Pharmacological sensitivity of ATP release triggered by photoliberation of inositol‐1,4,5‐trisphosphate and zero extracellular calcium in brain endothelial cells

Abstract

Recently, ATP has gained much interest as an extracellular messenger involved in the communication of calcium signals between cells. The mechanism of ATP release is, however, still a matter of debate. In the present study we investigated the possible contribution of connexin hemichannels or ion channels in the release of ATP in GP8, a rat brain endothelial cell line. Release of ATP was triggered by photoactivation of InsP(3) or by reducing the extracellular calcium concentration. Both trigger protocols induced ATP release significantly above baseline. InsP(3)-triggered ATP release was completely blocked by alpha-glycyrrhetinic acid (alpha-GA), the connexin mimetic peptides gap 26 and 27, and the trivalent ions gadolinium and lanthanum. ATP release triggered by zero calcium was, in addition to these substances, also blocked by flufenamic acid (FFA), niflumic acid, and NPPB. Gap 27 selectively blocked zero calcium-triggered ATP release in connexin-43 transfected HeLa cells, while having no effect in wild-type and connexin-32 transfected cells. Of all the agents used, only alpha-GA, FFA and NPPB significantly reduced gap junctional coupling. In conclusion, InsP(3) and zero calcium-triggered ATP release show major similarities but also some differences in their sensitivity to the agents applied. It is suggested that both stimuli trigger ATP release through the same mechanism, which is connexin-dependent, permeable in both directions, potently blocked by connexin mimetic peptides, and consistent with the opening of connexin hemichannels.

ATP release through connexin hemichannels and gap junction transfer of second messengers propagate Ca 2+ signals across the inner ear

“…In particular, we compared calcein efflux rates in WT, Cx26 KO, and Cx30 KO cultures and we also tested the effects of CBX and lanthanum (La 3ϩ ). The latter has been reported to block IP 3 -triggered ATP release in a rat brain endothelial cell line (40). La 3ϩ also impairs dye uptake in rat cortical astrocytes expressing Cx43 (41) and blocks hemichannel currents evoked by depolarization of HeLa cells transfected with cDNA encoding rat Cx43 (42).…”

Role of Gap Junction Channel in the Development of Beat-to-Beat Action Potential Repolarization Variability and Arrhythmias

“…Partial uncoupling of gap junctions prior to ischaemia by ischemic-preconditioning preserves the electrical coupling of cells during a subsequent ischemic insult, indicating that a partial closure of gap junctions may play a trigger role in the protection [94,96,154,155]. GAP26 and GAP27 blocks calcium-triggered ATP release mediated by Cx43 hemichannels [156][157][158][159]. Hemichannels are not engaged to gap junctions, and they are open under several physiological and pathophysiological conditions [160].…”

Connexin 43 Hemichannels and Pharmacotherapy of Myocardial Ischemia Injury

“…The conventional gap junction blockers (as heptanol) that are available to date act on both whole gap junction channels and unapposed Hc. Interestingly, Cx-mimetic peptides have recently emerged as powerful tools capable of blocking primarily the unapposed Cx43Hc by mimicking short amino acids sequences on the extracellular loops of Cx (references (Braet et al, 2003a;Evans et al, 2006;Leybaert et al, 2003;Martin et al, 2005;Pearson et al, 2005) testing ATP release from various non-cardiac cell types). Here, we investigated the consequent effect of two Cx43-mimetic peptides, Gap26 and Gap 27, on the electrophysiology of Cx43Hc exogenously expressed in the tsA201 cells using the patch clamp technique.…”

“…Initially, the Cx-mimetic peptides were designed with the intention of selectively blocking gap junction channels by intercepting the apposition of pairs of Hc from adjacent cells (Dahl et al, 1994;Warner et al, 1995), but have subsequently emerged as potent blockers for the unapposed CxHc with little or no immediate effect on gap junction (Braet et al, 2003a;Evans et al, 2006;Leybaert et al, 2003;Martin et al, 2005;Pearson et al, 2005).…”

“…However, under ischemic stress, the amount of non-phosphorylated Cx43 increases (Beardslee et al, 2000). Phosphorylation causes the unapposed Cx43Hc to close whereas, conversely, Cx43 dephosphorylation increases Hc conductance and permeability (Bao et al, 2007;Contreras et al, 2002;John et al, 1999b;Kondo et al, 2000;Li et al, 2001), an effect that can result in abnormal elevation of intracellular sodium and calcium loads (Li et al, 2001), release of ATP (Braet et al, 2003a;Braet et al, 2003b;Contreras et al, 2002), osmotic imbalance (John et al, 1999b;Li et al, 2001;Quist et al, 2000), swelling of myocytes Steenbergen et al, 1985;Tranum-Jensen et al, 1981) and lead to irreversible tissue injury (Shintani-Ishida et al, 2007) and cell death. Several kinases are involved in Cx43 phosphorylation (Cottrell et al, 2003;Duncan & Fletcher, 2002;Shi et al, 2001;TenBroek et al, 2001;Warn-Cramer et al, 1998), including protein kinase C (PKC) .…”