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or they may dock with each other in macromolecular arrays joining two adjacent
cells and so create a cluster of transmembrane pores (Gap junctions) that link the
cytosols of the adjacent cells. Investigation of the possible involvement of the 37, 40,
and 43 Kd isoforms that are most commonly seen in endothelial cells [15] revealed
only the clear presence of the 43 Kd isoform by Western blot. Furthermore, CX43
was detectable at both native and elevated apparent molecular weight [37], implying
the CX43 could be subject to phosphorylation and so potential regulation by kinases.
Confirmation that the CX proteins expressed in UAEC were indeed forming true
Gap junctions rather than hemichannels was demonstrated by the finding that scrape
loading of a high density cell monolayer with Lucifer yellow dye promoted dye
entry alongside the cut in the cell monolayer, but not generally across all the cells
[37]. Hemichannels on the surface of every cell alone would simply have allowed
uniform dye entry to the entire monolayer independent of the cut, while only true
Gap junctions would give the result observed with dye entry only to cells adjacent to
the cut site. Further evidence for the formation of true Gap junctions by CX43 itself
was achieved using peptides of sequences encoding the extracellular loop regions
of the different CX isoforms. In docking to make Gap junctions, CX proteins bind
each other via extracellular loops, and peptides made from copies of these same
sequences can competitively interfere with the coupling process. Communication
via a true Gap junction is then lost, while function through a pore on the surface of
the cell to release agents to the extracellular space remains unaltered. We have found
that a peptide to the CX43 sequence (SRPTEKTIFII - termed GAP27) is completely
successful in preventing cell synchronization and further has a profound effect in
preventing long term [Ca
2+
]i burst responses to ATP at high cell density. In contrast,
other classes of GAP peptides encoding sequences of CX40 or 37, or a scrambled
version of CX43 GAP27 were ineffective [37]. Thus CX43 alone mediates inter-cell
communication of relevance to [Ca
2+
]i signaling, and further that such communi-
cation in the form of true Gap junctions is critical to long term [Ca
2+
]i bursts (see
Fig. 11.5b). In addition, GAP27 will inhibit the burst pattern of both P-UAEC and
NP-UAEC down to a common lower level. It is not clear at this time if the CX43
Gap junctions transport Ca
2+
or IP3 itself, but either way there are two important
conclusions that can be drawn concerning pregnancy adaptation of cell signaling
in UAEC, namely: (1) while such bursts of capacitative entry may be a function of
TRPC/IP3R interaction, this process is in turn regulated by CX43 Gap junctions,
and (2) pregnancy specific enhancement of this burst activity is not regulated at the
level of CX43 expression but instead is regulated by pregnancy specific enhance-
ment of Gap junction function. Given the additional finding that this same GAP27
peptide also inhibits the activation of eNOS in P-UAEC and NP-UAEC down to a
common level [12], it is possible to conclude that while the molecular means by
which pregnancy enhances NO production is indeed through an associated increase
in capacitative entry in UAEC at the level of IP3R2-TRPC3 interaction, this process
is only increased because of the pregnancy enhancement of CX43 GAP junction
communication.
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