Nitric oxide, endothelium-derived hyperpolarizing factor, and smooth muscle-dependent mechanisms contribute to magnesium-dependent vascular relaxation in mouse arteries.

AIM: Magnesium (Mg2+ ) is a vasorelaxant. The underlying physiological mechanisms driving this vasorelaxation remain unclear. Studies were designed to test the hypothesis that multiple signaling pathways including nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) in endothelial cells as well as Ca2+ antagonization and TRPM7 channels in vascular smooth muscle cells mediate Mg2+ -dependent vessel relaxation. METHODS: To uncover these mechanisms, force development was measured ex vivo in aorta rings from mice using isometric wire myography. Concentration responses to Mg2+ were studied in intact and endothelium-denuded aortas. Key findings were confirmed in second-order mesenteric resistance arteries perfused ex vivo using pressure myography. Effects of Mg2+ on NO formation were measured in Chinese Hamster Ovary (CHO) cells, isolated mesenteric vessels, and mouse urine. RESULTS: Mg2+ caused a significant concentration-dependent relaxation of aorta rings. This relaxation was attenuated significantly in endothelium-denuded aortas. The endothelium-dependent portion was inhibited by NO and cGMP blockade but not by cyclooxygenase inhibition. Mg2+ stimulated local NO formation in CHO cells and isolated mesenteric vessels without changing urinary NOx levels. High extracellular Mg2+ augmented acetylcholine-induced relaxation. SKCa and IKCa channel blockers apamin and TRAM34 inhibited Mg2+ -dependent relaxation. The endothelium-independent relaxation in aorta rings was inhibited by high extracellular Ca2+ . Combined blockade of NO, SKCa , and IKCa channels significantly reduced Mg2+ -dependent dilatation in mesenteric resistance vessels. CONCLUSIONS: In mouse conductance and resistance arteries Mg2+ -induced relaxation is contributed by endothelial NO formation, EDHF pathways, antagonism of Ca2+ in smooth muscle cells, and additional unidentified mechanisms.

Dynamics of Podospora anserina Genome Evolution in a Long-Term Experiment.

The Podospora anserina long-term evolution experiment (PaLTEE) is the only running filamentous fungus study, which is still going on. The aim of our work is to trace the evolutionary dynamics of the accumulation of mutations in the genomes of eight haploid populations of P. anserina. The results of the genome-wide analysis of all of the lineages, performed 8 years after the start of the PaLTEE, are presented. Data analysis detected 312 single nucleotide polymorphisms (SNPs) and 39 short insertion-deletion mutations (indels) in total. There was a clear trend towards a linear increase in the number of SNPs depending on the experiment duration. Among 312 SNPs, 153 were fixed in the coding regions of P. anserina genome. Relatively few synonymous mutations were found, exactly 38; 42 were classified as nonsense mutations; 72 were assigned to missense mutations. In addition, 21 out of 39 indels identified were also localized in coding regions. Here, we also report the detection of parallel evolution at the paralog level in the P. anserina model system. Parallelism in evolution at the level of protein functions also occurs. The latter is especially true for various transcription factors, which may indicate selection leading to optimization of the wide range of cellular processes under experimental conditions.

[Effects derived from occupational exposure to hazardous substances in pregnant working women: systematic review]. / Efectos derivados de la exposición laboral en las mujeres trabajadoras embarazadas expuestas a sustancias peligrosas: revisión sistemática.

OBJECTIVE: To review the effects derived from occupational exposure on pregnant working women exposed to hazardous substances. METHODS: Critical analysis of the works retrieved by systematic review in MEDLINE (PubMed), EMBASE, Cochrane Library, Scopus, Web of Science, LILACS and MEDES, up to January 2021. The Search Equation was developed by means of the descriptors «Pregnancy¼, «Hazardous Substances¼ and «Occupational Exposure¼, as well as using related Entry Terms and the filters «Humans¼ and «Adult: 19+ years¼. The quality of the articles was evaluated via the STROBE questionnaire and the level of evidence and the grade of recommendation via the SIGN criteria. RESULTS: Out of the 420 references retrieved (366 digitally and 54 manually) and after applying the inclusion and exclusion criteria, 35 articles were selected. The STROBE evaluation obtained a median of 15.32, and the SIGN criteria, a 2+ level of evidence and a C grade of recommendation. Article obsolescence was high (Burton-Kebler half-life: 22.0; Price index: 0.00%). In 25 out of 35 articles, the exposure is to organic solvents. In 22 articles, congenital anomalies were notified. CONCLUSIONS: The reviewed articles exhibited high obsolescence and a degree of evidence and recommendation that did not allow to fully ensure the validity and reliability of the observations made. The results manifested the association between the exposure to dangerous substances and the occurrence of different damages regarding the health of a pregnant worker and her offspring. The most common exposure was to organic solvents and the most observed effect was congenital alterations.

Objetivo: Revisar los efectos derivados de la exposición laboral en las mujeres trabajadoras embarazadas expuestas a sustancias peligrosas. Métodos: Análisis crítico de los trabajos recuperados mediante revisión sistemática en MEDLINE (PubMed), EMBASE, Cochrane Library, Scopus, Web of Science, LILACS y MEDES, hasta enero 2021. La ecuación de búsqueda se formuló́ mediante los descriptores «Pregnancy¼, «Hazardous Substances¼ y «Occupational Exposure¼, utilizando también los Entry Terms relacionados y los filtros: «Humans¼ y «Adult: 19+ years¼. La calidad de los artículos se evaluó mediante el cuestionario STROBE y el nivel de evidencia y grado de recomendación mediante los criterios SIGN. Resultados: De las 420 referencias recuperadas (366 de forma digital y 54 manual), aplicando los criterios de inclusión y exclusión, se seleccionaron 35 artículos. La evaluación mediante STROBE dio mediana de 15,32 y según los criterios SIGN se obtuvo grado de evidencia 2+ y recomendación C. La obsolescencia fue elevada (semiperiodo de Burton-Kebler: 22,00; índice de Price: 0,00%). En 25 de los 35 estudios revisados la exposición fue a disolventes orgánicos. En 22 artículos se notificó alteraciones congénitas. Conclusiones: Los trabajos revisados presentaron un alto índice de obsolescencia y un grado de evidencia y recomendación que no permitió asegurar por completo la validez y fiabilidad de las observaciones realizadas. Los resultados mostraron la asociación entre la exposición a sustancias peligrosas con la aparición de diferentes daños para la salud de la trabajadora embarazada y su descendencia. La mayor exposición fue a los disolventes orgánicos y el efecto más observado las alteraciones congénitas.

Efectos derivados de la exposición laboral en las mujeres trabajadoras embarazadas expuestas a sustancias peligrosas: revisión sistemática / Effects derived from occupational exposure to hazardous substances in pregnant working women: systematic review

Objetivo: Revisar y analizar los efectos derivados de la exposición laboral en las mujeres trabajadoras embarazadas expuestas a sustancias peligrosas. Métodos: Análisis crítico de los trabajos recuperados mediante revisión sistemática en MEDLINE (PubMed), EMBASE, Cochrane Library, Scopus, Web of Science, LILACS y MEDES, hasta enero 2021. La ecuación de búsqueda se formuló́ mediante los descriptores «Pregnancy», «Hazardous Substances» y «Occupational Exposure», utilizando también los Entry Terms relacionados y los filtros: «Humans» y «Adult: 19+ years». La calidad de los artículos se evaluó mediante el cuestionario STROBE y el nivel de evidencia y grado de recomendación mediante los criterios SIGN. Resultados: De las 420 referencias recuperadas (366 de forma digital y 54 manual), tras aplicar los criterios de inclusión y exclusión, se seleccionaron 35 artículos. La evaluación mediante STROBE dio una mediana de 15,32 y según los criterios SIGN se obtuvo un grado de evidencia 2+ y recomendación C. La obsolescencia de las publicaciones fue elevada (semiperiodo de Burton-Kebler: 22,00; índice de Price: 0%). En 25 de los 35 estudios revisados la exposición fue a los disolventes orgánicos. En 22 artículos se notificó la aparición de alteraciones congénitas. Conclusiones: Los trabajos revisados presentaron un alto índice de obsolescencia y un grado de evidencia y recomendación que no permitió asegurar por completo la validez y fiabilidad de las observaciones realizadas. Los resultados mostraron la asociación entre la exposición a sustancias peligrosas con la aparición de diferentes daños para la salud de la trabajadora embarazada y su descendencia. La mayor exposición fue a los disolventes orgánicos y el efecto más observado las alteraciones congénitas (AU)

Objective: To systematically review and analyze the health effects derived from occupational exposure to hazardous substances in pregnant working women. Methods: Critical analysis of studies retrieved by systematic review of MEDLINE (PubMed), EMBASE, Cochrane Library, Scopus, Web of Science, LILACS and MEDES, through January 2021. The search strategy was developed by means of the descriptors «pregnancy», «hazardous substances» and «occupational exposure», as well as by using related entry terms and the filters «humans» and «adult: 19+ years». Study quality was assessed using the STROBE questionnaire, and the level of evidence and grade of recommendation via the SIGN criteria. Results: Out of 420 references identified (366 digitally and 54 manually) and after applying the inclusion and exclusion criteria, 35 articles were selected. The STROBE evaluation yielded a median score of 15.32, and the SIGN criteria a 2+ level of evidence and a C grade of recommendation. Article obsolescence was high (Burton-Kebler half-life: 22.0; Price index: 0%). In 25 out of 35 studies, the exposure evaluated was to organic solvents. In 22 articles, congenital abnormalities were identified as a health effect. Conclusions: This systematic review revealed a high level of obsolescence and a degree of evidence and recommendation that limit the validity and reliability of the observations. Results indicated an association between exposure to hazardous substances and the occurrence of different adverse health effects in pregnant workers and their offspring. The most common exposure was to organic solvents and the most common observed effect were congenital abnormalities (AU)

Nitrogen-obtaining and -conserving strategies in xylotrophic basidiomycetes.

Nitrogen in sufficient quantities is strictly necessary for all living organisms. In this study, the ability of some xylotrophic basidiomycetes to grow extremely long on a solid growth medium full of carbon nutrition but lacking a nitrogen source in its composition was discovered. The nitrogen oligotrophy of wood-decaying fungi is associated with their adaptation to live in a wood substrate, which is also deficient in nitrogen content. This nitrogen-depleted cultural growth is called "pseudo-foraging" and can be used as a simplified model of wood-decaying growth. Four main nitrogen-obtaining and -conserving strategies (nitrogen concentration, using alternative sources of nitrogen, economy of growth, and nutritional recycling), which are attributed to wood-colonizing xylotrophs in the literature, were revised studying the pseudo-foraging model. Based on the results, some aspects of the behavior of xylotrophs deep in undecomposed wood were predicted. For example, one of the results is that for pseudo-foraging xylotrophs, the main way to obtain nitrogen is its concentration in their mycelium from the nutrient medium in which nitrogen is contained in the impurities of the components of the medium. The result suggests that in bulk solid wood, the nitrogen concentration strategy also dominates the strategy of using diazotrophic and other alternative nitrogen. In addition, three individual unprecedented mechanisms, which supposedly help the xylotrophic fungi to colonize wood in nature (generation of fine mycelium, macrovesicular endocytosis, formation and conversion of super-elongated mitochondria), were investigated in the laboratory.

Genetics of Adaptation of the Ascomycetous Fungus Podospora anserina to Submerged Cultivation.

Podospora anserina is a model ascomycetous fungus which shows pronounced phenotypic senescence when grown on solid medium but possesses unlimited lifespan under submerged cultivation. In order to study the genetic aspects of adaptation of P. anserina to submerged cultivation, we initiated a long-term evolution experiment. In the course of the first 4 years of the experiment, 125 single-nucleotide substitutions and 23 short indels were fixed in eight independently evolving populations. Six proteins that affect fungal growth and development evolved in more than one population; in particular, in the G-protein alpha subunit FadA, new alleles fixed in seven out of eight experimental populations, and these fixations affected just four amino acid sites, which is an unprecedented level of parallelism in experimental evolution. Parallel evolution at the level of genes and pathways, an excess of nonsense and missense substitutions, and an elevated conservation of proteins and their sites where the changes occurred suggest that many of the observed fixations were adaptive and driven by positive selection.

Prostaglandin induced changes in the tone of porcine retinal arterioles in vitro involve other factors than calcium activity in perivascular cells.

The cellular basis for the regulation of retinal blood flow is unknown, but recently a new type of perivascular cell (PVC) with pericyte characteristics was identified in the retinal arterial vascular wall located immediately external to the vascular smooth muscle cells. A possible involvement of this cell type in the regulation of retinal vascular tone might be elucidated by studying differences in the response after the addition of compounds stimulating respectively relaxation and contraction. The effects of PGE2 and PGF2α on vascular tone and calcium activity in PVCs in porcine retinal arterioles were studied in a confocal myograph after the addition of the ryanodine receptor blocker ryanodine, the L-type Ca(2+) channel blocker nifedipine, the non-specific cation channel blocker LOE908, the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) blocker CPA, and the inositol triphosphate receptor (IP3R) and transient receptor potential (TRP) ion channel blocker 2-APB. The Ca(2+) channel blockers nifedipine and LOE908 induced significant relaxation of retinal arterioles. After the addition of both PGE2 and PGF2α calcium activity in the PVCs was significantly reduced by both the SERCA inhibitor CPA and the IP3R antagonist 2-APB, but the changes in calcium activity were unrelated to the changes in tone induced by PGE2 and PGF2α. Changes in the tone of porcine retinal arterioles in vitro induced by PGE2 and PGF2α involve other factors than calcium activity in the perivascular cells.

Association between endothelial dysfunction and depression-like symptoms in chronic mild stress model of depression.

OBJECTIVE: Cardiovascular diseases have high comorbidity with major depression. Endothelial dysfunction may explain the adverse cardiovascular outcome in depression; therefore, we analyzed it in vitro. In the chronic mild stress model, some rats develop depression-like symptoms (including "anhedonia"), whereas others are stress resilient. METHODS: After 8 weeks of chronic mild stress, anhedonic rats reduced their sucrose intake by 55% (7%), whereas resilient rats did not. Acetylcholine-induced endothelium-dependent relaxation of norepinephrine-preconstricted mesenteric arteries was analyzed in nonstressed, anhedonic, and resilient rat groups. RESULTS: Small resistance arteries from anhedonic rats were less sensitive to acetylcholine than those of the nonstressed and resilient groups (p = .029). Pathways of endothelium-dependent relaxation were altered in arteries from anhedonic rats. Nitric oxide (NO)-dependent relaxation and endothelial NO synthase expression were increased in arteries from anhedonic rats (0.235 [0.039] arbitrary units and 155.7% [8.15%]) compared with the nonstressed (0.135 [0.012] arbitrary units and 100.0% [8.08%]) and resilient (0.152 [0.018] arbitrary units and 108.1% [11.65%]) groups (p < .001 and p = .002, respectively). Inhibition of cyclooxygenase (COX) activity revealed increased COX-2-dependent relaxation in the anhedonic group. In contrast, endothelial NO synthase- and COX-independent relaxation to acetylcholine (endothelium-dependent hyperpolarization-like response) was reduced in anhedonic rats (p < .001). This was associated with decreased transcription of intermediate-conductance Ca-activated K channels. CONCLUSIONS: Our findings demonstrate that depression-like symptoms are associated with reduced endothelium-dependent relaxation due to suppressed endothelium-dependent hyperpolarization-like relaxation despite up-regulation of the NO and COX-2-dependent pathways in rat mesenteric arteries. These changes could affect peripheral resistance and organ perfusion in major depression.

Downregulation of L-type Ca2+ channel in rat mesenteric arteries leads to loss of smooth muscle contractile phenotype and inward hypertrophic remodeling.

L-type Ca(2+) channels (LTCCs) are important for vascular smooth muscle cell (VSMC) contraction, as well as VSMC differentiation, as indicated by loss of LTCCs during VSMC dedifferentiation. However, it is not clear whether loss of LTCCs is a primary event underlying phenotypic modulation or whether loss of LTCCs has significance for vascular structure. We used small interference RNA (siRNA) transfection in vivo to investigate the role of LTCCs in VSMC phenotypic expression and structure of rat mesenteric arteries. siRNA reduced LTCC mRNA and protein expression in rat mesenteric arteries 3 days after siRNA transfection to 12.7 ± 0.7% and 47.3 ± 13%, respectively: this was associated with an increased resting intracellular Ca(2+) concentration ([Ca(2+)]i). Despite the high [Ca(2+)]i, the contractility was reduced (tension development to norepinephrine was 3.5 ± 0.2 N/m and 0.8 ± 0.2 N/m for sham-transfected and downregulated arteries respectively; P < 0.05). Expression of contractile phenotype marker genes was reduced in arteries downregulated for LTCCs. Phenotypic changes were associated with a 45% increase in number of VSMCs and a consequent increase of media thickness and media area. Ten days after siRNA transfection arterial structure was again normalized. The contractile responses of LTCC-siRNA transfected arteries were elevated in comparison with matched controls 10 days after transfection. The study provides strong evidence for causal relationships between LTCC expression and VSMC contractile phenotype, as well as novel data addressing the complex relationship between VSMC contractility, phenotype, and vascular structure. These findings are relevant for understanding diseases, associated with phenotype changes of VSMC and vascular remodeling, such as atherosclerosis and hypertension.

Vascular smooth muscle cell phenotype is defined by Ca2+-dependent transcription factors.

Ca(2+) is an important second messenger in vascular smooth muscle cells (VSMCs). Therefore, VSMCs exercise tight control of the intracellular Ca(2+) concentration ([Ca(2+)]i) by expressing a wide repertoire of Ca(2+) channels and transporters. The presence of several pathways for Ca(2+) influx and efflux provides many possibilities for controlling [Ca(2+)]i in a spatial and temporal manner. Intracellular Ca(2+) has a dual role in VSMCs; first, it is necessary for VSMC contraction; and, second, it can activate multiple transcription factors. These factors are cAMP response element-binding protein, nuclear factor of activated T lymphocytes, and serum response factor. Furthermore, it was recently reported that the C-terminus of voltage-dependent L-type Ca(2+) calcium channels can regulate transcription in VSMCs. Transcription regulation in VSMCs modulates the expression patterns of genes, including genes coding for contractile and cytoskeleton proteins, and those promoting proliferation and cell growth. Depending on their gene expression, VSMCs can exist in different functional states or phenotypes. The majority of healthy VSMCs show a contractile phenotype, characterized by high contractile ability and a low proliferative rate. However, VSMCs can undergo phenotypic modulation with different physiological and pathological stimuli, whereby they start to proliferate, migrate, and synthesize excessive extracellular matrix. These events are associated with injury repair and angiogenesis, but also with the development of cardiovascular pathologies, such as atherosclerosis and hypertension. This review discusses the currently known Ca(2+)-dependent transcription factors in VSMCs, their regulation by Ca(2+) signalling, and their role in the VSMC phenotype.

K(Ca)3.1 channel downregulation and impaired endothelium-derived hyperpolarization-type relaxation in pulmonary arteries from chronically hypoxic rats.

Calcium-activated potassium channels of small (K(Ca)2, SK) and intermediate (K(Ca)3.1, IK) conductance are involved in endothelium-dependent relaxation of pulmonary arteries. We hypothesized that the function and expression of K(Ca)2 and K(Ca)3.1 increase as a compensatory mechanism to counteract hypoxia-induced pulmonary hypertension in rats. For functional studies, pulmonary arteries were mounted in microvascular myographs for isometric tension recordings. The K(Ca) channel expression was evaluated by immunoblotting and quantitative PCR. Although ACh induced similar relaxations, the ACh-induced relaxations were abolished by the combined inhibition of nitric oxide synthase (by L-nitro-arginine, L-NOARG), cyclo-oxygenase (by indomethacin) and soluble guanylate cyclase (by ODQ) in pulmonary arteries from hypoxic rats, whereas 20 ± 6% (n = 8) maximal relaxation in response to ACh persisted in arteries from normoxic rats. Inhibiting Na(+),K(+)-ATPase with ouabain or blocking K(Ca)2 and K(Ca)3.1 channels reduced the persisting ACh-induced relaxation. In the presence of L-NOARG and indomethacin, a novel K(Ca)2 and K(Ca)3.1 channel activator, NS4591, induced concentration- and endothelium-dependent relaxations, which were markedly reduced in arteries from chronically hypoxic rats compared with arteries from normoxic rats. The mRNA levels of K(Ca)2.3 and K(Ca)3.1 were unaltered, whereas K(Ca)2.3 protein expression was upregulated and K(Ca)3.1 protein expression downregulated in pulmonary arteries from rats exposed to hypoxia. In conclusion, endothelium-dependent relaxation was conserved in pulmonary arteries from chronically hypoxic rats, while endothelium-derived hyperpolarization (EDH)-type relaxation was impaired in chronically hypoxic pulmonary small arteries despite upregulation of K(Ca)2.3 channels. Since impaired EDH-type relaxation was accompanied by K(Ca)3.1 channel protein downregulation, these findings suggest that K(Ca)3.1 channels are important for the maintenance of EDH-type relaxation.

Intracellular Ca²âº signalling and phenotype of vascular smooth muscle cells.

Vascular smooth muscle cells (VSMCs) express considerable plasticity in their phenotype and even can change their phenotype in vivo depending on the functional demand. In addition to contractile phenotype, VSMCs can be proliferative, migrating and/or synthetic. Importantly, contractile and non-contractile phenotypes differ significantly in their intracellular Ca²âº signalling, which is a consequence of difference in expression of Ca²âº transport proteins. Contractile VSMCs express Ca²âº transporters, including voltage-gated L-type Ca²âº channels and SERCA2a pump, which maintain low resting cytosolic Ca²âº and allow dynamic changes of Ca²âº in the spatial and temporal domain, while non-contractile VSMCs have significantly reduced voltage dependence of Ca²âº entry. These changes associated with phenotypic switch are consequences of changes in gene expression programmes, where the expression of phenotype-specific proteins and other proteins is suppressed. Importantly, Ca²âº -sensitive transcription factors, including serum response factor, cAMP response element-binding protein and nuclear factor of activated T lymphocytes, which are important for this phenotype switch, can be activated by different types of Ca²âº signalling. Thus, different Ca²âº transport proteins not only control averaged intracellular Ca²âº but also through their differences in the character of the Ca²âº signal modulate the activity of transcription factors and thus initiate phenotype switch. The essential stimuli for phenotype switch are unknown, but intracellular Ca²âº is an important second messenger in the cell transcription programme. This article reviews the relationship between intracellular Ca²âº signalling and VSMC phenotype.