Introduction: Gases, such as nitric oxide (NO), carbon monoxide (CO), or hydrogen sulfide (H2S), termed gasotransmitters, play an increasingly important role in understanding of how electrical signaling of cells is modulated. H2S is well-known to act on various ion channels and receptors. In a previous study we reported that H2S increased calcium-activated potassium (BK) channel activity.Aims: The goal of the present study is to investigate the modulatory effect of BK channel phosphorylation on the action of H2S on the channel as well as to recalculate and determine the H2S concentrations in aqueous sodium hydrogen sulfide (NaHS) solutions.Methods: Single channel recordings of GH3, GH4, and GH4 STREX cells were used to analyze channel open probability, amplitude, and open dwell times. H2S was measured with an anion selective electrode.Results: The concentration of H2S produced from NaHS was recalculated taking pH, temperature salinity of the perfusate, and evaporation of H2S into account. The results indicate that from a concentration of 300 μM NaHS, only 11–13%, i.e., 34–41 μM is effective as H2S in solution. GH3, GH4, and GH4 STREX cells respond differently to phosphorylation. BK channel open probability (Po) of all cells lines used was increased by H2S in ATP-containing solutions. PKA prevented the action of H2S on channel Po in GH4 and GH4 STREX, but not in GH3 cells. H2S, high significantly increased Po of all PKG pretreated cells. In the presence of PKC, which lowers channel activity, H2S increased channel Po of GH4 and GH4 STREX, but not those of GH3 cells. H2S increased open dwell times of GH3 cells in the absence of ATP significantly. A significant increase of dwell times with H2S was also observed in the presence of okadaic acid.Conclusions: Our results suggest that phosphorylation by PKG primes the channels for H2S activation and indicate that channel phosphorylation plays an important role in the response to H2S.
Potassium channels are ubiquitous in cells and serve essential functions in physiology and pathophysiology. Potassium channel blockers have been shown to block tumour growth by arresting cells at the G 0 /G 1 checkpoint of the cell cycle. We investigated the effect of quinidine and caesium (Cs + ) on cell proliferation, LDH (lactate dehydrogenase) release, free internal calcium, membrane potential, polyamine concentration, ODC (ornithine decarboxylase) activity and polyamine uptake in C6 glioma cells. The EC 50 for reducing cell proliferation was 112 µM for quinidine, whereas Cs + was less effective with an EC 50 of 4.75 mM. KCl or sucrose did not affect proliferation. LDH release was augmented by quinidine. Quinidine caused a transient increase in free internal calcium but decreased calcium after a 48 h incubation period. Further 300 µM quinidine depolarized the cell membrane in a similar range as did 30 mM KCl. Quinidine decreased cellular putrescine beyond detection levels while spermidine and spermine remained unaffected. ODC activity was reduced. Addition of putrescine could not override the antiproliferative effect owing to a reduced activity of the polyamine transporter. Our study indicates that the antiproliferative effect of quinidine is not due to a simple membrane depolarization but is caused by a block of ODC activity.
Background: Padma Lax (PL) is a multi-component herbal laxative, derived from traditional Tibetan medicine. It has been used in the treatment of constipation dominant irritable bowel syndrome. Beyond its purgative and bowel-regulating properties we found it to exhibit antiproliferative properties. Materials and Methods: C6 tumor cells were incubated with either an ethanolic or aqueous extract of PL. Cell proliferation, cell cycle, percentage of apoptotic cells, caspase-3/-7 activity as well as mitochondrial membrane potential were determined. Results: Ethanolic extracts of PL inhibited cell proliferation in a dose- and time-dependent manner (half max concentration: 384.4 µg/ml after 48 h of incubation). Aqueous extracts were less effective. Ginger and elecampane were the active components of PL in respect to its antiproliferative action and were found to act synergistically. Supplementing the culture medium with polyamines could not override the cytostatic action of PL. Incubation of C6 cells with PL in the presence of catalase proved that the PL effect was specific and not due to oxidative stress. PL had no effects on the cell cycle at a low dose but arrested cells in G1 at high concentrations. Reduction of cell numbers was found to be due to apoptosis. The caspase- 3/-7 pathway was not involved in the PL-induced cell death. However, mitochondrial membrane potential was lost during the course of incubation with PL indicating a mitochondrial- but not caspase-mediated induction of apoptosis. Conclusion: PL exhibits antiproliferative properties which may be beneficial to prevent constipation-related cancer. This study may also contribute to a future development of a new herbal-based antiproliferative treatment.
Background: Lipedema, diagnosed most often in women, is a progressive disease characterized by the disproportionate and symmetrical distribution of adipose tissue, primarily in the extremities. Although numerous results from in vitro and in vivo studies have been published, many questions regarding the pathology and genetic background of lipedema remain unanswered. Methods: In this study, adipose tissue–derived stromal/stem cells were isolated from lipoaspirates derived from nonobese and obese donors with or without lipedema. Growth and morphology, metabolic activity, differentiation potential, and gene expression were evaluated using quantification of lipid accumulation, metabolic activity assay, live-cell imaging, reverse transcription polymerase chain reaction, quantitative polymerase chain reaction, and immunocytochemical staining. Results: The adipogenic potential of lipedema and nonlipedema adipose tissue–derived stromal/stem cells did not rise in parallel with the donors’ body mass index and did not differ significantly between groups. However, in vitro differentiated adipocytes from nonobese lipedema donors showed significant upregulation of adipogenic gene expression compared with nonobese controls. All other genes tested were expressed equally in lipedema and nonlipedema adipocytes. The adiponectin/leptin ratio was significantly reduced in adipocytes from obese lipedema donors compared with their nonobese lipedema counterparts. Increased stress fiber–integrated smooth muscle actin was visible in lipedema adipocytes compared with nonlipedema controls and appeared enhanced in adipocytes from obese lipedema donors. Conclusions: Not only lipedema per se but also body mass index of donors affect adipogenic gene expression substantially in vitro. The significantly reduced adiponectin/leptin ratio and the increased occurrence of myofibroblast-like cells in obese lipedema adipocyte cultures underscores the importance of attention to the co-occurrence of lipedema and obesity. These are important findings toward accurate diagnosis of lipedema. Clinical Relevance Statement: Our study highlights not only the difficulty in lipedema diagnostics but also the tremendous need for further studies on lipedema tissue. Although lipedema might seem to be an underestimated field in plastic and reconstructive surgery, the power it holds to provide better treatment to future patients can not be promoted enough.
1. Using immunological methods, we have identified parvalbumin-like material in the triclade flatworms, Polycelis nigra, Polycelis auriculata, Crenobia alpina, Dugesia tahitiensis, and Dugesia polychroa. 2. Western immunoblot analysis of these five species revealed heat stable parvalbumin-immunoreactive proteins between 32 kD and 44 kD. 3. Proteins at 19 and 32 kD revealed intense labeling with 45Ca2+. 4. Double immunodiffusion of planarian supernatants showed complete fusion of precipitates, indicating immunological relatedness of the parvalbumin-like material among the species investigated. 5. Immunocytochemical studies exhibit parvalbumin-immunoreactive material exclusively in neurons supporting the notion of an early evolutionary appearance of these proteins in the nervous system.
The reversibility of the effect of gossypol on testicular histology and fertility was studied in rats. Adult males of proven fertility were treated orally with gossypol acetic acid (15 mg/kg) for 9 or 16 weeks (groups 1 and 2, respectively). Another groups of animals (group 3) was given gossypol (15 mg/kg) for 16 weeks and killed 6 weeks after the end of treatment. Control animals (group 4) were given the vehicle only by oral intubation. In the mating studies, although only 33% of the animals in group 1 were infertile, 100% infertility was observed following 16 weeks of gossypol treatment (group 2). All animals in group 3 regained their fertility 6 weeks after cessation of drug treatment. Damage was observed to 15.7% of the seminiferous tubules after 9 weeks of drug treatment, and to 78% after 16 weeks of treatment. Extensive vacuolization, increased numbers of lipid droplets, degeneration of germ cells, loosening of the epithelium, and a significant decrease in the number of pachytene spermatocytes (stages VII-X) and spermatids (steps 7-10 at stages VII-X) were observed after gossypol treatment. There was a decrease in the diameter of only stage VIII seminiferous tubules after 9 weeks of treatment, whereas a reduction was observed in the tubules of all stages after 16 weeks of gossypol treatment. In the recovery phase, the diameter of seminiferous tubules was similar to that of controls, except for tubules at stage VIII. No change in the area of the lumen of the seminiferous tubules and lipid bodies was observed after 9 weeks of drug treatment, but a marked reduction in the area of the lumen (stages II-X) and an increase in lipid bodies (all stages) was observed after 16 weeks of gossypol treatment. Six weeks after cessation of treatment, the area of the lumen and the number of lipid bodies were comparable to values in controls. A reduction in the area of the epithelium was restricted to just a few stages (VIII-XIV) in treated animals at 9 weeks, whereas after 16 weeks the area of the epithelium was decreased in all tubules. In the recovery phase, except for tubules at stage VIII, the area of the seminiferous epithelium was comparable to that in controls.
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