ABSTRACT
Incubation of [3H]-tyrosine methionine5-enkephalin (MET) with human brain preparations (100,000g supernatant; sections of the limbic system, thalamus, basal ganglia, cerebellum, and cortex) results in its rapid and complete degradation; over 95% of the initial labeled tyrosine is recovered as the free aminoacid within 10 min. Results show a considerable range in the peptide initial velocity (Iv) and half-life (t1/2) degradation values obtained from different brain sections of individual brains, either from the same or from different main brain areas. This relatively wide range of values was scattered, failing to identify consistent differences between the various brains areas studied. Differences in brain tissue storage time or repeated sample freezing and thawing failed to alter significantly either of these kinetic parameters of MET metabolism. Peptide degradation rate (optimum pH and temperature of 7.4 and 37 degrees C, respectively) was concentration-dependent inhibited by known aminopeptidase inhibitors (puromycin, bacitracin, and bestatin, and to a lesser extent by thioridazine). However, it was not significantly affected by either N-carboxymethyl phenyl leucine, captopril or thiorphan [dipeptidyl peptidase(s) or peptidyl dipeptidase(s) inhibitors, respectively]. A better understanding of the mechanisms regulating brain MET metabolism may contribute to the rational design of pharmacological strategies based in the modulation of its bioavailability.
Subject(s)
Brain/metabolism , Enkephalin, Methionine/metabolism , Aged , Half-Life , Humans , Kinetics , Male , Middle AgedABSTRACT
Leptin is a cytokine produced by white adipose tissue that circulates in direct proportion to adiposity and is an important signal of energy balance. Leptin inhibits food intake in rodents by inhibiting the orexigenic neuropetides neuropeptide Y (NPY) and agouti regulated peptide (AgRP) and stimulating the anorexigenic neuropeptides alpha-melanocyte-stimulating hormone (alpha-MSH) and cocaine-amphetamine-regulated transcript (CART). In order to extend our understanding of neuroendocrine regulation of appetite in the primate, we determined the effect of a metabolic challenge on CART, NPY, and leptin receptor (Ob-R) messenger ribonucleic acid (mRNA) in the nonhuman primate (NHP) hypothalamus. Ten adult female rhesus monkeys were either maintained on a regular diet or fasted for two days before euthanasia. CART, NPY, and Ob-R mRNA were measured by in situ hybridization histochemistry (ISHH). A 2-day fast decreased CART expression in the ARC, increased NPY gene expression in the supraoptic nucleus (SON) and paraventricular nucleus (PVN), and increased Ob-R expression in the ventromedial nucleus (VMN). This is the first report that fasting inhibits CART expression and stimulates Ob-R expression in monkeys. Increased NPY expression in the SON and PVN, but not the ARC of fasted monkeys also is novel. With some exceptions, our observations are confirmatory of findings in rodent studies. Similarities in the neuroendocrine responses to a metabolic challenge in monkeys and rodents support extending existing hypotheses of neuroendocrine control of energy homeostasis to primates.
Subject(s)
Fasting/metabolism , Hypothalamus/metabolism , Macaca mulatta/metabolism , Nerve Tissue Proteins/metabolism , Neuropeptide Y/metabolism , Receptors, Cell Surface/metabolism , Animals , Energy Metabolism/genetics , Energy Metabolism/physiology , Female , Nerve Tissue Proteins/genetics , Neuropeptide Y/genetics , RNA, Messenger/analysis , Receptors, Cell Surface/genetics , Receptors, Leptin , Statistics, NonparametricABSTRACT
Drug addiction results from a subversion of neural circuits that control motivation. Although the hedonic and addictive properties of psychostimulants and drugs of abuse are predominantly attributed to dopamine and glutamate, it is appreciated that other signaling molecules in the brain are important. This study suggests that cocaine- and amphetamine-regulated transcript (CART) peptides modulate the locomotor and motivational properties of psychostimulants. The behavioral effects of cocaine and amphetamine were examined in Carttm1Amgen knockout (Cart KO) and wild-type (WT) mice. Acute amphetamine administration increased in locomotor activity in WT mice, but this response was attenuated in Cart KO mice. Repeated amphetamine produced locomotor sensitization in WT mice but hardly any in Cart KO mice. Amphetamine elicited conditioned place preference in both genotypes, but amphetamine's potency was reduced in the Cart KO mice. Intravenous cocaine self-administration was observed in both genotypes, but Cart KO mice consumed less cocaine and responded less for cocaine than WT mice. The behavioral effects of psychostimulants were reduced in the mutant Cart KO mice. By contrast, open field activity and sucrose preference of drug-naive mice WT and Cart KO mice were not significantly different. The attenuated effects of amphetamine and cocaine in Cart KO mice suggest a positive neuromodulatory role for CART peptides in the locomotor and motivational properties of psychostimulants and implicate CART peptides in psychostimulant addiction.
Subject(s)
Central Nervous System Stimulants/pharmacology , Conditioning, Operant/drug effects , Motor Activity/drug effects , Nerve Tissue Proteins/administration & dosage , Neurotransmitter Agents/administration & dosage , Amphetamine/administration & dosage , Animals , Choice Behavior/drug effects , Cocaine/administration & dosage , Drug Administration Schedule , Drug Interactions , Injections, Intraperitoneal , Male , Mice , Mice, Knockout , Motivation , Peptide Fragments/administration & dosageABSTRACT
CART (Cocaine- and Amphetamine-Regulated Transcript) peptides modulate food intake and psychostimulant drug actions. Several CART peptides that contain multiple disulfide bonds were produced by overexpression in Escherichia coli bacteria as fusion products with a C-terminal histidine tag. Since these peptides were found denatured in inclusion bodies, in vitro refolding was used to reconstitute their biological activity. These CART peptides were tested for their ability to inhibit food intake in rats. Of recombinant rat CART peptides 1-102 long, 1-102 short, and 55-102, only CART peptide 55-102 dose-dependently inhibited neuropeptide Y stimulated food intake in rats. This effect was dependent on refolding of the peptide since the unfolded version was unable to inhibit food intake. Unfolded CART peptide 55-102 HIS tag, but not CART peptide 1-102 HIS tag, stimulated food intake in rats treated with NPY or vehicle. This effect of unfolded CART peptide 55-102 was consistent with that of an antagonist and is the first description of one. These studies showed that production of CART peptides by bacterial expression is a viable alternative to current methods.
