Tissue-specific regulation of IRS-1 in unilaterally nephrectomized rats

Insulin stimulates the tyrosine kinase activity of its receptor, resulting in the phosphorylation of its cytosolic substrate, insulin receptor substrate 1 (IRS-1). IRS-1 is also a substrate for different peptides and growth factors, and a transgenic mouse "knockout" for this protein does not have normal growth. However, the role of IRS-1 in kidney hypertrophy and/or hyperplasia was not investigated. In the present study we investigated IRS-1 protein and tyrosine phosphorylation levels in the remnant kidney after unilateral nephrectomy (UNX) in 6-week-old male Wistar rats. After insulin stimulation the levels of insulin receptor and IRS-1 tyrosine phosphorylation were reduced to 79 ± 5% (P<0.005) and 58 ± 6% (P<0.0001), respectively, of the control (C) levels, in the remnant kidney. It is possible that a circulating factor and/or a local (paracrine) factor playing a role in kidney growth can influence the early steps of insulin action in parallel. To investigate the hypothesis of a circulating factor, we studied the early steps of insulin action in liver and muscle of unilateral nephrectomized rats. There was no change in pp185 tyrosine phosphorylation levels in liver (C 100 ± 12% vs UNX 89 ± 9%, NS) and muscle (C 100 ± 22% vs UNX 91 ± 17%, NS), and also there was no change in IRS-1 phosphorylation levels in both tissues. These data demonstrate that after unilateral nephrectomy there is a decrease in insulin-induced insulin receptor and IRS-1 tyrosine phosphorylation levels in kidney but not in liver and muscle. It will be of interest to investigate which factors, probably paracrine ones, regulate these early steps of insulin action in the contralateral kidney of unilaterally nephrectomized rats.

The C-peptide response to a standard mixed meal in a group of Brazilian type 1 diabetic patients

In order to analyze the different parameters used in the interpretation of C-peptide response in a functional test, we compared a group of 26 type 1 diabetics aged 21.1 ± 8.2 years, with a diabetes duration of 7.9 ± 6.7 months, with a group of 24 non-diabetic subjects aged 25.0 ± 4.4 years. A standard mixed meal of 317 kcal was used as a stimulus. Blood sampling for C-peptide determinations was performed at regular intervals. Although all the studied C-peptide variables were significantly lower in the diabetic group (P<0.0001), some overlapping of parameters was observed between the two groups. The highest degree of overlapping was found for basal value (BV) (30.8%) and percent increase (42.31%), and the lowest for incremental area, absolute increase, peak value (PV) (3.8%), and total area (7.7%) (c2 = 31.6, P<0.0001). We did not observe a definite pattern in the time of maximum response among the 21 diabetics who showed an increase in C-peptide levels after the stimulus. In this group, however, there was a highly significant number of late responses (120 min) (c2 = 5.7, P<0.002). Although BV showed a significant correlation with PV (rS = 0.95, P<0.0001), the basal levels of C-peptide did not differentiate the groups with and without response to the stimulus. We conclude that the diabetic group studied showed delayed and reduced C-peptide responses, and that the functional test can be an important tool for the evaluation of residual ß cell function.

Lung tissue mechanics in the early stages of induced paracoccidioidomycosis in rats

Pulmonary dysfunction represents the most important cause of death in patients with paracoccidioidomycosis (PBM). In order to investigate the functional changes of the lungs in the early stages of PBM, a model of benign disease was developed by intratracheal challenge of 12-week old isogenic Wistar rats with 1 x 106 yeast forms of Paracoccidioides brasiliensis. Animals were studied 30 and 60 days after infection, when fully developed granulomas were demonstrable in the lungs. Measurements of airway resistance, lung elastance and tissue hysteresis were made during sinusoidal deformations (100 breaths/min, tidal volume = 2 ml) with direct measurement of alveolar pressure using the alveolar capsule technique. Infection caused a significant increase in hysteresis (infected: 1.69, N = 13; control: 1.13, N = 12, P = 0.024, ANOVA), with no alterations in airway resistance or lung elastance. Histopathological analysis revealed the presence of fully developed granulomas located in the axial compartment of the lung interstitial space. These results suggest that alterations of tissue mechanics represent an early event in experimental PBM

Repair of the anophthalmic cavity of rats with synthetic hydroxyapatite

We placed spheres of synthetic hydroxyapatite (calcium chloride combined with sodium phosphate) in the eviscerated or enucleated orbital cavity of rats in order to evaluate the biocompatibility of this material with the orbital cavity. The study was conducted on 50 albino rats, 25 of which were submitted to enucleation and 25 to evisceration of one eye. The animals were sacrificed 7, 15, 21, 30 and 60 days after surgery and the orbital content was submitted to histopathological examination. A reaction of the young granulation tissue type was observed first. The hydroxyapatite was gradually surrounded by a granulomatous macrophage inflammatory response and covered with dense connective tissue that formed a sort of" mesh" septating and supporting progressively smaller blocks of the substance. The same type of reaction was observed in the enucleated and eviscerated cavities. We conclude that synthetic hydroxyapatite is an inert nonallergenic material which is appropriate for volume replacement in the anophthalmic cavity

Rotavirus and reovirus interaction with mouse peritoneal resident phagocytic cells

Rotaviruses and reoviruses are involved in human and animal diseases. It is known that both viruses penetrate the gastrointestinal tract but their interaction with phagocytic cells is unknown. To study this interaction, peritoneal resident phagocytic cells were used and rotavirus and reovirus replication in peritoneal phagocytic cells was observed. However, rotavirus replication in these cells led to the production of defective particles since MA-104 cells inoculated with rotavirus phagocytic cell lysate did not show any evidence of virus replication. On the basis of these results, we suggest that, although reovirus dissemination may be helped by these phagocytic cells, these cells may control rotavirus infection and probably contribute to the prevention of its dissemination.

Clearance-inducing antibodies are responsible for protection against the acute phase of Trypanosoma cruzi infection in mice

A study was conducted on mice infected with strains Y and CL of Trypanosoma cruzi. The ability of anti-Y and anti-CL sera to induce complement-mediated lysis, immune clearance and protection against the acute phase of the infection was studied using homologous anti-Y or anti-CL serum tested with the Y or CL strain, or heterologous anti-Y serum tested with the CL strain or anti-CL serum tested with the Y strain. Complement-mediated lysis was induced by both homologous and heterologous antisera but protection was afforded only by homologous antisera. Immune clearance was induced by homologous but not by heterologous antisera. Antisera with high clearance ability were able to confer protection whereas antisera with high lytic ability were not. These results show a high correlation between the antibody ability to induce clearance and to confer protection and suggest that clearance rather than lysis is responsible for protection against the acute phase of the infection. The mechanism of antibody protection against the acute phase of the infection is discussed.

Thalidomide protects mice against LPS-induced shock

Thalidomide has been shown to selectively inhibit TNF-a production in vitro by lipopolysaccharide (LPS)-stimulated monocytes. TNF-a has been shown to play a pivotal role in the pathophysiology of endotoxic shock. Using a mouse model of LPS-induced shock, we investigated the effects of thalidomide on the production of TNF-a and other cytokines and on animal survival. After injection of 100-350 µg LPS into mice, cytokines including TNF-a, IL-6, IL-10, IL-1ß, GM-CSF and IFN-g were measured in the serum. Administration of 200 mg/kg thalidomide to mice before LPS challenge modified the profile of LPS-induced cytokine secretion. Serum TNF-a levels were reduced by 93%, in a dose-dependent manner, and TNF-a mRNA expression in the spleens of mice was reduced by 70%. Serum IL-6 levels were also inhibited by 50%. Thalidomide induced a two-fold increase in serum IL-10 levels. Thalidomide treatment did not interfere with the production of GM-CSF, IL-1ß or IFN-g. The LD50 of LPS in this model was increased by thalidomide pre-treatment from 150 µg to 300 µg in 72 h. Thus, at otherwise lethal doses of LPS, thalidomide treatment was found to protect animals from death

Distribution of the a2, a3, and a5 nicotinic acetylcholine receptor subunits in the chick brain

Nicotinic acetylcholine receptors (nAChRs) are ionotropic receptors comprised of a and ß subunits. These receptors are widely distributed in the central nervous system, and previous studies have revealed specific patterns of localization for some nAChR subunits in the vertebrate brain. In the present study we used immunohistochemical methods and monoclonal antibodies to localize the a2, a3, and a5 nAChR subunits in the chick mesencephalon and diencephalon. We observed a differential distribution of these three subunits in the chick brain, and showed that the somata and neuropil of many central structures contain the a5 nAChR subunit. The a2 and a3 subunits, on the other hand, exhibited a more restricted distribution than a5 and other subunits previously studied, namely a7, a8 and ß2. The patterns of distribution of the different nAChR subunits suggest that neurons in many brain structures may contain several subtypes of nAChRs and that in a few regions one particular subtype may determine the cholinergic nicotinic responses

Acetylcholinesterase activity in the pons and medulla oblongata of rats after chronic electroconvulsive shock

An imbalance between cholinergic and noradrenergic neurotransmission has been proposed for the etiology of affective disorders. According to this hypothesis, depression would be the result of enhanced cholinergic and reduced noradrenergic neurotransmission. Repeated electroconvulsive shock (ECS) is an effective treatment for depression; moreover, in laboratory animals it induces changes in brain noradrenergic neurotransmission similar to those obtained by chronic treatment with antidepressant drugs (down-regulation of beta-adrenergic receptors). The aim of the present study was to determine whether repeated ECS in rats changes acetylcholinesterase (Achase) activity. Achase controls the level of acetylcholine (Ach) in the synaptic cleft and its levels seem to be regulated by the interaction between Ach and its receptor. Thus, a decrease in Achase activity would suggest decreased cholinergic activity. Adult male Wistar rats received one ECS (80 mA, 0.2 s, 60 Hz) daily for 7 days. Control rats were handled in the same way without receiving the shock. Rats were sacrificed 24 h after the last ECS and membrane-bound and soluble Achase activity was assayed in homogenates obtained from the pons and medulla oblongata. A statistically significant decrease in membrane-bound Achase activity (nmol thiocholine formed min-1 mg protein-1) (control 182.6 ± 14.8, ECS 162.2 ± 14.2, P<0.05) and an increase in soluble Achase activity in the medulla oblongata (control 133.6 ± 4.2, ECS 145.8 ± 12.3, P<0.05) were observed. No statistical differences were observed in Achase activity in the pons. Although repeated ECS induced a decrease in membrane-bound Achase activity, the lack of changes in the pons (control Achase activity: total 231.0 ± 34.5, membrane-bound 298.9 ± 18.5, soluble 203.9 ± 30.9), the region where the locus coeruleus, the main noradrenergic nucleus, is located, does not seem to favor the existence of an interaction between cholinergic and noradrenergic neurotransmission after ECS treatment

Participation of kinins in the inhibitory action of captopril on acute hypertension induced by L-NAME in anesthetized rats

The aim of the present study was to investigate the role of bradykinin in the inhibitory action of captopril in hypertension induced by L-NAME in anesthetized rats. Male Wistar rats (260-320 g) were anesthetized with chloralose and arterial blood pressure was recorded with a polygraph pressure transducer. The hypertensive effect of L-NAME was studied in rats pretreated with saline, captopril or HOE 140 plus captopril. The effect of captopril was also studied during the sustained pressor effect of L-NAME. The acute pressor effect of L-NAME (10 mg/kg, iv) was significantly reduced by iv pretreatment with 2 mg/kg captopril (D increase of 49 ± 4.9 mmHg reduced to 20 ± 5.4 mmHg, P = 0.01). The pressor effect of L-NAME (D increase of 38 ± 4.8 mmHg) observed in rats pretreated with captopril and HOE 140 (0.1 mg/kg, iv) was not significantly different from that induced by L-NAME in rats pretreated with saline (P = 0.09). During the sustained pressor effect induced by L-NAME (D increase of 49 ± 4.9 mmHg) captopril induced a significant (P<0.05) reduction in arterial blood pressure (D decrease of 22 ± 3.0 mmHg). The present results demonstrate that the acute pressor effect of L-NAME is reduced by captopril and this inhibitory effect may be partly dependent on the potentiation of the vasodilator actions of bradykinin

Antinociceptive effect of intrathecal neostigmine evaluated in rats by two different pain models

The analgesic efficacy of cholinergic agonists and anticholinesterase agents has been widely recognized. The analgesic effect obtained by activating cholinergic mechanisms, however, seems to depend on the experimental pain model utilized for its evaluation. The antinociceptive effect of intraspinal neostigmine was examined in rats submitted concurrently to the tail flick and formalin tests. Neostigmine (8.25 and 16.5 nmol) produced a dose-dependent antinociceptive effect in the tail flick test (a model of phasic pain) and reduced the first phase (phasic pain) of the animal response to formalin also in a dose-dependent manner. The second phase (tonic pain) of the response to formalin, however, was slightly reduced after a longer period of time only by the higher dose of the anticholinesterase. The effect of neostigmine was not significantly different when the drug was injected into rats submitted exclusively to the tail flick test. The second phase of the animal response to formalin was slightly reduced by neostigmine (8.25 nmol) and strongly inhibited by the higher dose of the anticholinesterase when injection was made after the first phase. We conclude that phasic and tonic pain can both be controlled by high doses of neostigmine. In addition, we show that inhibition by a lower dose of neostigmine of the formalin-induced phasic pain did not prevent the subsequent occurrence of tonic pain produced by the irritant

Computerized analysis of isometric tension studies provides important additional information about vasomotor activity

Concentration-response curves of isometric tension studies on isolated blood vessels are obtained traditionally. Although parameters such as Imax, EC50 and pA2 may be readily calculated, this method does not provide information on the temporal profile of the responses or the actual nature of the reaction curves. Computerized data acquisition systems can be used to obtain average data that represent a new source of otherwise inaccessible information, since early and late responses may be observed separately in detail

Is the Thoroughbred race-horse under chronic stress?

Thoroughbred fillies were divided into three groups according to age: group 1, 7 fillies aged 1 to 2 years (G1) starting the training program; group 2, 9 fillies aged 2 to 3 years (G2) in a full training program; group 3, 8 older fillies 3 to 4 years of age (G3) training and racing. Blood samples were collected weekly from July to December. Cortisol was quantified using a solid phase DPC kit. The intra- and interassay coefficients of variation were 12.5% and 15.65% and sensitivity was 1.9 ± 0.2 nmol/l. The semester average of cortisol levels varied between groups: G1 = 148.8 ± 6.7, G2 = 125.7 ± 5.8, G3 = 101.1 ± 5.4 nmol/l, with G3 differing statistically from the other groups. The lower cortisol levels observed in the older fillies lead us to propose that the stress stimulus, when maintained over a long period of time, may become chronic and result in a reduction of hypophyseal corticotropin-releasing hormone receptors. The secretion of endogenous opioids may also lead to low serum cortisol levels.

Pulmonary emphysema induced by passive smoking: an experimental study in rats

We describe a short time model for inducing experimental emphysema in rats by chronic tobacco smoke inhalation. Three groups of male Wistar rats (6 months old) were studied: controls (N = 8), rats intoxicated for 45 days (s-45, N = 7) or for 90 days (s-90, N = 8). The exposed animals were intoxicated 3 times a day (10 cigarettes per exposure period), 5 days a week. Pulmonary damage was assessed by means of functional tests and quantitative pathological examination of the airways and lung parenchyma. The s-45 and s-90 animals were similar in terms of functional residual capacity (FRC) corrected for body weight (FRC/kg) but both groups of smoking rats exhibited significantly higher FRC/kg values than the controls (s-45 = 6.33; s-90 = 6.46; controls = 3.78; P<0.05). When the two groups of smoking rats were pooled together and compared to controls, they showed decreased lung elastance (1.6 vs 2.19; P = 0.046) and increased mean linear intercept (Lm) (85.14 vs 66.44; P = 0.025). The s-90 animals presented higher inflammation and muscular hypertrophy at the level of the axial bronchus than the controls (P<0.05). When smoking groups were pooled and compared to controls, they presented significantly higher inflammation at the lateral level (P = 0.028), as well as airway secretory hyperplasia (P = 0.024) and smooth muscle hypertrophy (P = 0.005) at the axial level. Due to its simplicity, low cost and short duration, this technique may be a useful model to obtain new information about airspace remodeling due to chronic tobacco consumption

Hemodynamic responses to acute aortic coarctation in conscious sinoaortic denervated rats

The hemodynamic responses to acute (45 min) partial aortic constriction were studied in conscious intact (N = 7) or sinoaortic denervated (SAD) adult male Wistar rats (280-350 g, N = 7) implanted with carotid and femoral arterial catheters, a pneumatic cuff around the abdominal aorta and a pulsed Doppler flow probe to measure changes in aortic resistance. In addition, the hypertensive response and the reflex bradycardia elicited by total (N = 8) vs partial (N = 7) aortic constriction (monitored by maintenance of the pressure distal to the cuff at 50 mmHg) were compared in two other groups of intact rats. Intact rats presented a smaller hypertensive response (26 to 40% above basal level) to partial aortic constriction than SAD rats (38 to 58%). The calculated change in aortic resistance imposed by constriction of the aorta increased progressively only in intact rats, but was significantly smaller (193 to 306%) than that observed (501 to 591%) in SAD rats. Intact rats showed a significant bradycardia (23 to 26% change in basal heart rate) throughout coarctation, whereas the SAD rats did not (1 to 3%). Partial or total occlusion of the aorta induced similar hypertensive responses (37-38% vs 24-30% for total constriction) as well as reflex bradycardia (-15 to -17% vs -22 to -33%) despite a greater gradient in pressure (97-98 vs 129-140 mmHg) caused by total constriction. The present data indicate that the integrity of the baroreflex in intact rats can cause the hypertensive response to level off at a lower value than in SAD rats despite a progressive increase in aortic resistance. In addition, they also indicate that the degree of partial aortic constriction by maintenance of the pressure distal to the cuff at 50 mmHg already elicits a maximal stimulation of the arterial baroreflex