Ashish K. Gadicherla

Paracrine signaling through plasma membrane hemichannels

Plasma membrane hemichannels composed of connexin (Cx) proteins are essential components of gap junction channels but accumulating evidence suggests functions of hemichannels beyond the communication provided by junctional channels. Hemichannels not incorporated into gap junctions, called unapposed hemichannels, can open in response to a variety of signals, electrical and chemical, thereby forming a conduit between the cells interior and the extracellular milieu. Open hemichannels allow the bidirectional passage of ions and small metabolic or signaling molecules of below 1-2kDa molecular weight. In addition to connexins, hemichannels can also be formed by pannexin (Panx) proteins and current evidence suggests that Cx26, Cx32, Cx36, Cx43 and Panx1, form hemichannels that allow the diffusive release of paracrine messengers. In particular, the case is strong for ATP but substantial evidence is also available for other messengers like glutamate and prostaglandins or metabolic substances like NAD(+) or glutathione. While this field is clearly in expansion, evidence is still lacking at essential points of the paracrine signaling cascade that includes not only messenger release, but also downstream receptor signaling and consequent functional effects. The data available at this moment largely derives from in vitro experiments and still suffers from the difficulty of separating the functions of connexin-based hemichannels from gap junctions and from pannexin hemichannels. However, messengers like ATP or glutamate have universal roles in the body and further defining the contribution of hemichannels as a possible release pathway is expected to open novel avenues for better understanding their contribution to a variety of physiological and pathological processes. This article is part of a Special Issue entitled: The Communicating junctions, roles and dysfunctions.

Endothelial calcium dynamics, connexin channels and blood-brain barrier function

Situated between the circulation and the brain, the blood-brain barrier (BBB) protects the brain from circulating toxins while securing a specialized environment for neuro-glial signaling. BBB capillary endothelial cells exhibit low transcytotic activity and a tight, junctional network that, aided by the cytoskeleton, restricts paracellular permeability. The latter is subject of extensive research as it relates to neuropathology, edema and inflammation. A key determinant in regulating paracellular permeability is the endothelial cytoplasmic Ca(2+) concentration ([Ca(2+)]i) that affects junctional and cytoskeletal proteins. Ca(2+) signals are not one-time events restricted to a single cell but often appear as oscillatory [Ca(2+)]i changes that may propagate between cells as intercellular Ca(2+) waves. The effect of Ca(2+) oscillations/waves on BBB function is largely unknown and we here review current evidence on how [Ca(2+)]i dynamics influence BBB permeability.

IP3, a small molecule with a powerful message.

Research conducted over the past two decades has provided convincing evidence that cell death, and more specifically apoptosis, can exceed single cell boundaries and can be strongly influenced by intercellular communication networks. We recently reported that gap junctions (i.e. channels directly connecting the cytoplasm of neighboring cells) composed of connexin43 or connexin26 provide a direct pathway to promote and expand cell death, and that inositol 1,4,5-trisphosphate (IP3) diffusion via these channels is crucial to provoke apoptosis in adjacent healthy cells. However, IP3 itself is not sufficient to induce cell death and additional factors appear to be necessary to create conditions in which IP3 will exert proapoptotic effects. Although IP3-evoked Ca(2+) signaling is known to be required for normal cell survival, it is also actively involved in apoptosis induction and progression. As such, it is evident that an accurate fine-tuning of this signaling mechanism is crucial for normal cell physiology, while a malfunction can lead to cell death. Here, we review the role of IP3 as an intracellular and intercellular cell death messenger, focusing on the endoplasmic reticulum-mitochondrial synapse, followed by a discussion of plausible elements that can convert IP3 from a physiological molecule to a killer substance. Finally, we highlight several pathological conditions in which anomalous intercellular IP3/Ca(2+) signaling might play a role. This article is part of a Special Issue entitled:12th European Symposium on Calcium.

Mitochondrial Cx43 hemichannels contribute to mitochondrial calcium entry and cell death in the heart

Mitochondrial connexin 43 (Cx43) plays a key role in cardiac cytoprotection caused by repeated exposure to short periods of non-lethal ischemia/reperfusion, a condition known as ischemic preconditioning. Cx43 also forms calcium (Ca2+)-permeable hemichannels that may potentially lead to mitochondrial Ca2+ overload and cell death. Here, we studied the role of Cx43 in facilitating mitochondrial Ca2+ entry and investigated its downstream consequences. To that purpose, we used various connexin-targeting peptides interacting with extracellular (Gap26) and intracellular (Gap19, RRNYRRNY) Cx43 domains, and tested their effect on mitochondrial dye- and Ca2+-uptake, electrophysiological properties of plasmalemmal and mitochondrial Cx43 channels, and cell injury/cell death. Our results in isolated mice cardiac subsarcolemmal mitochondria indicate that Cx43 forms hemichannels that contribute to Ca2+ entry and may trigger permeability transition and cell injury/death. RRNYRRNY displayed the strongest effects in all assays and inhibited plasma membrane as well as mitochondrial Cx43 hemichannels. RRNYRRNY also strongly reduced the infarct size in ex vivo cardiac ischemia–reperfusion studies. These results indicate that Cx43 contributes to mitochondrial Ca2+ homeostasis and is involved in triggering cell injury/death pathways that can be inhibited by RRNYRRNY peptide.

Inhibiting connexin channels protects against cryopreservation-induced cell death in human blood vessels

OBJECTIVES Cryopreserved blood vessels are being increasingly employed in vascular reconstruction procedures but freezing/thawing is associated with significant cell death that may lead to graft failure. Vascular cells express connexin proteins that form gap junction channels and hemichannels. Gap junction channels directly connect the cytoplasm of adjacent cells and may facilitate the passage of cell death messengers leading to bystander cell death. Two hemichannels form a gap junction channel but these channels are also present as free non-connected hemichannels. Hemichannels are normally closed but may open under stressful conditions and thereby promote cell death. We here investigated whether blocking gap junctions and hemichannels could prevent cell death after cryopreservation. MATERIALS AND METHODS Inclusion of Gap27, a connexin channel inhibitory peptide, during cryopreservation and thawing of human saphenous veins and femoral arteries was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assays and histological examination. RESULTS We report that Gap27 significantly reduces cell death in human femoral arteries and saphenous veins when present during cryopreservation/thawing. In particular, smooth muscle cell death was reduced by 73% in arteries and 71% in veins, while endothelial cell death was reduced by 32% in arteries and 51% in veins. CONCLUSIONS We conclude that inhibiting connexin channels during cryopreservation strongly promotes vascular cell viability.

Electroporation Loading of Membrane-Impermeable Molecules to Investigate Intra- and Intercellular Ca2+ Signaling

Electroporation is a technique that temporarily induces pores in the plasma membranes of cells, thereby allowing plasma membrane-impermeable substances to enter the cells. This loading method requires an electrical drive circuit providing an electroporation signal, an electrode to apply the signal to a localized zone in a cell monolayer, and a special solution that has a low electrical conductivity. To avoid impairment of cell function and cell death from the electroporation procedure itself, the applied electrical signal should ideally be a high-frequency oscillating signal (50 kHz) without any direct current (DC) component. Here, we describe the detailed procedure of electroporation loading.

Reduced Tyrosine Nitration of VDAC and Decreased Apoptosis by Mitochondria-Directed Therapy After Cardiac Ischemia Reperfusion in Isolated Hearts

Superoxide (O2•-) produced during cardiac ischemia-reperfusion (IR) injury reacts with nitric oxide to form peroxynitrite (ONOO-). ONOO- induces protein tyrosine nitration (tyrN) that causes protein structural alteration and dysfunction. The mitochondrial voltage-dependent anion channel (VDAC) plays an important role in regulating the metabolic and energetic functions of mitochondria and contributes to mitochondrial-mediated apoptosis. It is not known if VDAC is nitrated by ONOO- during IR or how this modification might compromise cardiac function after IR. Because of the importance of VDAC modification, we hypothesized that the clinically used anti-anginal drug ranolazine (RAN), which also reduces cardiac IR injury, does so via a mitochondrial mechanism, i.e., in part by decreasing VDAC tyrN. To test this, isolated guinea pig hearts were perfused with KR buffer for 40 min (time control, TC), or for 30 min of ischemia plus 10 min of reperfusion, with or without 10 µM RAN infused before ischemia. Mitochondria were isolated at the end of each treatment. VDAC tyrN was determined by IP with anti-nitrotyrosine antibody (NTab), followed by Western blotting (WB) with anti-VDAC antibody. The effect of RAN on VDAC tyrN was also examined. Cytochrome c release was checked as the marker for apoptosis. We found that enhanced VDAC tyrN was increased by 108% after IR vs. TC and cytochrome c was higher in the cytosol after IR than after TC. RAN treatment decreased VDAC tyrN by 31%, while decreasing cytochrome c release by 38%, compared to IR. These results indicate that VDAC tyrN and a concomitant increase in cytochrome c release occur during IR injury, and importantly, that cardioprotection by RAN occurs in part by reducing VDAC tyrN, which may impede activation of apoptotic pathways during IR injury.

Differential Decreases in Respiratory Complex I and II Expression and Activity after Cardiac Ischemia and Reperfusion and Modulation by Mitochondria-Targeted Therapy

Mitochondrial respiratory complexes are variably susceptible to structural and functional changes during ischemia reperfusion injury (IR). Complex I may be most susceptible to IR and complex II (SQR), both an electron donor and TCA cycle intermediate, relatively less susceptible. We tested for differential expression and activities of complexes I and II during IR +/- a mitochondria-targeted therapy (MTT). Guinea pig hearts were exposed to one of 5 protocols: 40 min time control (TC), 30 min global ischemia (I30), 30 min ischemia with 10 min reperfusion (IR10), and 10 µM ranolazine (Ran), as a MTT, given 1 min before I30 (Ran+I30) or IR (Ran+IR10). Mitochondria were isolated and assayed for complex I (Ndufa9) and complex II (FP) protein expression (western blotting), or complex I and II enzyme activities (absorbance spectrophotometry) after hypotonic freeze-thaw rupture. Complex I expression at I30 and activity at I30 and IR10 were markedly decreased; Ran treatment nearly restored complex I expression and activity after I30 and IR10. Complex II expression was not altered by I or IR, +/- Ran, complex II activity was decreased less than in complex I, and Ran increased activity only with I30. Differences in complex I and II expression and activities may result from more deleterious post-translational modifications triggered by I and IR in complex I than II. A more compromised function in complex I may reduce forward electron flux to complex II, allowing increased electron flux from succinate to ubiquinone because of less compromised SQR activity. Further studies will ascertain the mechanism behind the greater functional impairment of complex I vs. complex II after IR and the partial restoration of complex I and II activity by Ran, a known cardioprotective drug.

Buffer Magnesium Limits Mitochondrial Calcium Uptake but not Matrix Calcium Buffering in Response to ADP

Mg2+ is known to limit Ca2+ uptake by mitochondria through the Ca2+ uniporter. Changes in matrix Ca2+ concentration are an important signaling pathway in mitochondrial function as well as in apoptosis. In a previous study we showed an increase in matrix free Ca2+ in response to added ADP in MgCl2 free buffer. Because of the presumed role of Mg2+ in mitochondrial regulation of Ca2+ we explored the effects of buffer Mg2+ on matrix Ca2+ uptake and buffering in isolated mitochondria. Guinea pig heart mitochondria were isolated by differential centrifugation, loaded with the fluorescent dye Indo 1 AM and then suspended in respiration media, containing 1 mM of EGTA, with or without added 1 mM MgCl2. To the mitochondrial suspension was added 0.5 mM pyruvic acid, either 0.25, 0.5 or 0.75 mM CaCl2, and 250 μM ADP. Adding 0.25, 0.5 and 0.75 mM Ca2+ caused a dose-dependent increase in matrix Ca2+ of 14, 35 and 45%, respectively, in the group without Mg2+ in the buffer, and 6, 18 and 42%, respectively, in the group with Mg2+ in the buffer. The differences in uptake between Mg2+ and no Mg2+ groups were significant in the 0.25 and 0.5 mM groups, but not in the 0.75 mM group. The additional increase in matrix free Ca2+ in response to ADP without Mg2+ was 9, 11 and 9% for the 0.25, 0.5 and 0.75 mM Ca2+ groups, respectively. These additional increases in matrix free Ca2+ with ADP were not significantly altered by Mg2+. We conclude that external Mg2+ alters the uptake of Ca2+ into the mitochondrial matrix, but does not alter the increase in matrix ionized Ca2+ after addition of ADP.