Alterations in gene expressions encoding preproET-1 and NOS in pulmonary tissue in endotoxemic rats

Septic shock is characterized by hypotension and a hyporeactive response to vasopressor agents. The pathogenesis is due to vascular leaks and an increased synthesis of cytokines and nitric oxide (NO). The present study examined the time-dependent alterations of endothelin-1 (ET-1) and the expression of NO synthase (NOS) in lung tissue in a septic rat model. Normal Sprague-Dawley (SD) rats aged 10 weeks received 15 mg/kg lipopolysaccharide (LPS) and then were sacrificed at different time points (1, 3, 6, and 10 hrs). Rats that did not receive LPS were considered to be controls. Both systolic and diastolic pressure decreased in SD rats after LPS administration. Time-dependent onset of features of acute lung injury, such as the infiltration of inflammatory cells and thickening of alveolar septa, were seen in rats that received LPS. A 2.8-fold increase in the expression of preproET-1 level was observed in lung tissue 6 hrs after LPS administration. The expression of endothelial NOS (eNOS) was also altered in lung tissue in a time-dependent fashion. After the administration of LPS, there was a 16-fold increase in the expression of eNOS mRNA. The peak expression of inducible NOS (iNOS) in lung tissue specimens obtained from rats that received LPS was 45-fold higher than that in control rats. ET-1 is a potent vasoconstrictor and thereby may play an important role in the pathogenesis of acute lung injury in a septic rat model. The increased expression of NOS may result in excess NO production and may also play a role in the pulmonary complications of endotoxemia.     

Pulmonary expression of preproET-1 and preproET-3 mRNAs is altered reciprocally in rats after inhalation of air pollutants

Perturbation of vascular homeostasis is an important mechanism related to the acute health effects of inhaled pollutants. Inhalation of urban particulate matter and ozone by rats has been shown to result in increased synthesis of the potent vasoactive peptide endothelin (ET)-1 in the lungs, with spillover into the circulation. In the present work, we have analyzed the interrelationships between responses of the three major endothelin isoforms, ET-1[1-21], ET-2[1-21], and ET-3[1-21], to inhaled pollutants at the peptide and gene expression levels. Fisher-344 rats were exposed for 4 hrs by nose-only route to clean air, urban particles EHC-93 (0, 50 mg/m3), ozone (0, 0.8 ppm), or ozone and particles together. Circulating levels of both the ET-1 [1-21] and ET-3[1-21] peptides were increased immediately after exposure to particulate matter or ozone. While expression of preproET-1 mRNA in the lungs increased, expression of preproET-3 mRNA decreased immediately after exposure. PreproET-2 mRNA was not detected in the lungs, and exposure to either pollutant did not affect plasma ET-2 levels. Co-exposure to ozone and particles, while altering lung preproET-1 and preproET-3 mRNA levels in a fashion similar to ozone alone, did not cause changes in the circulating levels of the two corresponding peptides. Thus, de novo synthesis of ET-3 in the lungs is not responsible for the increase of circulating plasma ET-3 after inhalation of pollutants, which implies regulation of preproET-3 at a remote site and, hence, systemic impacts of the pollutants. Upregulation of preproET-1 coupled with down-regulation of preproET-3 in the lungs of animals exposed to air pollutants implies a mismatch of local ET-1/ET(A) receptor-mediated vasoconstriction and ET-3/ET(B) receptor-mediated vasodilation.     

Alterations in brain dopaminergic receptors in developing hypo- and hyperthyroid rats

The administration of an iodide-rich diet to pregnant and lactating rats, and to their weaned litters, or the anti-thyroid drug methimazole injected into high-iodide pups induced a mild and a severe hypothyroidism, respectively. Treatment of newborn rats with triiodothyronine provoked neonatal hyperthyroidism.The binding of [³H]spiroperidol to dopaminergic receptors in striatal membranes from 31 day-old, hypo- and hyperthyroid rats was significantly decreased, compared to euthyroids. These alterations appeared to involve the density of dopaminergic receptors, and not their affinity for the radioligand, the only exception being a hyperthyroidism-caused decrease in the dissociation constant.     

Hetero-oligomerization of adenosine A1 receptors with P2Y1 receptors in rat brains

Adenosine and ATP modulate cellular and tissue functions via specific P1 and P2 receptors, respectively. Although, in general, adenosine inhibits excitability and ATP functions as an excitatory transmitter in the central nervous system, little is known about the direct interaction between P1 and P2 receptors. We recently demonstrated that the G(i/o)-coupled adenosine A1 receptor (A1R) and G(q/11)-coupled P2Y1 receptor (P2Y1R) form a heteromeric complex with a unique pharmacology in cotransfected HEK293T cells using the coimmunoprecipitation of differentially epitope-tagged forms of the receptor [Yoshioka et al. (2001) Proc. Natl. Acad. Sci. USA 98, 7617-7622], although it remained to be determined whether this hetero-oligomerization occurs in vivo. In the present study, we first demonstrated a high degree of colocalization of A1R and P2Y1R by double immunofluorescence experiments with confocal laser microscopy in rat cortex, hippocampus and cerebellum in addition to primary cultures of cortical neurons. Then, a direct association of A1R with P2Y1R was shown in coimmunoprecipitation studies using membrane extracts from these regions of rat brain. Together, these results suggest the widespread colocalization of A1R and P2Y1R in rat brain, and both receptors can exist in the same neuron, and therefore associate as hetero-oligomeric complexes in the rat brain.     

Alterations in central and peripheral adrenergic receptors in deoxycorticosterone/salt hypertensive rats

The treatment of uninephrectomized rats with deoxycorticosterone (DOCA) and salt for 6 weeks caused a significant systolic hypertension and cardiac and renal hypertrophy. There was a significant decrease in the density of cardiac α₁- and β-, and renal α₁-adrenoceptors in DOCA/salt hypertensive rats, as compared to uninephrectomized salt loaded control rats. In contrast, the cerebral cortex, corpus striatum, hippocampus and hypothalamus/thalamus of hypertensive rats showed a significant increase in adrenoceptor binding in these hypertensive rats. In contrast, muscarinic cholinergic receptors and [³H]yohimbine binding sites were not altered in most tissues of the hypertensive rats. The present study suggests an important role for central and peripheral α₁- and β-adrenoceptors in the pathogenesis of hypertension.     

Obesity reduced the gene expressions of leptin receptors in hypothalamus and liver.

The expression of leptin receptor (OB-R) is downregulated by leptin in some cell lines. This study investigated the expressions of leptin receptors at central nerve system and peripheral site in a dietary model of obesity. Rats in the 8 week high-diet and control group were classified based on body weight gain into obese and control groups. Serum leptin and insulin concentrations were measured and gene expressions of short form of leptin receptor (OB-Ra) and long form (OB-Rb) in hypothalamus and liver were detected by RT-PCR. The levels of serum leptin in obese rats were increased compared with control rats (p<0.05). The levels of OB-Ra and OB-Rb gene expressions in both hypothalamus and liver in obese rats were reduced significantly (p<0.01). Serum leptin concentrations of obese rats had a significant negative relationship with both of OB-Ra or OB-Rb gene expression levels in hypothalamus and liver (p<0.01). On the other hand, serum insulin levels had no relationship with OB-Ra or OB-Rb gene expression levels in neither liver nor hypothalamus. Rats with diet-induced obesity have hyperleptinemia and reduced expressions of leptin receptors in hypothalamus and liver. The results suggest that a leptin downregulated OB-R expression is one of leptin resistant mechanisms for maintaining obesity.     

Alterations in the binding characteristics of glucocorticoid receptors from obese Zucker rats

Obese Zucker rats appear to lack a circadian rhythm of serum corticosterone and maintain relatively high concentrations throughout the 24-h day. The binding characteristics of glucocorticoid receptors in lean and obese Zucker rats were examined in three tissues suggested to be involved in the feedback inhibition of corticosterone: the anterior pituitary, hypothalamus and hippocampus. Hepatic glucocorticoid receptors were also examined to determine if receptor alterations exist in a peripheral tissue. The dissociation constant (Kd) of glucocorticoid receptors in the anterior pituitary of obese rats was 50% greater than the Kd of receptors derived from lean rats. This suggests a decrease in the affinity of these receptors and could indicate a reduced feedback inhibition of corticosterone at the anterior pituitary. Hepatic glucocorticoid receptors of obese rats also showed an increase (150%) in the Kd of binding and a reduction (40%) in the number of receptors. No difference was observed in the Kd or maximal binding of receptors from the hypothalamus or hippocampus of lean and obese rats. It appears that glucocorticoid receptor alterations exist in obese Zucker rats and that these alterations may affect the drive of the pituitary-adrenal axis and possibly the expression of obesity.     

Role of ET(A) receptors in experimental ANG II-induced hypertension in rats

The objectives were to determine if ANG II-induced hypertension is maintained by activation of endothelin type A (ET(A)) receptors by endogenous ET-1 and if this effect is influenced by salt intake. Male rats were maintained on high sodium intake (HS; 6 meq/day) or on normal sodium intake (NS; 2 meq/day). Hypertension was produced by intravenous infusion of ANG II (5 ng/min) for 15 days. Five-day oral dosing with the selective ET(A)-receptor antagonist ABT-627 (~2 mg. kg(-1). day(-1)) reduced mean arterial pressure (MAP) to baseline levels in rats on HS receiving ANG II infusion, but it did not affect MAP in normotensive HS controls. In rats on NS, ABT-627 only transiently decreased MAP in rats receiving ANG II and slightly reduced MAP in normotensive controls. ABT-627 produced mild retention of sodium and water in NS rats receiving ANG II, but not in any other group. These results indicate that ET-1 plays a role in ANG II-induced hypertension via activation of ET(A) receptors and that this role is more prominent in rats on HS.     

The relationship between glucagon receptors and metabolic profile in two strains of rats: Wistar-Furth and Sprague-Dawley

We studied glucagon and insulin binding to isolated hepatocyte receptors in Wistar-Furth (WF) and Sprague-Dawley (SD) rats, using 125I-labeled hormones. Hepatocytes from WF rats bound more glucagon than hepatocytes from SD rats. There were no differences in insulin binding. These observations prompted us to investigate other strain differences. Fasting and nonfasting serum glucose, glucagon, insulin, and growth hormone were measured. WF animals had a lower fasting glucose and higher fasting glucagon than SD animals, while SD rats had higher nonfasting insulin levels and a higher hepatic glycogen content. Total hepatic glucose production in response to glucagon (10(-8) M) was greater in WF than in SD rats, while glucagon-stimulated gluconeogenesis from alanine was the same in the two groups of animals. We concluded that the decreased glucagon binding does not play a significant role in the maintenance of serum glucose or in the gluconeogenetic response glucagon, and that neither these responses nor the serum glucagon levels appears to be correlated with the number of glucagon receptors. We conclude further that different animal strains of the same species may differ in their biologic responses.     

Food consumption and mouse-killing in Sprague-Dawley rats

The effects of daily 10-min mouse exposure tests on food consumption were studied in killer and non-killer Sprague-Dawley rats. The results showed both mouse-killers and non-killers significantly to decrease their food consumption when tested daily for mouse-killing. The effect was more pronounced in mouse-killers than in non-killers and the day-to-day individual fluctuation of food consumption was greater in mouse-killers during the mouse confrontation period.     

Postnatal developmental expressions of neurotransmitters and receptors in various brain stem nuclei of rats.

Previously, we reported that the expression of cytochrome oxidase in a number of brain stem nuclei exhibited a plateau or reduction at postnatal day (P) 3-4 and a dramatic decrease at P12, against a general increase with age. The present study examined the expression of glutamate, N-methyl-D-aspartate receptor subunit 1 (NMDAR1), GABA, GABAB receptors, glycine receptors, and glutamate receptor subunit 2 (GluR2) in the ventrolateral subnucleus of the solitary tract nucleus, nucleus ambiguus, hypoglossal nucleus, medial accessory olivary nucleus, dorsal motor nucleus of the vagus, and cuneate nucleus, from P2 to P21 in rats. Results showed that 1) the expression of glutamate increased with age in a majority of the nuclei, whereas that of NMDAR1 showed heterogeneity among the nuclei; 2) GABA and GABAB expressions decreased with age, whereas that of glycine receptors increased with age; 3) GluR2 showed two peaks, at P3-4 and P12; and 4) glutamate and NMDAR1 showed a significant reduction, whereas GABA, GABAB receptors, glycine receptors, and GluR2 exhibited a concomitant increase at P12. These features were present but less pronounced in hypoglossal nucleus and dorsal motor nucleus of the vagus and were absent in the cuneate nucleus. These data suggest that brain stem nuclei, directly or indirectly related to respiratory control, share a common developmental trend with the pre-Botzinger complex in having a transient period of imbalance between inhibitory and excitatory drives at P12. During this critical period, the respiratory system may be more vulnerable to excessive exogenous stressors.     

Retinal dystrophy in Sprague-Dawley rats.

A spontaneous retinal dystrophy was found in 4 percent of Sprague-Dawley-derived rats examined. The lesion occurred both unilaterally and bilaterally in equal frequency, but the incidence in the females was 2 times greater than in males. Retinal change consisted of focal or diffuse absence of the outer layers of the retina, but frank degenerative changes or progression of the lesion was not observed. The cause of the dystrophy was not determined, but its increased occurrence with increasing age of the rats suggests an age-related lesion.     

Endothelin ET(A) and ET(B) receptors in postnatal intestine

We aimed to characterize endothelin (ET) receptors in the swine intestinal vasculature and to determine ischemia-reperfusion (I/R) effects on these receptors. Saturation and competitive binding assays were performed on mesenteric artery protein membranes from 1- and 40-day-old animals, both control and those subjected to 1 h of partial ischemia followed by 6 h of reperfusion in vivo. Scatchard analysis of saturation binding with (125)I-labeled ET-1 in membranes from endothelium-denuded (E(-)) vessels revealed that the maximum number of binding sites was greater in younger animals. Competitive (125)I-ET-1 binding was significant for a one-site model with ET-1, ET-3, and sarafotoxin S6c (S6c) in membranes from endothelium-intact (E(+)) and E(-) vessels in both age groups. The maximum number of ET-1 binding sites was significantly greater in younger animals. In the presence of the ET(A) receptor antagonist BQ-123, competitive (125)I-ET-1 binding was significant for a one-site model with ET-1 and S6c in membranes from E(+) vessels in both age groups. The maximum number of ET-1 binding sites was significantly greater in younger animals. After I/R, the maximum number of ET-1 binding sites was unchanged. In the presence of BQ-123, specific binding by ET-1 and S6c was eliminated in both age groups after I/R. These results suggest that both ET receptor populations are expressed to a greater degree in younger animals and I/R significantly affects the ET(B) receptor.     

Opposite regulation of hypothalamic orexin and neuropeptide Y receptors and peptide expressions in obese Zucker rats

Many hyothalamic neuropeptides are involved in the regulation of food intake and body weight. The orexins (OX) which are synthesized in the lateral hypothalamus are among the most recently characterized whereas neuropeptide Y (NPY) belongs to a group of "older" peptides extensively studied for their effects on feeding behavior. Both stimulate food ingestion in rodents. In this experiment, we measured the expressions of these peptides as well as of their receptors (OX1-R and OX2-R, Y1 and Y5) in the hypothalamus of obese hyperphagic and lean Zucker rats by real-time RT-PCR using the TaqMan apparatus. NPY mRNA expression in the obese rats was significantly increased by a factor of 10 (P < 0.002) whereas expressions of the Y1 and Y5 receptors were decreased by 25% (P < 0.01) and 50% (P < 0.002), respectively. Their prepro-orexin mRNA expression was more than twofold decreased (P < 0.01) and expressions of their OX receptors 1 and 2 mRNA were five- and fourfold increased (P < 0.05), respectively. An inverse phenomenon was therefore noted between the two peptides: for NPY, increased levels and downregulation of receptors; and for OX, diminished levels with upregulation of receptors. The reasons for these changes might be linked to the absence of leptin signaling as similar profiles are found in the ob/ob mice. For orexins at least, other factors such as hyperglycemia might be involved. Based on anatomical considerations, a direct effect of NPY or of other brain peptides such as CRH cannot be excluded. We conclude that the diminution in the OX tone might participate in a counterregulatory system necessary to limit the noxious effects of NPY on food intake and body weight.     

硫化氢与内毒素血症大鼠心肌损伤的关系

为探讨硫化氢（hydrogen sulfide,H2S）与内毒素血症大鼠心肌损伤的关系,采用静脉注射脂多糖（lipopolysaccharide,LPS）的方法制备内毒素血症大鼠模型,将雄性Wistar大鼠随机分为正常对照组、LPS组、LPS＋炔丙基甘氨酸（propargylglycine,PPG,H2S代谢酶抑制剂）组、LPS＋NaHS（H2S供体）组。观察给药后4h内大鼠平均动脉压（mean arterial pressure,MAP）的变化,测定血浆肿瘤坏死因子-α（tumor necrosis factor-α,TNF-α）和H2S含量,光学显微镜观察心肌组织形态学变化并测定心肌组织中TNF-α、H2S含量及乳酸脱氢酶（lactate dehydrogenase,LDH）和髓过氧化物酶（myeloperoxidase,MPO）的活性。结果如下：（1）与正常对照组相比,LPS组大鼠血压迅速下降,血浆TNF-α、H2S含量显著增高,且血浆中H2S含量与血压呈显著负相关,LPS注射后1、2、4h时相关系数分别为-0．936、-0．913和-0．908（均P〈0．05）;心肌组织TNF-α、H2S含量及LDH、MPO活性也明显升高,并出现组织损伤;（2）给予PPG能显著抑制血浆TNF-α、H2S含量的增高,并可显著减轻LPS所致的血压下降（均P〈0．05）和心肌组织损伤,降低心肌组织中TNF-α、H2S含量及LDH、MPO活性;（3）给予NaHS后,与LPS组相比,大鼠血浆TNF-α、H2S含量增高,血压明显下降（均P〈0．05）,心肌组织损伤加重,心肌组织中TNF-α、H2S含量及LDH、MPO活性增高。结果提示,内毒素血症大鼠低血压和心肌损伤的部分原因是由于H2S生成增多。     

Alterations in the accumulation patterns of polyamines in brains of myelin-deficient mice

Quaking mutants and jimpy mutants of mice have known deficiencies of myelination of the central nervous system, as well as lesser involvement of the peripheral nervous system. Both mutants also have altered polyamine synthesis and accumulation, particularly in the hindbrain and spinal column. The ratio of spermidine/spermine, which generally is higher in tissues with high rates of biosynthetic activity, was significantly lower in the mutants as compared to their normal siblings. In quaking mutants, 5 months of age, the spermidine concentration of hindbrain and spinal column was 60% that of controls. In contrast, the decreased spermidine/spermine ratio in jimpy mutants resulted from a marked increase in the spermine concentration in both forebrain and hindbrain. Alterations in the spermidine/spermine ratio could lead to reductions in the biosynthetic potential of the brain during development.