Calcium Wave Propagation Triggered by Local Mechanical Stimulation as a Method for Studying Gap Junctions and Hemichannels.

Intercellular communication is essential for the coordination and synchronization of cellular processes. Gap junction channels play an important role to communicate between cells and organs, including the brain, lung, liver, lens, retina, and heart. Gap junctions enable a direct route for ions like calcium and potassium, and low molecular weight compounds, such as inositol 1,4,5-trisphosphate, cyclic adenosine monophosphate, and various kinds of metabolites to pass between cells.

Nutrient Starvation Decreases Cx43 Levels and Limits Intercellular Communication in Primary Bovine Corneal Endothelial Cells.

Connexin (Cx) proteins form large conductance channels which function as regulators of communication between neighboring cells via gap junctions and/or hemichannels. Intercellular communication is essential to coordinate cellular responses in tissues and organs, thereby fulfilling an essential role in the spreading of signaling, survival and death processes. Connexin 43 (Cx43), a major connexin isoform in brain and heart, is rapidly turned over.

Cx43-hemichannel function and regulation in physiology and pathophysiology: insights from the bovine corneal endothelial cell system and beyond.

Intercellular communication in primary bovine corneal endothelial cells (BCECs) is mainly driven by the release of extracellular ATP through Cx43 hemichannels. Studying the characteristics of Ca(2+)-wave propagation in BCECs, an important form of intercellular communication, in response to physiological signaling events has led to the discovery of important insights in the functional properties and regulation of native Cx43 hemichannels. Together with ectopic expression models for Cx43 hemichannels and truncated/mutated Cx43 versions, it became very clear that loop/tail interactions play a key role in controlling the activity of Cx43 hemichannels.

Mechanical stimulation-induced calcium wave propagation in cell monolayers: the example of bovine corneal endothelial cells.

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca(2+)-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca(2+) that initiate the propagation of the Ca(2+)-wave concentrically from the mechanically stimulated cell to the neighboring cells.

Regulation of connexin- and pannexin-based channels by post-translational modifications.

Connexin (Cx) and pannexin (Panx) proteins form large conductance channels, which function as regulators of communication between neighbouring cells via gap junctions and/or hemichannels. Intercellular communication is essential to coordinate cellular responses in tissues and organs, thereby fulfilling an essential role in the spreading of signalling, survival and death processes. The functional properties of gap junctions and hemichannels are modulated by different physiological and pathophysiological stimuli.

Peptides and peptide-derived molecules targeting the intracellular domains of Cx43: gap junctions versus hemichannels.

About a decade ago, the molecular determinants controlling the opening and closing of Cx43 gap junction channels have been identified. Advanced biophysical approaches revealed a critical role for structural rearrangements in the cytoplasmic loop and dimerization of the C-terminal tail, resulting in binding of the C-terminal tail to the cytoplasmic loop and Cx43 gap junction channel closure during cellular acidosis. This has spurred the development of Cx43-mimetic peptides and peptidomimetics that interfere with these loop/tail interactions, thereby preventing the closure of Cx43 gap junctions, e.

Negatively charged residues (Asp378 and Asp379) in the last ten amino acids of the C-terminal tail of Cx43 hemichannels are essential for loop/tail interactions.

Connexin 43 (Cx43)-hemichannel activity is controlled by intramolecular interactions between cytoplasmic loop and C-terminal tail. We previously identified the last 10 amino acids of the C-terminal tail of Cx43 as essential for Cx43-hemichannel activity. We developed a cell-permeable peptide covering this sequence (TAT-Cx43CT).

RhoA GTPase switch controls Cx43-hemichannel activity through the contractile system.

ATP-dependent paracrine signaling, mediated via the release of ATP through plasma membrane-embedded hemichannels of the connexin family, coordinates a synchronized response between neighboring cells. Connexin 43 (Cx43) hemichannels that are present in the plasma membrane need to be tightly regulated to ensure cell viability. In monolayers of bovine corneal endothelial cells (BCEC),Cx43-mediated ATP release is strongly inhibited when the cells are treated with inflammatory mediators, in particular thrombin and histamine.

The contractile system as a negative regulator of the connexin 43 hemichannel.

The molecular mechanisms underlying the regulation of gap junction (GJ) channels based on the 43-kDa connexin isoform (Cx43) have been studied extensively. GJ channels are formed by the docking of opposed hemichannels in adjacent cells. Mounting data indicate that unopposed Cx43 hemichannels are also functional in the plasma membrane.

Non-channel functions of connexins in cell growth and cell death.

Cellular communication mediated by gap junction channels and hemichannels, both composed of connexin proteins, constitutes two acknowledged regulatory platforms in the accomplishment of tissue homeostasis. In recent years, an abundance of reports has been published indicating functions for connexin proteins in the control of the cellular life cycle that occur independently of their channel activities. This has yet been most exemplified in the context of cell growth and cell death, and is therefore as such addressed in the current paper.

Pannexin channels in ATP release and beyond: an unexpected rendezvous at the endoplasmic reticulum.

The pannexin (Panx) family of proteins, which is co-expressed with connexins (Cxs) in vertebrates, was found to be a new GJ-forming protein family related to invertebrate innexins. During the past ten years, different studies showed that Panxs mainly form hemichannels in the plasma membrane and mediate paracrine signalling by providing a flux pathway for ions such as Ca²(+), for ATP and perhaps for other compounds, in response to physiological and pathological stimuli. Although the physiological role of Panxs as a hemichannel was questioned, there is increasing evidence that Panx play a role in vasodilatation, initiation of inflammatory responses, ischemic death of neurons, epilepsy and in tumor suppression.

Intramolecular loop/tail interactions are essential for connexin 43-hemichannel activity.

Connexin-assembled gap junctions (GJs) and hemichannels coordinate intercellular signaling processes. Although the regulation of connexins in GJs has been well characterized, the molecular determinants controlling connexin-hemichannel activity are unresolved. Here we investigated the regulation of Cx43-hemichannel activity by actomyosin contractility and intracellular [Ca(2+)] ([Ca(2+)](i)) using plasma membrane-permeable TAT peptides (100 μM) designed to interfere with interactions between the cytoplasmic loop (CL) and carboxy-terminal (CT) in primary bovine corneal endothelial cells and HeLa, C6 glioma, and Xenopus oocytes ectopically expressing Cx43.

Reduced intercellular communication and altered morphology of bovine corneal endothelial cells with prolonged time in cell culture.

Purpose: Mechanical stimulation induces intercellular Ca(2 +) waves in the corneal endothelium. The extent of the wave propagation is dependent on the activity of gap junctions, hemichannels, and ectonucleotidases. To further establish the use of a cell culture model to investigate intercellular communication, in this study, we have characterized the changes in the Ca(2 +) wave propagation in bovine corneal endothelial cells with prolonged time in culture.

Pannexins, distant relatives of the connexin family with specific cellular functions?

Intercellular communication (IC) is mediated by gap junctions (GJs) and hemichannels, which consist of proteins. This has been particularly well documented for the connexin (Cx) family. Initially, Cxs were thought to be the only proteins capable of GJ formation in vertebrates.

The myosin II ATPase inhibitor blebbistatin prevents thrombin-induced inhibition of intercellular calcium wave propagation in corneal endothelial cells.

Purpose: Thrombin inhibits intercellular Ca(2+) wave propagation in bovine corneal endothelial cells (BCECs) through a mechanism dependent on myosin light chain (MLC) phosphorylation. In this study, blebbistatin, a selective myosin II ATPase inhibitor, was used to investigate whether the effect of thrombin is mediated by enhanced actomyosin contractility.

Methods: BCECs were exposed to thrombin (2 U/mL) for 5 minutes.

Nature of the nuclear inclusions formed by PQBP1, a protein linked to neurodegenerative polyglutamine diseases.

PQBP1, for polyglutamine tract-binding protein-1, has been linked to progressive neurodegenerative diseases, such as spinocerebellar ataxia, that are caused by the expansion of a polyglutamine repeat in a key regulatory protein. The overexpression of PQBP1 results in the formation of nuclear inclusions, reminiscent of the protein aggregates that are detected in polyglutamine diseases. We show here that the occurrence of PQBP1-induced nuclear inclusions is dramatically increased by the co-expression of the pre-mRNA splicing factor SIPP1, a protein ligand of PQBP1.

Adenosine opposes thrombin-induced inhibition of intercellular calcium wave in corneal endothelial cells.

Purpose: In corneal endothelial cells, intercellular Ca(2+) waves elicited by a mechanical stimulus involve paracrine intercellular communication, mediated by ATP release via connexin hemichannels, as well as gap junctional intercellular communication. Both mechanisms are inhibited by thrombin, which activates RhoA and hence results in myosin light chain phosphorylation. This study was conducted to examine the effects of adenosine, which is known to oppose thrombin-induced RhoA activation, thereby leading to myosin light chain dephosphorylation, on gap junctional intercellular communication and paracrine intercellular communication in cultured bovine corneal endothelial cells.

Thrombin inhibits intercellular calcium wave propagation in corneal endothelial cells by modulation of hemichannels and gap junctions.

Purpose: Thrombin, a serine protease, breaks down the barrier integrity of corneal endothelial cells by phosphorylation of the regulatory light chain of myosin II (myosin light chain; MLC), which induces contractility of the actin cytoskeleton. This study was undertaken to investigate the effect of thrombin on gap junctional (GJIC) and paracrine (PIC) intercellular communication in cultured bovine corneal endothelial cells (BCECs).

Methods: An intercellular Ca(2+) wave, a form of cell-cell communication, was elicited by applying a mechanical stimulus to a single cell in a confluent monolayer.

Gap junctional intercellular communication in bovine corneal endothelial cells.

Gap junctions and/or paracrine mediators, such as ATP, mediate intercellular communication (IC) in non-excitable cells. This study investigates the contribution of gap junctions toward IC during propagation of Ca(2+) waves in cultured bovine corneal endothelial cells (BCEC) elicited by applying a point mechanical stimulus to a single cell in a confluent monolayer. Changes in [Ca(2+)](i) were visualized using the fluorescent dye Fluo-4.

Determinants of the nucleolar targeting of protein phosphatase-1.

The ubiquitously expressed protein Ser/Thr phosphatase-1 isoforms PP1alpha, PP1beta and PP1gamma1 are dynamically targeted to distinct, but overlapping cellular compartments by associated proteins. Within the nucleus of HeLa cells, EGFP-tagged PP1gamma1 and PP1beta were predominantly targeted to the nucleoli, while PP1alpha showed a more diffuse distribution. Using PP1 chimaeras and point mutants we show here that a single N-terminal residue, i.

ATP release through connexin hemichannels in corneal endothelial cells.

Purpose: Intercellular Ca(2+) wave propagation is a distinct form of cell-cell communication. In corneal endothelial cells, intercellular Ca(2+) wave propagation evoked by a point mechanical stimulus (PMS) is partially mediated by adenosine triphosphate (ATP) release and subsequent activation of P2Y receptors. This study was conducted to investigate the possibility that extrajunctional connexons (hemichannels) play a role in ATP release during PMS-induced Ca(2+) wave propagation in bovine corneal endothelial cells (BCECs).

ATP-dependent paracrine intercellular communication in cultured bovine corneal endothelial cells.

Purpose: Intercellular communication (IC) in nonexcitable cells is mediated through gap junctions and/or through the release of paracrine mediators. This study was conducted to investigate adenosine-5' triphosphate (ATP)-dependent paracrine IC in the propagation of Ca2+ waves in confluent monolayers of cultured bovine corneal endothelial cells (BCECs).

Methods: A Ca2+ wave was induced by point mechanical stimulation (PMS) of a single cell by indentation with a glass micropipette (approximately 1 microm tip) for <1 second.

Interactor-mediated nuclear translocation and retention of protein phosphatase-1.

Protein Ser/Thr phosphatase-1 (PP1) is a ubiquitous eukaryotic enzyme that controls numerous cellular processes by the dephosphorylation of key regulatory proteins. PP1 is expressed in various cellular compartments but is most abundant in the nucleus. We have examined the determinants for the nuclear localization of enhanced green fluorescent protein-tagged PP1 in COS1 cells.

Microtubule-dependent redistribution of the type-1 inositol 1,4,5-trisphosphate receptor in A7r5 smooth muscle cells.

In A7r5 vascular smooth muscle cells, the two expressed inositol 1,4,5-trisphosphate receptor (IP(3)R) isoforms were differentially localized. IP(3)R1 was predominantly localized in the perinuclear region, whereas IP(3)R3 was homogeneously distributed over the cytoplasm. Prolonged stimulation (1-5 hours) of cells with 3 microM arginine-vasopressin induced a redistribution of IP(3)R1 from the perinuclear region to the entire cytoplasm, whereas the localization of IP(3)R3 appeared to be unaffected.