Subunit Gene Expression Research Articles

Neuroadaptations in D2‐like autoreceptor function following AMPH self‐administration

Exposure to amphetamine (AMPH) self‐administration (0.187 mg/kg/inj, 5 days) results in neuroadaptations in the mesolimbic dopamine system. Dopamine D2‐like autoreceptors are critical modulators of dopaminergic signaling but an understudied topic in drug addiction research. We report a marked subsensitivity of D2 autoreceptors following chronic AMPH. After escalation of AMPH self‐administration in rats, we found that the ability of D2 autoreceptors to inhibit dopamine release was reduced in AMPH rats (IC50 = 200 nM) compared to controls (30 nM), as assessed by voltammetry in the nucleus accumbens core. The reduced function was not due to reduced expression, because AMPH increased D2 short form (presynaptic D2 form) mRNA (46%). To determine the mechanism of D2 subsensitivity, we examined gene expression of the inhibitory subunit of G protein (Gαo) and regulator of G protein signaling 4 (RGS4). Gαo couples to the D2 autoreceptor for signaling, and RGS4 negatively regulates Gαo‐mediated D2 receptor function. We found decreased Gαo mRNA (23%) and increased RGS4 mRNA (27%) in the ventral tegmental area, which likely contribute to the decreased function of D2 autoreceptors following repeated AMPH administration. It is possible that changes in Gαo and RGS4 signaling may be involved in early stages of addiction, increasing dopamine release and contributing to compulsive AMPH‐taking behavior.

N‐acetylcysteine administration modulates NADPH oxidase in skeletal muscle of rats with heart failure

This study aimed to evaluate the influence of antioxidant N‐acetylcysteine on NADPH oxidase (NOX) activity and gene expression in soleus muscle of rats with myocardial infarctioninduced heart failure.MethodsMyocardial infarction (MI) was induced by left coronary occlusion. Four months later, rats were assigned to three groups: Sham (n=8), MI‐C (MI without treatment, n=8), and MI‐NAC (MI treated with N‐acetylcysteine, 120 mg/kg/day, n=8). Rats with small MI were excluded. Six months after surgery, NOX activity was assessed by HPLC analysis of dihydroethidium oxidation fluorescent products in soleus muscle. Gene expression of NOX complex subunits was evaluated by RT‐PCR.ResultsMI size did not differ between MI‐C and MI‐NAC groups (p>;0.05, Student's t test). NADPH oxidase activity was not different between MI‐C and Sham, but it was reduced in MI‐NAC compared to MI‐C group. Table shows the RT‐PCR results (ANOVA and Bonferroni; *p<0.05 vs. Sham, #p<0.05 vs. MI‐C):ConclusionN‐acetylcysteine administration reduces NADPH oxidase activity and gene expression in soleus muscle during heart failure. Support: FAPESP and CNPq.

ASSA13-03-37 Adriamycin Induced Cardiac Dysfunction is Attenuated by Selenium Via Restoring Oxidative Stress Mediated Expression of ATP Sensitive Potassium Channels in Rats

<h3>Objective</h3> Cardiac toxicity is an important complication of chemotherapy. Selenium (Se) acts as antioxidant agent to protect myocytes from oxidative damage. Lack of Se would lead to enhancement of oxidative stress. It has been mentioned that impairment of ATP sensitive potassium channels (K_ATP) which are expressed in myocytes are correlated with oxidative mediated cardiac dysfunction. The possible mechanism of adriamycin (ADR) and/or Se deficiency induced cardiac dysfunction, and cardioprotective effects of Se against ADR induced cardiac toxicity were investigated in this study. <h3>Methods</h3> Animal model of cardiac dysfunction was induced by adriamycin inraperitoneal injection or/and Se deficit diet. Then by intraperitoneal injections, Se supplementation was applied to rats. We assessed cardiac function by serum brain natriuretic peptide (BNP) level, echocardiographic and hemodynamic parameters. Cardiac glutathione peroxidase (GPx) activity which mirrors oxidative stress indirectly was measured spectrophotometrically. Expression levels of K_ATP channels were evaluated by real-time polymerase chain reaction (PCR) and western blotting. <h3>Results</h3> The results showed that cardiac function and cardiac GPx activity decreased remarkably after administration of ADR or Se deficiency; more dramatic impairment of cardiac function and cardiac GPx activity were observed after co-administration of ADR and Se deficiency. Mechanically, down-regulation of KATP subunits gene expression in cardiac tissue was found after administration of ADR or Se deficiency; more significant inhibition of KATP subunits gene expression was identified after co-administration of ADR and Se deficiency. However, cardiac toxicity of ADR was alleviated by Se supplementation, accompanied by restoring of cardiac GPx activity and KATP subunits gene expression. <h3>Conclusions</h3> These results indicate that decreased oxidative mediated expression of KATP is involved in adriamycin and/or Se deficiency induced cardiac dysfunction; Se deficiency exacerbates adriamycin induced cardiac dysfunction by future inhibition of oxidative mediated KATP expression; Se supplementation seems to protect against adriamycin induced cardiac dysfunction by alleviating oxidative stress and then restoring KATP expression, showing potential clinical application in cancer chemotherapy.

Gene Expression of Catalytic Proteasome Subunits and Resistance Toward Proteasome Inhibition of B Lymphocytes from Patients with Primary Sjögren Syndrome

Dysregulation of proteasome subunit β1i expression has been shown in total blood mononuclear cells (PBMC) from patients with primary Sjögren syndrome (pSS), a B cell-driven systemic autoimmune disorder. Proteasome activation was investigated in sorted blood cells from patients with pSS and controls by measuring transcript levels of constitutive (β1/β2/β5) and corresponding immunoproteasome catalytic subunits (β1i/β2i/β5i) using real-time PCR. At protein level, β1i protein expression was analyzed by immunoblotting. Functional effects of proteasome inhibition on proteolytic activity and induction of apoptosis were also evaluated in cellular subsets. The proteasome was found to be activated in pSS, with upregulation of gene expression of catalytic proteasome subunits. Western blot analysis revealed decreased β1i protein expression in pSS B lymphocytes, with decreased protein despite increased messenger RNA (mRNA) levels. After proteasome inhibition in vitro, proteolytic activity was less reduced and resistance to apoptosis was increased in B lymphocytes compared to other cells. In pSS, catalytic subunits of the proteasome are upregulated at the mRNA level, while dysregulation of subunit β1i is attributed to B lymphocytes. B cell resistance after proteasome inhibition differs from the classical concept of increased susceptibility toward inhibition in activated cells, supporting the novel notion that susceptibility depends on cellular intrinsic factors and on proteasome activation.

Synergistic Activity of Bortezomib and HDACi in Preclinical Models of B-cell Precursor Acute Lymphoblastic Leukemia via Modulation of p53, PI3K/AKT, and NF-κB

Relapse of disease and subsequent resistance to established therapies remains a major challenge in the treatment of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). New therapeutic options, such as proteasome and histone deacetylase inhibitors (HDACi) with a toxicity profile differing from that of conventional cytotoxic agents, are needed for these extensively pretreated patients. Antiproliferative and proapoptotic effects of combined HDACi/proteasome inhibitor treatments were analyzed using BCP-ALL monocultures, cocultures with primary mesenchymal stroma cells from patients with ALL, and xenograft mouse models. The underlying molecular mechanisms associated with combined treatment were determined by gene expression profiling and protein validation. We identified the proteasome inhibitor bortezomib as a promising combination partner for HDACi due to the substantial synergistic antileukemic activity in BCP-ALL cells after concomitant application. This effect was maintained or even increased in the presence of chemotherapeutic agents. The synergistic effect of combined HDACi/BTZ treatment was associated with the regulation of genes involved in cell cycle, JUN/MAPK, PI3K/AKT, p53, ubiquitin/proteasome, and NF-κB pathways. We observed an activation of NF-κB after bortezomib treatment and the induction of apoptosis-related NF-κB target genes such as TNFαRs after concomitant treatment, indicating a possible involvement of NF-κB as proapoptotic mediator. In this context, significantly lower NF-κB subunits gene expression was detected in leukemia cells from patients who developed a relapse during frontline chemotherapy, compared with those who relapsed after cessation of frontline therapy. These results provide a rationale for the integration of HDACi/BTZ combinations into current childhood BCP-ALL treatment protocols.

Effects of time of estrogen deprivation on anxiety-like behavior and GABAA receptor plasticity in ovariectomized rats

Ovariectomized animals have frequently been used to study the effects of estrogen deficiency on mood disorders, particularly anxiety disorder. However, a range of results including anxiolytic, anxiogenic, and no behavioral effects have been reported. One discrepancy was the different in behavioral testing time following ovariectomized; therefore, the aim of this study was to examine the effects of time of estrogen deprivation on anxiety-like behavior and on GABAA receptor subunit gene expressions in ovariectomized rats. The GABAA receptor was of special interest as it had been shown to be influenced by estrogen. In this study, adult female Wistar rats were ovariectomized and randomly assigned into 2 groups: ovariectomized-rat (Ovx) and ovariectomized-rat treated with estrogen (E2) at the dosage of 1μg/kg BW. At 7, 14, 21 and 28 days after ovariectomy, the rats were tested with elevated T-maze (ETM) and open field. We found that after ovariectomy, the Ovx showed signs of anxiety as demonstrated by the increased in inhibitory avoidance latency in the ETM with significant effect at day 21 and even higher at day 28. On the other hand, the escape latency was not differed between each time point. These behavioral data implied that the anxiety in term of generalized anxiety disorder as interpreted from impaired inhibitory avoidance in the ETM was affected by estrogen depletion; while, the anxiety disorder in term of panic disorder as shown by escape latency was unaffected. For gene expression analysis, the GABAA receptor α2-, α3- and α4-subunits in Ovx groups were significantly increased in the midbrain compared to E2 groups; whereas, in the amygdala, the gene expressions were not different between Ovx and E2 groups. In conclusion, these results indicated that ovariectomized as early as 21 day can induce anxiety and the altered GABAA receptor subunit may be partially responsible for anxiety following estrogen deprivation.

Selenium Attenuates Adriamycin-Induced Cardiac Dysfunction via Restoring Expression of ATP-Sensitive Potassium Channels in Rats

The possible mechanism of adriamycin (ADR) and/or selenium (Se) deficiency-induced cardiac dysfunction, and cardioprotective effects of Se against ADR-induced cardiac toxicity were investigated in this study. Cardiac function was evaluated by plasma brain natriuretic peptide level and echocardiographic and hemodynamic parameters. Cardiac glutathione peroxidase (GPx) activity was assessed spectrophotometrically. Expression of ATP-sensitive potassium channels (KATP) subunits-SUR2A and Kir6.2-were examined by real-time PCR and Western blotting. The results showed that cardiac function and cardiac GPx activity decreased remarkably after administration of ADR or Se deficiency; more dramatic impairment of cardiac function and cardiac GPx activity were observed after co-administration of ADR and Se deficiency. Mechanically, it is novel for us to find down-regulation of KATP subunits gene expression in cardiac tissue after administration of ADR or Se deficiency, and more significant inhibition of cardiac KATP gene expression was identified after co-administration of ADR and Se deficiency. Furthermore, cardiac toxicity of ADR was found alleviated by Se supplementation, accompanied by restoring of cardiac GPx activity and cardiac KATP gene expression. These results indicate that decreased expression of cardiac KATP is involved in adriamycin and/or Se deficiency-induced cardiac dysfunction; Se deficiency exacerbates adriamycin-induced cardiac dysfunction by future inhibition of KATP expression; Se supplementation seems to protect against adriamycin-induced cardiac dysfunction via restoring KATP expression, showing potential clinical application in cancer chemotherapy.

The gene expression of different integrin subunits in rat nucleus pulposus cells under periodic mechanical stress

Objective To evaluate the gene expression of different integrinsubunits in rat nucleus pulposus cells under periodic mechanical stress.Methods Nucleus pulposus cells obtained from intervetebral disc cells of 4-week-old rats were planted on glass slides.All slides were randomized into two groups:the control group and the stress group.The cells in the stress group were cultured under periodic mechanical stress (0-200 kPa,0.1 Hz) for 6 h,then the gene expression of type Ⅱ collagen,aggrecan and different integrin subunits was detected by using real-time fluorescent quantitative polymerase chain reaction (FQ-PCR).Results The gene expression levels of typeⅡ collagen,aggrecan and integin α1 were upgraded to 5.04,4.52 and 1.71 times higher in stress group than in control group respectively (P ＜ 0.05).Conclusion Periodic mechanical stress can promote the metabolism of rat nucleus pulposus cells.Integrin α1 may play important roles in the mechanical transductions. Key words: Integrins; Periodic mechanical stress; Nucleus pulposus cell

Exercise-induced morphine insensitivity is accompanied with a decrease in specific G-protein subunits gene expression in rats

Numerous studies have shown a decreased analgesic potency of morphine in exercise trained animals. Since G-protein subunits have a critical role in opioid effects at the cellular level and the exact mechanism(s) of exercise-induced morphine insensitivity has not been fully clarified, the present study was designed to determine the changes in the levels of Gαi and Gβ proteins gene expression involved in this phenomenon. All experiments were carried out on male Wistar rats. Nociceptive thermal threshold was determined by the tail-flick method. Physical training was performed using treadmill apparatus. Semiquantitative RT-PCR and Western blot techniques were used to estimate Gai and Gβ mRNA and protein levels, respectively and normalized to an internal standard (β-actin).The antinociceptive effect of intrathecal morphine (5, 10 and 15μg) was significantly suppressed in trained rats. Following physical training, a significant decrease in the mRNA and protein levels of Gαi (P<0.001 and P<0.05 respectively) and Gβ (P<0.01 and P<0.001 respectively) were observed in the dorsal portion of the lumbar spinal cord.In conclusion, our results reveal that the expression patterns of the opioid receptor signaling elements change in exercise training animals. This may be, at least partly, responsible for exercise-induced morphine insensitivity.

Acetylcholine receptor subunit and P-glycoprotein transcription patterns in levamisole-susceptible and -resistant Haemonchus contortus

The mechanism of resistance to the anthelmintic levamisole in parasitic nematodes is poorly understood, although there is some evidence implicating changes in expression of nicotinic acetylcholine receptor (nAChR) subunit genes. Hence, in order to define levamisole resistance mechanisms in some Australian field-derived isolates of Haemonchus contortus we examined gene expression patterns and SNPs in nAChR subunit genes, as well as expression levels for P-glycoprotein (P-gp) and receptor ancillary protein genes, in various life stages of one levamisole-sensitive and three levamisole-resistant isolates of this species. Larvae of two isolates showed high-level resistance to levamisole (resistance ratios at the IC50>600) while the third isolate showed a degree of heterogeneity, with a resistance factor of only 1.1-fold at the IC50 alongside the presence of a resistant subpopulation. Transcription patterns for nAChR subunit genes showed a great degree of variability across the different life stages and isolates. The most consistent observation was the down-regulation of Hco-unc-63a in adults of all resistant isolates. Transcription of this gene was also reduced in the L3 stage of the two most resistant isolates, highlighting its potential as a resistance marker in the readily accessible free-living stages. There was down regulation of all four Hco-unc-29 paralogs in adults of one resistant isolate. There were no consistent changes in expression of P-gps or ancillary protein genes across the resistant isolates. The present study has demonstrated a complex pattern of nAChR subunit gene expression in H. contortus, and has highlighted several instances where reduced expression of subunit genes (Hco-unc-63a, Hco-unc-29) may be associated with the observed levamisole resistance. The data also suggests that it will be difficult to detect resistance using gene transcription-based methods on pooled larval samples from isolates containing only a resistant subpopulation due to the averaging of gene expression data across the whole population.

Expression and activity of PARP family members in the hippocampus during systemic inflammation: Their role in the regulation of prooxidative genes

Poly(ADP-ribose)polymerase-1 (PARP-1) plays an important role in DNA repair processes during oxidative/genotoxic stress, in regulation of transcription factors and in cell death mechanisms. However, little is known about the physiopathological role of other PARP family members. In this study we analyzed, for the first time, expression of PARP family genes in the hippocampus of mice subjected to lipopolysaccharide (LPS)-evoked systemic inflammatory response (SIR). Moreover, the effect of SIR on PARP activity and on its role in gene expression was evaluated.Our data indicated that SIR enhances PARP-1, -3, -9, -12 and -14 gene expressions in mouse hippocampus. PARP activity in the hippocampus was also considerably elevated during SIR with a maximum value at 12h after LPS administration, indicating significant formation and accumulation of poly(ADP-ribose) (PAR) – this reaction’s product. Concomitantly, higher expression of genes for inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), TNF-α and NADPH oxidase subunits occurred in the hippocampus during the first hours of SIR. The inhibitor of PARP, i.e. 3-aminobenzamide (3-AB; 30mg/kg b.w.), protected the hippocampal cells against PAR formation and expression of the gene for iNOS. However, PARP activity had no effect on the mRNA level of COX-2, TNF-α and NADPH oxidase in the hippocampus. Bioinformatic analysis predicted that interaction between PARP/PAR and 41 transcription factors may be important for regulation of expression of 139 genes from the group of 262 that changed expression in the hippocampus during SIR.Summarizing, enhancement of gene expression and activity of PARP family members by SIR could lead, through modification of the protein poly(ADP-ribosylation) process, to changes in the proteins’ activity. A higher level of PAR and PARP/PAR protein interaction may affect the functioning of several transcription factors. PARP activity plays a role in regulation of expression of iNOS, the main enzyme responsible for oxidative/nitrosative stress in SIR.

Effect of salsolinol on GnRH receptor and LHβ subunit gene expression in the anterior pituitary of lactating sheep

Effect of salsolinol on GnRH receptor and LHβ subunit gene expression in the anterior pituitary of lactating sheep

The antioxidants in oils heated at frying temperature, whether natural or added, could protect against postprandial oxidative stress in obese people

We have investigated the effects of the intake of oils heated at frying temperature in order to find an oil model for deep-frying that prevents postprandial oxidative stress. Twenty obese people received four breakfasts following a randomised crossover design consisting of different oils (virgin olive oil (VOO), sunflower oil (SFO), and a mixed seed oil (SFO/canola oil) with added dimethylpolysiloxane (SOX) or natural antioxidants from olives (SOP)), which were subjected to 20 heating cycles. The intake of SFO-breakfast reduced plasma GSH levels and the GSH/GSSG ratio, increased protein carbonyl levels, and induced a higher gene expression of the different NADPH-oxidase subunits, Nrf2-Keap1 activation, gene expression of the antioxidant enzymes in peripheral blood mononuclear cells and antioxidant plasma activities than the intake of the breakfasts prepared with VOO, SOP and SOX. Oils with phenolic compounds, whether natural (VOO) or artificially added (SOP), or with artificial antioxidant (SOX), could reduce postprandial oxidative stress compared with sunflower oil.

MicroRNA-mediated GABAAα-1 receptor subunit down-regulation in adult spinal cord following neonatal cystitis-induced chronic visceral pain in rats

The nociceptive transmission under pathological chronic pain conditions involves transcriptional and/or translational alteration in spinal neurotransmitters, receptor expressions, and modification of neuronal functions. Studies indicate the involvement of microRNA (miRNA) – mediated transcriptional deregulation in the pathophysiology of acute and chronic pain. In the present study, we tested the hypothesis that long-term cross-organ colonic hypersensitivity in neonatal zymosan-induced cystitis is due to miRNA-mediated posttranscriptional suppression of the developing spinal GABAergic system. Cystitis was produced by intravesicular injection of zymosan (1% in saline) into the bladder during postnatal (P) days P14 through P16 and spinal dorsal horns (L6–S1) were collected either on P60 (unchallenged groups) or on P30 after a zymosan re-challenge on P29 (re-challenged groups). miRNA arrays and real-time reverse transcription–polymerase chain reaction (RT-PCR) revealed significant, but differential, up-regulation of mature miR-181a in the L6–S1 spinal dorsal horns from zymosan-treated rats compared with saline-treated controls in both the unchallenged and re-challenged groups. The target gene analysis demonstrated multiple complementary binding sites in miR-181a for GABAA receptor subunit GABAAα-1 gene with a miRSVR score of −1.83. An increase in miR-181a concomitantly resulted in significant down-regulation of GABAAα-1 receptor subunit gene and protein expression in adult spinal cords from rats with neonatal cystitis. Intrathecal administration of the GABAA receptor agonist muscimol failed to attenuate the viscero-motor response (VMR) to colon distension in rats with neonatal cystitis, whereas in adult zymosan-treated rats the drug produced significant decrease in VMR. These results support an integral role for miRNA-mediated transcriptional deregulation of the GABAergic system in neonatal cystitis-induced chronic pelvic pain.

Analysis of differential expression of Mediator subunit genes in Arabidopsis

Mediator is a conserved eukaryotic multiprotein complex required by RNA polymerase II for transcription of its target genes. Till date, there is no report explaining the signals that affect the overall concentration of individual Med subunits. In this report, we have analyzed the effect of different phytohormones and stresses on the transcript level of Med genes in Arabidopsis. Hormones like auxin and JA, and cold stress did not show significant effect. ABA moderately increased the transcript abundance of more than 70% of AtMed genes analyzed in this study. However, there was noticeable change in the transcript level of several AtMed genes in response to BR. Stresses like high light, dark and salt also caused significant change in the transcript abundance of many AtMed genes. These data reveal that different environmental cues can affect stoichiometric concentration of Med subunits by affecting the transcription of their respective genes. This may, in turn, affect the overall arrangement of functional Mediator complex. This also suggests that some subunits may have some specific functions to play in response different signals.

Molecular pathways controlling inhibitory receptor expression

Epilepsy is a disease of complex etiology, and multiple molecular mechanisms contribute to its development. Temporal lobe epilepsy (TLE) may result from an initial precipitating event such as hypoxia, head injury, or prolonged seizure (i.e., status epilepticus [SE]), that is followed by a latent period of months to years before spontaneous seizures occur. γ-Aminobutyric acid (GABA)(A) receptor (GABA(A) R) subunit changes occur during this latent period and may persist following the onset of spontaneous seizures. Research into the molecular mechanisms regulating these changes and potential targets for intervention to reverse GABA(A) R subunit alterations have uncovered seizure-induced pathways that contribute to epileptogenesis. Several growth or transcription factors are known to be activated by SE, including (but not limited to): brain-derived neurotrophic factor (BDNF), cAMP response element binding protein (CREB), inducible cAMP early repressor (ICER), and early growth response factors (Egrs). Results of multiple studies suggest that these factors transcriptionally regulate GABA(A) R subunit gene expression in a way that is pertinent to the development of epilepsy. This article focuses on these signaling elements and describes their possible roles in gene regulatory pathways that may be critical in the development of chronic epilepsy.

Neuronal cell-type specific DNA methylation patterns of the Cacna1c gene

Gene expression of the alpha-1 subunit of the L-type voltage-gated calcium channel, CACNA1C, is known to be complexly regulated. Because CACNA1C is not only a crucial gene in normal brain function but also a promising candidate risk gene for psychiatric disorders such as bipolar disorder and schizophrenia, elucidating the molecular basis of transcriptional regulatory mechanism will be critically important. Here we examined DNA methylation status of CpG islands and a CpG island shore on mouse Cacna1c in neuronal and non-neuronal nuclei, which were separated with a fluorescent activated cell sorting technique. We found that neurons and non-neurons showed differential DNA methylation profile on a CpG island shore. This difference was evolutionarily conserved in human neuronal and non-neuronal nuclei in the prefrontal cortex, suggesting that DNA methylation status on the CpG island shore of Cacna1c may have an important role in transcript regulation.

Mouse zygote-specific proteasome assembly chaperone important for maternal-to-zygotic transition

SummaryDuring the maternal-to-zygotic transition (MZT), maternal proteins in oocytes are degraded by the ubiquitin–proteasome system (UPS), and new proteins are synthesized from the zygotic genome. However, the specific mechanisms underlying the UPS at the MZT are not well understood. We identified a molecule named zygote-specific proteasome assembly chaperone (ZPAC) that is specifically expressed in mouse gonads, and expression of ZPAC was transiently increased at the mouse MZT. ZPAC formed a complex with Ump1 and associated with precursor forms of 20S proteasomes. Transcription of ZPAC genes was also under the control of an autoregulatory feedback mechanism for the compensation of reduced proteasome activity similar to Ump1 and 20S proteasome subunit gene expression. Knockdown of ZPAC in early embryos caused a significant reduction of proteasome activity and decrease in Ump1 and mature proteasomes, leading to accumulation of proteins that need to be degraded at the MZT and early developmental arrest. Therefore, a unique proteasome assembly pathway mediated by ZPAC is important for progression of the mouse MZT.

Diuretic Effect of Compounds from Hibiscus sabdariffa by Modulation of the Aldosterone Activity

Recent studies of Hibiscus sabdariffa Linn.have demonstrated that it presents diuretic, natriuretic, and potassium sparing effects. However, the mechanism that induces these effects has not yet been elucidated. The aim of this study was to explore the possible mechanism of action for the diuretic effect of Hibiscus sabdariffa extract and its fractions.The aqueous extract from this plant and the fractions obtained with solvents of different polarities were administered to adrenalectomized rats, and the diuretic effect was measured in the presence of deoxycorticosterone acetate (aldosterone analog).The effect on renal filtration was also evaluated in an in situ kidney model, and finally, the effect of diuretic active extracts on gene expression of the alpha subunit from the transporter (αENaC) of renal epithelial cell was quantified. The subsequent results were obtained: The aqueous extract of Hibiscus sabdariffa presented the following chemical composition, 32.4 mg/g delphinidin-3-O-sambubioside, 11.5 mg/g cyanidin-3-O-sambubioside, 11.5 mg/g quercetin, and chlorogenic acid 2.7 mg/g. The concentration of anthocyanins was diminished until disappearance due to decrease of the polarity of the solvents used in the extraction process, in contrast to the flavonoids and chlorogenic acid, which had their concentration increased. The diuretic effect caused by adrenalectomy in rats was reversed by deoxycorticosterone acetate activity. However, the effect of deoxycorticosterone acetate was antagonized by spironolactone, the aqueous extract of Hibiscus sabdariffa, and the acetonitrile : methanol 5 : 5 mixture extract, administered orally. A similar effect was observed on renal filtration obtained from the isolated kidney model.When the gene expression levels of αENaC was measured in adrenalectomized rats, it was observed that spironolactone, the aqueous extract of Hibiscus sabdariffa, the acetonitrile : methanol 5 : 5 mixture, as well as the acetonitrile extract significantly decreased the expression of this protein.The conclusion of this work is that the diuretic, natriuretic, and potassium sparing effects of Hibiscus sabdariffa are due in part to the modulation of aldosterone activity by the presence in the extract of this plant of compounds potentially responsible for this modulation, as anthocyanins, flavonoids, and chlorogenic acid.

IMPAIRED VASCULAR RESPONSES TO CALCITONIN GENE-RELATED PEPTIDE IN THE AGEING α-CGRP KNOCKOUT MOUSE

Calcitonin gene-related peptide (CGRP) is a potent microvascular vasodilator derived predominantly from perivascular sensory neurones. It has been suggested that vascular reactivity to CGRP is diminished with increasing age whilst...