Muscarinic Agonists Cause Calcium Influx and Calcium Mobilization in Forebrain Neurons In Vitro

1989 ◽  
Vol 53 (1) ◽  
pp. 226-233 ◽  
Author(s):  
Ian J. Reynolds ◽  
Richard J. Miller
Keyword(s):  
Calcium Influx ◽  
Calcium Mobilization ◽  
Muscarinic Agonists ◽  
Forebrain Neurons

scholarly journals Strategies for Developing Functional Secretory Epithelia from Porcine Salivary Gland Explant Outgrowth Culture Models

Biomolecules ◽  
2019 ◽  
Vol 9 (11) ◽  
pp. 657 ◽  
Author(s):  
Urkasemsin ◽  
Castillo ◽  
Rungarunlert ◽  
Klincumhom ◽  
Ferreira
Keyword(s):  
Salivary Gland ◽  
Parotid Gland ◽  
Calcium Influx ◽  
Amylase Activity ◽  
Smooth Muscle Actin ◽  
Dry Mouth ◽  
Muscarinic Agonists ◽  
Proliferation Markers ◽  
Epithelial Barrier Function

Research efforts have been made to develop human salivary gland (SG) secretory epithelia for transplantation in patients with SG hypofunction and dry mouth (xerostomia). However, the limited availability of human biopsies hinders the generation of sufficient cell numbers for epithelia formation and regeneration. Porcine SG have several similarities to their human counterparts, hence could replace human cells in SG modelling studies in vitro. Our study aims to establish porcine SG explant outgrowth models to generate functional secretory epithelia for regeneration purposes to rescue hyposalivation. Cells were isolated and expanded from porcine submandibular and parotid gland explants. Flow cytometry, immunocytochemistry, and gene arrays were performed to assess proliferation, standard mesenchymal stem cell, and putative SG epithelial stem/progenitor cell markers. Epithelial differentiation was induced and different SG-specific markers investigated. Functional assays upon neurostimulation determined α-amylase activity, trans-epithelial electrical resistance, and calcium influx. Primary cells exhibited SG epithelial progenitors and proliferation markers. After differentiation, SG markers were abundantly expressed resembling epithelial lineages (E-cadherin, Krt5, Krt14), and myoepithelial (α-smooth muscle actin) and neuronal (β3-tubulin, Chrm3) compartments. Differentiated cells from submandibular gland explant models displayed significantly greater proliferation, number of epithelial progenitors, amylase activity, and epithelial barrier function when compared to parotid gland models. Intracellular calcium was mobilized upon cholinergic and adrenergic neurostimulation. In summary, this study highlights new strategies to develop secretory epithelia from porcine SG explants, suitable for future proof-of-concept SG regeneration studies, as well as for testing novel muscarinic agonists and other biomolecules for dry mouth.

scholarly journals 68Ga-Radiolabeling and Pharmacological Characterization of a Kit-Based Formulation of the Gastrin-Releasing Peptide Receptor (GRP-R) Antagonist RM2 for Convenient Preparation of [68Ga]Ga-RM2

Pharmaceutics ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1160
Author(s):  
Adrien Chastel ◽  
Delphine Vimont ◽  
Stephane Claverol ◽  
Marion Zerna ◽  
Sacha Bodin ◽  
...  
Keyword(s):  
Calcium Mobilization ◽  
Pharmacological Characterization ◽  
Peptide Receptor ◽  
Gastrin Releasing Peptide ◽  
Production Route ◽  
Membrane Bound ◽  
One Year ◽  
Calcium Mobilization Assay

Background: [68Ga]Ga-RM2 is a potent Gastrin-Releasing Peptide-receptor (GRP-R) antagonist for imaging prostate cancer and breast cancer, currently under clinical evaluation in several specialized centers around the world. Targeted radionuclide therapy of GRP-R-expressing tumors is also being investigated. We here report the characteristics of a kit-based formulation of RM2 that should ease the development of GRP-R imaging and make it available to more institutions and patients. Methods: Stability of the investigated kits over one year was determined using LC/MS/MS and UV-HPLC. Direct 68Ga-radiolabeling was optimized with respect to buffer (pH), temperature, reaction time and shaking time. Conventionally prepared [68Ga]Ga-RM2 using an automated synthesizer was used as a comparator. Finally, the [68Ga]Ga-RM2 product was assessed with regards to hydrophilicity, affinity, internalization, membrane bound fraction, calcium mobilization assay and efflux, which is a valuable addition to the in vivo literature. Results: The kit-based formulation, kept between 2 °C and 8 °C, was stable for over one year. Using acetate buffer pH 3.0 in 2.5–5.1 mL total volume, heating at 100 °C during 10 min and cooling down for 5 min, the [68Ga]Ga-RM2 produced by kit complies with the requirements of the European Pharmacopoeia. Compared with the module production route, the [68Ga]Ga-RM2 produced by kit was faster, displayed higher yields, higher volumetric activity and was devoid of ethanol. In in vitro evaluations, the [68Ga]Ga-RM2 displayed sub-nanomolar affinity (Kd = 0.25 ± 0.19 nM), receptor specific and time dependent membrane-bound fraction of 42.0 ± 5.1% at 60 min and GRP-R mediated internalization of 24.4 ± 4.3% at 30 min. The [natGa]Ga-RM2 was ineffective in stimulating intracellular calcium mobilization. Finally, the efflux of the internalized activity was 64.3 ± 6.5% at 5 min. Conclusion: The kit-based formulation of RM2 is suitable to disseminate GRP-R imaging and therapy to distant hospitals without complex radiochemistry equipment.

scholarly journals A sand fly salivary protein acts as a neutrophil chemoattractant

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Anderson B. Guimaraes-Costa ◽  
John P. Shannon ◽  
Ingrid Waclawiak ◽  
Jullyanna Oliveira ◽  
Claudio Meneses ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Calcium Influx ◽  
Parasite Burden ◽  
Salivary Protein ◽  
Sand Fly ◽  
Human Neutrophils ◽  
Chemotactic Protein ◽  
The Impact

AbstractApart from bacterial formyl peptides or viral chemokine mimicry, a non-vertebrate or insect protein that directly attracts mammalian innate cells such as neutrophils has not been molecularly characterized. Here, we show that members of sand fly yellow salivary proteins induce in vitro chemotaxis of mouse, canine and human neutrophils in transwell migration or EZ-TAXIScan assays. We demonstrate murine neutrophil recruitment in vivo using flow cytometry and two-photon intravital microscopy in Lysozyme-M-eGFP transgenic mice. We establish that the structure of this ~ 45 kDa neutrophil chemotactic protein does not resemble that of known chemokines. This chemoattractant acts through a G-protein-coupled receptor and is dependent on calcium influx. Of significance, this chemoattractant protein enhances lesion pathology (P < 0.0001) and increases parasite burden (P < 0.001) in mice upon co-injection with Leishmania parasites, underlining the impact of the sand fly salivary yellow proteins on disease outcome. These findings show that some arthropod vector-derived factors, such as this chemotactic salivary protein, activate rather than inhibit the host innate immune response, and that pathogens take advantage of these inflammatory responses to establish in the host.

scholarly journals Contractile and Endothelium-Dependent Dilatory Responses of Cerebral Arteries at Various Extracellular Magnesium Concentrations

1991 ◽  
Vol 11 (1) ◽  
pp. 161-164 ◽  
Author(s):  
Mária Faragó ◽  
Csaba Szabó ◽  
Eörs Dóra ◽  
Ildikó Horváth ◽  
Arisztid G. B. Kovách
Keyword(s):  
Smooth Muscle ◽  
Vascular Reactivity ◽  
Calcium Influx ◽  
Cerebral Arteries ◽  
Inhibitory Effect ◽  
Contractile Responses ◽  
Middle Cerebral Arteries ◽  
The Right ◽  
Endothelial Receptor

To clarify the effect of extracellular magnesium (Mg2+) on the vascular reactivity of feline isolated middle cerebral arteries, the effects of slight alterations in the Mg2+ concentration on the contractile and endothelium-dependent dilatory responses were investigated in vitro. The contractions, induced by 10−8-10−5 M norepinephrine, were significantly potentiated at low Mg2+ (0.8 m M v. the normal, 1.2 m M). High (1.6 and 2.0 m M) Mg2+ exhibited an inhibitory effect on the contractile responses. No significant changes, however, in the EC50 values for norepinephrine were found. The endothelium-dependent relaxations induced by 108–10−5 M acetylcholine were inhibited by high (1.6 and 2.0 m M) Mg2+. Lowering of the Mg2+ concentration to 0.8 m M or total withdrawal of this ion from the medium failed to alter the dilatory potency of acetylcholine. The changes in the dilatory responses also shifted the EC50 values for acetylcholine to the right. The present results show that the contractile responses of the cerebral arteries are extremely susceptible to the changes of Mg2+ concentrations. In response to contractile and endothelium-dependent dilatory agonists, Mg2+ probably affects both the calcium influx into the endothelial and smooth muscle cells as well as the binding of acetylcholine to its endothelial receptor. Since Mg2+ deficiency might facilitate the contractile but not the endothelium-dependent relaxant responses, the present study supports a role for Mg2+ deficiency in the development of the cerebral vasospasm.

Levobupivacaine-induced contraction of isolated rat aorta is calcium dependent

2011 ◽  
Vol 89 (7) ◽  
pp. 467-476 ◽  
Author(s):  
Ji Seok Baik ◽  
Ju-Tae Sohn ◽  
Seong-Ho Ok ◽  
Jae-Gak Kim ◽  
Hui-Jin Sung ◽  
...  
Keyword(s):  
Methylene Blue ◽  
Smooth Muscle ◽  
Calcium Influx ◽  
Rat Aorta ◽  
Isometric Tension ◽  
Guanosine Monophosphate ◽  
Response Curves ◽  
Calcium Dependent ◽  
Isolated Rat

Levobupivacaine is a long-acting local anesthetic that intrinsically produces vasoconstriction in isolated vessels. The goals of this study were to investigate the calcium-dependent mechanism underlying levobupivacaine-induced contraction of isolated rat aorta in vitro and to elucidate the pathway responsible for the endothelium-dependent attenuation of levobupivacaine-induced contraction. Isolated rat aortic rings were suspended to record isometric tension. Cumulative levobupivacaine concentration–response curves were generated in either the presence or absence of the antagonists verapamil, nifedipine, SKF-96365, 2-aminoethoxydiphenylborate, Gd3+, NW-nitro-l-arginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), and methylene blue, either alone or in combination. Verapamil, nifedipine, SKF-96365, 2-aminoethoxydiphenylborate, low calcium concentrations, and calcium-free Krebs solution attenuated levobupivacaine-induced contraction. Gd3+ had no effect on levobupivacaine-induced contraction. Levobupivacaine increased intracellular calcium levels in vascular smooth muscle cells. L-NAME, ODQ, and methylene blue increased levobupivacaine-induced contraction in endothelium-intact aorta. SKF-96365 attenuated calcium-induced contraction in a previously calcium-free isotonic depolarizing solution containing 100 mmol/L KCl. Levobupivacaine-induced contraction of rat aortic smooth muscle is mediated primarily by calcium influx from the extracellular space mainly via voltage-operated calcium channels and, in part, by inositol 1,4,5-trisphosphate receptor-mediated release of calcium from the sarcoplasmic reticulum. The nitric oxide – cyclic guanosine monophosphate pathway is involved in the endothelium-dependent attenuation of levobupivacaine-induced contraction.

scholarly journals Signal transduction mechanisms involved in in vitro ram sperm capacitation

Reproduction ◽  
2006 ◽  
Vol 132 (5) ◽  
pp. 721-732 ◽  
Author(s):  
Patricia Grasa ◽  
José Álvaro Cebrián-Pérez ◽  
Teresa Muiño-Blanco
Keyword(s):  
Tyrosine Phosphorylation ◽  
Calcium Influx ◽  
Calcium Entry ◽  
Calcium Entry Blocker ◽  
Protein Tyrosine Phosphorylation ◽  
Sperm Capacitation ◽  
Protein Tyrosine ◽  
Entry Blocker ◽  
Ram Sperm

We validate the chlortetracycline (CTC) technique for the evaluation of capacitation and acrosome reaction-like changes in ram sperm, carrying out a double estimation of the acrosome status after treatment with lysophosphatidylcholine, using fluorescein isocyanate (FITC)-RCA/ethidium homodimer 1 (EthD-1) and CTC/EthD-1. Highly consistent results and a positive correlation between the results of acrosome-reacted sperm evaluated with both techniques were obtained. In this study, we evaluate the effects of ram sperm capacitation of BSA, Ca2+, NaHCO3and cAMP agonists and their influence on the associated protein tyrosine phosphorylation. We found a time-dependent increase in capacitation related to protein tyrosine phosphorylation, either in the absence or the presence of BSA. The addition of an increasing concentration of cholesterol to samples containing BSA did not influence results. The effect of bicarbonate was concentration-dependent, with a significantly lowered value of non-capacitated sperm in the presence 18 and 25 mM. The addition of extracellular calcium did not significantly increase either the proportion of capacitated sperm or the protein tyrosine phosphorylation signalling, although a significantly higher value of acrosome-reacted sperm was found in samples containing 4 mM Ca2+. cAMP agonists increased capacitated sperm and protein tyrosine phosphorylation signalling. The inhibition of protein kinase A by H-89 caused a decrease in sperm capacitation. Addition of a calcium-entry blocker (Verapamil; Sigma) did not influence results, which suggests that the calcium entry blocker was unable to inhibit the calcium influx associated with capacitation in ram sperm. Our findings might benefit our understanding of the biochemical mechanisms involved in mammalian sperm capacitation and ultimately, fertility.

scholarly journals Nanobody-Mediated Inhibition of P2X7 on Microglia Improves Stroke Outcome

2021 ◽  
Author(s):  
Maximilian Wilmes ◽  
Carolina Pinto Espinoza ◽  
Peter Ludewig ◽  
Arthur Liesz ◽  
Annette Nicke ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Transgenic Mice ◽  
Tissue Damage ◽  
Calcium Influx ◽  
Atp Release ◽  
Artery Occlusion ◽  
Membrane Pores ◽  
Caspase 1 ◽  
Ischemic Tissue

Abstract BackgroundPrevious studies have demonstrated that purinergic receptors could be therapeutic targets to modulate the inflammatory response in multiple brain disease models. However, tools for the selective and efficient targeting of these receptors are scarce. The new development of P2X7-specific nanobodies (nbs) enables us to effectively block the P2X7-channel.MethodsTemporary middle cerebral artery occlusion (tMCAO) in wildtype and P2X7-transgenic mice was used as a model for ischemic stroke. ATP release was assessed in transgenic ATP sensor mice. Stroke size was measured without treatment and after injection of P2X7-specific nbs i.v. and i.c.v. directly before tMCAO-surgery. P2X7-GFP expressing transgenic mice were used to show immunhistochemically P2X7 distribution in the brain. In vitro cultured microglia were used to investigate calcium-influx, pore-formation via DAPI uptake, caspase 1 activation and IL-1b release after incubation with P2X7-specific nbs. ResultsATP sensor mice showed an increase of ATP-release in the ischemic hemisphere compared to the contralateral hemisphere or sham mice up to 24 h after stroke. We could further verify the role of the ATP-P2X7 axis in P2X7-overexpressing mice, which showed significantly greater stroke volumes after 24 h. In vitro experiments with primary microglia cells showed that P2X7-specific nanobodies were capable of dampening the ATP-trigged calcium-influx and formation of membrane pores measured by Fluo4 fluorescence or DAPI uptake. We found a lower caspase 1 activity and a subsequently lower IL-1b release. However, the intravenous (i.v.) injection of P2X7-specific nanobodies compared to isotype controls before the tMCAO-surgery did not result in smaller stroke size compared to isotype controls. As demonstrated by FACS, nbs had only reached brain infiltrating macrophages but not microglia. To reach microglia, we injected the P2X7-spezific nbs or the isotype directly intraventricularly (icv). 30 mg of P2X7-specific nbs proved efficient for microglial targeting, reducing post-stroke microglia activation and stroke size significantly.ConclusionHere, we demonstrate the importance of locally produced ATP for the tissue damage observed in ischemic stroke and we show the potential of icv injected P2X7-specific nbs to reduce ischemic tissue damage.

Abstract 11710: Poloxamer 188 Protects Coronary Artery Endothelial Cells After Extended Hypoxia and Reoxygenation in an in vitro Model of Simulated Ischemia/Reperfusion Injury

Circulation ◽  
2021 ◽  
Vol 144 (Suppl_2) ◽  
Author(s):  
zhu li ◽  
Matthew J Hampton ◽  
Matthew B Barajas ◽  
Matthias L Riess
Keyword(s):  
Endothelial Cells ◽  
Coronary Artery ◽  
Myocardial Ischemia ◽  
Ischemia Reperfusion Injury ◽  
Calcium Influx ◽  
Ischemia Reperfusion ◽  
Cell Types ◽  
Membrane Function ◽  
Ldh Release

Reperfusion restores blood flow after myocardial ischemia but can cause additional cellular injury by the sudden reintroduction of oxygen and nutrients. There is still no effective remedy for myocardial ischemia/reperfusion (IR) injury. Our previous study using cardiomyocytes (CMs) found that, after 3 hrs hypoxia followed by 2 hrs reoxygenation, viability decreased, and release of lactate dehydrogenase (LDH), calcium influx, membrane leakage (insertion of fluorescent probe FM1-43) significantly increased, indicating that cell membrane function was negatively affected. This was attenuated by the triblock copolymer Poloxamer (P)188. Here, we first hypothesized that endothelial cells are also susceptible to simulated IR injury, albeit requiring longer hypoxia times. We further hypothesized that P188 can also attenuate simulated IR injury in endothelial cells when given upon reoxygenation. Mouse coronary artery endothelial cells (MCAECs) were exposed to different durations of hypoxia (2, 3, 12 and 24 hrs) in serum- and glucose-free media +/- reoxygenation for 2 hrs in regular media. P188 was administered upon reoxygenation at 0, 100, 300 or 1,000 μM in experiments of 24 hrs hypoxia / 2 hrs reoxygenation. LDH release was measured and compared to appropriately timed normoxic control experiments. Reoxygenation and hypoxia times significantly longer than 3 hrs were required to elicit sufficient injury (panel A). When P188 was given upon reoxygenation after 24 hrs hypoxia, it dose-dependently attenuated LDH release (panel B). These findings contrast to the higher susceptibility of CMs to IR injury that only allowed shorter hypoxia durations. They also confirm a protective effect of P188 on the endothelium, not just on CMs. These findings have important implications for co-culture models with MCAECs and CMs to elucidate the interplay of both cell types on each other when studying mechanisms of cardioprotective strategies and compounds like P188.

Sign in / Sign up

Export Citation Format

Share Document