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International Journal of Molecular... Aug 2022Cell-cell communication via gap junction channels is known to be inhibited by the anesthetics heptanol, halothane and isoflurane; however, despite numerous studies, the... (Review)
Review
Cell-cell communication via gap junction channels is known to be inhibited by the anesthetics heptanol, halothane and isoflurane; however, despite numerous studies, the mechanism of gap junction channel gating by anesthetics is still poorly understood. In the early nineties, we reported that gating by anesthetics is strongly potentiated by caffeine and theophylline and inhibited by 4-Aminopyridine. Neither Ca channel blockers nor 3-isobutyl-1-methylxanthine (IBMX), forskolin, CPT-cAMP, 8Br-cGMP, adenosine, phorbol ester or H7 had significant effects on gating by anesthetics. In our publication, we concluded that neither cytosolic Ca nor pH were involved, and suggested a direct effect of anesthetics on gap junction channel proteins. However, while a direct effect cannot be excluded, based on the potentiating effect of caffeine and theophylline added to anesthetics and data published over the past three decades, we are now reconsidering our earlier interpretation and propose an alternative hypothesis that uncoupling by heptanol, halothane and isoflurane may actually result from a rise in cytosolic Ca concentration ([Ca]) and consequential activation of calmodulin linked to gap junction proteins.
Topics: Anesthetics; Anesthetics, Inhalation; Caffeine; Calcium; Calmodulin; Cell Communication; Connexins; Gap Junctions; Halothane; Heptanol; Ion Channels; Isoflurane; Theophylline
PubMed: 36012286
DOI: 10.3390/ijms23169017 -
IScience Jul 2023Protein kinase C (PKC) isoforms regulate many important signaling pathways. Here, we report that PKC activation by phorbol 12-myristate 13-acetate (PMA) enhanced A...
Protein kinase C (PKC) isoforms regulate many important signaling pathways. Here, we report that PKC activation by phorbol 12-myristate 13-acetate (PMA) enhanced A adenosine receptor (AR)-mediated, but not β-adrenergic receptor-mediated, cAMP accumulation, in H9C2 cardiomyocyte-like and HEK293 cells. In addition to enhancement, PKC (PMA-treatment) also activated AAR with low E (H9C2 and NIH3T3 cells endogenously expressing AAR), or with high E (AAR-overexpressing HEK293 cells) to induce cAMP accumulation. AAR activation induced by PKC was inhibited by AAR and PKC inhibitors but enhanced by AAR overexpression. Gαi isoforms and PKCγ isoform were found to be involved in both enhancement of AAR function and AAR activation. Thus, we establish PKC as an endogenous modulator and activator of AAR, involving G and PKCγ. Depending on signaling pathway, PKC could activate and enhance, or alternatively inhibit AAR activity. These findings are relevant to common functions of AAR and PKC, e.g. cardioprotection and cancer progression/treatment.
PubMed: 37404375
DOI: 10.1016/j.isci.2023.107178 -
Cells May 2023Previously established immune-responsive co-culture models with macrophages have limitations due to the dedifferentiation of macrophages in long-term cultures. This...
Previously established immune-responsive co-culture models with macrophages have limitations due to the dedifferentiation of macrophages in long-term cultures. This study is the first report of a long-term (21-day) triple co-culture of THP-1 macrophages (THP-1m) with Caco-2 intestinal epithelial cells and HT-29-methotrexate (MTX) goblet cells. We demonstrated that high-density seeded THP-1 cells treated with 100 ng/mL phorbol 12-myristate 13-acetate for 48 h differentiated stably and could be cultured for up to 21 days. THP-1m were identified by their adherent morphology and lysosome expansion. In the triple co-culture immune-responsive model, cytokine secretions during lipopolysaccharide-induced inflammation were confirmed. Tumor necrosis factor-alpha and interleukin 6 levels were elevated in the inflamed state, reaching 824.7 ± 130.0 pg/mL and 609.7 ± 139.5 pg/mL, respectively. Intestinal membrane integrity was maintained with a transepithelial electrical resistance value of 336.4 ± 18.0 Ω·cm. Overall, our findings suggest that THP-1m can be effectively employed in models of long-term immune responses in both normal and chronic inflammatory states of the intestinal epithelium, making them a valuable tool for future research on the association between the immune system and gut health.
Topics: Humans; Caco-2 Cells; Macrophages; Intestinal Mucosa; Tumor Necrosis Factor-alpha; Coculture Techniques; Inflammation
PubMed: 37408263
DOI: 10.3390/cells12101427 -
Neurotoxicology Dec 2022Amphetamine (AMPH) causes the degeneration of dopamine terminals in the central nervous system. The mechanisms for this damage are unclear. We found AMPH reduced level...
Amphetamine (AMPH) causes the degeneration of dopamine terminals in the central nervous system. The mechanisms for this damage are unclear. We found AMPH reduced level of GAP-43 in the striatum of rats that receives rich dopaminergic terminals. Using PC12 cells as dopaminergic neuronal models, we further found that AMPH inhibited GAP-43 and GAP-43 phosphorylation in PC12 cells. The reduced GAP-43 was correlated with neurite injury of PC12 cells. The PKCβ1, an upstream molecule of GAP-43, was also inhibited by AMPH. Phorbol 12-myristate 13-acetate (PMA) as a specific activator of PKC increased levels of PKCβ1 and GAP-43, and efficiently prevented neurite degeneration of PC12 cells induced by AMPH. On the other side, enzastuarin, an inhibitor of PKC, decreased levels of PKCβ1 and GAP-43, and caused neurite injury of PC12 cells. Together, our results suggest that AMPH induces neurite injury in PC12 cells through inhibiting PKCβ1/GAP-43 pathway.
Topics: Animals; Rats; Amphetamine; PC12 Cells; Neurites; GAP-43 Protein; Tetradecanoylphorbol Acetate; Dopamine
PubMed: 36150536
DOI: 10.1016/j.neuro.2022.09.004 -
Scientific Reports May 2021Understanding the platelet activation molecular pathways by characterizing specific protein clusters within platelets is essential to identify the platelet activation...
Understanding the platelet activation molecular pathways by characterizing specific protein clusters within platelets is essential to identify the platelet activation state and improve the existing therapies for hemostatic disorders. Here, we employed various state-of-the-art super-resolution imaging and quantification methods to characterize the platelet spatiotemporal ultrastructural change during the activation process due to phorbol 12-myristate 13-acetate (PMA) stimuli by observing the cytoskeletal elements and various organelles at nanoscale, which cannot be done using conventional microscopy. Platelets could be spread out with the guidance of actin and microtubules, and most organelles were centralized probably due to the limited space of the peripheral thin regions or the close association with the open canalicular system (OCS). Among the centralized organelles, we provided evidence that granules are fused with the OCS to release their cargo through enlarged OCS. These findings highlight the concerted ultrastructural reorganization and relative arrangements of various organelles upon activation and call for a reassessment of previously unresolved complex and multi-factorial activation processes.
Topics: Actin Cytoskeleton; Humans; Organelles; Platelet Activation; Tetradecanoylphorbol Acetate
PubMed: 34006947
DOI: 10.1038/s41598-021-89799-9 -
American Journal of Physiology. Renal... Sep 2019Macrophage-mediated inflammation plays a critical role in hypertensive kidney disease. Here, we investigated the role of transient receptor potential ankyrin 1 (TRPA1),...
Macrophage-mediated inflammation plays a critical role in hypertensive kidney disease. Here, we investigated the role of transient receptor potential ankyrin 1 (TRPA1), a sensor of inflammation, in angiotensin II (ANG II)-induced renal injury. Subcutaneous infusion of ANG II (600 ng·min·kg) for 28 days was used to induce hypertension and renal injury in mice. The results showed that ANG II-induced hypertensive mice have decreased renal Trpa1 expression ( < 0.01), whereas ANG II receptor type 1a-deficient hypotensive mice have increased renal Trpa1 expression ( < 0.05) compared with their normotensive counterparts. ANG II induced similar elevations of systolic blood pressure in and wild-type (WT) mice but led to higher levels of blood urea nitrogen ( < 0.05), serum creatinine ( < 0.05), and renal fibrosis ( < 0.01) in mice than WT mice. Similarly, ANG II increased both CD68/inducible nitric oxide synthase M1 and CD68/arginase 1 M2 macrophages in the kidneys of both and WT mice (all < 0.01), with higher extents in mice (both < 0.01). Compared with WT mice, mice had significantly increased expression levels of inflammatory cytokines and their receptors in the kidney. Cultured murine macrophages were stimulated with phorbol 12-myristate 13-acetate, which downregulated gene expression of TRPA1 ( < 0.01). A TRPA1 agonist, cinnamaldehyde, significantly inhibited phorbol 12-myristate 13-acetate-stimulated expression of IL-1β and chemokine (C-C motif) ligand 2 in macrophages, which were attenuated by pretreatment with a TRPA1 antagonist, HC030031. Furthermore, activation of TRPA1 with cinnamaldehyde induced apoptosis of macrophages. These findings suggest that TRPA1 may play a protective role in ANG II-induced renal injury, likely through inhibiting macrophage-mediated inflammation.
Topics: Angiotensin II; Animals; Apoptosis; Biomarkers; Blood Pressure; Blood Urea Nitrogen; Creatinine; Cytokines; Disease Models, Animal; Fibrosis; Gene Knockdown Techniques; Hypertension; Kidney; Kidney Diseases; Macrophage Activation; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; RAW 264.7 Cells; Receptor, Angiotensin, Type 1; TRPA1 Cation Channel
PubMed: 31339777
DOI: 10.1152/ajprenal.00069.2019 -
PloS One 2020Intense dance training leads to inflammation, which may impair the health and performance of the practitioners. Herein, we evaluate the effect of a single street dancing...
Intense dance training leads to inflammation, which may impair the health and performance of the practitioners. Herein, we evaluate the effect of a single street dancing class on the profile of muscle enzymes, lymphocyte activation, and cell surface CD62L expression. We also investigated the correlation between muscle enzymes, adhesion molecules, and lymphocyte activation in dancers. Fifteen male participants (mean ± standard error: age 22.4 ± 1.08 years, body mass index 24.8 ± 0.69 kg/m2, body fat 12.3 ± 1.52%), who were amateur dancers, had blood samples collected previously and subsequent to a high-intensity street dance class. After the class, dancers showed an increase in total lymphocyte count (2.0-fold), creatine kinase (CK)-NAC (4.87%), and CK-MB (3.36%). We also observed a decrease (2.5-fold) in reactive oxygen species (ROS) produced by lymphocytes, under phorbol myristate acetate-stimulated environments. Following the dance class, CD62L expression in lymphocytes decreased (51.42%), while there was a negative correlation between the intensity of the exercise and CD62L expression (r = -0.73; p = 0.01). Lymphocytes were less responsive to stimuli after a single bout of street dancing, indicating transient immunosuppression.
Topics: Creatine Kinase, MB Form; Dancing; Heart Rate; Humans; Inflammation; L-Selectin; Lymphocyte Activation; Lymphocyte Count; Male; Reactive Oxygen Species; Tetradecanoylphorbol Acetate; Young Adult
PubMed: 32956398
DOI: 10.1371/journal.pone.0239516 -
Antioxidants (Basel, Switzerland) Feb 2021Dairy calves are unable to mount an effective immune response during their first weeks of life, which contributes to increased disease susceptibility during this period....
Dairy calves are unable to mount an effective immune response during their first weeks of life, which contributes to increased disease susceptibility during this period. Oxidative stress (OS) diminishes the immune cell capabilities of humans and adult cows, and dairy calves also experience OS during their first month of life. However, the impact that OS may have on neonatal calf immunity remains unexplored. Thus, we aimed to evaluate the impact of OS on newborn calf lymphocyte functions. For this, we conducted two experiments. First, we assessed the association of OS status throughout the first month of age and the circulating concentrations of the cytokines interferon-gamma (IFN-γ) and interleukin (IL) 4, as well as the expression of cytokine-encoding genes and in peripheral mononuclear blood cells (PBMCs) of 12 calves. Subsequently, we isolated PBMCs from another 6 neonatal calves to investigate in vitro the effect of OS on immune responses in terms of activation of lymphocytes, cytokine expression, and antibody production following stimulation with phorbol 12-myristate 13-acetate or bovine herpesvirus-1. The results were compared statistically through mixed models. Calves exposed to high OS status in their first month of age showed higher concentrations of IL-4 and expression of and and lower concentrations of IFN-γ and expression of and than calves exposed to lower OS. In vitro, OS reduced lymphocyte activation, production of antibodies, and protein and gene expression of key cytokines. Collectively, our results demonstrate that OS can compromise some immune responses of newborn calves. Hence, further studies are needed to explore the mechanisms of how OS affects the different lymphocyte subsets and the potential of ameliorating OS in newborn calves as a strategy to augment the functional capacity of calf immune cells, as well as enhance calves' resistance to infections.
PubMed: 33562350
DOI: 10.3390/antiox10020255 -
Scientific Reports Sep 2023Neutrophils can release neutrophil extracellular traps (NETs) containing DNA fibres and antimicrobial peptides to immobilize invading pathogens. NET formation (NETosis)...
Neutrophils can release neutrophil extracellular traps (NETs) containing DNA fibres and antimicrobial peptides to immobilize invading pathogens. NET formation (NETosis) plays a vital role in inflammation and immune responses. In this study we investigated the impact of surgical trauma on NETosis of neutrophils. Nine patients undergoing "Transcatheter/percutaneous aortic valve implantation" (TAVI/PAVI, mild surgical trauma), and ten undergoing "Aortocoronary bypass" (ACB, severe surgical trauma) were included in our pilot study. Peripheral blood was collected before, end of, and after surgery (24 h and 48 h). Neutrophilic granulocytes were isolated and stimulated in vitro with Phorbol-12-myristate-13-acetate (PMA). NETosis rate was examined by microscopy. In addition, HLA-DR surface expression on circulating monocytes was analysed by flow-cytometry as a prognostic marker of the immune status. Both surgical procedures led to significant down regulation of monocytic HLA-DR surface expression, albeit more pronounced in ACB patients, and there was a similar trend in NETosis regulation over the surgical 24H course. Upon PMA stimulation, no significant difference in NETosis was observed over time in TAVI/PAVI group; however, a decreasing NETosis trend with a significant drop upon ACB surgery was evident. The reduced PMA-induced NETosis in ACB group suggests that the inducibility of neutrophils to form NETs following severe surgical trauma may be compromised. Moreover, the decreased monocytic HLA-DR expression suggests a post-operative immunosuppressed status in all patients, with a bigger impact by ACB, which might be attributed to the extracorporeal circulation or tissue damage occurring during surgery.
Topics: Humans; Extracellular Traps; Pilot Projects; Neutrophils; Down-Regulation; Granulocytes
PubMed: 37709941
DOI: 10.1038/s41598-023-42565-5 -
Journal of Natural Products Dec 2023To investigate the role of the secondary 5-hydroxy group in the activity of the anticancer drug tigilanol tiglate () (Stelfonta), oxidation of this epoxytigliane...
To investigate the role of the secondary 5-hydroxy group in the activity of the anticancer drug tigilanol tiglate () (Stelfonta), oxidation of this epoxytigliane diterpenoid from the Australian rainforest plant was attempted. Eventually, 5-dehydrotigilanol tiglate () proved too unstable to be characterized in terms of biological activity and, therefore, was not a suitable tool compound for bioactivity studies. On the other hand, a series of remarkable skeletal rearrangements associated with the presence of a 5-keto group were discovered during its synthesis, including a dismutative ring expansion of ring A and a mechanistically unprecedented dyotropic substituent swap around the C-4/C-10 bond. Taken together, these observations highlight the propensity of the α-hydroxy-β-diketone system to trigger complex skeletal rearrangements and pave the way to new areas of the natural products chemical space.
Topics: Phorbols; Australia; Diterpenes; Antineoplastic Agents; Biological Products
PubMed: 37991924
DOI: 10.1021/acs.jnatprod.3c00834