Age-dependent changes in adenosine A(1) receptor distribution and density within the nucleus tractus solitarii of normotensive and hypertensive rats

This study shows the distribution and density of adenosine A1 receptor (A(1)R) within the nucleus tractus solitarii (NTS) of Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) from birth to adulthood (1, 15, 30 and 90 days old). The NTS shows heterogeneous distribution of A(1)R in dorsomedial/dorsolateral, subpostremal and medial/intermediate subnuclei. A(1)R decrease from rostral to caudal within dorsomedial/dorsolateral subnucleus in 15-, 30- and 90-day-old WKY and SHR. A(1)R increase from rostral to caudal subpostremal subnucleus in 30- and 90-day-old WKY, and in 15-, 30- and 90-day-old SHR. Furthermore, A(1)Rs are increased in SHR as compared with WKY within dorsomedial/dorsolateral in 30- and 90-day-old and within subpostremal of 15-, 30- and 90-day-old rats. Finally, A(1)Rs increase from 1- to 30-day-old rats. Medial/intermediate did not show any changes in A(1)R from rostral to caudal levels, age or strain. In summary, our result highlights the importance of A1 adenosine system regarding the neural control of blood pressure and the development of hypertension.

Prolonged consumption of soy or fish-oil-enriched diets differentially affects the pattern of hypothalamic neuronal activation induced by refeeding in rats

We used c-Fos immunoreactivity to estimate neuronal activation in hypothalamic feeding-regulatory areas of 3-month-old rats fed control or oil-enriched diets (soy or fish) since weaning. While no diet effect was observed in c-Fos immunoreactivity of 24-h fasted animals, the acute response to refeeding was modified by both hyperlipidic diets but with different patterns. Upon refeeding, control-diet rats had significantly increased c-Fos immunoreactivity only in the paraventricular hypothalamic nucleus (PVH, 142%). In soy-diet rats, refeeding with the soy diet increased c-Fos immunoreactivity in dorsomedial hypothalamic nucleus (DMH, 271%) and lateral hypothalamic area (LH, 303%). Refeeding fish-diet rats with the fish diet increased c-Fos immunoreactivity in PVH (161%), DMH (177%), VMH (81%), and ARC (127%). Compared to the fish-diet, c-Fos immunoreactivity was increased in LH by the soy-diet while it was decreased in ventromedial hypothalamic nucleus (VMH) and arcuate hypothalamic nucleus (ARC). Based on the known roles of the activated nuclei, it is suggested that, unlike the fish-diet, the soy-diet induced a potentially obesogenic profile, with high LH and low VMH/PVH activation after refeeding.

Fasting differentially regulates plasma corticosterone-binding globulin, glucocorticoid receptor, and cell cycle in the gastric mucosa of pups and adult rats

Ogias D, de Andrade Sa ER, Kasai A, Moisan M, Alvares EP, Gama P. Fasting differentially regulates plasma corticosterone-binding globulin, glucocorticoid receptor, and cell cycle in the gastric mucosa of pups and adult rats. Am J Physiol Gastrointest Liver Physiol 298: G117-G125, 2010. First published October 15, 2009; doi:10.1152/ajpgi.00245.2009.-The nutritional status influences gastric growth, and interestingly, whereas cell proliferation is stimulated by fasting in suckling rats, it is inhibited in adult animals. Corticosterone takes part in the mechanisms that govern development, and its effects are regulated in particular by corticosterone-binding globulin (CBG) and glucocorticoid receptor (GR). To investigate whether corticosterone activity responds to fasting and how possible changes might control gastric epithelial cell cycle, we evaluated different parameters during the progression of fasting in 18- and 40-day-old rats. Food restriction induced higher corticosterone plasma concentration at both ages, but only in pups did CBG binding increase after short-and long-term treatments. Fasting also increased gastric GR at transcriptional and protein levels, but the effect was more pronounced in 40-day-old animals. Moreover, in pups, GR was observed in the cytoplasm, whereas, in adults, it accumulated in the nucleus after the onset of fasting. Heat shock protein (HSP) 70 and HSP 90 were differentially regulated and might contribute to the stability of GR and to the high cytoplasmic levels in pups and elevated shuttling in adult rats. As for gastric epithelial cell cycle, whereas cyclin D1 and p21 increased during fasting in pups, in adults, cyclin E slowly decreased, concomitant with higher p27. In summary, we demonstrated that corticosterone function is differentially regulated by fasting in 18-and 40-day-old rats, and such variation might attenuate any possible suppressive effects during postnatal development. We suggest that this mechanism could ultimately increase cell proliferation and allow regular gastric growth during adverse nutritional conditions.

EGFR is involved in control of gastric cell proliferation through activation of MAPK and Src signalling pathways in early-weaned rats

Objectives: Early weaning (EW) increases proliferation of the gastric epithelium in parallel with higher expression of transforming growth factor alpha and its receptor epidermal growth factor receptor (EGFR). The primary objective of the present study was to examine involvement of EGFR signalling in regulating mucosal cell proliferation during the early weaning period. Materials and methods: Fifteen-day-old rats were split into two groups: suckling (control) and EW, in which pups were separated from the dam. Animals were killed daily until the 18th day, 3 days after onset of treatment. To investigate the role of EGFR in proliferation control, EW pups were injected with AG1478, an EGFR inhibitor; signalling molecules, proliferative indices and cell cycle-related proteins were evaluated. Results: EW increased ERK1/2 and Src phosphorylation at 17 days, but p-Akt levels were unchanged. Moreover, at 17 days, AG1478 administration impaired ERK phosphorylation, whereas p-Src and p-Akt were not altered. AG1478 treatment reduced mitotic and DNA synthesis indices, which were determined on HE-stained and BrdU-labelled sections. Finally, AG1478 injection decreased p21 levels in the gastric mucosa at 17 days, while no changes were detected in p27, cyclin E, CDK2, cyclin D1 and CDK4 concentrations. Conclusions: EGFR is part of the mechanism that regulates cell proliferation in rat gastric mucosa during early weaning. We suggest that such responses might depend on activation of MAPK and/or Src signalling pathways and regulation of p21 levels.

In vivo effects of TGF beta 1 on the the growth of gastric epithelium in suckling rats

As the content of Transforming Growth Factor-beta (TGF beta) wanes in the milk of lactating rat, an increase in TGF beta is observed in the gastric epithelia concomitant with differentiation of the glands upon weaning. Whereas TGF beta has been shown to inhibit the proliferation of gastrointestinal cells in vitro, its functional significance and mechanisms of action have not been studied in vivo. Therefore, we administered TGF beta 1 (1 ng/g body wt.) to 14-day-old rats in which the gastric epithelium was induced to proliferate by fasting, and determined the involvement of signaling through Smads and the impact on epithelial cell proliferation and apoptosis. After the gavage, we observed the progressive increase of active TGF beta 1 while T beta RII-receptor remained constant in the gastric mucosa. By immunohistochemistry, we showed Smad2/3 increase at 60 min (p < 0.05) and Smad2 phosphorylation/activation and translocation to the nucleus most prominently between 0 and 30 min after treatment (p < 0.05). Importantly, TGF beta 1 inhibited cell proliferation (p < 0.05), which was estimated by BrDU pulse-labeling 12 h after gavage. Lower proliferation was reflected by increased p27(kip1) at 2 h (p < 0.05). Also, TGF beta 1 increased apoptosis as measured by M30 labeling at 60 and 180 min (p < 0.001), and by morphological features at 12 h (p < 0.05). In addition, we observed higher levels of activated caspase 3 (17 kDa) from 0 to 30 min. Altogether, these data indicate a direct effect of TGF beta 1 signaling through Smads on both inhibiting proliferation, through alteration of cycle proteins, and inducing apoptosis of gastric epithelial cells in vivo. Further, the studies suggest a potential role for both milk and tissue-expressed TG beta 1 in gastric growth during postnatal development, (C) 2007 Elsevier B.V. All rights reserved.

Effects of Cobalt Chloride in Junctional Epithelium and Reduced Enamel Epithelium of the Rat Maxillary First Molar

Cobalt is one of the main components of cast metal alloys broadly used in dentistry. It is the constituent of 45 to 70% of numerous prosthetic works. There are evidences that metal elements cause systemic and local toxicity. The purpose of the present study was to evaluate the effects of cobalt on the junctional epithelium and reduced enamel epithelium of the first superior molar in rats, during lactation. To do this, 1-day old rats were used, whose mothers received 300mg of cobalt chloride per liter of distilled water in the drinker, during lactation. After 21 days, the rat pups were killed with an anesthetic overdose. The heads were separated, fixed in ""alfac"", decalcified and embedded in paraffin. Frontal sections stained with hematoxylin and eosin were employed. Karyometric methods allowed to estimate the following parameters: biggest, smallest and mean diameters, D/d ratio, perimeter, area, volume, volume/area ratio, eccentricity, form coefficient and contour index. Stereologic methods allow to evaluate: cytoplasm/nucleus ratio, cell and cytoplasm volume, cell number density, external surface/basal membrane ratio, thickness of the epithelial layers and surface density. All the collected data were subjected to statistic analysis by the non-parametric Wilcoxon-Mann-Whitney test. The nuclei of the studied tissues showed smaller values after karyometry for: diameters; perimeter, area, volume and volume/area ratio. Stereologically, it was observed, in the junctional epithelium and in the reduced enamel epithelium, smaller cells with scarce cytoplasm, reflected in the greater number of cells per mm3 of tissue. In this study, cobalt caused epithelial atrophy, indicating a direct action on the junctional and enamel epithelium.

Differential regulation of the renin-angiotensin system by nicotine in WKY and SHR glia

Given that (1) the renin-angiotensin system (RAS) is compartmentalized within the central nervous system in neurons and glia (2) the major source of brain angiotensinogen is the glial cells, (3) the importance of RAS in the central control of blood pressure, and (4) nicotine increases the probability of development of hypertension associated to genetic predisposition; the objective of the present study was to evaluate the effects of nicotine on the RAS in cultured glial cells from the brainstem and hypothalamus of Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. Ligand binding, real-time PCR and western blotting assays were used to compare the expression of angiotensinogen, angiotensin converting enzyme, angiotensin converting enzyme 2 and angiotensin II type1 receptors. We demonstrate, for the first time, that there are significant differences in the basal levels of RAS components between WKY and SHR rats in glia from 1-day-old rats. We also observed that nicotine is able to modulate the renin-angiotensin system in glial cells from the brainstem and hypothalamus and that the SHR responses were more pronounced than WKY ones. The present data suggest that nicotine effects on the RAS might collaborate to the development of neurogenic hypertension in SHR through modulation of glial cells.

Intra-strain variations of baroreflex sensitivity in young Wistar-Kyoto rats

Purpose: To compare baroreflex sensitivity among conscious rats of the same strain. Methods: Male WKY rats (eight weeks old) were studied. Cannulas were inserted into the abdominal aortic artery through the right femoral artery to measure mean arterial pressure (MAP) and heart rate (HR). Baroreflex gain was calculated as the ratio between variation of HR in function of the MAP variation (Delta HR/Delta MAP) tested with a depressor dose of sodium nitroprusside (SNP, 50 mu g/kg, iv) and with a pressor dose of phenylephrine (PE, 8 mu g/kg, iv). We divided the rats into four groups: 1) Low bradycardic baroreflex (LB), BG between -1 and -2 bpm/mmHg tested with PE; 2) High bradycardic baroreflex (HB), BG < -2 bpm/mmHg tested with PE; 3) Low tachycardic baroreflex (LT), BG between -1 and -2 bpm/mmHg tested with SNP and; 4) High tachycardic baroreflex (HT), BG < -2 bpm/mmHg tested with SNP. Significant differences were considered for p<0.05. Results: Approximately 82% of the rats presented reduced bradycardic reflex while 22 showed attenuated tachycardic reflex. No alterations were noted regarding basal MAP and HR, tachycardic and bradycardic peak and HR range. Conclusions: There was alteration in baroreflex sensitivity among rats of the same strain. Care should be taken when interpreting studies employing WKY as a control for the SHR.

The variability of baroreflex sensitivity in juvenile, spontaneously hypertensive rats

In this study the baroreflex sensitivity of conscious, juvenile, spontaneously hypertensive rats (SHRs) was compared. The study population consisted of 19 eight-week-old male SHRs. The baroreflex sensitivity was quantified as the derivative of the variation in heart rate (HR) and the variation of mean arterial pressure (baroreflex sensitivity = Delta HR/Delta MAP). MAP was manipulated with sodium nitroprusside (SNP) and phenylephrine (PHE), administered via an inserted cannula in the right femoral vein. The SHRs were divided into four groups: (1) low bradycardic baroreflex (LB) where the baroreflex gain (BG) was between 0 and 1 bpm/mmHg with PHE; (2) high bradycardic baroreflex (HB), where the BG was < -1 bpm/mmHg with PHE; (3) low tachycardic baroreflex (LT) where the BC was between 0 and 3 bpm/mmHg with SNP; (4) high tachycardic baroreflex (HT) where the BG was > 3 bpm/mmHg with SNP. We noted that 36.8% of the rats presented with an increased bradycardic reflex, while 27.8% demonstrated an attenuated tachycardic reflex. No significant alterations were noted regarding the basal MAP and HR. There were significant differences in the baroreflex sensitivity between SHRs in the same laboratory. One should be careful when interpreting studies employing the SHR as a research model.

Cramoll 1,4 lectin increases ROS production, calcium levels, and cytokine expression in treated spleen cells of rats

This study reports the in vivo stimulatory effects of Cramoll 1,4 on rat spleen lymphocytes as evidenced by an increase in intracellular reactive oxygen species (ROS) production, Ca(2+) levels, and interleukin (IL)-1 beta expression. Cramoll 1,4 extracted from seeds of the Leguminosae Cratylia mollis Mart., is a lectin with antitumor and lymphocyte mitogenic activities. Animals (Nine-week-old male albino Wistar rats, Rattus norvegicus) were treated with intraperitoneal injection of Cramoll 1,4 (235 mu g ml(-1) single dose) and, 7 days later, spleen lymphocytes were isolated and analyzed for intracellular ROS, cytosolic Ca(2+), and IL-6, IL-10, and IL-1 mRNAs. Cell viability was investigated by annexin V-FITC and 7-amino-actinomycin D staining. The data showed that in lymphocytes activated by Cramoll 1,4 the increase in cytosolic and mitochondrial ROS was related to higher cytosolic Ca(2+) levels. Apoptosis and necrosis were not detected in statistically significant values and thus the lectin effector activities did not induce lymphocyte death. In vivo Cramoll 1,4 treatment led to a significant increase in IL-1 beta but IL-6 and -10 levels did not change. Cramoll 1,4 had modulator activities on spleen lymphocytes and stimulated the Th2 response.

Effects of exercise on the morphology of the myenteric neurons of the duodenum of Wistar rats during the ageing process

This study aimed to evaluate the effects of regular physical activity on the morphology of the myenteric plexus of the duodenum in rats during the ageing process. To this end, 45 Wistar rats were divided into three groups: C (sedentary - 6 months old), S (sedentary - 12 months old) and T (trained - 12 months old). The animals of group S were given with a physical activity programme consisting of a 10-min-treadmill workout once a week. The animals of group T were submitted to the physical activity programme five times a week. Their duodenums were collected and submitted to the techniques of nicotinamide adenine dinucleotide (NADH)-diaphorase enzyme histochemistry for whole-mount preparations and transmission electron microscopy. No differences in the constitution of the myenteric plexuses were found when the sedentary and trained groups were compared with the control group. The ultrastructural features were similar for the three groups. However, it was verified that the physical activity of the trained animals resulted in a similar myenteric neuron morphology to that of the adult animals (6 months old), thereby confirming its beneficial effect, as the sedentary animals had larger alterations in the collagen fibrils and the basal membrane that occur through ageing. The quantitative analysis showed that the NADH-diaphorase positive neurons decreased with ageing and increased with physical activity (P > 0.05). No significant alteration (P > 0.05) in the neuronal profile area of the NADH-diaphorase positive neurons has been observed with ageing.

Obesity induced by neonatal treatment with monosodium glutamate impairs microvascular reactivity in adult rats: Role of NO and prostanoids

Background and aim: given that obesity is an independent risk factor for the development of cardiovascular diseases we decided to investigate the mechanisms involved in microvascular dysfunction using a monosodium glutamate (MSG)-induced model of obesity, which allows us to work on both normotensive and normoglycemic conditions. Methods and results: Male offspring of Wistar rats received MSG from the second to the sixth day after birth. Sixteen-week-old MSG rats displayed higher Lee index, fat accumulation, dyslipidemia and insulin resistance, with no alteration in glycemia and blood pressure. The effect of norepinephrine (NE), which was increased in MSG rats, was potentiated by L-nitro arginine methyl ester (L-NAME) or tetraethylammonium (TEA) and was reversed by indomethacin and NS-398. Sensitivity to acetylcholine (ACh), which was reduced in MSG rats, was further impaired by L-NAME or TEA, and was corrected by indomethacin, NS-398 and tetrahydrobiopterin (BH4). MSG rats displayed increased endothelium-independent relaxation to sodium nitroprusside. A reduced prostacyclin/tromboxane ratio was found in the mesenteric beds of MSG rats. Mesenteric arterioles of MSG rats also displayed reduced nitric oxide (NO) production along with increased reactive oxygen species (ROS) generation; these were corrected by BH4 and either L-NAME or superoxide dismutase, respectively. The protein expression of eNOS and cyclooxygenase (COX)-2 was increased in mesenteric arterioles from MSG rats. Conclusion: Obesity/insulin resistance has a detrimental impact on vascular function. Reduced NO bioavailability and increased ROS generation from uncoupled eNOS and imbalanced release of COX products from COX-2 play a critical role in the development of these vascular alterations (C) 2010 Elsevier B.V. All rights reserved.

Chlorpropamide treatment restores the reduced carrageenan-induced paw edema and pleural exudate volume in diabetic rats

Objective and design: Knowing that hyperglycemia is a hallmark of vascular dysfunction in diabetes and that neonatal streptozotocin-induced diabetic rats (n-STZ) present reduced inflammatory response, we decided to evaluate the effect of chlorpropamide-lowered blood glucose levels on carrageenan-induced rat paw edema and pleural exudate in n-STZ. Materials: Diabetes was induced by STZ injection (160 mg/kg, ip) in neonates (2-day-old) Wistar rats. Treatment: n-STZ diabetic rats were treated with chlorpropamide (200 mg/kg, 15 d, by gavage) 8 weeks after STZ injection. Methods: Carrageenan-induced paw edema and pleural exudate volumes were assessed concomitantly with peripheral and exudate leukocyte count. We also evaluated the expression of inducible nitric oxide synthase (iNOS) in lungs of all experimental groups. Results: Chlorpropamide treatment improved glucose tolerance, beta-cell function (assessed by HOMA-beta), corrected paw edema, and pleural exudate volume in n-STZ. Neither leukocyte count nor iNOS expression were affected by diabetes or by chlorpropamide treatment. Conclusion: Chlorpropamide treatment by restoring beta-cell function, reducing blood sugar levels, and improving glucose tolerance might be contributing to the correction of the reduced inflammatory response tested as paw edema and pleural exudate in n-STZ diabetic rats.

Dipeptidyl peptidase IV inhibition attenuates blood pressure rising in young spontaneously hypertensive rats

Objectives The present study aimed to assess the effect of the specific dipeptidyl peptidase IV (DPPIV) inhibitor sitagliptin on blood pressure and renal function in young prehypertensive (5-week-old) and adult spontaneously hypertensive rats (SHRs; 14-week-old). Methods Sitagliptin (40 mg/kg twice daily) was given by oral gavage to young (Y-SHR + IDPPIV) and adult (A-SHR R IDPPIV) SHRs for 8 days. Kidney function was assessed daily and compared with age-matched vehicle-treated SHR (Y-SHR and A-SHR) and with normotensive Wistar-Kyoto rats (Y-WKY and A-WKY). Arterial blood pressure was measured in these animals at the end of the experimental protocol. Additionally, Na(+)/H(+) exchanger isoform 3 (NHE3) function and expression in microvilli membrane vesicles were assessed in young animals. Results Mean arterial blood pressure of Y-SHR + IDPPIV was significantly lower than that of Y-SHR (104 +/- 3 vs. 123 +/- 5 mmHg, P < 0.01) and was similar to Y-WKY (94 +/- 4 mmHg, P > 0.05). Compared to Y-SHR, Y-SHR + IDPPIV exhibited enhanced cumulative urinary flow and sodium excretion and decreased NHE3 activity and expression in proximal tubule microvilli. In the A-SHR, sitagliptin treatment had no significant effect on either renal function or arterial blood pressure. Conclusion Our data suggest that DPPIV inhibition attenuates blood pressure rising in young prehypertensive SHRs, partially by inhibiting NHE3 activity in renal proximal tubule. J Hypertens 29:520-528 (C) 2011 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.