ATP induces contraction of cultured brain capillary pericytes via activation of P2Y-type purinergic receptors.

Brain capillary pericytes have been suggested to play a role in the regulation of cerebral blood flow under physiological and pathophysiological conditions. ATP has been shown to cause constriction of capillaries under ischemic conditions and suggested to be involved in the "no-reflow" phenomenon. To investigate the effects of extracellular ATP on pericyte cell contraction, we studied purinergic receptor activation of cultured bovine brain capillary pericytes. We measured intracellular Ca2+ concentration ([Ca2+]i) responses to purinergic agonists with the fluorescent indicators fura-2 and Cal-520 and estimated contraction of pericytes as relative change in cell area, using real-time confocal imaging. Addition of ATP caused an increase in cytosolic calcium and contraction of the brain capillary pericytes, both reversible and inhibited by the purinergic receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). Furthermore, we demonstrated that ATP-induced contraction could be eliminated by intracellular calcium chelation with BAPTA, indicating that the contraction was mediated via purinergic P2-type receptor-mediated [Ca2+]i signaling. ATP stimulation induced inositol triphosphate signaling, consistent with the notion of P2Y receptor activation. Receptor profiling studies demonstrated the presence of P2Y1 and P2Y2 receptors, using ATP, UTP, ADP, and the subtype specific agonists MRS2365 (P2Y1) and 2-thio-UTP (P2Y2). Addition of specific P2X agonists only caused an [Ca2+]i increase at high concentrations, attributed to activation of inositol triphosphate signaling. Our results suggest that contraction of brain capillary pericytes in vitro by activation of P2Y-type purinergic receptors is caused by intracellular calcium release. This adds more mechanistic understanding of the role of pericytes in vessel constriction and points toward P2Y receptors as potential therapeutic targets.NEW & NOTEWORTHY The study concerns brain capillary pericytes, which have been suggested to play a role in the regulation of cerebral blood flow. We show that extracellular ATP causes contraction of primary brain pericytes by stimulation of purinergic receptors and subsequent release of intracellular Ca2+ concentration ([Ca2+]i). The contraction is mainly mediated through activation of P2Y-receptor subtypes, including P2Y1 and P2Y2. These findings add more mechanistic understanding of the role of pericytes in regulation of capillary blood flow. ATP was earlier suggested to be involved in capillary constriction in brain pathologies, and our study gives a detailed account of a part of this important mechanism.

Apoptotic properties of the type 1 interferon induced family of human mitochondrial membrane ISG12 proteins.

BACKGROUND INFORMATION: Interferons are a family of cytokines with growth inhibitory and antiviral functions, which exert their biological actions through the expression of interferon-stimulated genes (ISGs). The human ISG12 family of proteins comprises ISG12A, ISG12B, ISG12C and ISG6-16. Due to differential splicing and a gene variation, the human ISG12A protein exists as a full-length ISG12A form and three ISG12A variants. ISG12 genes have been found transcriptionally dysregulated in many disorders. High levels of ISG12A mRNA have been found in breast and ovarian cancers. Loss of heterozygosity at the position of the ISG12 genes often occurs in ovarian carcinomas and lymphoblastic leukemias. Both ISG12A and ISG6-16 are up-regulated in psoriasis. RESULTS: We demonstrate here that expression of the human full-length ISG12A protein sensitises cells for TNFα and the BH3 mimetic gossypol induced apoptosis, and the other ISG12A variants as well as ISG12B and ISG12C can induce apoptosis directly in HEK293 cells. Also ISG6-16 sensitises HEK293 cells for gossypol-induced apoptosis. In the ISG12 motif, two putative Bcl-2 homology (BH)3 like motifs were found, which may be decisive for the apoptotic properties of the ISG12 proteins. A series of BH3 mutants was made in ISG12AΔ-S, the smallest apoptosis-inducing ISG12A variant and our results indicate that ISG12AΔ-S indeed possesses features resembling those of BH3-only proteins. Supporting this notion are our findings that the full-length ISG12A co-immunoprecipitates with the Bcl-2 protein, and the apoptotic properties of the ISG12A variants are reduced in Bcl-2 expressing HEK293 cells. In addition, full-length ISG12A is able to form homodimers, which suggests a possible involvement in pore formation during apoptosis. The full-length ISG12A, the three ISG12A variants and the ISG12B proteins were found to be localised in the mitochondria. CONCLUSIONS: Our results suggest that the ISG12 family of proteins has an important role for the apoptotic properties induced by type 1 interferon. SIGNIFICANCE: The ISG12 family constitute small hydrophic proteins involved in apoptosis. This is the first comparison of the apoptotic potentials of the full-length ISG12A protein and the three ISG12A variants. The differential apoptotic potentials of these proteins might have an impact on the strategies to monitor and interpret their dysregulation associated with many disorders.

Combined nasal- and oropharyngeal self-swab provides equivalent performance compared to professionally collected oropharyngeal swabs in detecting SARS-CoV-2 in a real-life setting.

PURPOSE: To investigate the performance of a combined nasal midturbinate- and oropharyngeal (NAOP) self-swab compared to a deep oropharyngeal (OP) swab by health care workers (HCW) in detecting SARS-CoV-2 in a real-life setting. METHODS: Paired swabs from 1119 participants were included. RT-PCR were used to detect SARS-CoV-2 in both swab samples. RESULTS: 330 participants tested positive. The sensitivity of the combined self-swab and OP swab was 96.9 % and 95.4 % respectively, whereas the Ct-values for self-swabs were significantly lower compared to OP swabs. CONCLUSION: The combined NAOP self-swab outperformed the OP swab and thus, the NAOP self-swab may be an alternative sampling method under the given circumstances.

Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies.

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte function is not fully known. Pericytes have been suggested to be involved in capillary development, regulation of endothelial barrier tightness and trancytosis activity, regulation of capillary tone and to play crucial roles in certain brain pathologies. Pericytes are challenging to investigate in the intact brain due to the difficulties in visualizing processes in the brain parenchyma, as well as the close proximity to the other cells of the NVU. The present protocol describes a method for isolation and culture of primary bovine brain capillary pericytes and their following usage in calcium imaging studies, where effects of agonists involved in brain signaling and pathologies can be investigated. Cortical capillary fragments are allowed to attach to the bottom of culture flasks and, after 6 days, endothelial cells and pericytes have grown out from the capillary fragments. The endothelial cells are removed by gentle trypsinization and pericytes are cultured for 5 additional days before passaging. Isolated pericytes are seeded in 96-well culture plates and loaded with the calcium indicator dye (Fura-2 acetoxymethyl (AM)) to allow for measurements of intracellular calcium levels in a plate reader setup. Alternatively, pericytes are seeded on coverslips and mounted in cell chambers. Following loading with the calcium indicator (Cal-520 AM), calcium live-imaging can be performed using confocal microscopy at an excitation wavelength of 488 nm and emission wavelength of 510-520 nm. The method described here has been used to obtain the first intracellular calcium measurements from primary brain capillary pericytes, demonstrating that pericytes are stimulated via ATP and are able to contract in vitro.

Evaluating the involvement of cerebral microvascular endothelial Na+/K+-ATPase and Na+-K+-2Cl- co-transporter in electrolyte fluxes in an in vitro blood-brain barrier model of dehydration.

The blood-brain barrier (BBB) is involved in brain water and salt homeostasis. Blood osmolarity increases during dehydration and water is osmotically extracted from the brain. The loss of water is less than expected from pure osmotic forces, due to brain electrolyte accumulation. Although the underlying molecular mechanisms are unresolved, the current model suggests the luminally expressed Na+-K+-2Cl- co-transporter 1 (NKCC1) as a key component, while the role of the Na+/K+-ATPase remains uninvestigated. To test the involvement of these proteins in brain electrolyte flux under mimicked dehydration, we employed a tight in vitro co-culture BBB model with primary cultures of brain endothelial cells and astrocytes. The Na+/K+-ATPase and the NKCC1 were both functionally dominant in the abluminal membrane. Exposure of the in vitro BBB model to conditions mimicking systemic dehydration, i.e. hyperosmotic conditions, vasopressin, or increased [K+]o illustrated that NKCC1 activity was unaffected by exposure to vasopressin and to hyperosmotic conditions. Hyperosmotic conditions and increased K+ concentrations enhanced the Na+/K+-ATPase activity, here determined to consist of the α1 ß1 and α1 ß3 isozymes. Abluminally expressed endothelial Na+/K+-ATPase, and not NKCC1, may therefore counteract osmotic brain water loss during systemic dehydration by promoting brain Na+ accumulation.