Endotoxin shock: prevented by naloxone in intact but not hypophysectomized rats

Previous studies established that naloxone reverses hypotension in endotoxin, hemorrhagic, and spinal shock. We studied endotoxin shock in hypophysectomized (Hx) rats, which have little circulating beta-endorphin. Hx or intact rats received surgically implanted jugular catheters for drug injection and aortic catheters for arterial blood pressure (MAP) recording. On the second day after implantation, rats were pretreated with either naloxone or saline. Two minutes later each rat received endotoxin. Following endotoxin, all rats showed a brief biphasic hypertensive-hypotensive response followed by stabilization of MAP near baseline. Within 20 min, all Hx rats, regardless of pretreatment, and the saline-treated intact rats, showed progressive hypotension (P less than 0.005). Only the naloxone-pretreated intact rats maintained a stable MAP. Plasma endorphin measured at 20 min was undetectable in Hx rats in contrast to intact rats (P less than 0.001); plasma corticosterone levels were likewise suppressed in the Hx rats (P less than 0.01). Thus (1) naloxone protected only the rats with an intact pituitary-adrenal-sympathetic system, and (2) pituitary endorphin is not required to generate endotoxin shock in hypophysectomized rats.     

Inhibition of mating in Aplysia fasciata by food stimuli in the environment, but not by post-ingestion stimuli causing satiation

Aplysia mating is inhibited when animals have steady-state access to food. We examined which stimuli provided by food inhibit mating. Pre-feeding animals to satiation caused a decrease in mating immediately after the meal. This effect could be mimicked by placing food into the water for the length of time of a meal just before the animals were allowed to mate, but not by filling the gut with non-nutritive bulk, which inhibits feeding. The presence of food in the water while animals were allowed to mate caused a stronger inhibition of mating. When food was maintained in the water for 24 h, animals adapted to this stimulus, indicating that the maintained presence of food could not alone account for the inhibition of mating in steady-state conditions of access to food. However, food in the water for 24 h, coupled with an occasional touch of food to the lips while animals had access to mates, caused strong inhibition of mating. We tested the hypothesis that food initiates a state of food arousal, which competes with sexual arousal, and thereby inhibits mating. If this hypothesis is correct, gut fill should facilitate mating, by inhibiting food arousal. However, this effect was not observed. The data support the hypothesis that food stimuli directly inhibit sexual arousal, rather than acting via an excitation of food arousal. Accepted: 14 June 1997     

Stress during adolescence enhances locomotor sensitization to nicotine in adulthood in female, but not male, rats

A wide body of research has indicated that perinatal exposure to stressors alters the organism, notably by programming behavioral and neuroendocrine responses and sensitivity to drugs of abuse in adulthood. Recent evidence suggests that adolescence also may represent a sensitive period of brain development, and yet there has been little research on the long-lasting effects of stressors during this period. We investigated the effects of pubertal social stress (PS; daily 1-h isolation followed by pairing with a new cage mate on postnatal days 33-48) on locomotor sensitization to injections of nicotine and corticosterone response to restraint stress when the rats were adults (approximately 3 weeks after PS). There were no differences among the groups in locomotor activity to injections of saline. However, PS females had enhanced locomotor sensitization to repeated doses of nicotine compared to control (non-stressed; NS) females, whereas PS males and NS males did not differ. PS enhanced the corticosterone response to restraint in male rats previously sensitized to nicotine and decreased the corticosterone response in nonsensitized male rats. In contrast, PS females and NS females did not differ in plasma corticosterone levels in response to restraint stress, but NS females showed enhanced corticosterone release to restraint after sensitization to nicotine. Thus, during adolescence, social stressors can have long-lasting effects, and the effects appear to differ for males and females.     

Recombinant human IFN-gamma, but not IFN-alpha or IFN-beta, enhances MHC- and non-MHC-encoded glycoproteins by a protein synthesis-dependent mechanism

Despite quantitative as well as qualitative differences, all three types of IFN (IFN-alpha, IFN-beta, and IFN-gamma) modulate the synthesis as well as the expression of class I and class II histocompatibility Ag and a melanoma-associated Ag located in the plasma membrane as well as the cytoplasm of human melanoma cells. By employing inhibitors of RNA and protein synthesis it was demonstrated that IFN-alpha and -beta increase the expression of histocompatibility products and this tumor-associated Ag by a process not requiring new protein synthesis. In contrast, IFN-gamma does require de novo protein synthesis for its modulatory activity. Thus, it appears that IFN might trigger various adaptive functions in different cell lineages by inducing at least two separate sets of responses specific for either IFN-alpha and -beta or IFN-gamma. Because the induction requirements for (2'-5')-oligoadenylate synthetase as well as for the development of a cellular antiviral state by different IFN also display a similar protein synthesis dependence pattern, the present results suggest that a similar set of cellular mediators may be involved in the modulation of antigenic expression by IFN-gamma in human melanoma cells.     

Mouse anaphylactic shock is caused by reduced cardiac output,but not by systemic vasodilatation or pulmonary vasoconstriction,via PAF and histamine

Aims

Systemic anaphylaxis is life-threatening, and its pathophysiology is not fully clarified. Mice are frequently used for experimental study on anaphylaxis. However, the hemodynamic features and mechanisms of mouse anaphylactic hypotension remain unknown. Therefore, we determined mechanisms of systemic and pulmonary vascular response to anaphylactic hypotension in anesthetized BALB/c mice by using receptor antagonists of chemical mediators.

Main methods

Anaphylaxis was actively induced by an intravenous injection of the ovalbumin antigen into open-chest artificially ventilated sensitized mice. Mean arterial pressure (MAP), pulmonary arterial pressure (PAP), left atrial pressure, central venous pressure, and aortic blood flow (ABF) were continuously measured.

Key findings

In sensitized control mice, MAP and ABF showed initial, transient increases, followed by progressive decreases after the antigen injection. Total peripheral resistance (TPR) did not decrease, while PAP initially and transiently increased to 18.5 ± 0.5 mm Hg and pulmonary vascular resistance (PVR) also significantly increased. The antigen-induced decreases in MAP and ABF were attenuated by pretreatment with either a platelet-activating factor (PAF) receptor antagonist, CV6209, or a histamine H₁ receptor antagonist, diphenhydramine, and were abolished by their combination. Diphenhydramine augmented the initial increases in PAP and PVR, but did not affect the decrease of the corresponding MAP fall. The antagonists of either leukotriene C₄ or serotonin, alone or in combination with CV6209, exerted no significant effects.

Significance

Mouse anaphylactic hypotension is caused by a decrease in cardiac output but not vasodilatation, via actions of PAF and histamine. The slight increase in PAP is not involved in mouse anaphylactic hypotension.     

Optimizing ventricular fibers: uniform strain or stress,but not ATP consumption,leads to high efficiency

The aim of this study was to investigate the influence of fiber orientation in the left ventricular (LV) wall on the ejection fraction, efficiency, and heterogeneity of the distributions of developed fiber stress, strain and ATP consumption. A finite element model of LV mechanics was used with active properties of the cardiac muscle described by the Huxley-type cross-bridge model. The computed variances of sarcomere length (SL(var)), developed stress (DS(var)), and ATP consumption (ATP(var)) have several minima at different transmural courses of helix fiber angle. We identified only one region in the used design space with high ejection fraction, high efficiency of the LV and relatively small SL(var), DS(var), and ATP(var). This region corresponds to the physiological distribution of the helix fiber angle in the LV wall. Transmural fiber angle can be predicted by minimizing SL(var) and DS(var), but not ATP(var). If ATP(var) was minimized, then the transverse fiber angle was considerably underestimated. The results suggest that ATP consumption distribution is not regulating the fiber orientation in the heart.     

White tea consumption slightly reduces iron absorption but not growth,food efficiency,protein utilization,or calcium,phosphorus, magnesium,and zinc absorption in rats

We investigated the antinutritional effect of white tea extract (0, 15, and 45 mg of the tea solid extract per kilogram body weight) incorporated in the drinking water of rats for 3 and 30 days. Gender-based differences were found for all these variables, except apparent protein digestibility and the apparent absorption of calcium, phosphorus, and iron. White tea extract consumption did not significantly change body weight gain, food intake, food efficiency, protein efficiency ratio, apparent protein digestibility, nitrogen balance, or the apparent absorption of calcium, phosphorus, magnesium, and zinc. Nevertheless, the apparent absorption of iron was slightly (15–18%) but significantly (P < 0.05) lower in rats that consumed white tea at the highest dose compared with the control groups at both 3 and 30 days. Our results suggest that the usual consumption of white tea is safe, although its effect on long-term iron absorption at high doses warrants more detailed investigation.     

Nitric oxide modulates salivary amylase and fluid, but not epidermal growth factor secretion in conscious rats

The involvement of the L-arginine/NO pathway in the control of salivary fluid, amylase and epidermal growth factor (EGF) secretion was investigated in conscious rats. For the collection of saliva, an oesophageal cannula was implanted. To obtain steady secretion, submaximal carbachol background infusion was given. Different treatments included NO synthase inhibitor N(G)-nitro-L-arginine (NOLA; with or without phentolamine, propranolol), L-arginine, D-arginine and NO donor 3-morpholinosydnonimine (SIN-1) administration. Volume, amylase activity and EGF output in the secreted fluid were determined in 30 min mixed saliva samples. Carbachol infusion alone produced a modest, sustained salivary fluid and amylase secretion. NOLA (30 mg/kg) further increased both fluid (p<0.001) and amylase outputs (p<0.001). These latter effects were prevented by L-arginine but not by D-arginine or by phentolamine. Propranolol administration decreased both fluid and amylase secretion below the carbachol plateau, and NOLA did not modify this suppressed secretory rate. SIN-1 did not alter either volume or amylase secretion. Interestingly, NOLA given without carbachol did not modify salivation. Neither carbachol nor NOLA changed salivary EGF output. The present results suggest that the L-arginine/NO pathway has a modulatory role in the cholinergic control of salivary amylase secretion, but not in EGF output. The mechanisms of inhibitory action of NO on salivary fluid and amylase secretion remain to be identified.     

Sex, but not maternal protein or folic acid intake, determines the fatty acid composition of hepatic phospholipids, but not of triacylglycerol, in adult rats

The aim of the study was to investigate whether the protein and folic acid content of the maternal diet and the sex of the offspring alter the polyunsaturated fatty acid content of hepatic phospholipids and triacylglycerol (TAG). Pregnant rats were fed diets containing 18% or 9% protein with either 1 or 5mg/kg folic acid. Maternal diet did not alter hepatic lipid composition in the adult offspring. Data from each maternal dietary group were combined and reanalysed. The proportion of 18:0, 20:4n-6 and 22:6n-3 in liver phospholipids was higher in females than in males, while hepatic TAG composition did not differ between sexes. Delta5 Desaturase expression was higher in females than in males. Neither Delta5 nor Delta6 desaturase expression was related to polyunsaturated fatty acid concentrations. These results suggest that sex differences in liver phospholipid fatty acid composition may reflect primary differences in the specificity of phospholipid biosynthesis.     

Lethality during continuous anthrax lethal toxin infusion is associated with circulatory shock but not inflammatory cytokine or nitric oxide release in rats

Although circulatory shock related to lethal toxin (LeTx) may play a primary role in lethality due to Bacillus anthracis infection, its mechanisms are unclear. We investigated whether LeTx-induced shock is associated with inflammatory cytokine and nitric oxide (NO) release. Sprague-Dawley rats with central venous and arterial catheters received 24-h infusions of LeTx (lethal factor 100 microg/kg; protective antigen 200 microg/kg) that produced death beginning at 9 h and a 7-day mortality rate of 53%. By 9 h, mean arterial blood pressure, heart rate, pH, and base excess were decreased and lactate and hemoglobin levels were increased in LeTx nonsurvivors compared with LeTx survivors and controls (diluent only) (P < or = 0.05 for each comparing the 3 groups). Despite these changes, arterial oxygen and circulating leukocytes and platelets were not decreased and TNF-alpha, IL-beta, IL-6, and IL-10 levels were not increased comparing either LeTx nonsurvivors or survivors to controls. Nitrate/nitrite levels and tissue histology also did not differ comparing LeTx animals and controls. In additional experiments, although 24-h infusions of LeTx and Escherichia coli LPS produced similar mortality rates (54 and 56%, respectively) and times to death (13.2 +/- 0.8 vs. 11.0 +/- 1.7 h, respectively) compared with controls, only LPS reduced circulating leukocytes, platelets, and IL-2 levels and increased TNF-alpha, IL-1 alpha and -1 beta, IL-6, IL-10, interferon-gamma, granulocyte macrophage-colony stimulating factor, RANTES, migratory inhibitory protein-1 alpha, -2, and -3, and monocyte chemotactic protein-1, as well as nitrate/nitrite levels (all P < or = 0.05 for the effects of LPS). Thus, in contrast to LPS, excessive inflammatory cytokine and NO release does not appear to contribute to the circulatory shock and lethality occurring with LeTx in this at model. Although therapies to modulate these host mediators may be applicable fo shock caused by LPS or other bacterial toxins, they may not with LeTx.     

Diterpene, 16-phyllocladanol enhances Th1 polarization induced by LPS-primed DC, but not TNF-alpha-primed DC

16-Phyllocladanol is diterpene isolated form the heartwood of Cryptomeria japonica. We demonstrate that the effect of 16-phyllocladanol on the phenotypic and functional maturation of human monocytes-derived DC in vitro. Human monocytes were exposed to 16-phyllocladanol alone, or in combination with LPS and thereafter co-cultured with naïve T cells. The expression levels of CD83 and HLA-DR on LPS-primed DC were enhanced by 16-phyllocladanol. 16-Phyllocladanol dose-dependently augmented the T cell stimulatory capacity in an allo MLR to LPS-primed DC and the production of IL-12p70 by LPS-primed DC. The cytokine production by 16-phyllocladanol-primed DC was not inhibited by anti-TLR2 and 4 mAbs. IFN-γ secretion from naïve T cells co-cultured with DC differentiated with LPS was also augmented by 16-phyllocladanol. These results suggest that the enhancement of Th1 cells polarization to LPS-primed DC induced by 16-phyllocladanol via the activation of IL-12p70 and independent on TLR2 or TLR4.     

Inhibition of oxygen consumption in skeletal muscle-derived mitochondria by pinacidil,diazoxide, and glibenclamide,but not by 5-hydroxydecanoate

Cell intermediary metabolism and energy production succeeds by means of mitochondria, whose activity is in relation to transmembrane potential and/or free radical production. Adenosine triphosphate (ATP)-dependent potassium channels (K_ATP) in several cell types have shown to couple cell metabolism to membrane potential and ATP production. In this study, we explore whether oxygen consumption in isolated skeletal-muscle mitochondria differs in the presence of distinct respiration substrates and whether these changes are affected by K_ATP-channel inhibitors such as glibenclamide, 5-Hydroxydecanoate (5-HD), and K_ATP channel activators (pinacidil and diazoxide). Results demonstrate a concentration-dependent diminution of respiration rate by glibenclamide (0.5–20 μM), pinacidil (1–50 μM), and diazoxide (50–200 μM), but no significant differences were found when the selective mitochondrial K_ATP-channel inhibitor (5-HD, 10–500 μM) was used. These results suggest that these K_ATP-channel agonists and antagonists exert an effect on mitochondrial respiration and that they could be acting on mito-K_ATP or other respiratory-chain components.     

Icaritin, an exogenous phytomolecule, enhances osteogenesis but not angiogenesis-an in vitro efficacy study

We found that Icaritin, an intestinal metabolite of Epimedium-derived flavonoids (EF) enhanced osteoblastic differentiation of mesenchymal stem cells (MSCs) only under osteogenic induction conditions. We also demonstrated its effect on inhibition of adipogenic differentiation of MSCs. Unlike the findings of others on EF compounds, we showed that Icaritin was unable to promote proliferation, migration and tube like structure formation by human umbilical vein endothelial cells (HUVECs) in vitro. These results suggested that the exogenous phytomolecule Icaritin possessed the potential for enhancing bone formation via its osteopromotive but not an osteoinductive mechanism. Though some flavonoids were shown to regulate the coupling process of angiogenesis and osteogenesis during bone repair, our results suggested that Icaritin did not have direct effect on enhancing angiogenesis in vitro.     

Transgenic expression of Bcl-xL or Bcl-2 by murine B cells enhances the in vivo antipolysaccharide, but not antiprotein, response to intact Streptococcus pneumoniae

IgG antipolysaccharide (PS) and antiprotein responses to Streptococcus pneumoniae (Pn) are both CD4(+) T cell dependent. However, the primary IgG anti-PS response terminates more quickly, uses a shorter period of T cell help, fails to generate memory, and is more dependent on membrane Ig (mIg) signaling. We thus determined whether this limited anti-PS response to Pn reflected a greater propensity of PS-specific B cells to undergo apoptosis. We used mice that constitutively expressed the antiapoptotic protein Bcl-x(L) or Bcl-2 as a B cell-specific transgene. Both transgenic (Tg) mice exhibited increased absolute numbers of splenic B-1 and peritoneal B-1b and B-2 cells, subsets implicated in anti-PS responses, but not in marginal zone B (MZB) cells. Both Tg mouse strains elicited, in an apparently Fas-independent manner, a more prolonged and higher peak primary IgM and IgG anti-PS, but not antiprotein, response to Pn, but without PS-specific memory. A similar effect was not observed using purified PS or pneumococcal conjugate vaccine. In vitro, both splenic MZB and follicular Tg B cells synthesized DNA at markedly higher levels than their wild-type counterparts, following mIg cross-linking. This was associated with increased clonal expansion and decreased apoptosis. Using Lsc(-/-) mice, the Pn-induced IgG response specific for the capsular PS was found to be almost entirely dependent on MZB cells. Collectively, these data suggest that apoptosis may limit mIg-dependent clonal expansion of PS-specific B cells during a primary immune response to an intact bacterium, as well as decrease the pool of PS-responding B cell subsets.     

Chronic resveratrol enhances endothelium-dependent relaxation but does not alter eNOS levels in aorta of spontaneously hypertensive rats

Spontaneously hypertensive rats (SHRs) were administered the red wine polyphenol resveratrol in drinking water at 0, 0.448, or 4.48 mg/l (control, low, or high, respectively) for 28 days. The low dosage was chosen to mimic moderate red wine consumption. After the treatment period, thoracic aorta rings were excised for in vitro assessment of vasomotor function. Chronic resveratrol significantly improved endothelium-dependent relaxation to acetylcholine (Ach), increasing maximal values to 80.8% +/- 5.2% and 80.8% +/- 5.0% in low and high groups, respectively, compared with 60.7% +/- 1.4% in controls (P<0.01). This treatment effect was eliminated in the presence of the endothelial nitric oxide synthase (eNOS) blocker N(omega)-nitro-L-arginine methyl ester. Resveratrol did not affect relaxation to sodium nitroprusside or systolic blood pressure in SHRs. In contrast to the SHR results, chronic resveratrol in Sprague Dawley rats did not affect vasomotor function in aorta rings in response to Ach. Hydrogen peroxide was reduced in the SHR thoracic aorta by a high dosage of resveratrol (P<0.05), but it was not significantly altered in other tissues tested. Thoracic aorta immunoblots revealed no significant treatment effects in SHRs on eNOS, superoxide dismutases 1 and 2, gp91phox, or Hsp90. Thus, these data provide novel evidence of improved endothelium-dependent vasorelaxation in hypertensive, but not normotensive, animals as a result of chronic resveratrol consumption mimicking dosages resulting from moderate red wine consumption. This response was not dependent on increases in eNOS expression but was dependent on improved NO bioavailability.     

Hypoleptinemia, but not hypoinsulinemia, induces hyperphagia in streptozotocin-induced diabetic rats

To assess the dominance between hypoinsulinemia and hypoleptinemia as factors in the development of hyperphagia in streptozotocin (STZ)-induced diabetes mellitus (STZ-DM) rodents with respect to hormone-neuropeptide interactions, changes in gene expression of agouti gene-related protein (AGRP) in the arcuate nucleus of the hypothalamus were investigated using STZ-DM rats, fasting Zucker fa/fa rats and STZ-DM agouti (STZ-DM A(y)/a) mice. AGRP mRNA and neuropeptide Y mRNA were both significantly up-regulated in STZ-DM rats, which are associated with body weight loss, hyperglycemia, hypoinsulinemia and hypoleptinemia. We proceeded to analyze whether insulin or leptin played the greater role in the regulation of AGRP using Zucker fa/fa rats. The AGRP mRNA did not differ significantly between fasted fa/fa rats, which have both leptin-insensitivity and hypoinsulinemia, and fed Zuckers, which have leptin-insensitivity and hyperinsulinemia. We further found that up-regulation of AGRP expression was normalized by infusion of leptin into the third cerebroventricle (i3vt), but not by i3vt infusion of insulin, although up-regulation of AGRP was partially corrected by systemic insulin infusion. The latter finding supports hypoleptinemia as a key-modulator of STZ-DM-induced hyperphagia because systemic insulin infusion, at least partially, restored hypoleptinemia through its acceleration of fat deposition, as demonstrated by the partial recovery of lost body weight. After STZ-DM induction, A(y)/a mice whose melanocortin-4 receptor (MC4-R) was blocked by ectopic expression of agouti protein additionally accelerated hyperphagia and up-regulated AGRP mRNA, implying that the mechanism is triggered by a leptin deficit rather than by the main action of the message through MC4-R. Hypoleptinemia, but not hypoinsulinemia per se, thus develops hyperphagia in STZ-DM rodents. These results are very much in line with evidence that hypothalamic neuropeptides are potently regulated by leptin as downstream targets of its actions.     

Detection of ovine lentivirus in the cumulus cells, but not in the oocytes or follicular fluid, of naturally infected sheep

The aim of this study was to examine the Maedi-Visna virus (MVV) infection status of oocytes, cumulus cells, and follicular fluid taken from 140 ewes from breeding flocks. MVV proviral-DNA and MVV RNA were detected using nested-PCR and RT-PCR MVV gene amplification, respectively in the gag gene. Nested-PCR analysis for MVV proviral-DNA was positive in peripheral blood mononuclear cells in 37.1% (52/140) of ewes and in 44.6% (125/280) of ovarian cortex samples. The examination of samples taken from ovarian follicles demonstrated that 8/280 batches of cumulus cells contained MVV proviral-DNA, whereas none of the 280 batches of oocytes taken from the same ovaries and whose cumulus cells has been removed, was found to be PCR positive. This was confirmed by RT-PCR analysis showing no MVV-viral RNA detection in all batches of oocytes without cumulus cells (0/280) and follicular fluid samples taken from the last 88 ovaries (0/88). The purity of the oocyte fraction and the efficacy of cumulus cell removal from oocytes was proved by absence of granulosa cell-specific mRNA in all batches of oocytes lacking the cumulus cells, using RT-PCR. This is the first demonstration that ewe cumulus cells harbor MVV genome and despite being in contact with these infected-cumulus cells, the oocytes and follicular fluid remain free from infection. In addition, the enzymatic and mechanical procedures we used to remove infected-cumulus cells surrounding the oocytes, are effective to generate MVV free-oocytes from MVV-infected ewes.     

Aggregation in quads but not pairs of rats exposed to cat odor or bright light

In many prey species aggregation of individuals is a defensive strategy commonly employed in response to predators and predator-related cues. However, very little work has explored this adaptive response in laboratory rats. It is known that individual rats show characteristic defensive responses to predator odors, such as hiding, avoidance, inhibition of foraging, feeding and reproduction, and risk assessment directed toward the odor source. However, whether these species-typical responses in individuals are altered in the presence of other conspecifics is yet to be characterized. The present study therefore examined the defensive response of groups of two rats (dyads) or four rats (quads) to two unconditioned stressors: bright ambient light and cat odor (a 2g ball of cat fur). The dyads and quads were formed from familiar cage mates and test sessions (20 min) occurred in a large open arena (1200 mm(2)) to which the rats had been extensively habituated under dark conditions. The results showed that when quads of rats were exposed to either cat odor or bright light in this arena, they showed characteristic increases in close social proximity, termed "huddling". A tight grouping of 3 (triplet) or 4 (quad) rats was commonly seen in response to cat fur, while triplets were more commonly seen in response to bright light. Interestingly there was no evidence for increased social proximity in dyads exposed to either stressor, only in quads. However, cat odor caused other signs of fear (such as decreased locomotor activity and increased defecation) in both quads and dyads. It is concluded that huddling is a rodent defensive strategy in rats when anxiogenic stimuli are encountered by larger groups of rats.