RESUMO
KEY POINTS: Skeletal muscle fatigue limits performance in various physical activities, with exercise intolerance being a key symptom in a broad spectrum of diseases. We investigated whether a small molecule fast skeletal troponin activator (FSTA), CK-2066260, can mitigate muscle fatigue by reducing the cytosolic free [Ca2+ ] required to produce a given submaximal force and hence decreasing the energy requirement. Isolated intact single mouse muscle fibres and rat muscles in-situ treated with CK-2066260 showed improved muscle endurance., which was accompanied by decreased ATP demand and reduced glycogen usage. CK-2066260 treatment improved in-vivo exercise capacity in healthy rats and in a rat model of peripheral artery insufficiency. In conclusion, we show that the FSTA CK-2066260 effectively counteracts muscle fatigue in rodent skeletal muscle in vitro, in situ, and in vivo. This may translate to humans and provide a promising pharmacological treatment to patients suffering from severe muscle weakness and exercise intolerance. ABSTRACT: Skeletal muscle fatigue limits performance during physical exercise and exacerbated muscle fatigue is a prominent symptom among a broad spectrum of diseases. The present study investigated whether skeletal muscle fatigue is affected by the fast skeletal muscle troponin activator (FSTA) CK-2066260, which increases myofibrillar Ca2+ sensitivity and amplifies the submaximal force response. Because more force is produced for a given Ca2+ , we hypothesized that CK-2066260 could mitigate muscle fatigue by reducing the energetic cost of muscle activation. Isolated single mouse muscle fibres were fatigued by 100 repeated 350 ms contractions while measuring force and the cytosolic free [Ca2+ ] or [Mg2+ ] ([Mg2+ ]i ). When starting fatiguing stimulation at matching forces (i.e. lower stimulation frequency with CK-2066260): force was decreased by â¼50% with and by â¼75% without CK-2066260; [Mg2+ ]i was increased by â¼10% with and â¼32% without CK-2066260, reflecting a larger decrease in [ATP] in the latter. The glycogen content in in situ stimulated rat muscles fatigued by repeated contractions at matching forces was about two times higher with than without CK-2066260. Voluntary exercise capacity, assessed by rats performing rotarod exercise and treadmill running, was improved in the presence of CK-2066260. CK-2066260 treatment also increased skeletal muscle fatigue resistance and exercise performance in a rat model of peripheral artery insufficiency. In conclusion, we demonstrate that the FSTA CK-2066260 mitigates skeletal muscle fatigue by reducing the metabolic cost of force generation.
Assuntos
Contração Muscular/fisiologia , Fadiga Muscular/fisiologia , Fibras Musculares de Contração Rápida/metabolismo , Troponina/metabolismo , Animais , Cálcio/metabolismo , Feminino , Glicogênio/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miofibrilas/metabolismo , Condicionamento Físico Animal/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
Heart failure-mediated skeletal myopathy, which is characterized by muscle atrophy and muscle metabolism dysfunction, often manifests as dyspnea and limb muscle fatigue. We have previously demonstrated that increasing Ca(2+) sensitivity of the sarcomere by a small-molecule fast skeletal troponin activator improves skeletal muscle force and exercise performance in healthy rats and models of neuromuscular disease. The objective of this study was to investigate the effect of a novel fast skeletal troponin activator, CK-2127107 (2-aminoalkyl-5-N-heteroarylpyrimidine), on skeletal muscle function and exercise performance in rats exhibiting heart failure-mediated skeletal myopathy. Rats underwent a left anterior descending coronary artery ligation, resulting in myocardial infarction and a progressive decline in cardiac function [left anterior descending coronary artery heart failure (LAD-HF)]. Compared with sham-operated control rats, LAD-HF rat hindlimb and diaphragm muscles exhibited significant muscle atrophy. Fatigability was increased during repeated in situ isokinetic plantar flexor muscle contractions. CK-2127107 produced a leftward shift in the force-Ca(2+) relationship of skinned, single diaphragm, and extensor digitorum longus fibers. Exercise performance, which was assessed by rotarod running, was lower in vehicle-treated LAD-HF rats than in sham controls (116 ± 22 versus 193 ± 31 seconds, respectively; mean ± S.E.M.; P = 0.04). In the LAD-HF rats, a single oral dose of CK-2127107 (10 mg/kg p.o.) increased running time compared with vehicle treatment (283 ± 47 versus 116 ± 22 seconds; P = 0.0004). In summary, CK-2127107 substantially increases exercise performance in this heart failure model, suggesting that modulation of skeletal muscle function by a fast skeletal troponin activator may be a useful therapeutic in heart failure-associated exercise intolerance.
Assuntos
Insuficiência Cardíaca Sistólica/fisiopatologia , Músculo Esquelético/efeitos dos fármacos , Condicionamento Físico Animal , Pirimidinas/farmacologia , Troponina/metabolismo , Animais , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Bovinos , Feminino , Insuficiência Cardíaca Sistólica/complicações , Insuficiência Cardíaca Sistólica/metabolismo , Contração Muscular/efeitos dos fármacos , Fibras Musculares de Contração Rápida/efeitos dos fármacos , Fibras Musculares de Contração Rápida/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatologia , Atrofia Muscular/etiologia , Atrofia Muscular/metabolismo , Atrofia Muscular/fisiopatologia , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/fisiopatologia , Miofibrilas/efeitos dos fármacos , Miofibrilas/metabolismo , Coelhos , Ratos Sprague-Dawley , Teste de Desempenho do Rota-RodRESUMO
In mice of normal weight and with diet-induced obesity, a high-fat, low-carbohydrate ketogenic diet (KD) causes weight loss, reduced circulating glucose and lipids, and dramatic changes in hepatic gene expression. Many of the effects of KD are mediated by fibroblast growth factor 21 (FGF21). We tested the effects of KD feeding on ob/ob mice to determine if metabolic effects would occur in obesity secondarily to leptin deficiency. We evaluated the effect of prolonged KD feeding on weight, energy homeostasis, circulating metabolites, glucose homeostasis, and gene expression. Subsequently, we evaluated the effects of leptin and fasting on FGF21 expression in ob/ob mice. KD feeding of ob/ob mice normalized fasting glycemia and substantially reduced insulin and lipid levels in the absence of weight loss. KD feeding was associated with significant increases in lipid oxidative genes and reduced expression of lipid synthetic genes, including stearoyl-coenzyme A desaturase 1, but no change in expression of inflammatory markers. In chow-fed ob/ob mice, FGF21 mRNA was elevated 10-fold compared with wild-type animals, and no increase from this elevated baseline was seen with KD feeding. Administration of leptin to chow-fed ob/ob mice led to a 24-fold induction of FGF21. Fasting also induced hepatic FGF21 in ob/ob mice. Thus, KD feeding improved ob/ob mouse glucose homeostasis without weight loss or altered caloric intake. These data demonstrate that manipulation of dietary macronutrient composition can lead to marked improvements in metabolic profile of leptin-deficient obese mice in the absence of weight loss.
Assuntos
Dieta com Restrição de Carboidratos , Dieta Cetogênica , Intolerância à Glucose/dietoterapia , Redução de Peso/fisiologia , Animais , Calorimetria Indireta , Fatores de Crescimento de Fibroblastos/biossíntese , Fatores de Crescimento de Fibroblastos/genética , Teste de Tolerância a Glucose , Homeostase/fisiologia , Mediadores da Inflamação/metabolismo , Resistência à Insulina/fisiologia , Interleucina-6/metabolismo , Leptina/sangue , Leptina/farmacologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/dietoterapia , RNA/biossíntese , RNA/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Triglicerídeos/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The gastrointestinal-derived hormone peptide YY (PYY) is released from intestinal L-cells post-prandially in proportion to calorie intake, and modulates food intake. Peripheral administration of PYY((3-36)) reduces food intake and body weight in rodents and suppresses appetite and food intake in humans. PYY((3-36)) is hypothesised to inhibit food intake via activation of the auto-inhibitory pre-synaptic neuropeptide Y (NPY) Y2 receptor (Y2R) present on arcuate (ARC) NPY neurons. We aimed to investigate the feeding effect of PYY((3-36)) following blockade of ARC Y2R, using the specific receptor antagonist BIIE0246, in the rat. We found that pre-treatment with BIIE0246 (1 nmol) into the ARC attenuated the reduction in feeding observed following intraperitoneal injection of PYY((3-36)) (7.5 nmol/kg) (0-1 h food intake: BIIE0246/PYY((3-36)): 3.8 +/- 0.4 g; vs. Vehicle/PYY((3-36)): 2.7 +/- 0.2 g; P < 0.05). We found plasma PYY levels to be maximal at 120 min post-initiation of feeding. On investigation of the endogenous role of the Y2R, we found that ARC administration of BIIE0246 alone significantly increased feeding in satiated rats compared to vehicle-injected controls (0-1 h food intake: BIIE0246: 4.1 +/- 0.7 g; vs. vehicle: 1.7 +/- 0.7 g; P < 0.05), suggesting that Y2R antagonism disinhibits the NPY neuron thus stimulating feeding in otherwise satiated rats. These studies suggest that the Y2R plays an important role in post-prandial satiety and provide further insight into the mechanisms of action of PYY((3-36)).
Assuntos
Arginina/análogos & derivados , Arginina/farmacologia , Benzazepinas/farmacologia , Ingestão de Alimentos/efeitos dos fármacos , Peptídeo YY/sangue , Peptídeo YY/farmacologia , Receptores de Neuropeptídeo Y/antagonistas & inibidores , Animais , Apetite/efeitos dos fármacos , Apetite/fisiologia , Núcleo Arqueado do Hipotálamo/citologia , Núcleo Arqueado do Hipotálamo/efeitos dos fármacos , Núcleo Arqueado do Hipotálamo/fisiologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Ingestão de Alimentos/fisiologia , Masculino , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Fragmentos de Peptídeos , Ratos , Ratos Wistar , Saciação/efeitos dos fármacos , Saciação/fisiologiaRESUMO
The cytokines IL-1 and IL-6 are able to induce prostaglandin (PG)-dependent activation of the hypothalamo-pituitary-adrenal axis (HPAA) and are thought to play key roles in immune-neuroendocrine interactions during inflammation. The present study shows that inflammation induced by im injection of turpentine (TPS) in the hind limb of mice causes an increase in the plasma concentration of IL-6, but not that of IL-1 alpha or IL-1 beta, together with a prolonged (>18-h) activation of the HPAA. IL-6 plays a causal role in the TPS-induced elevation in HPAA activity, because the sustained (8-18 h) increases in 1) plasma corticosterone, 2) plasma ACTH, and 3) induction of c-Fos in the hypothalamic paraventricular nucleus are all markedly blunted in IL-6-deficient (IL-6(-/-)) mice. Peripheral administration of a neutralizing IL-6 antiserum inhibited the plasma corticosterone response of normal (C57BL/6) mice to hind limb inflammation to an extent similar to that seen in IL-6(-/-) mice, suggesting that the IL-6 responsible for the increased HPAA activity is produced, or acts, on the blood side of the blood-brain barrier. We also show that IL-6 in the circulation is induced almost exclusively at the local inflammatory site, where IL-1 beta is produced. Induction of IL-6 and activation of the HPAA are dependent upon prior activation of an IL-1 type I receptor, as both are inhibited in type I IL-1 receptor-deficient mice. Furthermore, hind limb inflammation induced cyclooxygenase-2 protein expression around the cerebrovasculature of normal (IL-6(+/+)), but not IL-6(-/-), mice. Based on these data, we propose that IL-6 is produced at the local inflammatory site under the control of IL-1 beta and is the circulating afferent signal that is in part responsible for elevated HPAA activity, possibly acting via eicosanoid production within the cerebrovasculature.
Assuntos
Membro Posterior , Sistema Hipotálamo-Hipofisário/fisiopatologia , Inflamação/fisiopatologia , Interleucina-6/metabolismo , Sistema Hipófise-Suprarrenal/fisiopatologia , Transdução de Sinais/fisiologia , Animais , Circulação Cerebrovascular , Ciclo-Oxigenase 2 , Endotélio Vascular/enzimologia , Indução Enzimática/fisiologia , Feminino , Membro Posterior/metabolismo , Inflamação/induzido quimicamente , Interleucina-1/metabolismo , Interleucina-6/deficiência , Irritantes , Isoenzimas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Prostaglandina-Endoperóxido Sintases/metabolismo , Ratos , Ratos Sprague-Dawley , TerebintinaRESUMO
The hypothalamic neuropeptide melanin-concentrating hormone (MCH) increases feeding when injected intracerebroventricularly in rats. To identify the hypothalamic nuclei responsible for the orexigenic effect, we injected the peptide into discrete hypothalamic nuclei known to express the MCH receptor, MCH1R. MCH (0.6 nmol) elicited a rapid and significant increase in feeding in satiated rats following injection into the arcuate nucleus (0-1 h: 421 +/- 60%; P < 0.01). An elevation in feeding was also observed following injection into the paraventricular nucleus, which was sustained up to 4 h post injection (0-4 h: 218 +/- 29%; P < 0.01). A significant increase in feeding during this time period was also observed following injection into the dorsomedial nucleus (0-4 h: 155 +/- 12%; P < 0.05). No significant alteration in feeding was observed following injection into the supraoptic nucleus, lateral hypothalamic area, medial preoptic area, anterior hypothalamic area, or ventromedial nucleus of the hypothalamus. To identify the neurotransmitters that may be potentially involved in this effect, we examined their release from hypothalamic explants in vitro following exogenous MCH administration. MCH (1 micro M) increased the release of the orexigenic neurotransmitters neuropeptide Y (37.8 +/- 6.0 fmol/explant vs. basal 30.2 +/- 4.3 fmol/explant; P < 0.05) and agouti-related peptide (4.1 +/- 0.6 fmol/explant vs. basal 2.4 +/- 0.2 fmol/explant; P < 0.05) and decreased the release of the anorectic neurotransmitters alpha-MSH (41.7 +/- 6.8 fmol/explant vs. basal 65.9 +/- 11.0 fmol/explant; P < 0.01) and cocaine- and amphetamine-regulated transcript (112.3 +/- 12.4 fmol/explant vs. basal 167.4 +/- 13.0 fmol/explant; P < 0.001). These studies suggest that the orexigenic effect of MCH may be mediated via activation or inhibition of these feeding circuits within the arcuate nucleus and paraventricular nucleus of the hypothalamus.
Assuntos
Núcleo Arqueado do Hipotálamo/metabolismo , Comportamento Alimentar/efeitos dos fármacos , Hormônios Hipotalâmicos/farmacologia , Melaninas/farmacologia , Núcleo Hipotalâmico Paraventricular/metabolismo , Hormônios Hipofisários/farmacologia , Proteína Relacionada com Agouti , Animais , Apetite/efeitos dos fármacos , Apetite/fisiologia , Núcleo Arqueado do Hipotálamo/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Ingestão de Alimentos/fisiologia , Comportamento Alimentar/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Microinjeções , Neuropeptídeo Y/metabolismo , Núcleo Hipotalâmico Paraventricular/efeitos dos fármacos , Proteínas/metabolismo , Ratos , Ratos Wistar , alfa-MSH/metabolismoRESUMO
BACKGROUND: The hypothalamic neuropeptide melanin-concentrating hormone (MCH) plays a critical role in energy homeostasis. Abundant expression of the MCH receptor is observed outside the hypothalamus, especially in the dorsal and the ventral striatum, raising the possibility that MCH modulates the function of the midbrain dopamine neurons and associated circuitry. METHODS: The MCH receptor 1 (MCHR1) expression was assessed by in situ hybridization. Expression of dopamine transporter (DAT) and the dopamine D1 and D2 receptor (D1R and D2R) subtypes in the caudate-putamen (CPu) and the nucleus accumbens (Acb) was evaluated by immunoblotting. Amperometry in ex vivo slices of the Acb was used to measure evoked-dopamine release in MCH-/ - mice. Catalepsy in MCH+/+ and MCH-/- mice was assessed by the bar test after haloperidol injection. Locomotor activity was measured after acute and chronic treatment with amphetamine and after dopamine reuptake inhibitor GBR 12909 administration. RESULTS: The psychostimulant amphetamine caused enhanced behavioral sensitization in MCH-/- mice. We found significantly elevated expression of the DAT in the Acb of MCH-/- mice. The DAT-mediated uptake of dopamine was also enhanced in MCH-/- mice consistent with increased expression of DAT. We also found that evoked dopamine release is significantly increased in the Acb shell of MCH-/- mice. The GBR 12909 administration increased the locomotor activity of MCH-/- mice significantly above that of MCH+/+ mice. CONCLUSIONS: These results demonstrate that MCH, in addition to its known role in feeding and weight regulation, plays a critical role in regulating Acb dopamine signaling and related behavioral responses.
Assuntos
Dopamina/metabolismo , Sistema Límbico/metabolismo , Receptores de Somatostatina/deficiência , Recompensa , Anfetamina/farmacologia , Análise de Variância , Animais , Comportamento Animal , Catalepsia/induzido quimicamente , Catalepsia/genética , Antagonistas de Dopamina/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Potenciais Evocados/efeitos dos fármacos , Potenciais Evocados/fisiologia , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/fisiologia , Haloperidol/farmacologia , Técnicas In Vitro , Sistema Límbico/anatomia & histologia , Sistema Límbico/efeitos dos fármacos , Camundongos , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Piperazinas/farmacologia , Radioimunoensaio/métodos , Receptores de Dopamina D1/genética , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismoRESUMO
Mice fed a high-fat, low-carbohydrate ketogenic diet (KD) exhibit marked changes in hepatic metabolism and energy homeostasis. Here, we identify liver-derived fibroblast growth factor 21 (FGF21) as an endocrine regulator of the ketotic state. Hepatic expression and circulating levels of FGF21 are induced by both KD and fasting, are rapidly suppressed by refeeding, and are in large part downstream of PPARalpha. Importantly, adenoviral knockdown of hepatic FGF21 in KD-fed mice causes fatty liver, lipemia, and reduced serum ketones, due at least in part to altered expression of key genes governing lipid and ketone metabolism. Hence, induction of FGF21 in liver is required for the normal activation of hepatic lipid oxidation, triglyceride clearance, and ketogenesis induced by KD. These findings identify hepatic FGF21 as a critical regulator of lipid homeostasis and identify a physiological role for this hepatic hormone.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Hepatócitos/metabolismo , Metabolismo dos Lipídeos/fisiologia , Fígado/metabolismo , PPAR alfa/fisiologia , Adenoviridae/genética , Animais , Células Cultivadas , Fenofibrato/farmacologia , Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Regulação da Expressão Gênica/efeitos dos fármacos , Hipolipemiantes/farmacologia , Immunoblotting , Cetose/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Caloric restriction, leanness and decreased activity of insulin/insulin-like growth factor 1 (IGF-1) receptor signaling are associated with increased longevity in a wide range of organisms from Caenorhabditis elegans to humans. Fat-specific insulin receptor knock-out (FIRKO) mice represent an interesting dichotomy, with leanness and increased lifespan, despite normal or increased food intake. To determine the mechanisms by which a lack of insulin signaling in adipose tissue might exert this effect, we performed physiological and gene expression studies in FIRKO and control mice as they aged. At the whole body level, FIRKO mice demonstrated an increase in basal metabolic rate and respiratory exchange ratio. Analysis of gene expression in white adipose tissue (WAT) of FIRKO mice from 6 to 36 months of age revealed persistently high expression of the nuclear-encoded mitochondrial genes involved in glycolysis, tricarboxylic acid cycle, beta-oxidation and oxidative phosphorylation as compared to expression of the same genes in WAT from controls that showed a tendency to decline in expression with age. These changes in gene expression were correlated with increased cytochrome c and cytochrome c oxidase subunit IV at the protein level, increased citrate synthase activity, increased expression of peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha) and PGC-1beta, and an increase in mitochondrial DNA in WAT of FIRKO mice. Together, these data suggest that maintenance of mitochondrial activity and metabolic rates in adipose tissue may be important contributors to the increased lifespan of the FIRKO mouse.
Assuntos
Tecido Adiposo Branco/metabolismo , Expressão Gênica , Genes Mitocondriais/genética , Longevidade/fisiologia , Mitocôndrias/metabolismo , Magreza/metabolismo , Tecido Adiposo Branco/química , Animais , Citrato (si)-Sintase/metabolismo , Ciclo do Ácido Cítrico/genética , Citocromos c/metabolismo , DNA Mitocondrial/análise , Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Glicólise/genética , Longevidade/genética , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/ultraestrutura , Análise de Sequência com Séries de Oligonucleotídeos , Oxirredução , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Receptor de Insulina/genética , Magreza/genética , Transativadores/metabolismo , Fatores de TranscriçãoRESUMO
Ketogenic diets have been used as an approach to weight loss on the basis of the theoretical advantage of a low-carbohydrate, high-fat diet. To evaluate the physiological and metabolic effects of such diets on weight we studied mice consuming a very-low-carbohydrate, ketogenic diet (KD). This diet had profound effects on energy balance and gene expression. C57BL/6 mice animals were fed one of four diets: KD; a commonly used obesogenic high-fat, high-sucrose diet (HF); 66% caloric restriction (CR); and control chow (C). Mice on KD ate the same calories as mice on C and HF, but weight dropped and stabilized at 85% initial weight, similar to CR. This was consistent with increased energy expenditure seen in animals fed KD vs. those on C and CR. Microarray analysis of liver showed a unique pattern of gene expression in KD, with increased expression of genes in fatty acid oxidation pathways and reduction in lipid synthesis pathways. Animals made obese on HF and transitioned to KD lost all excess body weight, improved glucose tolerance, and increased energy expenditure. Analysis of key genes showed similar changes as those seen in lean animals placed directly on KD. Additionally, AMP kinase activity was increased, with a corresponding decrease in ACC activity. These data indicate that KD induces a unique metabolic state congruous with weight loss.
Assuntos
Dieta com Restrição de Carboidratos , Gorduras na Dieta/administração & dosagem , Corpos Cetônicos/biossíntese , Cetose/metabolismo , Acetil-CoA Carboxilase/antagonistas & inibidores , Adenilato Quinase/metabolismo , Animais , Restrição Calórica , Gorduras na Dieta/farmacologia , Metabolismo Energético , Expressão Gênica , Teste de Tolerância a Glucose , Cetose/genética , Cetose/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Redução de PesoRESUMO
Intracerebroventricular (ICV) injection of prolactin-releasing peptide (PrRP) is known to increase plasma adrenocorticotropin (ACTH) and cause c-fos expression in the hypothalamic paraventricular nucleus (PVN). We hypothesize that this is the site at which PrRP acts to increase plasma ACTH. We have used ICV injection and direct intranuclear injection of PrRP into the PVN to investigate the sites important in the stimulation of ACTH release in vivo. To investigate the mechanism of action by which PrRP increases ACTH, we have used primary culture of pituitary cells and measured neuropeptide release from in vitro hypothalamic incubations. ICV administration of PrRP increased plasma ACTH 10 min post-injection (PrRP 5 nmol 81.0 +/- 23.5 pg/ml vs. saline 16.8 +/- 14.1 pg/ml, p < 0.05). Intra-PVN injection of PrRP increased ACTH 5 min post-injection (PrRP 1 nmol 22.9 +/- 5.0 pg/ml vs. saline 10.3 +/- 1.4 pg/ml, p < 0.05). This effect continued until 40 min post-injection (PrRP 1 nmol 9.9 +/- 1.5 pg/ml vs. saline 6.2 +/- 0.5 pg/ml, p < 0.05). In vitro PrRP (1-100 nmol/l) did not effect basal or corticotropin-releasing hormone (CRH)-stimulated ACTH release from dispersed anterior pituitary cells. PrRP increased hypothalamic release of CRH (PrRP 100 nmol/l 1.4 +/- 0.2 nmol/explant vs. the basal 1.1 +/- 0.2 nmol/explant, p < 0.05) but not arginine vasopressin. PrRP also stimulated neuropeptide Y release (PrRP 100 nmol/l 56.5 +/- 11.8 pmol/explant vs. basal 24.0 +/- 1.9 pmol/explant, p < 0.01), a neuropeptide known to stimulate the hypothalamo-pituitary-adrenal axis. Our data suggest that in vitro PrRP does not have a direct action on the corticotrope but increases plasma ACTH via the PVN and this effect involves the release of hypothalamic neuropeptides including CRH and neuropeptide Y.