Content of endogenous opiates and adrenocorticotropic hormone in the brain structures of rats of different ages

The contents of beta-endorphin (BE), methionine-enkephalin (MEK), and adrenocorticotropic hormone (ACTH) in the hypophysis and hypothalamus of intact 4- to 6-week-old and 16-week-old Wistar rats was studied. The maximum BE concentration was found in the hypophysis, whereas the maximum MEK and ACTH concentrations were found in the hypothalamus. Aging was followed by a decrease in the concentrations of all above substances, except BE, whose concentration in the hypophysis of the older rat group was markedly higher than in the hypophysis of 4- to 6-week-old animals.     

Receptor-mediated uptake of hypertriglyceridemic very low density lipoproteins by normal human fibroblasts

Our previous studies showed that very low density lipoproteins, Sf 60-400 (VLDL), from hypertriglyceridemia subjects, but not VLDL from normolipemic subjects, suppress HMG-CoA reductase activity in normal human fibroblasts. To determine if this functional abnormality of hypertriglyceridemic VLDL resulted from differences in uptake of the VLDL by the low density lipoprotein (LDL) receptor pathway, we isolated VLDL subclasses from the d less than 1.006 g/ml fraction of normal and hypertriglyceridemic plasma by flotation through a discontinuous salt gradient for direct and competitive binding studies in cultured human fibroblasts. VLDL from the plasma of subjects with hypertriglyceridemia types 4 and 5 were at least as effective as normal LDL in competing for 125I-labeled LDL binding, uptake, and degradation when compared either on the basis of protein content or on a particle basis. By contrast, normolipemic Sf 60-400 VLDL were ineffective in competing with the degradation of 125I-labeled LDL, and Sf 20-60 VLDL (VLDL3) were less effective in reducing specific 125I-labeled LDL degradation than were LDL, consistent with their effects on HMG-CoA reductase activity. In direct binding studies, radiolabeled VLDL from hypertriglyceridemic but not normolipemic subjects were bound, internalized, and degraded with high affinity and specificity by normal fibroblasts. Uptake and degradation of iodinated hypertriglyceridemic VLDL Sf 100-400 showed a saturable dependence on VLDL concentration. Specific degradation plateaued at approximately 25 micrograms VLDL protein/ml, with a half maximal value at 6 micrograms/ml. The most effective competitor of hypertriglyceridemic VLDL uptake and degradation was hypertriglyceridemic VLDL itself. LDL were effective only at high concentrations. Uptake of normal VLDL by normal cells was a linear rather than saturable function of VLDL concentration. By contrast, cellular uptake of the smaller normal VLDL3 was greater than uptake of larger VLDL and showed saturation dependence. After incubation of normal VLDL with 125I-labeled apoprotein E, reisolated 125I-E-VLDL were as effective as LDL in suppression of HMG-CoA reductase activity, suggesting that apoE is involved in receptor-mediated uptake of large suppressive VLDL. We conclude that 1) hypertriglyceridemic VLDL Sf 60-400 are bound, internalized, and degraded by normal fibroblasts primarily by the high affinity LDL receptor-mediated pathway; 2) by contrast, normal VLDL, Sf 60-400 are bound, internalized, and degraded by normal fibroblasts primarily by nonspecific, nonsaturable routes; and 3) of the normal VLDL subclasses, only the smallest Sf 20-60 fraction is bound and internalized via the LDL pathway.     

Theophylline stimulation of gluconeogenesis from alanine in normal and sympathectomized rats.

The influence of theophylline ethylenediamine (100 mg/kg i.p.) on gluconeogenesis was studied in normal and in adrenodemedullated and reserpinized rats after overnight fasting by measuring the time-course of Alanine-14C incorporation into Glucose-14C. In the normal rat, theophylline produced a moderate hyperglycemia associated with an increased conversion of alanine to glucose at all time intervals. In addition, a marked rise of plasma levels of insulin and glucagon was observed. In sympathetctomized rats, plasma glucose and gluconeogenesis were again enhanced by theophylline, but the pattern of these modifications differed from that of normal rats since the peak values occurred earlier. Subsequently, both parameters rapidly declined reaching values lower than controls at the end of the experiment. Insulin response to theophylline was higher in sympathectomized animals in comparison to normal rats, while glucagon response was approximately of the same magnitude in the two groups. From these findings it was concluded that theophylline is able to stimulate gluconeogenesis from alanine both in the normal and sympathectomized rat. The different pattern of alanine conversion to glucose seems to depend on the different participation of insulin and catecholamines in the two groups.     

The stimulation of normal human melanocyte proliferation in vitro by melanocyte growth factor from bovine brain

Cell culture conditions for the selective growth and serial propagation of normal human melanocytes from epidermal tissue are described. In addition to the presence of 2% fetal bovine serum, the human melanocyte cell culture environment contains the following growth factor supplements: epidermal growth factor (10 ng/ml), triiodothyronine (10(-9) M), hydrocortisone, (5 X 10(-5) M), insulin (10 micrograms/ml), transferrin (10 micrograms/ml), 7S nerve growth factor (100 ng/ml) cholera toxin (10(-10) M), and bovine brain extract (150 micrograms/ml). The ability to establish selectively the human melanocyte in vitro has been attributed to the contrast between human epidermal keratinocytes and melanocytes for attachment to fibronectin, while the growth of the human melanocyte has been attributed to the mitogenic activity of the growth factor-supplemented medium. Human melanocytes can be cultivated for at least 15 cumulative population doublings and are capable of [3H]-Dopa incorporation. The growth factor-supplemented medium contains a neutral extract from bovine brain that is a potent source of a human melanocyte mitogen. The biological activity of melanocyte growth factor is described as a heat and alkaline-labile mitogen with an estimated molecular weight of 30,000 by gel exclusion chromatography and a weakly cationic isoelectric point. The mitogen is capable of stimulating the growth of quiescent populations of human melanocytes in vitro. The ability to isolate and propagate normal human melanocytes in vitro permitted an examination of the expression of fibronectin and tissue plasminogen activator. Human epidermal melanocytes established in culture do not contain either tissue plasminogen activator or fibronectin. In contrast, human melanoma cell lines contain immunologically detectable fibronectin and tissue plasminogen activator. The absence of tissue plasminogen activator and fibronectin in normal human melanocytes also occurs under conditions of co-cultivation with human melanoma cells. These contrasts between normal human melanocytes and human melanoma cells may be relevant to the metastatic capabilities of human melanoma.     

Insulin stimulation of hepatic malic enzyme activity in normal and diabetic rats controlled by different regulatory processes

A comparison of the processes controlling the increase in hepatic malic enzyme activity in insulin-treated normal and diabetic rats indicated the existence of two distinct regulatory mechanisms. Livers were removed at 12, 36, and 60 h after insulin treatment of normal and alloxan-diabetic rats, and the activity, quantity, and specific activity (units/nmol), of malic enzyme was determined. In normal rats, a significant increase in activity occurred 12 h after insulin, whereas 36 h of insulin treatment was required for diabetic rats to show an increase in enzyme activity. This suggested that the return of malic enzyme activity from the depleted levels measured in diabetic rats probably involved a different sequence of events. A malic enzyme specific radioimmunoassay confirmed this. The increase in activity in insulin-treated normal rats was due to an increase in the quantity of malic enzyme. In insulin-treated diabetic rats, the increase in activity resulted from increases in both enzyme quantity and the specific activity of the enzyme, which returned to levels observed in normal rats.     

Progressive brain metabolic changes under deep brain stimulation of subthalamic nucleus in parkinsonian rats

Deep brain stimulation (DBS) of the subthalamic nucleus (STN) is an efficient neurosurgical treatment for advanced Parkinson's disease. Non‐invasive metabolic neuroimaging during the course of DBS in animal models may contribute to our understanding of its action mechanisms. Here, DBS was adapted to in vivo proton magnetic resonance spectroscopy at 11.7 T in the rat to follow metabolic changes in main basal ganglia structures, the striatum, and the substantia nigra pars reticulata (SNr). Measurements were repeated OFF and ON acute and subchronic (7 days) STN‐DBS in control and parkinsonian (6‐hydroxydopamine lesion) conditions. Acute DBS reversed the increases in glutamate, glutamine, and GABA levels induced by the dopamine lesion in the striatum but not in the SNr. Subchronic DBS normalized GABA in both the striatum and SNr, and glutamate in the striatum. Taurine levels were markedly decreased under subchronic DBS in the striatum and SNr in both lesioned and unlesioned rats. Microdialysis in the striatum further showed that extracellular taurine was increased. These data reveal that STN‐DBS has duration‐dependent metabolic effects in the basal ganglia, consistent with development of adaptive mechanisms. In addition to counteracting defects induced by the dopamine lesion, prolonged DBS has proper effects independent of the pathological condition.

     

Receptor-related interactions of opiates with PGE-induced adenylate cyclase in brain

The inhibition by opiates of the PGE2-induced formation of cAMP in slices from rat brain striatum was investigated. A maximal, 3.5-fold increase over the basal level of cAMP was obtained with an EC50 for PGE2 of 3 microM. Opiate agonists of both mu and kappa type were inhibitory. The IC50 values for morphine, levorphanol and ethylketocyclazocine (EKC) were 110 nM, 80 nM and 25 nM, respectively. These values were similar to the potencies of the compounds in displacing stereospecifically bound 3H-etorphine in rat brain membranes. As evidenced by the inactivity of dextrorphan, the inhibition of PGE2-dependent cAMP formation was stereospecific. Also ineffective were the opiate antagonists naloxone, naltrexone and MR 2266. These compounds did, however, reverse the inhibition by agonists, displaying thereby selectivity toward the putative mu and kappa opiates. Thus, the inhibition by morphine was antagonized to a greater degree by naloxone than by MR 2266, and the action of EKC was blocked more effectively by MR 2266 relative to naloxone.     

Acute stimulation by glucocorticoids of gluconeogenesis from lactate/pyruvate in isolated hepatocytes from normal and adrenalectomized rats

Dexamethasone stimulated gluconeogenesis from lactate/pyruvate in suspensions of hepatocytes isolated from both adrenalectomized and normal fasted rats. This stimulation was observed in incubations with 1 mM pyruvate and at a lactate/pyruvate ratio of 25 but not at a ratio of 10-13. At a lactate/pyruvate ratio of 10-13, the stimulation by dexamethasone was progressively enhanced as the pyruvate concentration was decreased to 0.25 mM. Concurrent administration of a maximally stimulating concentration of dexamethasone with angiotensin II or glucagon yielded an additive stimulation at all concentrations of the peptide hormones tested. No potentiating or permissive actions of acute glucocorticoid administration were observed using hepatocytes from either normal or adrenalectomized animals. The acute stimulation by dexamethasone was antagonized by prior addition of progesterone or cortexolone to the hepatocyte suspensions. Triamcinolone and corticosterone also stimulated gluconeogenesis. Concentrations of the active glucocorticoids needed to elicit half-maximal stimulations (Kact) were approximately 100 nM for dexamethasone and triamcinolone and 400 nM for corticosterone. Deoxycorticosterone, 17 alpha-methyltestosterone, and 5 beta-dihydrocortisol did not stimulate. Stimulation of gluconeogenesis by dexamethasone was seen following a lag averaging 9 min after the time of steroid addition. Preliminary evidence suggests that this effect was not dependent upon a stimulation of protein synthesis, but the observed stimulation and inhibition of control rates of gluconeogenesis by cycloheximide and cordycepin, respectively, demonstrate the difficulties of working with such inhibitors in attempting to answer this question.     

Levels of manganese and iron in the brain of normal and manganese-poisoned rats

Forty rats were poisoned by manganese inhalation and were sacrificed after six and nine months. Tissue concentrations (in mg/g dry weight) were measured in brain, cerebellum and stem-brain of the experimental animals and the twenty control rats, for atomic absorption spectometry. The increase in tissue manganese concentrations were very pronounced in cerebellum, and minor in brain and stem-brain. The tissue iron concentrations undergo a light increase in cerebellum and stem-brain.     

Adenosine-receptor-mediated stimulation of low-Km GTPase in guinea-pig cerebral cortex.

Inhibition of receptor-coupled adenylate cyclase by hormones is proposed to be associated with GTP hydrolysis. Since adenosine inhibits cerebral-cortical adenylate cyclase via A1 adenosine receptors, the present study attempts to verify this mechanism for A1-selective adenosine derivatives. In guinea-pig cortical membranes N6-(phenylisopropyl)adenosine (PIA) increased the Vmax. of the low-Km GTPase, with an EC50 (concentration causing 50% of maximal stimulation) of about 0.1 microM, and the stimulatory effect was competitively antagonized by 5 microM-8-phenyltheophylline. The rank order of potency of the stereoisomers of PIA and of 5-(N-ethylcarboxamido)adenosine (NECA) to stimulate GTPase correlated with their ability to inhibit adenylate cyclase activity (R-PIA greater than NECA greater than S-PIA). Competition binding studies with (-)-N6- ([125I]iodo-4-hydroxyphenylisopropyl)adenosine suggest that adenylyl imidodiphosphate (p[NH]ppA), an essential component of the GTPase assay system, is a more potent A1-receptor agonist than ATP, with an IC50 (concentration giving half-maximal displacement of radioligand binding) of 7.9 microM. On the basis of the p[NH]ppA concentration used in the GTPase assay (1.25 mM), enzyme stimulation by adenosine seems to be highly underestimated. Nevertheless, adenosine-induced GTP hydrolysis reflects an increased turnover of guanine nucleotides at the Ni regulatory site and appears to be a crucial step in the sequence of events processing the inhibitory signal to adenylate cyclase.     

Direct neural effect of lateral hypothalamic stimulation on insulin secretion by pancreases of normal and obese rats

Perfusion of CNS intact pancreases with 200 mg/dl glucose with concomitant lateral hypothalamic area (LHA) stimulation significantly inhibited insulin secretion both in normal and obese rats. Sprague-Dawley, Zucker lean (FaFa) and Zucker obese (fafa) rats all responded in a similar manner, suggesting a general effect unrelated to metabolic state. Insulin secretion during mins 25-40 of perfusion was inhibited in Sprague Dawley, lean Zucker and obese Zucker rats by 31%, 42% and 33%, even though LHA stimulation took place from mins 20-25. Thus, the duration of inhibition was greater than the period of LHA stimulation, indicating that this pathway can induce prolonged changes in the responsiveness of the pancreas. The data presented in this study demonstrate that LHA stimulation, in the absence of humoral factors, results in a direct CNS-mediated suppression of insulin secretion which is relatively long lasting. This effect may illustrate a basic control mechanism by the CNS to regulate the endocrine pancreas.     

刺激大鼠穹窿下器官诱发饮水和脑内c—fos表达的胆碱能机制

实验以饮水行为脑内c-fos表达为指标,,观察刺激大鼠穹窿下器官（SFO）的效应,结果显示,刺激SFO能诱发明显的饮水行为,与此同时,前脑8个部位（终板血管器官,正中视前核,室旁核,视上核,下丘脑外侧区,穹窿周核背侧区,丘脑联合核和无名质）和后脑3个部位（最后区,孤束核和壁旁外侧核）的Fos蛋白表达明显增强,免疫组化双重染色结果显示,刺激SFO能诱导视上核和室旁核中部分神经元呈Fos蛋白和加压素共同表达。脑室注射阿托品能部分阻断刺激SFO诱发的饮水行为,脑内上述各部位所诱导的Fos蛋白表达也明显减弱,以上结果提示,M胆碱能机制参与 刺激SFO诱发的饮水行为和脑内Fos蛋白的表达。     

Catecholamine concentration in discrete brain areas following the withdrawal of barbital dependent rats

Noradrenaline (NA) and dopamine (DA) concentrations were measured in 5 discrete brain areas of barbital dependent rats following 0, 1 or 2 days of drug withdrawal. Statistically significant decreases in NA concentration were observed in the cerebral cortex and the thalamus of 1 day withdrawn rats while NA concentration in the hypothalamus was significantly reduced during the second day of withdrawal. The concentration of DA was significantly elevated in barbital dependent rats but declined following barbital withdrawal. Compared to control or nonwithdrawn rats, the concentration of DA in the thalamus was elevated by the second day of withdrawal. The changes in catecholamine concentration presumably reflect underlying effects of chronic barbital consumption or subsequent withdrawal on the synthesis, metabolism or utilization of these neurohumors.