Pannexin-1 channels show distinct morphology and no gap junction characteristics in mammalian cells.

Pannexins (Panx) are proteins with a similar membrane topology to connexins, the integral membrane protein of gap junctions. Panx1 channels are generally of major importance in a large number of system and cellular processes and their function has been thoroughly characterized. In contrast, little is known about channel structure and subcellular distribution. We therefore determine the subcellular localization of Panx1 channels in cultured cells and aim at the identification of channel morphology in vitro. Using freeze-fracture replica immunolabeling on EYFP-Panx1-overexpressing HEK 293 cells, large particles were identified in plasma membranes, which were immunogold-labeled using either GFP or Panx1 antibodies. There was no labeling or particles in the nuclear membranes of these cells, pointing to plasma membrane localization of Panx1-EYFP channels. The assembly of particles was irregular, this being in contrast to the regular pattern of gap junctions. The fact that no counterparts were identified on apposing cells, which would have been indicative of intercellular signaling, supported the idea of Panx1 channels within one membrane. Control cells (transfected with EYFP only, non-transfected) were devoid of both particles and immunogold labeling. Altogether, this study provides the first demonstration of Panx1 channel morphology and assembly in intact cells. The identification of Panx1 channels as large particles within the plasma membrane provides the knowledge required to enable recognition of Panx1 channels in tissues in future studies. Thus, these results open up new avenues for the detailed analysis of the subcellular localization of Panx1 and of its nearest neighbors such as purinergic receptors in vivo.

A Monoclonal Human Alveolar Epithelial Cell Line ("Arlo") with Pronounced Barrier Function for Studying Drug Permeability and Viral Infections.

In the development of orally inhaled drug products preclinical animal models regularly fail to predict pharmacological as well as toxicological responses in humans. Models based on human cells and tissues are potential alternatives to animal experimentation allowing for the isolation of essential processes of human biology and making them accessible in vitro. Here, the generation of a novel monoclonal cell line "Arlo," derived from the polyclonal human alveolar epithelium lentivirus immortalized cell line hAELVi via single-cell printing, and its characterization as a model for the human alveolar epithelium as well as a building block for future complex in vitro models is described. "Arlo" is systematically compared in vitro to primary human alveolar epithelial cells (hAEpCs) as well as to the polyclonal hAELVi cell line. "Arlo" cells show enhanced barrier properties with high transepithelial electrical resistance (TEER) of ≈3000 Ω cm2 and a potential difference (PD) of ≈30 mV under air-liquid interface (ALI) conditions, that can be modulated. The cells grow in a polarized monolayer and express genes relevant to barrier integrity as well as homeostasis as is observed in hAEpCs. Successful productive infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a proof-of-principle study offers an additional, attractive application of "Arlo" beyond biopharmaceutical experimentation.

TGF-Beta Modulates the Integrity of the Blood Brain Barrier In Vitro, and Is Associated with Metabolic Alterations in Pericytes.

The blood-brain barrier (BBB) is a selectively permeable boundary that separates the circulating blood from the extracellular fluid of the brain and is an essential component for brain homeostasis. In glioblastoma (GBM), the BBB of peritumoral vessels is often disrupted. Pericytes, being important to maintaining BBB integrity, can be functionally modified by GBM cells which induce proliferation and cell motility via the TGF-ß-mediated induction of central epithelial to mesenchymal transition (EMT) factors. We demonstrate that pericytes strengthen the integrity of the BBB in primary endothelial cell/pericyte co-cultures as an in vitro BBB model, using TEER measurement of the barrier integrity. In contrast, this effect was abrogated by TGF-ß or conditioned medium from TGF-ß secreting GBM cells, leading to the disruption of a so far intact and tight BBB. TGF-ß notably changed the metabolic behavior of pericytes, by shutting down the TCA cycle, driving energy generation from oxidative phosphorylation towards glycolysis, and by modulating pathways that are necessary for the biosynthesis of molecules used for proliferation and cell division. Combined metabolomic and transcriptomic analyses further underscored that the observed functional and metabolic changes of TGF-ß-treated pericytes are closely connected with their role as important supporting cells during angiogenic processes.

Transient receptor potential channel 6 in human skeletal muscle fibers: Investigation in fresh and conserved tissue samples.

Transient receptor potential channel 6 (TRPC6) channels constitute non-selective cation channels that are localized in the plasmalemma or sarcolemma, and have a leading permeability for the bivalent calcium ion. Animal models indicate an involvement of TRPC6 in malignant hyperthermia. The expression of TRPC6 in the sarcolemma has been demonstrated in the skeletal muscle fibers of mice. The importance of TRPC6 channels for the influx of calcium into the muscle cell has also been established. The presence of TRPC6 in tissues of human skeletal muscle is surmised. In order to confirm the presence of TRPC6 in human skeletal muscle, tissue samples of various skeletal muscles (Musculus deltoideus, pectoralis major, trizeps brachii and rectus femoris) from eight different human donors (n=8; six preserved cadavers and two non-preserved cadavers) were examined using immunohistochemistry. TRPC6 was found in all muscle fibers of all investigated bodies. Appropriate controls yielded the expected results. As demonstrated in animal studies and in studies of human cells, the presented results confirmed the presence of TRPC6 in human skeletal muscle tissue. Thus, TRPC6 is most likely important for calcium homeostasis and the proper function of human muscle fibers and may be a target for treatment in various muscular diseases.

First Responders to Hyperosmotic Stress in Murine Astrocytes: Connexin 43 Gap Junctions Are Subject to an Immediate Ultrastructural Reorganization.

In a short-term model of hyperosmotic stress, primary murine astrocytes were stimulated with a hyperosmolar sucrose solution for five minutes. Astrocytic gap junctions, which are mainly composed of Connexin (Cx) 43, displayed immediate ultrastructural changes, demonstrated by freeze-fracture replica immunogold labeling: their area, perimeter, and distance of intramembrane particles increased, whereas particle numbers per area decreased. Ultrastructural changes were, however, not accompanied by changes in Cx43 mRNA expression. In contrast, transcription of the gap junction regulator zonula occludens (ZO) protein 1 significantly increased, whereas its protein expression was unaffected. Phosphorylation of Serine (S) 368 of the Cx43 C-terminus has previously been associated with gap junction disassembly and reduction in gap junction communication. Hyperosmolar sucrose treatment led to enhanced phosphorylation of Cx43S368 and was accompanied by inhibition of gap junctional intercellular communication, demonstrated by a scrape loading-dye transfer assay. Taken together, Cx43 gap junctions are fast reacting elements in response to hyperosmolar challenges and can therefore be considered as one of the first responders to hyperosmolarity. In this process, phosphorylation of Cx43S368 was associated with disassembly of gap junctions and inhibition of their function. Thus, modulation of the gap junction assembly might represent a target in the treatment of brain edema or trauma.

Intercellular communication between alveolar epithelial cells and macrophages.

BACKGROUND: The alveolus in the lung tissue is an extremely vulnerable site. Alveolar macrophages control this micro-environment both in states of health and illnesssuch as acute lung injury and infection. It has been reported in mice in vivo that intercellular communication between alveolar macrophages and alveolar epithelial cells is mediated by gap junctions. However, little is known about thismicro-environment in human cells. METHODS: Since this gap junctional intercellular communication is hard to investigate in human tissues, a co-culture model of two human cell lines, one of epithelial and one of macrophage origin, was used. Immunoblot analysis, freeze fracture replica immunolabeling and electron microscopy were performed. RESULTS: Connexin (Cx) 43 protein expression as well as ultrastructurally defined Cx43 gap junctions were detected in co-cultures, yielding evidence of intercellular gap junctions between human alveolar cells of two distinct entities. CONCLUSION: Alveolar macrophages possibly have direct access to the alveolar epithelium via gap junctions in humans, enabling the orchestration of the microenvironment in physiology and disease states.

A hydrogel-based in vitro assay for the fast prediction of antibiotic accumulation in Gram-negative bacteria.

The pipeline of antibiotics has been for decades on an alarmingly low level. Considering the steadily emerging antibiotic resistance, novel tools are needed for early and easy identification of effective anti-infective compounds. In Gram-negative bacteria, the uptake of anti-infectives is especially limited. We here present a surprisingly simple in vitro model of the Gram-negative bacterial envelope, based on 20% (w/v) potato starch gel, printed on polycarbonate 96-well filter membranes. Rapid permeability measurements across this polysaccharide hydrogel allowed to correctly predict either high or low accumulation for all 16 tested anti-infectives in living Escherichia coli. Freeze-fracture TEM supports that the macromolecular network structure of the starch hydrogel may represent a useful surrogate of the Gram-negative bacterial envelope. A random forest analysis of in vitro data revealed molecular mass, minimum projection area, and rigidity as the most critical physicochemical parameters for hydrogel permeability, in agreement with reported structural features needed for uptake into Gram-negative bacteria. Correlating our dataset of 27 antibiotics from different structural classes to reported MIC values of nine clinically relevant pathogens allowed to distinguish active from nonactive compounds based on their low in vitro permeability specifically for Gram-negatives. The model may help to identify poorly permeable antimicrobial candidates before testing them on living bacteria.

Oxygen-Glucose Deprivation in Mouse Astrocytes is Associated with Ultrastructural Changes in Connexin 43 Gap Junctions.

In the intact brain, astrocytes play an important role in a number of physiological functions like spatial buffering of potassium, maintenance of calcium homeostasis, neurotransmitter release, regulation of the cerebral blood flow, and many more. As pathophysiological events upon hypoxic-ischemic brain injury include excitotoxicity by glutamate release as well as oxidative stress, astrocytes and their gap junction-based syncytium are of major relevance for regulating the extent of resulting brain damage. The gap junction protein Connexin (Cx) 43 contributes mainly to the astrocytic intercellular communication. As little is known about the ultrastructural assemblage of Cx43 and its changes in response to hypoxic events, we chose temporary oxygen and glucose deprivation with subsequent reoxygenation (OGD-R) as a metabolic inhibition model of hypoxia in primary murine astrocytes. Gap junction morphology and assembly/disintegration were analyzed at the ultrastructural level using freeze-fracture replica immunolabeling. The exposure of cultured astrocytes to short-term OGD-R resulted in the activation of ERK1/2 (p44/p42), downregulation of Cx43 protein expression, and the rearrangement of Cx43 particles within the cell membrane and within gap junctions. These changes in gap junction morphology were associated with phosphorylation of Cx43 at Serine 368. Analysis of the nearest-neighbor distance within gap junction plaques revealed the loosening of Cx43 particle clusters. Together with the observation of additional connexons being present in the vicinity of gap junction plaques after OGD-R treatment, our study indicates that changes in gap junction assembly are associated with the early phase of hypoxic cell damage.

Silica nanoparticles of microrods enter lung epithelial cells.

A novel type of microparticle has recently been engineered. It consists of amorphous silica nanoparticles and has a corncob-like shape. It has already been demonstrated in vivo that alveolar macrophages in the lung are able to engulf this particulate carrier and that it also functions successfully as a gene delivery system. This subsequently raises the question as to whether epithelial cells may also be possible targets for these microrods. For this purpose, the alveolar epithelial cell line A549 was used presently. The epithelial character of these confluent cells was documented by the presence of tight junctions using a freeze-fracture technique and transmission electron microscopy. A toxic effect of the particles incubated with these cells was largely excluded. The interaction of the microparticles with the epithelial cells was observed using confocal microscopy and live cell imaging. Interestingly, the particles entered the epithelial cells within hours. After 1 day, the intracellular particles began to disaggregate and release the silica nanoparticles. Thus, even epithelial cells may serve as targets for this novel carrier and gene delivery system. This is particularly important since safe and effective gene delivery remains an unsolved problem. In addition, delivery of anti-cancer and anti-infective drugs may be an application of this novel particulate carrier.