Hydrocortisone inhibits antigen-induced rise in intracellular free calcium concentration and abolishes leukotriene C4 production in leukemic basophils

Antigenic stimulation of rat basophilic leukemia cells (RBL-3H3) elevates intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and induces production of leukotriene C₄ (LTC₄). This model was used to examine the role of Ca²⁺ in LTC₄ formation, and inhibition by hydrocortisone (HC). HC, at a physiological concentration (2×10⁻⁷M), selectively prevented the stimulatory effect of the antigen on LTC₄ production whereas the response to calcium inophore (A23187) remained unimpaired. The inhibition by HC was time-dependent: half maximal response was reached at 2 hour and maximal response at 3 hours. Addition of arachidonic acid (3 μg/ml) did not overcome the inhibitory action of HC. An elevated [Ca²⁺]_i is known to be essential for the activation ob both 5-lipoxygenase and phospholipase A₂. The stimulatory effect of the antigen on LTC₄ production was abolished when the cells were incubated in Ca²⁺-deficient medium. Likewise, calcium ionophore stimulation shows dependence on extracellular Ca²⁺. Half maximal stimulation by the antigen and calcium ionophore was observed at external Ca²⁺ concentration of 150 μM and 40 μM respectively. Treatment with HC largely prevented the antigen-induced rise in [Ca²⁺]_i, measured by Quin 2. In addition, HC reduced by 70% the accumulation of ⁴⁵Ca²⁺ induced by the antigen. Collectively, these results demonstrate for the first time that HC reduces antigen-induced elevation of [Ca²⁺]_i, and this may be associated with the inhibitory action of HC on LTC₄ formation. This property could be partly responsible for the antiallergic and antiinflammatory activities of HC.     

A novel action of glucocorticosteroid in inhibition of leukotriene C4 production by rat basophilic leukemia cells: suppression of the elevation of cytosolic free Ca2+ induced by antigen

Rat basophilic leukemia (RBL-2H3) cells serve as a model to examine the role of elevated internal Ca2+ concentration ([Ca2+]i), following antigen (DNP10BSA)-induced stimulation of leukotriene C4 (LTC4) formation. A novel action of hydrocortisone (HC), to reduce increased [Ca2+]i and consequently inhibit LTC4 formation is assessed. Half-maximal time for elevation of [Ca2+]i induced by antigen was less than 1 min, and maximal elevation of [Ca2+]i (3-fold increase) was reached within 2-3 min. This high [Ca2+]i level waned gradually by 27% during 20 min of incubation. For induction of LTC4 formation, however, there was a refractory period of about 2 min, and half-maximal elevation was at 11 min. Following pretreatment with HC, the antigen-stimulated increase in [Ca2+]i was stunted by 41% at 2-3 min and by 73% at 20 min. LTC4 formation was almost abolished. There was a lag period of at least 2 h to observe any inhibition in both parameters, and the maximal inhibition was about 4 h. Cycloheximide, and receptor antagonist to glucocorticosteroid (RU486) completely prevented the inhibitory effects of HC on elevated [Ca2+]i and LTC4 formation. Estradiol and aldosterone (each at 2.10(-6) M) were virtually inactive, while another glucocorticosteroid, dexamethasone (2.10(-7) M) markedly suppressed antigen induction in both parameters. It is proposed that the inhibitory effect of HC on the formation of LTC4 could be attributed mainly to its ability to reduce elevated [Ca2+]i.     

Refractoriness of ovarian adenylate cyclase to continued hormonal stimulation.

Culture of preovulatory rat follicles with luteinizing hormone, follicle-stimulating hormone or prostaglandin E2 for 24 h reduced the subsequent response of adenylate cyclase to the homologous by 80, 50 and 90%, respectively; yet follicles refractory to luteinizing hormone fully responded to follicle-stimulating hormone responded to luteinizing hormone and prostaglandin E2, and those refractory to prostaglandin E2 could be stimulated by either gonadotropin. Desensitization of the adenylate cyclase system by luteinizing hormone was achieved by hormone concentrations of 0.8--2.0 mug/ml in the medium; a lower dose of luteinizing hormone (0.4 mug/ml), though effective in stimulating adenylate cyclase, did not induce refractoriness. Prostaglandin E2 caused partial refractoriness at dose levels of 0.1--0.25 mug/ml; higher dose levels were more effective. These findings suggest that continued exposure to the preovulatory follicle to elevated levels of hormones may cause perturbations in either the interaction between the hormone and its specific receptor or in a subsequent step essential for activation of adenylate cyclase.     

Mechanism of steroid action in inflammation: Inhibition of prostaglandin synthesis and release

Acute arthritis was induced by injection of cell-free extract of group A Streptococci into the knee joints of mature male rats. Slices of control and inflamed synovia were incubated for 30 to 240 minutes and the rate of prostaglandin E (PGE) released into the medium was measured by radioimmunoassay. PGE release from inflamed synovia was 5–8 fold higher than that in normal tissue. Incubation of inflamed synovia with corticosterone acetate, dexamethasone or prednisone (100 μg/ml) for one or four hours reduced PGE release by 33% and 55% respectively. Lower concentrations of corticosterone (10 – 30 μg/ml) were ineffective. Aldosterone and progesterone (100 μg/ml) had no effect on PGE release throughout the incubation period. Chloroquine (10 μg/ml) inhibited PGE release from inflamed synovia by 50%. Indomethacin (1 μg/ml) abolished PGE release by 90%. Corticosterone, dexamethasone and prednisone reduced PGE content of inflamed synovia by approximately 45% during a 4-h incubation period. Aldosterone and progesterone were ineffective, while indomethacin reduced PGE content by 70%. The suppressive action of corticosterone on PGE release was prevented by addition to the medium of arachidonic acid (2 μg/ml). By contrast, the inhibitory action of indomethacin was not affected by provision of exogenous substrate. We suggest that glucocorticosteroids reduce PGE release by limiting the availability of the substrate for prostaglandin biosynthesis, and this may well explain some of their anti-inflammatory properties.     

Cdk1 is essential for mammalian cyclosome/APC regulation

The cyclosome/APC (anaphase-promoting complex), the major component of cell-cycle-specific ubiquitin-mediated proteolysis of mitotic cyclins and of other cell cycle proteins, is essential for sister chromatid separation and for exit from mitosis. Cyclosome activity and substrate specificity are modulated by phosphorylation and by transient interactions with Fizzy/cdc20 (Fzy) and Fizzy-related/Hct1/Cdh1 (Fzr). This regulation has been studied so far in Drosophila embryos, in yeast, and in cell-free extracts in vitro. Studying cyclosome regulation in mammalian cells in vivo we found that both Fzr overexpression and Cdk1 inhibition can override the prometaphase checkpoint. We further show that Fzr activation of the cyclosome is negatively regulated by Cdk1. Finally, we show that the mammalian cdc14 phosphatase, like its budding yeast homologue, plays a role in cyclosome pathway regulation. These results suggest that Cdk1 is essential for coupling various activities of the cyclosome and in particular for preventing Fzr from short-circuiting the spindle pole checkpoint. Cdk1-cyclin B is thus an inhibitor, activator, and substrate of the cyclosome.     

Identification of Elements That Dictate the Specificity of Mitochondrial Hsp60 for Its Co-Chaperonin

Type I chaperonins (cpn60/Hsp60) are essential proteins that mediate the folding of proteins in bacteria, chloroplast and mitochondria. Despite the high sequence homology among chaperonins, the mitochondrial chaperonin system has developed unique properties that distinguish it from the widely-studied bacterial system (GroEL and GroES). The most relevant difference to this study is that mitochondrial chaperonins are able to refold denatured proteins only with the assistance of the mitochondrial co-chaperonin. This is in contrast to the bacterial chaperonin, which is able to function with the help of co-chaperonin from any source. The goal of our work was to determine structural elements that govern the specificity between chaperonin and co-chaperonin pairs using mitochondrial Hsp60 as model system. We used a mutagenesis approach to obtain human mitochondrial Hsp60 mutants that are able to function with the bacterial co-chaperonin, GroES. We isolated two mutants, a single mutant (E321K) and a double mutant (R264K/E358K) that, together with GroES, were able to rescue an E. coli strain, in which the endogenous chaperonin system was silenced. Although the mutations are located in the apical domain of the chaperonin, where the interaction with co-chaperonin takes place, none of the residues are located in positions that are directly responsible for co-chaperonin binding. Moreover, while both mutants were able to function with GroES, they showed distinct functional and structural properties. Our results indicate that the phenotype of the E321K mutant is caused mainly by a profound increase in the binding affinity to all co-chaperonins, while the phenotype of R264K/E358K is caused by a slight increase in affinity toward co-chaperonins that is accompanied by an alteration in the allosteric signal transmitted upon nucleotide binding. The latter changes lead to a great increase in affinity for GroES, with only a minor increase in affinity toward the mammalian mitochondrial co-chaperonin.     

Sequential Extraction as Novel Approach to Compare 12 Medicinal Plants From Kenya Regarding Their Potential to Release Chromium,Manganese, Copper,and Zinc

Studies have shown the participation of minerals in mechanisms involved in the pathogenesis of insulin resistance. Zinc, in particular, seems to play an important role in the secretion and action of this hormone. Therefore, the aim of this review is to understand the role of zinc in increasing insulin sensitivity. We conducted a search of articles published in the PubMed and ScienceDirect database selected from March 2016 to February 2018, using the keywords “zinc,” “insulin,” “insulin resistance,” “insulin sensitivity,” and “supplementation.” Following the eligibility criteria were selected 53 articles. The scientific evidences presented in this review show the importance of zinc and their carrier proteins in the synthesis and secretion of insulin, as well as in the signaling pathway of action of this hormone. Zinc deficiency is associated with glucose intolerance and insulin resistance; however, the effectiveness of the intervention with the zinc supplementation is still inconclusive.