Modulation of Tonic GABA Currents by Anion Channel and Connexin Hemichannel Antagonists

Anion channels and connexin hemichannels are permeable to amino acid neurotransmitters. It is hypothesized that these conductive pathways release GABA, thereby influencing ambient GABA levels and tonic GABAergic inhibition. To investigate this, we measured the effects of anion channel/hemichannel antagonists on tonic GABA currents of rat hippocampal neurons. In contrast to predictions, blockade of anion channels and hemichannels with NPPB potentiated tonic GABA currents of neurons in culture and acute hippocampal slices. In contrast, the anion channel/hemichannel antagonist carbenoxolone (CBX) inhibited tonic currents. These findings could result from alterations of ambient GABA concentration or direct effects on GABA_A receptors. To test for effects on GABA_A receptors, we measured currents evoked by exogenous GABA. Coapplication of NPPB with GABA potentiated GABA-evoked currents. CBX dose-dependently inhibited GABA-evoked currents. These results are consistent with direct effects of NPPB and CBX on GABA_A receptors. GABA release from hippocampal cell cultures was directly measured using HPLC. Inhibition of anion channels with NPPB or CBX did not affect GABA release from cultured hippocampal neurons. NPPB reduced GABA release from pure astrocytic cultures by 21%, but the total GABA release from astrocytes was small compared to that of mixed cultures. These data indicate that drugs commonly used to antagonize anion channels and connexin hemichannels may affect tonic currents via direct effects on GABA_A receptors and have negligible effects on ambient GABA concentrations. Interpretation of experiments using NPPB or CBX should include consideration of their effects on tonic GABA currents.     

Connexin 43 hemichannels contribute to cytoplasmic Ca2+ oscillations by providing a bimodal Ca2+-dependent Ca2+ entry pathway

Many cellular functions are driven by changes in the intracellular Ca(2+) concentration ([Ca(2+)](i)) that are highly organized in time and space. Ca(2+) oscillations are particularly important in this respect and are based on positive and negative [Ca(2+)](i) feedback on inositol 1,4,5-trisphosphate receptors (InsP(3)Rs). Connexin hemichannels are Ca(2+)-permeable plasma membrane channels that are also controlled by [Ca(2+)](i). We aimed to investigate how hemichannels may contribute to Ca(2+) oscillations. Madin-Darby canine kidney cells expressing connexin-32 (Cx32) and Cx43 were exposed to bradykinin (BK) or ATP to induce Ca(2+) oscillations. BK-induced oscillations were rapidly (minutes) and reversibly inhibited by the connexin-mimetic peptides (32)Gap27/(43)Gap26, whereas ATP-induced oscillations were unaffected. Furthermore, these peptides inhibited the BK-triggered release of calcein, a hemichannel-permeable dye. BK-induced oscillations, but not those induced by ATP, were dependent on extracellular Ca(2+). Alleviating the negative feedback of [Ca(2+)](i) on InsP(3)Rs using cytochrome c inhibited BK- and ATP-induced oscillations. Cx32 and Cx43 hemichannels are activated by <500 nm [Ca(2+)](i) but inhibited by higher concentrations and CT9 peptide (last 9 amino acids of the Cx43 C terminus) removes this high [Ca(2+)](i) inhibition. Unlike interfering with the bell-shaped dependence of InsP(3)Rs to [Ca(2+)](i), CT9 peptide prevented BK-induced oscillations but not those triggered by ATP. Collectively, these data indicate that connexin hemichannels contribute to BK-induced oscillations by allowing Ca(2+) entry during the rising phase of the Ca(2+) spikes and by providing an OFF mechanism during the falling phase of the spikes. Hemichannels were not sufficient to ignite oscillations by themselves; however, their contribution was crucial as hemichannel inhibition stopped the oscillations.     

Biophysical properties of connexin-45 gap junction hemichannels studied in vertebrate cells

Human HeLa cells transfected with mouse Cx45 and rat RIN cells transfected with chicken Cx45 were used to study the electrical and permeability properties of Cx45 gap junction hemichannels. With no extracellular Ca(2+), whole-cell recording revealed currents arising from hemichannels in both transfected cell lines. Multichannel currents showed a time-dependent activation or deactivation sensitive to voltage, V(m). These currents did not occur in non-transfected cells. The hemichannel currents were inhibited by raising extracellular Ca(2+) or by acidification with CO(2). The unitary conductance exhibited V(m) dependence (i.e., gamma(hc,main) increased/decreased with hyperpolarization/depolarization). Extrapolation to V(m) = 0 mV led to a gamma(hc,main) of 57 pS, roughly twice the conductance of an intact Cx45 gap junction channel. The open channel probability, P(o), was V(m)-dependent, declining at negative V(m) (P(o) < 0.11, V(m) < -50 mV), and increasing at positive V(m) (P(o) approximately 0.76, V(m) > 50 mV). Moreover, Cx45 nonjunctional hemichannels appeared to mediate lucifer yellow (LY) and propidium iodide (PI) dye uptake from the external solution when extracellular Ca(2+) level was reduced. Dye uptake was directly proportional to the number of functioning hemichannels. No significant dye uptake was detected in non-transfected cells. Cx45 transfected HeLa and RIN cells also allowed dye to leak out when preloaded with LY and then incubated in Ca(2+)-free external solution, whereas little or no dye leakage was observed when these cells were incubated with 2 mM external Ca(2+). Intact Cx45 gap junction channels allowed passage of either LY or PI dye, but their respective flux rates were different. Comparison of LY diffusion through Cx45 hemichannels and intact gap junction channels revealed that the former is more permeable, suggesting that gap junction channel pores exhibit more allosterical restriction to the dye molecules than the unopposed hemichannel. The data demonstrate the opening of Cx45 nonjunctional hemichannels in vertebrate cells when the external Ca(2+) concentration is reduced.     

Hemichannel-mediated inositol 1,4,5-trisphosphate (IP3) release in the cochlea: a novel mechanism of IP3 intercellular signaling

Inositol 1,4,5-trisphosphate (IP(3)) is an important second messenger that can trigger a Ca(2+) wave prolongated between cells. This intercellular signaling was found defective in some gap junction connexin deafness mutants. In this study, the mechanism underlying IP(3) intercellular signaling in the cochlea was investigated. A gap junction channel is composed of two hemichannels. By using a fluorescence polarization technique to measure IP(3) concentration, the authors found that IP(3) could be released by gap junction hemichannels in the cochlea. The IP(3) release was increased about three- to fivefold by the reduction of extracellular Ca(2+) concentration or by mechanical stress. This incremental release could be blocked by gap junction blockers but not eliminated by a purinergic P2x receptor antagonist and verapamil, which is a selective P-glycoprotein inhibitor inhibiting the ATP-binding cassette transporters. The authors also found that IP(3) receptors were extensively expressed in the cochlear sensory epithelium, including on the cell surface. Extracellular application of IP(3) could trigger cellular Ca(2+) elevation. This Ca(2+) elevation was eliminated by the gap junction hemichannel blocker. These data reveal that IP(3) can pass through hemichannels acting as an extracellular mediator to participate in intercellular signaling. This hemichannel-mediated extracellular pathway may play an important role in long-distance intercellular communication in the cochlea, given that IP(3) only has a short lifetime in the cytoplasm.     

Purinergic receptors mediate two distinct glutamate release pathways in hippocampal astrocytes

The purinergic P2X(7) receptor (P2X(7)R) can mediate glutamate release from cultured astrocytes. Using patch clamp recordings, we investigated whether P2X(7)Rs have the same action in hippocampal astrocytes in situ. We found that 2- and 3-O-(4-benzoylbenzoyl)ATP (BzATP), a potent, although unselective P2X(7)R agonist, triggers two different glutamate-mediated responses in CA1 pyramidal neurons; they are transient inward currents, which have the kinetic and pharmacological properties of previously described slow inward currents (SICs) due to Ca(2+)-dependent glutamate release from astrocytes, and a sustained tonic current. Although SICs were unaffected by P2X(7)Rs antagonists, the tonic current was inhibited, was amplified in low extracellular Ca(2+), and was insensitive to glutamate transporter and hemichannel inhibitors. BzATP triggered in astrocytes a large depolarization that was inhibited by P2X(7)R antagonists and amplified in low Ca(2+). In low Ca(2+) BzATP also induced lucifer yellow uptake into a subpopulation of astrocytes and CA3 neurons. Our results demonstrate that purinergic receptors other than the P2X(7)R mediate glutamate release that evokes SICs, whereas activation of a receptor that has features similar to the P2X(7)R, mediates a sustained glutamate efflux that generates a tonic current in CA1 neurons. This sustained glutamate efflux, which is potentiated under non-physiological conditions, may have important pathological actions in the brain.     

Intracellular calcium changes trigger connexin 32 hemichannel opening

Connexin hemichannels have been proposed as a diffusion pathway for the release of extracellular messengers like ATP and others, based on connexin expression models and inhibition by gap junction blockers. Hemichannels are opened by various experimental stimuli, but the physiological intracellular triggers are currently not known. We investigated the hypothesis that an increase of cytoplasmic calcium concentration ([Ca2+]i) triggers hemichannel opening, making use of peptides that are identical to a short amino-acid sequence on the connexin subunit to specifically block hemichannels, but not gap junction channels. Our work performed on connexin 32 (Cx32)-expressing cells showed that an increase in [Ca2+]i triggers ATP release and dye uptake that is dependent on Cx32 expression, blocked by Cx32 (but not Cx43) mimetic peptides and a calmodulin antagonist, and critically dependent on [Ca2+]i elevation within a window situated around 500 nM. Our results indicate that [Ca2+]i elevation triggers hemichannel opening, and suggest that these channels are under physiological control.     

Oxygen-glucose deprivation induces ATP release via maxi-anion channels in astrocytes

ATP represents a major gliotransmitter that serves as a signaling molecule for the cross talk between glial and neuronal cells. ATP has been shown to be released by astrocytes in response to a number of stimuli under nonischemic conditions. In this study, using a luciferin-luciferase assay, we found that mouse astrocytes in primary culture also exhibit massive release of ATP in response to ischemic stress mimicked by oxygen-glucose deprivation (OGD). Using a biosensor technique, the local ATP concentration at the surface of single astrocytes was found to increase to around 4 muM. The OGD-induced ATP release was inhibited by Gd(3+) and arachidonic acid but not by blockers of volume-sensitive outwardly rectifying Cl(-) channels, cystic fibrosis transmembrane conductance regulator (CFTR), multidrug resistance-related protein (MRP), connexin or pannexin hemichannels, P2X(7) receptors, and exocytotic vesicular transport. In cell-attached patches on single astrocytes, OGD caused activation of maxi-anion channels that were sensitive to Gd(3+) and arachidonic acid. The channel was found to be permeable to ATP(4-) with a permeability ratio of P(ATP)/P(Cl) = 0.11. Thus, it is concluded that ischemic stress induces ATP release from astrocytes and that the maxi-anion channel may serve as a major ATP-releasing pathway under ischemic conditions.     

Regulation of connexin hemichannels by monovalent cations

Opening of connexin hemichannels in the plasma membrane is highly regulated. Generally, depolarization and reduced extracellular Ca2+ promote hemichannel opening. Here we show that hemichannels formed of Cx50, a principal lens connexin, exhibit a novel form of regulation characterized by extraordinary sensitivity to extracellular monovalent cations. Replacement of extracellular Na+ with K+, while maintaining extracellular Ca2+ constant, resulted in >10-fold potentiation of Cx50 hemichannel currents, which reversed upon returning to Na+. External Cs+, Rb+, NH4+, but not Li+, choline, or TEA, exhibited a similar effect. The magnitude of potentiation of Cx50 hemichannel currents depended on the concentration of extracellular Ca2+, progressively decreasing as external Ca2+ was reduced. The primary effect of K+ appears to be a reduction in the ability of Ca2+, as well as other divalent cations, to close Cx50 hemichannels. Cx46 hemichannels exhibited a modest increase upon substituting Na+ with K+. Analyses of reciprocal chimeric hemichannels that swap NH2- and COOH-terminal halves of Cx46 and Cx50 demonstrate that the difference in regulation by monovalent ions in these connexins resides in the NH2-terminal half. Connexin hemichannels have been implicated in physiological roles, e.g., release of ATP and NAD+ and in pathological roles, e.g., cell death through loss or entry of ions and signaling molecules. Our results demonstrate a new, robust means of regulating hemichannels through a combination of extracellular monovalent and divalent cations, principally Na+, K+, and Ca2+.     

Air-Stimulated ATP Release from Keratinocytes Occurs through Connexin Hemichannels

Cutaneous ATP release plays an important role in both epidermal stratification and chronic pain, but little is known about ATP release mechanisms in keratinocytes that comprise the epidermis. In this study, we analyzed ATP release from cultured human neonatal keratinocytes briefly exposed to air, a process previously demonstrated to trigger ATP release from these cells. We show that exposing keratinocytes to air by removing media for 15 seconds causes a robust, long-lasting ATP release. This air-stimulated ATP release was increased in calcium differentiated cultures which showed a corresponding increase in connexin 43 mRNA, a major component of keratinocyte hemichannels. The known connexin hemichannel inhibitors 1-octanol and carbenoxolone both significantly reduced air-stimulated ATP release, as did two drugs traditionally used as ABC transporter inhibitors (glibenclamide and verapamil). These same 4 inhibitors also prevented an increase in the uptake of a connexin permeable dye induced by air exposure, confirming that connexin hemichannels are open during air-stimulated ATP release. In contrast, activity of the MDR1 ABC transporter was reduced by air exposure and the drugs that inhibited air-stimulated ATP release had differential effects on this transporter. These results indicate that air exposure elicits non-vesicular release of ATP from keratinocytes through connexin hemichannels and that drugs used to target connexin hemichannels and ABC transporters may cross-inhibit. Connexins represent a novel, peripheral target for the treatment of chronic pain and dermatological disease.     

Interferon-γ and high glucose-induced opening of Cx43 hemichannels causes endothelial cell dysfunction and damage

Both IFN-γ or high glucose have been linked to systemic inflammatory imbalance with serious repercussions not only for endothelial function but also for the formation of the atherosclerotic plaque. Although the uncontrolled opening of connexin hemichannels underpins the progression of various diseases, whether they are implicated in endothelial cell dysfunction and damage evoked by IFN-γ plus high glucose remains to be fully elucidated. In this study, by using live cell imaging and biochemical approaches, we demonstrate that IFN-γ plus high glucose augment endothelial connexin43 hemichannel activity, resulting in the increase of ATP release, ATP-mediated Ca²⁺ dynamics and production of nitric oxide and superoxide anion, as well as impaired insulin-mediated uptake and intercellular diffusion of glucose and cell survival. Based on our results, we propose that connexin 43 hemichannel inhibition could serve as a new approach for tackling the activation of detrimental signaling resulting in endothelial cell dysfunction and death caused by inflammatory mediators during atherosclerosis secondary to diabetes mellitus.     

Exocytotic release of ATP from cultured astrocytes

Astrocytes appear to communicate with each other as well as with neurons via ATP. However, the mechanisms of ATP release are controversial. To explore whether stimuli that increase [Ca(2+)](i) also trigger vesicular ATP release from astrocytes, we labeled ATP-containing vesicles with the fluorescent dye quinacrine, which exhibited a significant co-localization with atrial natriuretic peptide. The confocal microscopy study revealed that quinacrine-loaded vesicles displayed mainly non-directional spontaneous mobility with relatively short track lengths and small maximal displacements, whereas 4% of vesicles exhibited directional mobility. After ionomycin stimulation only non-directional vesicle mobility could be observed, indicating that an increase in [Ca(2+)](i) attenuated vesicle mobility. Total internal reflection fluorescence (TIRF) imaging in combination with epifluorescence showed that a high percentage of fluorescently labeled vesicles underwent fusion with the plasma membrane after stimulation with glutamate or ionomycin and that this event was Ca(2+)-dependent. This was confirmed by patch-clamp studies on HEK-293T cells transfected with P2X(3) receptor, used as sniffers for ATP release from astrocytes. Glutamate stimulation of astrocytes was followed by an increase in the incidence of small transient inward currents in sniffers, reminiscent of postsynaptic quantal events observed at synapses. Their incidence was highly dependent on extracellular Ca(2+). Collectively, these findings indicate that glutamate-stimulated ATP release from astrocytes was most likely exocytotic and that after stimulation the fraction of quinacrine-loaded vesicles, spontaneously exhibiting directional mobility, disappeared.     

ATP release by way of connexin 36 hemichannels mediates ischemic tolerance in vitro

Spreading depression (SD) is a self-propagating wave of neuronal and glial depolarization that may occur in virtually any gray matter region in the brain. One consequence of SD is an increased tolerance to ischemia. It has been shown that during cortical SD ATP is released into the extracellular space and activation of purinergic receptors leads to the induction of ischemic tolerance. In the present study we show that depolarization of cultured neurons induces ischemic tolerance which is mediated by purinergic receptor activation. Depolarization causes the release of ATP into the extracellular medium, which may be prevented by treatment with the connexin hemichannel blockers flufenamic acid and quinine, but not the pannexin hemichannel blocker carbenoxolone. Knockdown of connexin 36 expression by siRNA greatly reduces the amount of ATP released during depolarization and the subsequent degree of ischemic tolerance. We conclude that during depolarization neurons release ATP by way of connexin 36 hemichannels.     

Gap Junction Intercellular Communication Mediated by Connexin43 in Astrocytes Is Essential for Their Resistance to Oxidative Stress

Oxidative stress induced by reactive oxygen species (ROS) is associated with various neurological disorders including aging, neurodegenerative diseases, as well as traumatic and ischemic insults. Astrocytes have an important role in the anti-oxidative defense in the brain. The gap junction protein connexin43 (Cx43) forms intercellular channels as well as hemichannels in astrocytes. In the present study, we investigated the contribution of Cx43 to astrocytic death induced by the ROS hydrogen peroxide (H₂O₂) and the mechanism by which Cx43 exerts its effects. Lack of Cx43 expression or blockage of Cx43 channels resulted in increased ROS-induced astrocytic death, supporting a cell protective effect of functional Cx43 channels. H₂O₂ transiently increased hemichannel activity, but reduced gap junction intercellular communication (GJIC). GJIC in wild-type astrocytes recovered after 7 h, but was absent in Cx43 knock-out astrocytes. Blockage of Cx43 hemichannels incompletely inhibited H₂O₂-induced hemichannel activity, indicating the presence of other hemichannel proteins. Panx1, which is predicted to be a major hemichannel contributor in astrocytes, did not appear to have any cell protective effect from H₂O₂ insults. Our data suggest that GJIC is important for Cx43-mediated ROS resistance. In contrast to hypoxia/reoxygenation, H₂O₂ treatment decreased the ratio of the hypophosphorylated isoform to total Cx43 level. Cx43 has been reported to promote astrocytic death induced by hypoxia/reoxygenation. We therefore speculate the increase in Cx43 dephosphorylation may account for the facilitation of astrocytic death. Our findings suggest that the role of Cx43 in response to cellular stress is dependent on the activation of signaling pathways leading to alteration of Cx43 phosphorylation states.     

Hemichannels in cardiomyocytes open transiently during ischemia and contribute to reperfusion injury following brief ischemia

The aim of this study was to investigate changes in hemichannel activity during in vitro simulated ischemia [oxygen-glucose deprivation (OGD)] and the contribution of hemichannels to ischemia-reperfusion injury in rat neonatal cardiomyocytes. Dye uptake assays showed that hemichannels opened as OGD progressed, peaking after 1 h, and then closed, returning to the pre-OGD state after 2 h of OGD. The increase in dye uptake after 1 h of OGD was inhibited by hemichannel blockers (lanthanum chloride and a connexin 43 mimetic peptide, Gap26). During OGD, intracellular Ca(2+) concentration ([Ca(2+)](i)) began to increase after 1 h and reached several micromolar after 2 h. After 1 h of OGD, Gap26 inhibited the increases in hemichannel activity and [Ca(2+)](i). In contrast, dantrolene [an endo(sarco)plasmic reticulum Ca(2+) release inhibitor] suppressed the increase in [Ca(2+)](i), but not in hemichannel activity. After 2 h of OGD, the combined administration of 2',4'-dichlorobenzamil and dantrolene reduced [Ca(2+)](i) to <1 microM and increased hemichannel activity to the level attained after 1 h of OGD. Simulated ischemia-reperfusion, induced by 1 h of OGD followed by 2 h of recovery, reduced cell viability to 54% of the control level. The addition of Gap26 to OGD medium improved viability to 80% of the control level. In conclusion, this study demonstrated that 1) hemichannels open transiently during OGD, 2) closure of hemichannels, but not their opening, is regulated by an increase in [Ca(2+)](i) during OGD, and 3) open hemichannels contribute to cell injury during recovery from OGD.     

Disruption in Connexin-Based Communication Is Associated with Intracellular Ca2+ Signal Alterations in Astrocytes from Niemann-Pick Type C Mice

Reduced astrocytic gap junctional communication and enhanced hemichannel activity were recently shown to increase astroglial and neuronal vulnerability to neuroinflammation. Moreover, increasing evidence suggests that neuroinflammation plays a pivotal role in the development of Niemann-Pick type C (NPC) disease, an autosomal lethal neurodegenerative disorder that is mainly caused by mutations in the NPC1 gene. Therefore, we investigated whether the lack of NPC1 expression in murine astrocytes affects the functional state of gap junction channels and hemichannels. Cultured cortical astrocytes of NPC1 knock-out mice (Npc1^−/−) showed reduced intercellular communication via gap junctions and increased hemichannel activity. Similarly, astrocytes of newborn Npc1^−/− hippocampal slices presented high hemichannel activity, which was completely abrogated by connexin 43 hemichannel blockers and was resistant to inhibitors of pannexin 1 hemichannels. Npc1^−/− astrocytes also showed more intracellular Ca²⁺ signal oscillations mediated by functional connexin 43 hemichannels and P2Y₁ receptors. Therefore, Npc1^−/− astrocytes present features of connexin based channels compatible with those of reactive astrocytes and hemichannels might be a novel therapeutic target to reduce neuroinflammation in NPC disease.     

ATP and glutamate released via astroglial connexin 43 hemichannels mediate neuronal death through activation of pannexin 1 hemichannels

Inflammation contributes to neurodegeneration in post-ischemic brain, diabetes, and Alzheimer's disease. Participants in this inflammatory response include activation of microglia and astrocytes. We studied the role of microglia treated with amyloid-β peptide (Aβ) on hemichannel activity of astrocytes subjected to hypoxia in high glucose. Reoxygenation after 3?h hypoxia in high glucose induced transient astroglial permeabilization via Cx43 hemichannels and reduction in intercellular communication via Cx43 cell-cell channels. Both responses were greater and longer lasting in astrocytes previously exposed for 24 h to conditioned medium from Aβ-treated microglia (CM-Aβ). The effects of CM-Aβ were mimicked by TNF-α and IL-1β and were abrogated by neutralizing TNF-α with soluble receptor and IL-1β with a receptor antagonist. Astrocytes under basal conditions protected neurons against hypoxia, but exposure to CM-Aβ made them toxic to neurons subjected to a sub-lethal hypoxia/reoxygenation episode, revealing the additive nature of the insults. Astrocytes exposed to CM-Aβ induced permeabilization of cortical neurons through activation of neuronal pannexin 1 (Panx1) hemichannels by ATP and glutamate released through astroglial Cx43 hemichannels. In agreement, inhibition of NMDA or P2X receptors only partially reduced the activation of neuronal Panx1 hemichannels and neuronal mortality, but simultaneous inhibition of both receptors completely prevented the neurotoxic response. Therefore, we suggest that responses to ATP and glutamate converge in activation of neuronal Panx1 hemichannels. Thus, we propose that blocking hemichannels expressed by astrocytes and/or neurons in the inflamed nervous system could represent a novel and alternative strategy to reduce neuronal loss in various pathological states including Alzheimer's disease, diabetes and ischemia.     

The gating effect of calmodulin and calcium on the connexin50 hemichannel

Gap junction channels formed by connexin50 (Cx50) are critical for the maintenance of eye lens transparency, which is sensitive to pH and external Ca2+ concentration, but the mechanism is still not clear. In this study we performed dye uptake-leakage assays, patch clamping and confocal co-localization experiments to confirm the function of calmodulin (CaM) and Ca2+ in the Cx50 hemichannel. Below pH 7.4, lucifer yellow (LY)-preloaded Cx50-HeLa cells allow dye to leak out when washed with Ca2+-free solution or incubated in solution containing 50 microg/ml W7 (CaM inhibitor) first, then washed in solution containing 2 mM Ca2+, whereas little or no dye leakage was observed when LY-preloaded Cx50-HeLa cells were incubated in solution containing 2 mM Ca2+. Moreover, in the absence of Ca2+, polarizing pulses applied to Cx50-HeLa activated outward transmembrane currents, which were inhibited by 2 mM external Ca2+. When Cx50-HeLa cells were incubated with 2 mM Ca2+ and 50 microg/ml W7, the transmembrane currents were activated again. This indicates that Ca2+ and CaM play a gating role in Cx50 hemichannels. Either the chelation of Ca2+ or the inhibition of CaM increased the permeability of Cx50 hemichannels. The same phenomena were observed below pH 6.5. Furthermore, CaM could be localized in gap junctions formed by Cx50 below pH 6.5. Our results demonstrate that CaM and Ca2+ can cooperate in the gating control of Cx50 hemichannels.     

Connexin channels, connexin mimetic peptides and ATP release

Connexin hemichannels, that is, half gap junction channels (not connecting cells), have been implicated in the release of various messengers such as ATP and glutamate. We used connexin mimetic peptides, which are, small peptides mimicking a sequence on the connexin subunit, to investigate hemichannel functioning in endothelial cell lines. Short exposure (30 min) to synthetic peptides mimicking a sequence on the first or second extracellular loop of the connexin subunit strongly supressed ATP release and dye uptake triggered by either intracellular InsP(3) elevation or exposure to zero extracellular calcium, while gap junctional coupling was not affected under these conditions. The effect was dependent on the expression of connexin-43 in the cells. Connexin mimetic peptides thus appear to be interesting tools to distinguish connexin hemichannel from gap junction channel functioning. In addition, they are well suited to further explore the role of connexins in cellular release or uptake processes, to investigate hemichannel gating and to reveal new unknown functions of the large conductance hemichannel pathway between the cell and its environment. Work performed up to now with these peptides should be re-interpreted in terms of these new findings.     

Thrombin-induced ATP release from human umbilical vein endothelial cells

ATP and its degradation products play an important role as signaling molecules in the vascular system, and endothelial cells are considered to be an important source of nucleotide release. To investigate the mechanism and physiological significance of endothelial ATP release, we compared different pharmacological stimuli for their ability to evoke ATP release from first passage cultivated human umbilical vein endothelial cells (HUVECs). Agonists known to increase intracellular Ca(2+) levels (A23187, histamine, thrombin) induced a stable, non-lytic ATP release. Since thrombin proved to be the most robust and reproducible stimulus, the molecular mechanism of thrombin-mediated ATP release from HUVECs was further investigated. ATP rapidly increased with thrombin (1 U/ml) and reached a steady-state level after 4 min. Loading the cells with BAPTA-AM to capture intracellular calcium suppressed ATP release. The thrombin-specific, protease-activated receptor 1 (PAR-1)-specific agonist peptide TFLLRN (10 μM) fully mimicked thrombin action on ATP release. To identify the nature of the ATP-permeable pathway, we tested various inhibitors of potential ATP channels for their ability to inhibit the thrombin response. Carbenoxolone, an inhibitor of connexin hemichannels and pannexin channels, as well as Gd(3+) were highly effective in blocking the thrombin-mediated ATP release. Specifically targeting connexin43 (Cx43) and pannexin1 (Panx1) revealed that reducing Panx1 expression significantly reduced ATP release, while downregulating Cx43 was ineffective. Our study demonstrates that thrombin at physiological concentrations is a potent stimulus of endothelial ATP release involving PAR-1 receptor activation and intracellular calcium mobilization. ATP is released by a carbenoxolone- and Gd(3+)- sensitive pathway, most likely involving Panx1 channels.     

Connexin Channels, Connexin Mimetic Peptides and ATP Release

Connexin hemichannels, that is, half gap junction channels (not connecting cells), have been implicated in the release of various messengers such as ATP and glutamate. We used connexin mimetic peptides, which are, small peptides mimicking a sequence on the connexin subunit, to investigate hemichannel functioning in endothelial cell lines. Short exposure (30 min) to synthetic peptides mimicking a sequence on the first or second extracellular loop of the connexin subunit strongly supressed ATP release and dye uptake triggered by either intracellular InsP₃ elevation or exposure to zero extracellular calcium, while gap junctional coupling was not affected under these conditions. The effect was dependent on the expression of connexin-43 in the cells. Connexin mimetic peptides thus appear to be interesting tools to distinguish connexin hemichannel from gap junction channel functioning. In addition, they are well suited to further explore the role of connexins in cellular release or uptake processes, to investigate hemichannel gating and to reveal new unknown functions of the large conductance hemichannel pathway between the cell and its environment. Work performed up to now with these peptides should be re-interpreted in terms of these new findings.