The expression of free fatty acid 1 receptors (FFA1R), activated by long chain fatty acids in human pancreatic beta-cells and enhancing glucose-stimulated insulin secretion are an attractive target... Show moreThe expression of free fatty acid 1 receptors (FFA1R), activated by long chain fatty acids in human pancreatic beta-cells and enhancing glucose-stimulated insulin secretion are an attractive target to treat type 2 diabetes. Yet several clinical studies with synthetic FFA1R agonists had to be discontinued due to cytotoxicity and/or so-called "liver concerns". It is not clear whether these obstructions are FFA1R dependent. In this context we used CHO-AEQ cells expressing the bioluminescent calcium-sensitive protein aequorin to investigate calcium signaling elicited by FFA1 receptor ligands alpha-linolenic acid (ALA), oleic acid (OLA) and myristic acid (MYA). This study revealed complex modulation of intracellular calcium signaling by these fatty acids. First these compounds elicited a typical transient increase of intracellular calcium via binding to FFA1 receptors. Secondly slightly higher concentrations of ALA substantially reduced ATP mediated calcium responses in CHO-AEQ cells and Angiotensin II responses in CHO-AEQ cells expressing human AT 1 receptors. This effect was less pronounced with MYA and OLA and was not linked to FFA1 receptor activation nor to acute cytotoxicity as a result of plasma membrane perturbation. Yet it can be hypothesized that, in line with previous studies, unsaturated long chain fatty acids such as ALA and OLA are capable of inactivating the G-proteins involved in purinergic and Angiotensin AT(1) receptor calcium signaling. Alternatively the ability of fatty acids to deplete intracellular calcium stores might underly the observed cross-inhibition of these receptor responses in the same cells. Show less
Stel, W. van der; Yang, H.; Le Dévédec, S.E.; Water, B. van de; Beltman, J.B.; Danen, E.H.J. 2022
Cells can adjust their mitochondrial morphology by altering the balance between mitochondrial fission and fusion to adapt to stressful conditions. The connection between a chemical perturbation,... Show moreCells can adjust their mitochondrial morphology by altering the balance between mitochondrial fission and fusion to adapt to stressful conditions. The connection between a chemical perturbation, changes in mitochondrial function, and altered mitochondrial morphology is not well understood. Here, we made use of high-throughput high-content confocal microscopy to assess the effects of distinct classes of oxidative phosphorylation (OXPHOS) complex inhibitors on mitochondrial parameters in a concentration and time resolved manner. Mitochondrial morphology phenotypes were clustered based on machine learning algorithms and mitochondrial integrity patterns were mapped. In parallel, changes in mitochondrial membrane potential (MMP), mitochondrial and cellular ATP levels, and viability were microscopically assessed. We found that inhibition of MMP, mitochondrial ATP production, and oxygen consumption rate (OCR) using sublethal concentrations of complex I and III inhibitors did not trigger mitochondrial fragmentation. Instead, complex V inhibitors that suppressed ATP and OCR but increased MMP provoked a more fragmented mitochondrial morphology. In agreement, complex V but not complex I or III inhibitors triggered proteolytic cleavage of the mitochondrial fusion protein, OPA1. The relation between increased MMP and fragmentation did not extend beyond OXPHOS complex inhibitors: increasing MMP by blocking the mPTP pore did not lead to OPA1 cleavage or mitochondrial fragmentation and the OXPHOS uncoupler FCCP was associated with OPA1 cleavage and MMP reduction. Altogether, our findings connect vital mitochondrial functions and phenotypes in a high-throughput high-content confocal microscopy approach that help understanding of chemical-induced toxicity caused by OXPHOS complex perturbing chemicals. Show less
Suleimanova, A.; Talanov, M.; Maagdenberg, A.M.J.M. van den; Giniatullin, R. 2021
Familial hemiplegic migraine type 3 (FHM3) is caused by gain-of-function mutations in the SCN1A gene that encodes the alpha 1 subunit of voltage-gated Na(V)1.1 sodium channels. The high level of... Show moreFamilial hemiplegic migraine type 3 (FHM3) is caused by gain-of-function mutations in the SCN1A gene that encodes the alpha 1 subunit of voltage-gated Na(V)1.1 sodium channels. The high level of expression of Na(V)1.1 channels in peripheral trigeminal neurons may lead to abnormal nociceptive signaling thus contributing to migraine pain. Na(V)1.1 dysfunction is relevant also for other neurological disorders, foremost epilepsy and stroke that are comorbid with migraine. Here we used computer modeling to test the functional role of FHM3-mutated Na(V)1.1 channels in mechanisms of trigeminal pain. The activation of A delta-fibers was studied for two algogens, ATP and 5-HT, operating through P2X3 and 5-HT3 receptors, respectively, at trigeminal nerve terminals. In WT A delta-fibers of meningeal afferents, Na(V)1.1 channels efficiently participate in spike generation induced by ATP and 5-HT supported by Na(V)1.6 channels. Of the various FHM3 mutations tested, the L263V missense mutation, with a longer activation state and lower activation voltage, resulted in the most pronounced spiking activity. In contrast, mutations that result in a loss of Na(V)1.1 function largely reduced firing of trigeminal nerve fibers. The combined activation of P2X3 and 5-HT3 receptors and branching of nerve fibers resulted in very prolonged and high-frequency spiking activity in the mutants compared to WT. We identified, in silico, key determinants of long-lasting nociceptive activity in FHM3-mutated A delta-fibers that naturally express P2X3 and 5-HT3 receptors and suggest mutant-specific correction options. Modeled trigeminal nerve firing was significantly higher for FHM3 mutations, compared to WT, suggesting that pronounced nociceptive signaling may contribute to migraine pain. Show less
Delayed graft function is the manifestation of ischemia reperfusion injury in the context of kidney transplantation. While hundreds of interventions successfully reduce ischemia reperfusion injury... Show moreDelayed graft function is the manifestation of ischemia reperfusion injury in the context of kidney transplantation. While hundreds of interventions successfully reduce ischemia reperfusion injury in experimental models, all clinical interventions have failed. This explorative clinical evaluation examined possible metabolic origins of clinical ischemia reperfusion injury combining data from 18 pre- and post-reperfusion tissue biopsies with 36 sequential arteriovenous blood samplings over the graft in three study groups. These groups included living and deceased donor grafts with and without delayed graft function. Group allocation was based on clinical outcome. Magic angle NMR was used for tissue analysis and mass spectrometry-based platforms were used for plasma analysis. All kidneys were functional at one-year. Integration of metabolomic data identified a discriminatory profile to recognize future delayed graft function. This profile was characterized by post-reperfusion ATP/GTP catabolism (significantly impaired phosphocreatine recovery and significant persistent (hypo)xanthine production) and significant ongoing tissue damage. Failing high-energy phosphate recovery occurred despite activated glycolysis, fatty-acid oxidation, glutaminolysis and autophagia, and related to a defect at the level of the oxoglutarate dehydrogenase complex in the Krebs cycle. Clinical delayed graft function due to ischemia reperfusion injury associated with a post-reperfusion metabolic collapse. Thus, efforts to quench delayed graft function due to ischemia reperfusion injury should focus on conserving metabolic competence, either by preserving the integrity of the Krebs cycle and/or by recruiting metabolic salvage pathways. Show less
Delayed graft function is the manifestation of ischemia reperfusion injury in the context of kidney transplantation. While hundreds of interventions successfully reduce ischemia reperfusion injury... Show moreDelayed graft function is the manifestation of ischemia reperfusion injury in the context of kidney transplantation. While hundreds of interventions successfully reduce ischemia reperfusion injury in experimental models, all clinical interventions have failed. This explorative clinical evaluation examined possible metabolic origins of clinical ischemia reperfusion injury combining data from 18 pre- and post-reperfusion tissue biopsies with 36 sequential arteriovenous blood samplings over the graft in three study groups. These groups included living and deceased donor grafts with and without delayed graft function. Group allocation was based on clinical outcome. Magic angle NMR was used for tissue analysis and mass spectrometry-based platforms were used for plasma analysis. All kidneys were functional at one-year. Integration of metabolomic data identified a discriminatory profile to recognize future delayed graft function. This profile was characterized by post-reperfusion ATP/GTP catabolism (significantly impaired phosphocreatine recovery and significant persistent (hypo)xanthine production) and significant ongoing tissue damage. Failing high-energy phosphate recovery occurred despite activated glycolysis, fatty-acid oxidation, glutaminolysis and autophagia, and related to a defect at the level of the oxoglutarate dehydrogenase complex in the Krebs cycle. Clinical delayed graft function due to ischemia reperfusion injury associated with a post-reperfusion metabolic collapse. Thus, efforts to quench delayed graft function due to ischemia reperfusion injury should focus on conserving metabolic competence, either by preserving the integrity of the Krebs cycle and/or by recruiting metabolic salvage pathways. Show less
Tubular ATP release is regulated by mechanosensation of fluid shear stress (FSS). Polycystin-1/polycystin-2 (PC1/PC2) functions as a mechanosensory complex in the kidney. Extracellular ATP is... Show moreTubular ATP release is regulated by mechanosensation of fluid shear stress (FSS). Polycystin-1/polycystin-2 (PC1/PC2) functions as a mechanosensory complex in the kidney. Extracellular ATP is implicated in polycystic kidney disease (PKD), where PC1/PC2 is dysfunctional. This study aims to provide new insights into the ATP signaling under physiological conditions and PKD. Microfluidics, pharmacologic inhibition, and loss-of-function approaches were combined to assess the ATP release in mouse distal convoluted tubule 15 (mDCT15) cells. Kidney-specific Pkd1 knockout mice (iKsp-Pkd1(-/-)) and zebrafish pkd2 morphants (pkd2-MO) were as models for PKD. FSS-exposed mDCT15 cells displayed increased ATP release. Pannexin-1 inhibition and knockout decreased FSS-modulated ATP release. In iKsp-Pkd1(-/-) mice, elevated renal pannexin-1 mRNA expression and urinary ATP were observed. In Pkd1(-/-) mDCT15 cells, elevated ATP release was observed upon the FSS mechanosensation. In these cells, increased pannexin-1 mRNA expression was observed. Importantly, pannexin-1 inhibition in pkd2-MO decreased the renal cyst growth. Our results demonstrate that pannexin-1 channels mediate ATP release into the tubular lumen due to pro-urinary flow. We present pannexin-1 as novel therapeutic target to prevent the renal cyst growth in PKD. Show less
Nowodworska, A.; Maagdenberg, A.M.J.M. van den; Nistri, A.; Fabbretti, E. 2017
