Nonsulfated cholecystokinins in the small intestine of pigs and rats

Cholecystokinin (CCK) is a gut hormone that acts via two receptors. The CCK_A-receptor requires the tyrosyl residue in the C-terminal bioactive site of CCK to be O-sulfated, whereas, the CCK_B-receptor binds irrespective of sulfation. Consequently, unsulfated CCK peptides – if present – may constitute a hormone system that acts only through the CCK_B-receptor. Therefore, we have now examined whether, CCK peptides occur in nonsulfated form in the small intestine of pigs and rats. The concentrations of sulfated and nonsulfated CCK were measured by RIAs, one specific for sulfated CCKs and a new two-step assay specific for nonsulfated CCK. For further characterization, the intestinal extracts were subjected to size- and ion exchange-chromatography.The intestinal concentrations of sulfated and nonsulfated CCK were highest in the duodenum and the proximal part of jejunum both in the pig and the rat. The porcine duodenal mucosa contained 193 ± 84 pmol/g sulfated CCK and 31 ± 10 pmol/g nonsulfated CCK, and the upper rat intestine 70 ± 19 pmol/g and 8 ± 2 pmol/g, respectively. The degree of sulfation correlated with the endoproteolytic proCCK processing. Thus, 38% of porcine CCK-58 was unsulfated, whereas, only 12% of CCK-8 was unsulfated.The results show that a substantial part of intestinal CCK peptides in rats and pigs are not sulfated, and that the longer peptides (CCK-58 and CCK-33) are less sulfated than the shorter (CCK-22 and CCK-8). Hence, the results demonstrate that proCCK in the gut is processed both to sulfated and unsulfated α-amidated peptides of which the latter are assumed to act via the CCK_B-receptor.     

Intravenous injection of urocortin 1 induces a CRF2 mediated increase in circulating ghrelin and glucose levels through distinct mechanisms in rats

Urocortins (Ucns) injected peripherally decrease food intake and gastric emptying through peripheral CRF₂ receptors in rodents. However, whether Ucns influence circulating levels of the orexigenic and prokinetic hormone, ghrelin has been little investigated. We examined plasma levels of ghrelin and blood glucose after intravenous (iv) injection of Ucn 1, the CRF receptor subtype involved and underlying mechanisms in ad libitum fed rats equipped with a chronic iv cannula. Ucn 1 (10 μg/kg, iv) induced a rapid onset and long lasting increase in ghrelin levels reaching 68% and 219% at 0.5 and 3 h post injection respectively and a 5-h hyperglycemic response. The selective CRF₂ agonist, Ucn 2 (3 μg/kg, iv) increased fasting acyl (3 h: 49%) and des-acyl ghrelin levels (3 h: 30%) compared to vehicle while the preferential CRF₁ agonist, CRF (3 μg/kg, iv) had no effect. <!-- no-mfc -->Ucn 1's<!-- /no-mfc --> stimulatory actions were blocked by the selective CRF₂ antagonist, astressin₂-B (100 μg/kg, iv). Hexamethonium (10 mg/kg, sc) prevented Ucn 1-induced rise in total ghrelin levels while not altering the hyperglycemic response. These data indicate that systemic injection of Ucns induces a CRF₂-mediated increase in circulating ghrelin levels likely via indirect actions on gastric ghrelin cells that involves a nicotinic pathway independently from the hyperglycemic response.     

Chronic central ghrelin infusion reduces blood pressure and heart rate despite increasing appetite and promoting weight gain in normotensive and hypertensive rats

Acute studies showed that ghrelin acts on the central nervous system (CNS) to reduce blood pressure (BP), heart rate (HR) and sympathetic activity. However, the long-term CNS cardiovascular actions of ghrelin are still unclear. We tested whether chronic intracerebroventricular (ICV) infusion of ghrelin causes sustained reductions in BP, HR and whether it alters baroreceptor sensitivity (BRS) and autonomic input to the heart. A cannula was placed in the lateral ventricle of male Sprague–Dawley (SD) rats for ICV infusions via osmotic minipump (0.5 μl/h). BP and HR were measured 24-h/day by telemetry. After 5 days of control measurements, ghrelin (0.21 nmol/h) or saline vehicle were infused ICV for 10 days followed by a 5-day post-treatment period. Chronic ICV ghrelin infusion increased food intake (22 ± 3 to 26 ± 1 g/day) leading to ∼50 g body weight gain. BP fell slightly during ghrelin infusion while HR decreased by ∼26 bpm. In control animals BP and HR increased modestly. ICV Ghrelin infusion caused a 50% reduction in sympathetic tone to the heart but did not alter BRS. We also tested if the depressor responses to ICV ghrelin infusion were enhanced in spontaneously hypertensive rats (SHR) due to their high basal sympathetic tone. However, we observed similar BP and HR responses compared to normotensive rats. These results indicate that ghrelin, acting via direct actions on the CNS, has a sustained effect to lower HR and a modest impact to reduce BP in normotensive and hypertensive animals despite increasing appetite and body weight.     

Oxytocin induces penile erection and yawning when injected into the bed nucleus of the stria terminalis: Involvement of glutamic acid,dopamine, and nitric oxide

Oxytocin (5–100 ng), but not Arg⁸-vasopressin (100 ng), injected unilaterally into the bed nucleus of the stria terminalis (BNST) induces penile erection and yawning in a dose-dependent manner in male rats. The minimal effective dose was 20 ng for penile erection and 5 ng for yawning. Oxytocin responses were abolished not only by the oxytocin receptor antagonist d(CH₂)₅Tyr(Me)²-Orn⁸-vasotocin (1 μg), but also by (+) MK-801 (1 μg), an excitatory amino acid receptor antagonist of the N-methyl-d-aspartic acid (NMDA) subtype, SCH 23390 (1 μg), a D1 receptor antagonist, but not haloperidol (1 μg), a D2 receptor antagonist, and SMTC (40 μg), an inhibitor of neuronal nitric oxide synthase, injected into the BNST 15 min before oxytocin. Oxytocin-induced penile erection, but not yawning, was also abolished by CNQX (1 μg), an excitatory amino acid receptor antagonist of the AMPA subtype. In contrast, oxytocin responses were not reduced by bicuculline (20 ng), a GABA_A receptor antagonist, phaclofen (5 μg), a GABA_B receptor antagonist, CP 376395, a CRF receptor-1 antagonist (5 μg), or astressin 2B, a CRF receptor-2 antagonist (150 ng). Considering the ability of NMDA (100 ng) to induce penile erection and yawning when injected into the BNST and the available evidence showing possible interaction among oxytocin, glutamic acid, and dopamine in the BNST, oxytocin possibly activates glutamatergic neurotransmission in the BNST. This in turn leads to the activation of neural pathways projecting back to the paraventricular nucleus, medial preoptic area, ventral tegmental area, and/or ventral subiculum/amygdala, thereby inducing penile erection and yawning.     

Interactive effect of galanin-like peptide (GALP) and spontaneous exercise on energy metabolism

Galanin-like peptide (GALP) is a neuropeptide involved in energy metabolism. The interactive effect of GALP and exercise on energy metabolism has not been investigated. The aim of this study was to determine if energy metabolism in spontaneously exercising mice could be promoted by intracerebroventricular (ICV) GALP administration. Changes in respiratory exchange ratio in response to GALP ICV administration indicated that lipids were primarily consumed followed by a continuous consumption of glucose throughout the dark period in non-exercising mice. In mice permitted to spontaneously exercise on a running-wheel, GALP ICV administration increased the consumed oxygen volume and heat production level from 5 to 11 h after administration. These effects occurred independently from the total running distance. The interaction between GALP ICV administration and spontaneous exercise decreased body weight within 24 h (F_(1,16) = 5.772, p < 0.05), with no significant interaction observed regarding food and water intake or total distance. Energy metabolism-related enzymes were assessed in liver and skeletal muscle samples, with a significant interaction on mRNA expression between GALP ICV administration and spontaneous exercise observed in phosphoenolpyruvate carboxykinase (F_(1,16) = 18.602, p < 0.001) that regulates gluconeogenesis and glucose transporter-4 (F_(1,16) = 21.092, p < 0.001). GALP significantly decreased the mRNA expression of sterol regulatory element-binding protein-1c (p < 0.05) that regulates fatty acid synthesis regardless of spontaneous exercise with no changes to acetyl-CoA carboxylase a and fatty acid synthetase. These results indicate the GALP ICV administration can further promote energy metabolism when administered to spontaneously exercising mice.     

Feeding-suppressive mechanism of sulfated cholecystokinin (26-33) in chicks

The anorexigenic effect of cholecystokinin (CCK) is well documented in mammals, but documentation in neonatal chicks is limited. Thus, the present study investigated the mechanism underlying the anorexigenic effect of CCK in neonatal chicks. Intraperitoneal (IP) injection of sulfated CCK(26-33) (CCK8S) significantly decreased food intake in chicks at 60 and 300 nmol/kg. Non-sulfated CCK(26-33) (CCK8) also significantly decreased food intake, but its anorexigenic effect was observed only at the highest dose (300 nmol/kg) and short-lived. However, CCK(30-33) (CCK4) had no effect on food intake. Also, the intracerebroventricular (ICV) injection of CCK8S (0.2 and 1 nmol) significantly decreased food intake in chicks. Similar to IP administration, the anorexigenic effect of CCK8 was weak and CCK4 did not affect food intake. IP and ICV injections of CCK8S caused conditioned aversion and increased plasma corticosterone concentrations, suggesting that their anorexigenic effects might be related to stress and/or malaise. This might be true in ICV-injected CCK8S because co-injection of astressin, a corticotropin-releasing hormone receptor antagonist, tended to attenuate the effect of CCK8S. The present study revealed that N-terminal amino acids and the sulfation of Tyr are important for the anorexigenic effect of CCK8S after IP and ICV administered in chicks. Additionally, the effect of central CCK8S might be related to stress and/or malaise.     

Inhibition of serotonin outflow by nociceptin/orphaninFQ in dorsal raphe nucleus slices from normal and stressed rats: Role of corticotropin releasing factor

In the dorsal raphe nucleus (DRN) many inputs converge and interact to modulate serotonergic neuronal activity and the behavioral responses to stress. The effects exerted by two stress-related neuropeptides, corticotropin releasing factor (CRF) and nociceptin/orphaninFQ (N/OFQ), on the outflow of [³H]5-hydroxytryptamine were investigated in superfused rat dorsal raphe nucleus slices.Electrical stimulation (100 mA, 1 ms for 2 min) evoked a frequency-dependent peak of [³H]5-hydroxytryptamine outflow, which was sodium and calcium-dependent. Corticotropin releasing factor (1–100 nM), concentration-dependently inhibited the stimulation (3 Hz)-evoked [³H]5-hydroxytryptamine outflow; the inhibition by 30 nM corticotropin releasing factor (to 68 ± 5.7%) was prevented both by the non selective CRF receptor antagonist alpha-helicalCRF(9-41) (α-HEL) (300 nM) and by the CRF₁ receptor antagonist antalarmin (ANT) (100 nM). The CRF₂ agonist urocortin II (10 nM) did not modify [³H]5-hydroxytryptamine outflow, ruling out the involvement of CRF₂ receptors. Bicuculline (BIC), a GABA_A antagonist (10 μM), prevented the inhibitory effect of corticotropin releasing factor (30 nM), supporting the hypothesis that the inhibition was mediated by increased γ-aminobutyric acid (GABA) release. Nociceptin/orphaninFQ (1 nM–1 μM) exerted an antalarmin- and bicuculline-insensitive inhibition on [³H]5-hydroxytryptamine outflow, with the maximum at 100 nM (to 63 ± 4.2%), antagonized by the NOP receptor antagonist UFP-101 (1 μM). Dorsal raphe nucleus slices prepared from rats exposed to 15 min of forced swim stress displayed a reduced [³H]5-hydroxytryptamine outflow, in part reversed by antalarmin and further inhibited by nociceptin/orphaninFQ. These findings indicate that (i) both corticotropin releasing factor and nociceptin/orphaninFQ exert an inhibitory control on dorsal raphe nucleus serotonergic neurons; (ii) the inhibition by corticotropin releasing factor involves γ-aminobutyric acid neurons; (iii) nociceptin/orphaninFQ inhibits dorsal raphe nucleus serotonin system in a corticotropin releasing factor- and γ-aminobutyric acid-independent manner; (iv) nociceptin/orphaninFQ modulation is still operant in slices prepared from stressed rats. The nociceptin/orphaninFQ-NOP receptor system could represent a new target for drugs effective in stress-related disorders.     

Feeding responses to central administration of several somatostatin analogs in chicks

Somatostatin is well known as an inhibitor of growth hormone release from the anterior pituitary. Its effects are exerted via 5 subtypes of receptors, which are named SSTR1 through 5. We recently reported that intracerebroventricular (ICV) injection of somatostatin stimulates feeding behavior in chicks. However, the specific receptors which mediate this orexigenic effect have not been identified in chicks. Thus, the purpose of the present study was to identify the receptor subtypes involved in somatostatin-induced feeding using 5 somatostatin analogs. Chicks that received vapreotide and octreotide (less than 3 nmol), which are agonist of SSTR2 and SSTR5, increased their food intake. Additionally, chicks ICV injected with BIM23056 or L-817,818 (SSTR3 and SSTR5 agonists, respectively) also had increased food intake. However, ICV injection of the SSTR4 agonist L-803,087 did not cause an orexigenic effect, suggesting that SSTR4 might not be important in somatostatin-induced feeding behavior. In summary, results from this study may be interpreted as SSTR2, SSTR3 and SSTR5 are related to somatostatin-associated feeding behavior in chicks.     

Discovery of a 7-arylaminobenzimidazole series as novel CRF1 receptor antagonists

A promising lead compound 1 of a benzimidazole series has been identified as a corticotropin-releasing factor 1 (CRF₁) receptor antagonist. In this study, we focused on replacement of a 7-alkylamino group of 1, predicted to occupy a large lipophilic pocket of a CRF₁ receptor, with an aryl group. During the course of this examination, we established new synthetic approaches to 2,7-diarylaminobenzimidazoles. The novel synthesis of 7-arylaminobenzimidazoles culminated in the identification of compounds exhibiting inhibitory activities comparable to the alkyl analog 1. A representative compound, p-methoxyanilino analog 16g, showed potent CRF binding inhibitory activity against a human CRF₁ receptor and human CRF₁ receptor antagonistic activity (IC₅₀ = 27 nM, 56 nM, respectively). This compound exhibited ex vivo ¹²⁵I-Tyr⁰ (¹²⁵I-CRF) binding inhibitory activity in mouse frontal cortex, olfactory bulb, and pituitary gland at 20 mg/kg after oral administration. In this report, we discuss the structure–activity-relationship of these 7-arylamino-1H-benzimidazoles and their synthetic method.     

Corticotropin-releasing factor (CRF) receptor subtypes in mediating neuronal activation of brain areas involved in responses to intracerebroventricular CRF and stress in rats

Corticotropin-releasing factor (CRF) plays an important role in stress responses through activation of its receptor subtypes, CRF1 receptor (CRF₁) and CRF2 receptor (CRF₂). The parvocellular paraventricular nucleus of the hypothalamus (PVNp), the central nucleus of the amygdala (CeA), and the oval nucleus of the bed nucleus of the stria terminalis (BNSTov), which are rich in CRF neurons with equivocal expression of CRF₁ and CRF₂, are involved in stress-related responses. In these areas, Fos expression is induced by various stimuli, although the functions of CRF receptor subtypes in stimuli-induced Fos expression are unknown. To elucidate this issue and to examine whether Fos is expressed in CRF or non-CRF neurons in these areas, the effects of antalarmin and antisauvagine-30 (AS-30), CRF₁- and CRF₂-specific antagonists, respectively, on intracerebroventricular (ICV) CRF- or 60 min-restraint-induced Fos expression were examined in rats. ICV CRF increased the number of Fos-positive CRF and non-CRF neurons in the PVNp, with the increases being inhibited by antalarmin in CRF and non-CRF neurons and by AS-30 in CRF neurons. Restraint also increased Fos-positive CRF and non-CRF neurons in the PVNp, with the increases being inhibited by antalarmin in the CRF neurons. ICV CRF also increased Fos-positive non-CRF neurons in the CeA and the BNSTov, which was inhibited by AS-30 in both areas, and inhibited by antalarmin in the BNSTov only. Restraint increased Fos-positive non-CRF neurons in the CeA and BNSTov, with the increases being almost completely inhibited by either antagonist. These results indicate that both ICV CRF and restraint activate both CRF and non-CRF neurons in the PVNp and non-CRF neurons in the CeA and BNSTov, and that the activation is mediated by CRF₁ and/or CRF₂. However, the manner of involvement for CRF₁ and CRF₂ in ICV CRF- and restraint-induced activation of neurons differs with respect to the stimuli and brain areas; being roughly equivalent in the CeA and BNSTov, but different in the PVNp. Furthermore, the non-CRF_1&2-mediated signals seem to primarily play a role in restraint-induced activation of non-CRF neurons in the PVNp since the activation was not inhibited by CRF receptor antagonists.     

Effects of intracerebroventricular administration of the NPY-Y1 receptor antagonist, 1229U91, on hyperphagic and glycemic responses to acute and chronic intermediate insulin-induced hypoglycemia in female rats

Neuropeptide Y (NPY) Y1 receptors are implicated in CNS regulation of food intake, but their role in hypoglycemic hyperphagia remains unclear. The present studies utilized a pharmacological approach to investigate the hypothesis that NPY acts via Y1 receptor-dependent mechanisms to regulate feeding and blood glucose profiles during intermediate insulin-induced hypoglycemia. Groups of ovariectomized, estradiol benzoate-treated female rats were injected subcutaneously with one or four doses of neutral protamine Hagedorn insulin (NPH), on as many days, or with diluent alone. Before final treatments on day four, the animals were pretreated by intracerebroventricular (icv) delivery of the NPY Y1 receptor antagonist, 1229U91, or the vehicle, artificial cerebrospinal fluid (acsf). Food intake during acute hypoglycemia was significantly diminished between t_o and + 2 h in animals pretreated with the Y1 receptor antagonist versus vehicle. Administration of 1229U91 prior to the fourth of four NPH doses suppressed hypoglycemic hyperphagia over a relatively longer interval, e.g. 4 h, after t_o relative to the acute insulin group. Blood glucose levels after a single NPH injection were similar in acsf- and antagonist-pretreated rats at + 2, + 4, and + 6 h, but were lower at + 9 h in the latter group. Pretreatment with 1229U91 did not modify glucose profiles between + 2 and + 9 h after multiple dosing with NPH, but prevented recovery from hypoglycemia at + 12 h. The present results show that central NPY Y1 receptor antagonism inhibits hypoglycemic hyperphagia, and that this suppressive effect on feeding was of greater duration during recurring hypoglycemia. The data also show that Y1 receptor blockade decreases glycemic responses to both single and serial NPH dosing, albeit at different post-injection time points. The current studies support the view that NPY Y1 receptors function within central neural pathways that govern feeding and glycemic responses to intermediate-acting insulin, and that Y1 receptor-mediated stimulation of food intake may habituate in a positive manner to repetitive insulin-induced hypoglycemia. Further research is needed to evaluate the impact of chronic insulin-induced hypoglycemia on neuropeptide Y neurotransmission and Y1 receptor expression within regulatory circuitries that control food intake and glucostasis.     

Postprandial profiles of CCK after high fat and high carbohydrate meals and the relationship to satiety in humans

ContextCCK is understood to play a major role in appetite regulation. Difficulties in measuring CCK have limited the potential to assess its profile in relation to food-induced satiety. Improvements in methodology and progress in theoretical understanding of satiety/satiation make it timely for this to be revisited.ObjectiveFirst, examine how physiologically relevant postprandial CCK8/33(s) profiles are influenced by fat (HF) or carbohydrate (HCHO) meals. Second, to examine relationships between postprandial CCK and profiles of satiety (hunger/fullness) and satiation (meal size).Participants and designSixteen overweight/obese adults (11 females/5 males) participated in a randomised-crossover study (46 years, 29.8 kg/m²) in a university research centre. Plasma was collected preprandially and for 180 min postprandially. Simultaneously, ratings of hunger/fullness were tracked for 180 min before an ad libitum lunch was provided.ResultsCCK8/33(s) levels increased more rapidly and reached a higher peak following HF compared to HCHO breakfast (F_(1,15) = 14.737, p < 0.01). Profiles of hunger/fullness did not differ between conditions (F_(1,15) = 0.505, p = 0.488; F_(1,15) = 2.277, p = 0.152). There was no difference in energy intake from the ad libitum meal (HF-3958 versus HCHO-3925 kJ; t₍₁₄₎ = 0.201, p = 0.844). CCK8/33(s) profiles were not associated with subjective appetite during early and late phases of satiety; nor was there an association between CCK8/33(s) and meal size.ConclusionsThese results demonstrate CCK levels were higher after HF meal compared to HCHO isocaloric meal. There was no association between CCK levels and intensity of satiety, or with meal size. Under these circumstances, CCK does not appear to play a unique independent role in satiety/satiation. CCK probably acts in conjunction with other peptides and the action of the stomach.     

In vivo characterization of the effects of ghrelin on the modulation of acute pain at the supraspinal level in mice

Ghrelin, an acylated peptide produced in the stomach, increases food intake and growth hormone secretion, inhibits pro-inflammatory cascade, etc. Ghrelin and its receptor (GHS-R1a) mRNA were found in the area related to the regions for controlling pain transmission, such as the hypothalamus, the midbrain, the spinal cord, etc. Ghrelin has been shown to have antinociceptive activity and also anti-inflammatory properties in inflammatory pain and chronic neuropathic pain. Therefore, the aim of the present study was to investigate the effects of ghrelin for the first time in the acute pain modulation at the supraspinal level, using the tail withdrawal test and hot-plate test in mice. Intracerebroventricular (i.c.v.) administration of ghrelin (mouse, 0.1–3 nmol) produced a dose- and time-related antinociceptive effect in the tail withdrawal test and hot-plate test, respectively. Antinociceptive effect elicited by ghrelin (i.c.v., 1 nmol) was significantly antagonized by opioid receptor antagonist naloxone (i.c.v., 10 nmol co-injection or i.p., 10 mg/kg, 10 min prior to ghrelin) in both tail withdrawal test and hot-plate test. At these doses, naloxone significantly antagonized the antinociceptive effect induced by morphine (i.c.v., 3 nmol). Ghrelin (i.c.v., 1 nmol)-induced antinociception was significantly antagonized by co-injection with 10 nmol [d-Lys3]-GHRP-6, the selective antagonist of GHS-R1a identified more recently, while [d-Lys3]-GHRP-6 (10 nmol) alone induced neither hyperalgesia nor antinociception. Overall this data indicate that ghrelin could produce antinociception through an interaction with GHS-R1a and with the central opioid system. Thus ghrelin may be a promising peptide for developing new analgesic drugs.     

PYY(3-36) into the arcuate nucleus inhibits food deprivation-induced increases in food hoarding and intake

Central administration of neuropeptide Y (NPY) increases food intake in laboratory rats and mice, as well as food foraging and hoarding in Siberian hamsters. The NPY-Y1 and Y5 receptors (Rs) within the hypothalamus appear sufficient to account for these increases in ingestive behaviors. Stimulation of NPY-Y2Rs in the Arcuate nucleus (Arc) has an anorexigenic effect as shown by central or peripheral administration of its natural ligand peptide YY (3-36) and pharmacological NPY-Y2R antagonism by BIIE0246 increases food intake. Both effects on food intake by NPY-Y2R agonism and antagonism are relatively short-lived lasting ∼4 h. The role of NPY-Y2Rs in appetitive ingestive behaviors (food foraging/hoarding) is untested, however. Therefore, Siberians hamsters, a natural food hoarder, were housed in a semi-natural burrow/foraging system that had (a) foraging requirement (10 revolutions/pellet), no free food (true foraging group), (b) no running wheel access, free food (general malaise control) or (c) running wheel access, free food (exercise control). We microinjected BIIE0246 (antagonist) and PYY_(3-36) (agonist) into the Arc to test the role of NPY-Y2Rs there on ingestive behaviors. Food foraging, hoarding, and intake were not affected by Arc BIIE0246 microinjection in fed hamsters 1, 2, 4, and 24 h post injection. Stimulation of NPY-Y2Rs by PYY_(3-36) inhibited food intake at 0–1 and 1–2 h and food hoarding at 1–2 h without causing general malaise or affecting foraging. Collectively, these results implicate a sufficiency, but not necessity, of the Arc NPY-Y2R in the inhibition of food intake and food hoarding by Siberian hamsters.     

The hypotensive effect of intrathecally injected (m)VD-hemopressin(α) in urethane-anesthetized rats

Previous studies suggest that cannabinoids system plays an important role in cardiovascular regulation. (m)VD-hemopressin(α) (VD-Hpα), an 11-residue peptide originating from the α₁ chain of hemoglobin, was recently reported as a selective agonist of cannabinoid CB₁ receptor. The present study was undertaken to investigate the intrathecal (i.t.) action of (m)VD-Hpα on blood pressure in urethane-anesthetized rats. Our results demonstrated that injections of (m)VD-Hpα (5–30 nmol, i.t.) produced a dose-dependent decrease in mean arterial pressure (MAP), similar to that of the non-peptidic cannabinoid receptor agonist WIN55212-2 (1.25–10 nmol, i.t.). The hypotensive effect of (m)VD-Hpα was not influenced by the CB₁ receptor antagonist AM251 (20 nmol, i.t.) or the CB₂ receptor antagonist AM630 (20 nmol, i.t.). However, WIN55212-2-induced hypotension was almost completely prevented by i.t. administration of AM251, not by AM630. The spinal hypotension of (m)VD-Hpα and WIN55212-2 was significantly reduced by pretreatment with the α-adrenoceptor antagonist phentolamine (1 mg/kg, i.v.), but not by the β-adrenoceptor antagonist propranolol (2 mg/kg, i.v.) or the muscarinic receptor antagonist atropine (2 mg/kg, i.v.). In addition, l-NAME (50 mg/kg, i.v.), the inhibitor of nitric oxide (NO) synthase, significantly reduced WIN55212-2-induced hypotension, but had no effect on the hypotensive response to (m)VD-Hpα. Collectively, the results show that i.t. administration of (m)VD-Hpα induces a decrease in MAP via a non-CB₁ and non-CB₂ mechanism.     

Antinociceptive effects of two deltorphins analogs in the tail-immersion test in rats

The antinociceptive effects of analogs of deltorphins: cyclo(Nδ,Nδ-carbonyl-d-Orn2, Orn4)deltorphin (DEL-6) and deltorphin II N-(ureidoethyl)amide (DK-4) after intracerebroventricular (i.c.v.) administration were investigated in the tail-immersion test in rats. Morphine, the most commonly used μ-opioid receptors (MOR) agonist, was employed as a reference compound. The contribution of the MOR, δ-(DOR) and κ-opioid receptors (KOR) in antinociceptive effects of the deltorphins analogs was studies using selective antagonists of these receptors. The results indicated that DK-4 (5, 10 and 20 nmol) and DEL-6 (5, 10 and 20 nmol) were the most effective in alleviating thermal pain at the dose of 20 nmol. The antinociceptive potency of DEL-6 at the dose of 20 nmol was approximately equal but DK-4 at the dose of 20 nmol was less effective than morphine at the dose of 13 nmol. DOR antagonist – naltrindole (NTI, 5 nmol) very strongly and, to the lower extent MOR antagonist – β-funaltrexamine (β-FNA, 5 nmol), inhibited antinociceptive effect of DK-4 (20 nmol). In turn, β-FNA was more potent than NTI in inhibition of the antinociceptive effects of DEL-6. Co-administration of DEL-6 and morphine at doses of 5 nmol, which do not produce measurable antinociception, generated additive antinociceptive effect. Chronic intraperitoneal (i.p.) injection of morphine (9 days) displayed a marked analgesic tolerance to the challenge dose of morphine and a slight cross-tolerance to challenge doses of DEL-6 and DK-4, given i.c.v. These findings indicate that the new deltorphin analogs recruit DOR and MOR to attenuate the nociceptive response to acute thermal stimuli.     

Interaction Between Oxytocin and Opioidergic System on Food Intake Regulation in Neonatal Layer Type Chicken

The aim of the current study was to determine possible interaction of central oxytocin and opioidergic system on food intake regulation in neonatal layer-type chicken. In experiment 1, FD₃ chicken ICV injected with control solution, oxytocin (10 µg), β-FNA (µ receptor antagonist, 5 µg) and oxytocin (10 µg)?+?β-FNA were injected. Experiments 2–6 were similar to experiments 1, except chicken injected with nor-BNI (κ receptor antagonist, 5 µg), NTI (δ receptor antagonist, 5 µg), DAMGO (µ receptor agonist, 62.25 pmol), U-50488H (κ receptor agonist, 10 nmol), DPDPE (δ receptor agonist, 20 pmol) instead of β-FNA. In experiment 7, control solution, DAMGO (125 pmol), d(CH2)5Tyr(Me)-[Orn8]-vasotocin (oxytocin antagonist, 5 µg) and DAMGO?+?d(CH2)5Tyr(Me)-[Orn8]-vasotocin were ICV injected to FD₃ chicken. Experiments 8 and 9 were similar to experiments 7, except chicken injected with U-50488H (30 nmol) and DPDPE (40 pmol) instead of DAMGO. Then, cumulative food intake was recorded at 30, 60 and 120 min after injection. According to the results, ICV injection of the oxytocin (10 µg) significantly decreased food intake compared to control group (P?<?0.05). Co-injection of the oxytocin?+?β-FNA and oxytocin?+?U-50488H significantly decreased hypophagic effect of the oxytocin (P?<?0.05). While, co-injection of the oxytocin?+?nor-BNI or oxytocin?+?DAMGO significantly amplified hypophagic effect of the oxytocin in chicken (P?<?0.05). In addition, ICV injection of DAMGO (125 pmol) significantly decreased cumulative food intake compared to control group (P?<?0.05). However, co-addministration of the DAMGO?+?(CH2)5Tyr(Me)-[Orn8]-vasotocin significantly decreased hypophagic effect of the DAMGO (P?<?0.05) in chicken. These results suggested there are interconnection between oxytocin and opioidergic system on central food intake regulation, which mediates via µ and κ opioidergic receptors in neonatal layer-type chicken.

     

Interaction Between Nociceptin/Orphanin FQ and Adrenergic System on Food Intake in Neonatal Chicken

The information emerging from the studies demonstrates adrenergic system and nociceptin/orphanin FQ (N/OFQ) play a crucial role on appetite regulation but there is no information for their interaction. The purpose of this study was to examine the effects of intracerebroventricular (ICV) injection of prazosin (α₁ receptor antagonist), yohimbine (α₂ receptor antagonist), metoprolol (β₁ adrenergic receptor antagonist), ICI 118,551 (β₂ adrenergic receptor antagonist) and SR59230R (β₃ adrenergic receptor antagonist) on N/OFQ-induced hyperphagia by 3-h food-deprived neonatal broiler chicken. In experiment 1, chicken injected with saline, prazosin (10 nmol), N/OFQ (16 nmol) and co-injection of prazosin + N/OFQ. In experiment 2, ICV injection of saline, yohimbine (13 nmol), N/OFQ (16 nmol) and yohimbine + N/OFQ applied to the birds. In experiment 3, injections were saline, metoprolol (24 nmol), N/OFQ (16 nmol) and metoprolol + N/OFQ. In experiment 4, the birds received ICV injection of saline, ICI 118,551 (5 nmol), (C) N/OFQ (16 nmol) and co-administration of ICI 118,551 + N/OFQ. In experiment 5, chicken injected with saline, SR59230R (20 nmol), N/OFQ (16 nmol) and SR59230R + N/OFQ. Then, cumulative food intake was recorded until 120 min after injection. According to the results, ICV injection of N/OFQ significantly increased food intake (P < 0.001). The effect of N/OFQ significantly amplified by co-injection of N/OFQ + β₂ adrenergic receptor antagonist (P < 0.001). Also, administration of β₁ or β₃ adrenergic receptor antagonist had no effect on N/OFQ-induced hyperphagia (P > 0.05). These results suggest that the effect of N/OFQ on cumulative food intake is mediated via β₂ adrenergic receptors in neonatal chicken.     

The stomach and/or upper duodenum contain sites of action that control meal size and intermeal interval length by exogenous rat gastrin releasing peptide

The site(s) of action that control the reduction of food intake in response to the amphibian skin peptide bombesin (Bn) has been determined to be the area supplied by the celiac artery (CA), i.e., the stomach and the upper duodenum. Here, we investigated the gastrointestinal site(s) of action which controls meal size (MS) (normal rat chow) and intermeal interval length (IMI) by the mammalian homologues of Bn gastrin releasing peptides (GRP-10, GRP-27 and GRP-29, 0.01, 0.05, 0.1, 0.2 and 0.5 nmol/kg) infused in the CA, the cranial mesenteric artery (CMA, supplying the small and large intestine), the femoral artery (FA, control) and the portal vein (PV, draining the gastrointestinal tract, control) in freely fed rats immediately prior to the onset of the dark cycle. We found that (1) GRP-29 (0.05, 0.1, 0.2 and 0.5 nmol/kg) and GRP-27 (0.2 and 0.5 nmol/kg) in the CA and GRP-29 (0.5 nmol/kg) in the CMA reduced the MS relative to saline, (2) GRP-29 (0.1, 0.2 and 0.5 nmol/kg) and GRP-27 (0.2 and 0.5 nmol/kg) in the CA prolonged the IMI, (3) GRP-29 (0.1, 0.2 and 0.5 nmol/kg) in the CA and GRP-29 (0.5 nmol/kg) in the CMA increased the satiety ratio (SR, IMI/MS – the amount of food consumed per a given unit of time) and (4) neither peptide nor route showed any effect on the second MS. These results support an upper gastrointestinal site of action for MS and IMI length by GRP-27 and GRP-29, which is most likely the stomach and/or the duodenum.