Signal Transduction, Plasma Membrane Calcium Movements, and Pigment Translocation in Freshwater Shrimp Chromatophores

Crustacean color change results from the differential translocation of chromatophore pigments, regulated by neurosecretory peptides like red pigment concentrating hormone (RPCH) that, in the red ovarian chromatophores of the freshwater shrimp Macrobrachium olfersi, triggers pigment aggregation via increased cytosolic cGMP and Ca(2+) of both smooth endoplasmatic reticulum (SER) and extracellular origin. However, Ca(2+) movements during RPCH signaling and the mechanisms that regulate intracellular [Ca(2+)] are enigmatic. We investigate Ca(2+) transporters in the chromatophore plasma membrane and Ca(2+) movements that occur during RPCH signal transduction. Inhibition of the plasma membrane Ca(2+)-ATPase by La(3+) and indirect inhibition of the Na(+)/Ca(2+) exchanger by ouabain induce pigment aggregation, revealing a role for both in Ca(2+) extrusion. Ca(2+) channel blockade by La(3+) or Cd(2+) strongly inhibits slow-phase RPCH-triggered aggregation during which pigments disperse spontaneously. L-type Ca(2+) channel blockade by gabapentin markedly reduces rapid-phase translocation velocity; N- or P/Q-type blockade by omega-conotoxin MVIIC strongly inhibits RPCH-triggered aggregation and reduces velocity, effects revealing RPCH-signaled influx of extracellular Ca(2+). Plasma membrane depolarization, induced by increasing external K(+) from 5 to 50 mM, produces Ca(2+)-dependent pigment aggregation, whereas removal of K(+) from the perfusate causes pigment hyperdispersion, disclosing a clear correlation between membrane depolarization and pigment aggregation; K(+) channel blockade by Ba(2+) also partially inhibits RPCH action. We suggest that, during RPCH signal transduction, Ca(2+) released from the SER, together with K(+) channel closure, causes chromatophore membrane depolarization, leading to the opening of predominantly N- and/or P/Q-type voltage-gated Ca(2+) channels, and a Ca(2+)/cGMP cascade, resulting in pigment aggregation. J. Exp. Zool. 313A:605-617, 2010. (C) 2010 Wiley-Liss, Inc.

Compensation for obesity-induced insulin resistance in dogs: Causal web analysis of the associations of leptin and GLP-1.

Obesity affects aspects of glucose homeostasis such as insulin secretion and insulin sensitivity. Hormones secreted by adipocytes like leptin mediate the metabolic consequences of obesity. Incretin hormones like glucagon-like peptide-1 (GLP-1) increase insulin secretion in response to changes in blood glucose concentration and have been proposed to regulate insulin secretion in fasting, overweight dogs. The aim of this study was to examine hormonal mechanisms by which adiposity alters glucose homeostasis, plasma insulin concentration, and insulin sensitivity in spontaneously overweight dogs.

Disruption of NaS1 sulfate transport function in mice leads to enhanced acetaminophen-induced hepatotoxicity

Sulfate is required for detoxification of xenobiotics such as acetaminophen (APAP), a leading cause of liver failure in humans. The NaS1 sulfate transporter maintains blood sulfate levels sufficiently high for sulforiation reactions to work effectively for drug detoxification. In the present study, we identified two loss-of-function polymorphisms in the human NaS1 gene and showed the Nas1-null mouse to be hypersensitive to APAP hepatotoxicity. APAP treatment led to increased liver damage and decreased hepatic glutathione levels in the hyposulfatemic Nas1-null mice compared with that in normosulfatemic wild-type mice. Analysis of urinary APAP metabolites revealed a significantly lower ratio of APAP-sulfate to APAP-glucuronide in the Nas1-null mice. These results suggest hyposulfatemia increases sensitivity to APAP-induced hepatotoxicity by decreasing the sulfonation capacity to metabolize APAP. In conclusion, the results of this study highlight the importance of plasma sulfate level as a key modulator of acetaminophen metabolism and suggest that individuals with reduced NaS1 sulfate transporter function would be more sensitive to hepatotoxic agents.

Bariatric Surgery Reverses Natural Killer (NK) Cell Activity and NK-Related Cytokine Synthesis Impairment Induced by Morbid Obesity

Background Obesity is related to a higher rate of infections and some types of cancer. Here we analyzed the impact of obesity and weight loss induced by Roux-en-Y gastric bypass (RYGB) on immunological parameters, i.e., cytokine productions and natural killer cell function. Methods We analyzed 28 morbidly obese patients before and 6 months after RYGB. Biochemical parameters were analyzed in plasma. The percent of natural killer (NK) cells, their cytotoxicity, and the production of cytokines by peripheral blood mononuclear cells were analyzed. The percent of NK cells was determined by flow cytometry and cytokine production determined by enzyme-linked immunosorbent assay. NK cytotoxicity was determined by the lactate dehydrogenase release assay. Results The weight loss 6 months following surgery was 35.3 +/- 4.5 kg. RYGB also improves biochemical parameters. No significant difference was found in the percent of NK cells after surgery. We found an increase in the production of interferon-gamma, interleukin (IL)-12 and IL-18, but not in IL-2, 6 months after RYGB. Cytotoxic activity of NK cells was significantly enhanced 6 months after RYGB [17.1 +/- 14.7% before RYGB vs 51.8 +/- 11.3% at 6 months after, at 40: 1 effector to target cell ratio; p<0.001]. We observed significant post-surgical improvement in the cytotoxic activity curve in 22 out of 28 patients (78.6%), irrespective of the target to effector cell ratio. Conclusions The weight loss induced by RYGB modifies the production of cytokines related with NK cell function and improves its activity.

Gut ischemia/reperfusion induced acute lung injury is an alveolar macrophage dependent event

Background:. Although the role of the lung alveolar macrophage (AM) as a mediator of acute lung injury (ALI) after lung ischemia/reperfusion (I/R) has been suggested by animal experiments, it has not been determined whether AMs mediate ALI after intestinal I/R. The objective of this study was to determine the effect of AM elimination on ALI after intestinal I/R in rats. Mwthods: Male Wistar rats (n = 90) were randomly divided into three groups: the clodronate-liposomes (CLOD-LIP) group received intratracheal treatment with CLOD-LIP; the liposomes (LIP) group received intratracheal treatment with LIP; and the nontreated (UNTREAT) group received no treatment. Twenty-four hours later each group was randomly divided into three subgroups: the intestinal I/R subgroup was subjected to 45-minute intestinal ischemia and 2-hour reperfusion; the laparotomy (LAP) subgroup was subjected to LAP and sham procedures; the control (CTR) subgroup received no treatment. At the end of reperfusion, ALI was quantitated in all the animals by the Evans blue dye (EBD) method. Results: ALI values are expressed as EBD lung leakage (mu g EBD/g dry lung weight). EBD lung leakage values in the CLOD-LIP group were 32.59 +/- 12.74 for I/R, 27.74 +/- 7.99 for LAP, and 33.52 +/- 10.17 for CTR. In the LIP group, lung leakage values were 58.02 +/- 18.04 for I/R, 31.90 +/- 8.72 for LAP, and 27.17 +/- 11.48 for CTR. In the UNTREAT group, lung leakage values were 55.60 +/- 10.96 for I/R, 35.99 +/- 6.89 for LAP, and 30.83 +/- 8.41 for CTR. Within each group, LAP values did not differ from CTR values. However, in the LIP and UNTREAT groups, values for both the LAP and CTR subgroups were lower than values for the I/R subgroup (p < 0.001). The CLOD-LIP I/R subgroup value was less (p < 0.001) than the I/R subgroup values in the LIP and UNTREAT groups. These results indicated that I/R provokes ALI that can be prevented by CLOD-LIP treatment, and further suggested that AMs are essential for ALI occurrence induced by intestinal I/R in rats.

Urine is necessary to provoke bladder inflammation in protamine sulfate induced urothelial injury

Purpose: The bladder is normally impermeable to possible hostile environmental factors and toxic urinary wastes. Any disruption of the permeability barrier would permit the leakage of urine constituents into the underlying cells layers and subsequent inflammation. Protamine sulfate, which increases urothelial permeability, is used in experimental models of cystitis. We examined whether protamine sulfate alone could cause bladder inflammation or if the association of protamine sulfate and urine is needed for this condition. Materials and Methods: Female Wistar rats (Center for the Development of Experimental Models for Medicine and Biology, Federal University of Sao Paulo, Sao Paulo, Brazil) had the bladder catheterized and instilled with protamine sulfate (10 mg) or sterile saline for 30 minutes. To exclude urine other groups of rats underwent bilateral nephrectomy and the same procedure was used. One day after instillation the bladders were removed for histopathology. Edema and vascular congestion were graded from 0-none to 3-severe. Polymorphonuclear and mast cells were counted. The Kruskal-Wallis test was performed for statistical analysis. Results: Intravesical instillation of protamine sulfate in nonnephrectomized rats led to inflammation, in contrast to findings in rats instilled with saline. On the other hand, nephrectomized rats showed no inflammatory changes following the instillation of protamine sulfate or saline. The mast cell count was similar in all groups. Conclusions: Bladder inflammation in this experimental model of urothelial injury was not due to protamine sulfate alone. The association of protamine sulfate and urine was necessary to trigger the inflammatory cascade. Thus, urine indeed has an important role in the development of bladder inflammation in an environment of higher urothelial permeability.

Dietary salt restriction increases plasma lipoprotein and inflammatory marker concentrations in hypertensive patients

Background: Dietary salt restriction has been reported to adversely modify the plasma lipoprotein profile in hypertensive and in normotensive subjects. We investigated the effects of the low sodium intake (LSI) on the plasma lipoprotein profile and on inflammation and thrombosis biomarkers during the fasting and postprandial periods. Methods: Non-obese, non-treated hypertensive adults (n=41) were fed strictly controlled diets. An initial week on a control diet (CID, Na=160 mmol/day) was followed by 3 weeks on LSI (Na=60mmol/day). At admission and on the last day of each period, the 24-h ambulatory blood pressure was monitored and blood was drawn after an overnight fasting period and after a fat-rich test meal. Results: The dietary adherence was confirmed by 24-h urinary sodium excretion. Fasting triglyceride (TG), chylomicron-cholesterol, hsC-reactive protein (CRP), tumor necrosis factor-a (TNF-alpha). interleukin-6 (IL-6) concentrations, renin activity, aldosterone, insulin, and homeostasis model assessment insulin resistance (HOMA-IR) Values were higher, but non-esterified fatty acids (NEFA) were lower on LSI than on CD. For LSI, areas under the curve (AUC) of TG, chylomicron-cholesterol, apoB and the cholesterol/apoB ratio were increased, whereas AUC-NEFA was lowered. LSI did not modify body weight, hematocrit, fasting plasma cholesterol, glucose, adiponectin, leptin, fibrinogen and factor VII (FVII), and AUC of lipoprotein lipase and of lipoprotein remnants. Conclusion: LSI induced alterations in the plasma lipoproteins and in inflammatory markers that are common features of the metabolic syndrome. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

The relationship between plasma potassium concentration and muscle torque during recovery following intense exercise

The present study investigated the relationship between plasma potassium ion concentration ([K+]) and skeletal muscle torque during three different 15-min recovery periods after fatigue induced by four 30-s sprints. Four males and one female completed the multiple sprint exercise on three separate days; recovery was passive, i.e. no cycling exercise (PRec), active cycling at 30% peak oxygen consumption (V) over dot(2peak) (30% Rec) and active cycling at 60% (V) over dot(2peak) (60% Rec). Plasma [K+] was measured from blood sampled from an antecubital vein of subjects at rest and at 0, 3, 5, 10 and 15 min into each recovery. Isokinetic leg strength was measured at rest and at 1, 6, 11 and 16 min during each recovery. Following the exhaustive sprints; [K+] increased significantly from an average mean (SEM) resting value of 3.81 (0.07) mmol.l(-1) to 4.48 (0.19) mmol.l(-1) (P < 0.01). In all recovery conditions, plasma [K+] returned to resting levels within 3 min following the fourth sprint. However, in the two active recovery conditions plasma [K+] increased over the remainder of the recovery periods to 4.36 (0.12) mmol.l(-1) in the 30% Rec condition and 4.62 (0.12) mmol.l(-1) in the 60% Rec condition, the latter being significantly higher than the former (P < 0.01). The maximum torque measured following the sprints decreased significantly, on average, to 61.1 (8.36)% of peak levels (P < 0.01). After 15 min of recovery, maximum torque was highest in the 30% Rec condition at 92.13 (3.06)% of peak levels (P < 0.01), compared to 85.23 (3.64)% and 85.71 (0.82)% for the PRec and 60% Rec conditions, respectively. In contrast to the significant differences in plasma [K+] across all three recovery conditions, muscle torque recovery was significantly different in only the 30% Rec condition. In summary, recovery of peak levels of muscle torque following fatiguing exercise does not appear to follow changes in plasma [K+].

Ethanol feeding enhances inflammatory cytokine expression in lipopolysaccharide-induced hepatitis

Elevated concentrations of plasma tumour necrosis factor (TNF)-alpha, interleukin (IL)-1 and IL-6 have been detected in patients with alcoholic hepatitis and have been implicated in the pathogenesis of hepatocyte necrosis. The present study used a rat model to conduct a detailed histological and biochemical examination of the expression of various pro-inflammatory cytokines and associated liver pathology in ethanol-potentiated lipopolysaccharide (LPS)-induced liver injury. Male Wistar rats were pair-fed either the control or ethanol-containing (36% of caloric intake as ethanol) form of the Lieber-DeCarli liquid diet for 6 weeks. Liver injury was induced by the i.v. injection of LPS (1 mu g/g bodyweight), with animals being killed at O, 1, 3, 6, 12 and 24 h after injection. At the later time points, plasma transaminase and transpeptidase activities were significantly elevated in ethanol-fed LPS-treated rats compared with control-fed LPS-treated animals. At these times after LPS treatment, hepatocytes in ethanol-fed animals displayed fatty change and necrosis with an associated neutrophil polymorph infiltrate. Time course analysis revealed that plasma TNF-alpha (1-3 h post-LPS) and IL-6 (3 h post-LPS) bioactivity was significantly elevated in ethanol-fed compared with control-fed animals. No difference was seen in plasma IL-1 alpha concentration (maximal in both groups 6 h post-LPS). The expression of TNF-alpha, IL-1 alpha, IL-1 beta and IL-6 mRNA were elevated between 1 and 6 h post-LPS in the livers of both control and ethanol-fed rats. However, ethanol-fed LPS-treated animals exhibited significantly higher maximal expression of IL-1 and IL-6 mRNA. Comparison of the appearance of cytokine mRNA and plasma bioactivity indicated an effect of ethanol feeding on post-transcriptional processing and/or the kinetics of the circulating cytokines. Elevated levels of both hepatic cytokine mRNA expression and the preceding plasma cytokines are presumably a necessary prerequisite for hepatic injury seen in this model and, therefore, possibly for the damage seen in human alcoholics. Further studies using this model may lead to significant advances in our understanding of the pathogenic mechanisms of alcoholic liver disease in humans.

AT(1) receptor participates in the cardiac hypertrophy induced by resistance training in rats

Resistance training is accompanied by cardiac hypertrophy, but the role of the renin-angiotensin system (RAS) in this response is elusive. We evaluated this question in 36 male Wistar rats divided into six groups: control (n = 6); trained (n = 6); control + losartan (10 mg.kg(-1).day(-1), n = 6); trained + losartan (n = 6); control + high-salt diet (1%, n = 6); and trained + high-salt diet (1%, n = 6). High salt was used to inhibit the systemic RAS and losartan to block the AT(1) receptor. The exercise protocol consisted of: 4 x 12 bouts, 5x/wk during 8 wk, with 65-75% of one repetition maximum. Left ventricle weight-to-body weight ratio increased only in trained and trained + high-salt diet groups (8.5% and 10.6%, P < 0.05) compared with control. Also, none of the pathological cardiac hypertrophy markers, atrial natriuretic peptide, and alpha MHC (alpha-myosin heavy chain)-to-beta MHC ratio, were changed. ACE activity was analyzed by fluorometric assay (systemic and cardiac) and plasma renin activity (PRA) by RIA and remained unchanged upon resistance training, whereas PRA decreased significantly with the high-salt diet. Interestingly, using Western blot analysis and RT-PRC, no changes were observed in cardiac AT(2) receptor levels, whereas the AT(1) receptor gene (56%, P < 0.05) and protein (31%, P < 0.05) expressions were upregulated in the trained group. Also, cardiac ANG II concentration evaluated by ELISA remained unchanged (23.27 +/- 2.4 vs. 22.01 +/- 0.8 pg/mg, P > 0.05). Administration of a subhypotensive dose of losartan prevented left ventricle hypertrophy in response to the resistance training. Altogether, we provide evidence that resistance training-induced cardiac hypertrophy is accompanied by induction of AT(1) receptor expression with no changes in cardiac ANG II, which suggests a local activation of the RAS consistent with the hypertrophic response.

Adult rats are more sensitive to the vascular effects induced by hyperhomocysteinemia than young rats

We aimed to investigate the vascular effects of hyperhomocysteinemia (HHcy) on carotid arteries from young and adult rats. With this purpose young and adult rats received a solution of DL-homocysteine-thiolactone (1 g/kg body weight/day) in the drinking water for 7, 14 and 28 days. Increase on plasma homocysteine occurred in young and adult rats treated with DL-homocysteine-thiolactone in all periods. Vascular reactivity experiments using standard muscle bath procedures showed that HHcy enhanced the contractile response of endothelium-intact, carotid rings to phenylephrine in both young and adult rats. However, in young rats, the increased phenylephrine-induced contraction was observed after hyperhomocysteinemia for 14 and 28 days, whereas in adult rats this response was already apparent after 7 day treatment. HHcy impaired acetylcholine-induced relaxation in arteries from adult but not young rats. The contraction induced by phenylephrine in carotid arteries in the presence of Y-27632 was reversed to control values in arteries from young but not adult rats with hyperhomocysteinemia. HHcy did not alter the contraction induced by CaCl(2) in carotid arteries from young rats, but enhanced CaCl(2)-induced contraction in the arteries from adult rats. HHcy increased the basal levels of superoxide anion in arteries from both groups. Finally, HHcy decreased the basal levels of nitrite in arteries from adult but not young rats. The major new finding of the present work is that arteries from young rats are more resistant to vascular changes evoked by HHcy than arteries from adult rats. Also, we verified that the enhanced vascular response to phenylephrine observed in carotid arteries of DL-homocysteine thiolactone-treated rats is mediated by different mechanisms in young and adult rats. (C) 2010 Published by Elsevier Inc.

Role of nitric oxide in hyperpnea-induced bronchoconstriction and airway microvascular permeability in guinea pigs

The present study aimed to evaluate the role of nitric oxide (NO) on hyperpnea-induced bronchoconstriction (HIB) and airway microvascular hyperpermeability (AMP). Sixty-four guinea pigs were anesthetized, tracheotonnized, cannulated, and connected to animal ventilator to obtain pulmonary baseline respiratory system resistance (Rrs). Animals were then submitted to 5 minutes hyperpnea and Rrs was evaluated during 15 minutes after hyperpnea. AMP was evaluated by Evans blue dye (25 mg/kg) extravasation in airway tissues. Constitutive and inductible NO was evaluated by pretreating animals with N(G)-nitro-1-arginine methyl ester (I-NAME) (50 mg/kg), aminoguadinine (AG) (50 mg/kg), and I-arginine (100 mg/kg) and exhaled NO (NOex) was evaluated before and after drug administration and hyperpnea. The results show that I-NAME potentiated (57%) HIB and this effect was totally reversed by I-arginine pretreatment, whereas AG did not have effect on HIB. I-NAME decreased basal AMP (48%), but neither I-NAME nor AG had any effect on hyperpnea-induced AMP. NOex levels were decreased by 50% with I-NAME, effect that was reversed by I-arginine treatment. These results suggest that constitutive but not inducible NO could have a bronchoprotective effect on HIB in guinea pigs. The authors also observed that neither constitutive nor inducible NO seems to have any effect on hyperpnea-induced AMP.

In familial hyperaldosteronism type I, hybrid gene-induced aldosterone production dominates that induced by wild-type genes

We compared the aldosterone-producing potency of the angiotensin II-sensitive wild-type aldosterone synthase genes and the ACTH-sensitive hybrid 11 beta-hydroxylase/aldosterone synthase gene by examining aldosterone, PRA, and cortisol day-curves (2-hourly levels over 24 h) in patients with familial hyperaldosteronism type I, before and during long-term (0.8-13.5 yr) glucocorticoid treatment. In 8 untreated patients, PRA levels were usually suppressed, and aldosterone correlated strongly with cortisol (r = 0.69-0.99). Fourteen studies were performed on 10 patients receiving glucocorticoid treatment that corrected hypertension, hypokalemia, and PRA suppression in all. ACTH was markedly and continuously suppressed in 6 studies, 3 of which demonstrated strong correlations between aldosterone and PRA (r = 0.77-0.92), ACTH was only partially suppressed in the remaining 8 studies; aldosterone correlated strongly: 1) with cortisol alone in 5 (r = 0.71-0.98); 2) with cortisol (r = 0.90) and PRA (r = 0.74) in one; 3) with PRA only in one (r = 0.80); and 4) with neither PRA nor cortisol in one. Unless ACTH is markedly and continuously suppressed, aldosterone is more responsive to ACTH than to renin/angiotensin II, despite the latter being unsuppressed. This is consistent with the hybrid gene being more powerfully expressed than the wild-type aldosterone synthase genes in familial hyperaldosteronism type I.

Changes in plasma free fatty acid levels in septic patients are associated with cardiac damage and reduction in heart rate variability

Free fatty acids (FFAs) have been shown to produce alteration of heart rate variability (HRV) in healthy and diabetic individuals. Changes in HRV have been described in septic patients and in those with hyperglycemia and elevated plasma FFA levels. We studied if sepsis-induced heart damage and HRV alteration are associated with plasma FFA levels in patients. Thirty-one patients with sepsis were included. The patients were divided into two groups: survivors(n = 12) and nonsurvivors (n = 19). The following associations were investigated: (a) troponin I elevation and HRV reduction and (b) clinical evolution and HRV index, plasma troponin, and plasma FFA levels. Initial measurements of C-reactive protein and gravity Acute Physiology and Chronic Health Evaluation scores were similar in both groups. Overall, an increase in plasma troponin level was related to increased mortality risk. From the first day of study, the nonsurvivor group presented a reduced left ventricular stroke work systolic index and a reduced low frequency (LF) that is one of HRV indexes. The correlation coefficient for LF values and troponin was r(2) = 0.75 (P < 0.05). All patients presented elevated plasma FFA levels on the first day of the study (5.11 +/- 0.53 mg/mL), and this elevation was even greater in the nonsurvivor group compared with the survivors (6.88 +/- 0.13 vs. 3.85 +/- 0.48 mg/mL, respectively; P < 0.05). Cardiac damage was confirmed by measurement of plasma troponin I and histological analysis. Heart dysfunction was determined by left ventricular stroke work systolic index and HRV index in nonsurvivor patients. A relationship was found between plasma FFA levels, LFnu index, troponin levels, and histological changes. Plasma FFA levels emerged as possible cause of heart damage in sepsis.

Systemic chemotherapy-induced microsatellite instability in the mononuclear cell fraction of women with breast cancer can be reproduced in vitro and abrogated by amifostine

Objectives Microsatellite instability (MSI) induction by alkylating agent-based chemotherapy (ACHT) may underlie both tumor resistance to chemotherapy and secondary leukaemias in cancer patients. We investigated if ACHT could induce MSI in tumor-derived plasma-circulating DNA (pfDNA) and in normal peripheral blood mononuclear (PBMN) cells. We also evaluated if amifostine could interfere with this process in an in-vitro model. Methods MSI was determined in pfDNA, PBMN cells and urine cell-free DNA (ufDNA) of 33 breast cancer patients before and after ACHT. MCF-7 cells and PBMN from normal donors were exposed in vitro to melphalan, with or without amifostine. Results We observed at least one MSI event in PBMN cells, pfDNA or ufDNA of 87, 80 and 80% of patients, respectively. In vitro, melphalan induced MSI in both MCF-7 and normal PBMN cells. In PBMN cells, ACHT-induced MSI occurred together with a significant decrease in the expression of the DNA mismatch repair gene hMSH2. Amifostine decreased hMSH2 expression and also prevented MSI induction only in normal PBMN cells. Conclusions ACHT induced MSI in PBMN cells and in tumour-derived pfDNA. Because of its protective effect against ACHT induction of MSI in normal PBMN cells in vitro, amifostine may be a potential agent for preventing secondary leukaemias in patients exposed to ACHT.