are no tools available to limit BBB opening and our work shows that endothelial connexin channels may serve as a novel target to counteract a BBB permeability increase.
Please use this url to cite or link to this publication:
author
promoter
UGent
organization
year
type
dissertation (monograph)
subject
keyword
Calcium, Connexin, Blood brain barrier
pages
257 pages
publisher
Ghent University. Faculty of Medicine and Health Sciences
place of publication
Ghent, Belgium
defense location
Gent : Het Pand (zaal rector Vermeylen)
defense date
2012-07-02 17:00
language
English
UGent publication?
yes
classification
D1
additional info
dissertation in part contains copyrighted material
copyright statement
I have transferred the copyright for this publication to the publisher
id
2980289
handle
http://hdl.handle.net/1854/LU-2980289
date created
2012-09-06 14:58:11
date last changed
2012-09-07 09:18:44
@phdthesis{2980289,
  abstract     = {A typical feature of multicellular organisms is their capacity to communicate with surrounding cells, coordinating organ function. In vertebrate tissues, the integration of cellular functions is mediated by gap junctions, i.e. cell-cell channels that connect the cytoplasm of two neigboring cells. These channels are composed of two half gap junction channels or {\textquoteleft}hemi{\textquoteright}channels that, on their turn, enclose 6 connexin proteins. Over the years it has become evident that hemichannels are not mere building blocks of gap junction channels, but function by themselves as a tightly regulated conduit between the cell{\textquoteright}s interior and the extracellular space. Connexin channels, both gap junctions and hemichannels, are implicated in many tissue functions and their contribution to, for instance, Ca2+ signaling (changes in the intracellular Ca2+ concentration [Ca2+]i) has been very well characterized. Ca2+ signals are highly organized in time and space, presenting as intracellular Ca2+ oscillations and intercellular Ca2+ waves respectively. Two mechanisms have been identified supporting the latter. The first mechanism relies on the diffusion of Ca2+ or Ca2+ mobilizing messengers through gap junction channels. Alternatively, paracrine signaling involves the release of a Ca2+ mobilizing messenger into the extracellular space where it binds on its corresponding receptor and activates downstream signaling pathways in neighboring cells. Connexin hemichannels are likely candidates for the release of these messengers. 
In addition to their proposed role in Ca2+ wave propagation, evidence is accruing that hemichannels may be involved in Ca2+ oscillations. The aim of this work was to define the mechanism by which hemichannels are involved in Ca2+ oscillations. Oscillations are based on positive and negative [Ca2+]i feedback on InsP3 receptor (InsP3R) opening and Ca2+ release from the endoplasmic reticulum. Connexin hemichannels too are Ca2+-permeable plasma membrane channels that are controlled by [Ca2+]i; therefore they may have an active contribution to the Ca2+ oscillation machinery as well. We applied different agonists that trigger Ca2+ oscillations and determined the involvement of connexin hemichannels in immortalized and long term cultured, primary brain endothelial cells that express Cx37 and Cx43. Bradykinin-triggered Ca2+ oscillations were inhibited by interfering with connexin channels making use of the pan-connexin channel inhibitor carbenoxolone, Cx37/Cx43 knockdown or Gap27, a peptide blocker of Cx37/Cx43 channels. Gap27 inhibition of the oscillations was rapid (within minutes), indicating the involvement of hemichannels (not gap junctions). Work with connexin hemichannel-permeable dyes provided evidence for bradykinin-triggered hemichannel opening and we furthermore found that a bradykinin-activated purinergic signaling loop contributes to the oscillations. In contrast, Ca2+ oscillations provoked by exposure to ATP were not affected by carbenoxolone or Gap27. In Madine Darby Canine Kidney (MDCK) cells expressing Cx32 and Cx43, bradykinin-induced oscillations were rapidly and reversibly inhibited by the connexin mimetic peptides 32Gap27/43Gap26 and by Cx43 gene silencing, while ATP-induced oscillations were again unaffected. These peptides also inhibited the bradykinin-triggered release of hemichannel-permeable dyes. Furthermore, bradykinin-induced oscillations, but not those induced by ATP, were sensitive to lowering extracellular Ca2+ to 0.5 mM. Alleviating the negative feedback of [Ca2+]i on InsP3Rs using CytC inhibited both bradykinin- and ATP-induced oscillations. Similar to the InsP3R, Cx32 and Cx43 hemichannels are activated by [Ca2+]i {\textlangle} 500 nM but are inhibited by higher concentrations. CT9 peptide (last 9 amino acids of the Cx43 C-terminal tail) removed the inhibition by [Ca2+]i {\textrangle} 500 nM. Unlike interfering with the bell-shaped dependence of InsP3Rs to [Ca2+]i with CytC, CT9 peptide only prevented bradykinin-induced oscillations. Furthermore, hemichannel opening was not sufficient to set off oscillations by itself but a contribution of hemichannels was crucial as their inhibition stopped the oscillations.
Repetitive changes in [Ca2+]i are documented to initiate a myriad of cellular processes, but little is known on the effect of [Ca2+]i dynamics on blood-brain barrier (BBB) function. This barrier is present between the systemic circulation and the brain, and protects the nervous tissue from potentially toxic, circulating substances while securing a specialized environment for proper neuronal signaling. The BBB is formed by capillary endothelial cells that are characterized by an extremely low pinocytotic activity thus limiting non-specific transcellular access to the brain tissue. BBB endothelial cells are furthermore equipped with a tight and complex junctional network which, aided by the actin cytoskeleton, results in a restriction of the paracellular permeability. The latter route has been the subject of extensive research as an increase in paracellular permeability is often associated with a wide range of central nervous system pathologies and underlies brain edema and inflammation. 
In second instance we aimed to define a functional link for connexin-based Ca2+ dynamics (oscillations and waves) on BBB function. In particular, we explored the effects of hemichannel-supported Ca2+ oscillations on BBB endothelial permeability. Bradykinin triggered Ca2+ oscillations and increased endothelial permeability in immortalized and long term cultured, primary BBB endothelial cells. This was prevented by buffering intracellular Ca2+ changes with BAPTA indicating that Ca2+ oscillations are crucial in the permeability changes. Moreover, Gap27 inhibited the bradykinin-triggered endothelial permeability increase in in vitro and in vivo experiments. ATP, which induced oscillations that did not require hemichannels, did not disturb endothelial permeability. At the protein level we found bradykinin-induced alterations in the intermediate filament vimentin, but not in the tight junction proteins occludin and ZO-1. Again, these changes could be counteracted by Gap27 and were not found in cells treated with ATP. Exposing brain endothelial cells to low extracellular Ca2+ conditions triggered intercellular Ca2+ waves in the endothelial cultures. These waves elicited an increase in endothelial permeability that was inhibited by buffering [Ca2+]i changes, indicating a crucial role for [Ca2+]i changes, and by the connexin channel blocker Gap27. Although, the cell mass participating in either Ca2+ oscillations or Ca2+ waves was comparable, the permeability-increase triggered by low extracellular Ca2+ conditions largely exceeded that brought about by bradykinin, suggesting that intercellular Ca2+ waves are more efficient in modulating barrier function. Inhibiting protein kinase C, Ca2+/calmodulin-dependent kinase II and actomyosin contraction interfered with the permeability-increase brought about by Ca2+-free solution but did not influence the permeability increase triggered by bradykinin. 
Collectively, our data indicate that connexin hemichannels contribute to bradykinin-induced oscillations by allowing Ca2+-entry and/or release of ATP that acts in an autocrine manner, and that such hemichannel-supported oscillations increase BBB permeability. Additionally, intercellular Ca2+ waves that propagate by means of the different connexin channels result in more pronounced changes in BBB permeability. Currently, there are no tools available to limit BBB opening and our work shows that endothelial connexin channels may serve as a novel target to counteract a BBB permeability increase.},
  author       = {De Bock, Marijke},
  keyword      = {Calcium,Connexin,Blood brain barrier},
  language     = {eng},
  pages        = {257},
  publisher    = {Ghent University. Faculty of Medicine and Health Sciences},
  school       = {Ghent University},
  title        = {Connexin channels provide a target to manipulate calcium dynamics and blood brain barrier permeability},
  year         = {2012},
}

Chicago
De Bock, Marijke. 2012. “Connexin Channels Provide a Target to Manipulate Calcium Dynamics and Blood Brain Barrier Permeability”. Ghent, Belgium: Ghent University. Faculty of Medicine and Health Sciences.
APA
De Bock, Marijke. (2012). Connexin channels provide a target to manipulate calcium dynamics and blood brain barrier permeability. Ghent University. Faculty of Medicine and Health Sciences, Ghent, Belgium.
Vancouver
1.
De Bock M. Connexin channels provide a target to manipulate calcium dynamics and blood brain barrier permeability. [Ghent, Belgium]: Ghent University. Faculty of Medicine and Health Sciences; 2012.
MLA
De Bock, Marijke. “Connexin Channels Provide a Target to Manipulate Calcium Dynamics and Blood Brain Barrier Permeability.” 2012 : n. pag. Print.