Steven L. Parker

Nicotine administration enhances NPY expression in the rat hypothalamus

Epidemiological studies have shown an inverse relationship between cigarette smoking and body weight. In rodents, a negative correlation between nicotine and body weight has been reported, but this observation was largely derived from studies where relatively high doses of nicotine ( approximately 12 mg/kg/day) were used. In the current study, we showed that a negative relationship also holds for low doses of nicotine that are comparable to that consumed by average human smokers (<6 mg/kg/day). We also demonstrated that 14 days of nicotine administration (4 mg/kg/day) reduced average daily food intake by 19.5% (P<0.01) in the free-feeding nicotine-treated group compared to saline controls. No significant differences in body weight were detected between the nicotine-treated and pair-fed groups. To determine whether the effects of nicotine on food intake and body weight were related to neuropeptide Y (NPY) expression, semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and radioimmunoassay were utilized to measure NPY mRNA and peptide levels in various regions of the hypothalamus. Significantly higher levels of NPY mRNA (ca. 20-50%) and peptide (ca. 24-69%) were only detected in the nicotine-treated groups. In addition, significantly higher NPY contents were also obtained in two hypothalamic areas of pair-fed control animals. In summary, our data suggest that the pharmacological effects of nicotine on food intake and body weight may be mediated by changes in hypothalamic NPY levels, a neuropeptide that is pivotal to the hypothalamic regulation of food intake.

Hypothalamic orexin-A binding sites are downregulated by chronic nicotine treatment in the rat.

Chronic nicotine treatment (4 mg/kg per day; 14 days) significantly reduced the affinity and density of orexin-A binding sites in the anterior hypothalamus of rat brain. There was a significantly lower sensitivity of orexin-A binding to orexin peptides, to the related secretin and pituitary adenylate cyclase activating peptide, and to unrelated neuropeptide Y (NPY). This change correlated with selective downregulation of a fraction of hypothalamic NPY Y(1) receptors. In previous studies, we have demonstrated an increase in the levels of orexin-A peptide and NPY in discrete hypothalamic areas upon nicotine treatment. This finding contradicts an expected increase in the production of these orexigenic peptides in a model where an inverse relationship is observed between food consumption and nicotine treatment. This study provides a possible explanation to this inconsistency in that a decrease in affinity of orexin-A binding could reduce neural orexin signaling, which may contribute to decreased food intake observed in smokers and animals chronically treated with nicotine.

Gestational nicotine exposure reduces nicotinic cholinergic receptor (nAChR) expression in dopaminergic brain regions of adolescent rats

Children of women who smoked during pregnancy are at increased risk of dependence when smoking is initiated during adolescence. We previously reported that gestational nicotine exposure attenuated dopamine release induced by nicotine delivered during adolescence. In this study, we determined the effects of gestational nicotine exposure on nicotinic cholinergic receptor (nAChR) expression. Timed pregnant rats received nicotine (2 mg/kg/day) or vehicle via mini‐osmotic pumps during gestation. Treatments continued in pups via maternal nursing during postnatal days (PN) 2–14 (equivalent to the human in utero third trimester). On PN35, 125I‐epibatidine binding to nAChR was measured. The Bmax values (fmol/mg) in prefrontal cortex (PFC), nucleus accumbens (NAcc), substantia nigra (SN) and ventral tegmental area (VTA) were reduced by 26.6% (P < 0.05), 32.6% (P < 0.01), 23.0% (P < 0.01) and 27.6% (P < 0.05), respectively. In addition, gender differences were found in vehicle‐treated groups; in SN and VTA, females were 79.3% (P < 0.005) and 82.9% (P = 0.08) of males, respectively. The expression of nAChR subunit mRNAs was measured using real‐time RT‐PCR on laser‐capture microdissected tissues. In adolescent VTA, gestational nicotine exposure reduced (P < 0.05) nAChR subunit mRNAs encoding α3 (53.0%), α4 (23.9%), α5 (46.7%) and β4 (61.4%). In NAcc core, the treatment increased α3 mRNA (75.8%). In addition, the number of neurons in VTA was reduced by 15.0% (P < 0.001). These studies indicate that gestational exposure to nicotine induces long‐lasting changes in nAChR expression that may underlie the vulnerability of adolescents to dependence on nicotine.

Prolactin Stimulates the Release of Oxytocin in Lactating Rats: Evidence for a Physiological Role via an Action at the Neural Lobe

The present studies were designed to investigate whether prolactin (PRL) influences the secretion of oxytocin (OT) in lactating rats, and to test whether the previously reported inhibitory and stimulatory effects of dopamine-2 (D-2) agonists and antagonists, respectively, on OT release might be secondary to their respective inhibitory and stimulatory effects on the release of PRL. Intravenous administration of either rat (r) or ovine (o) PRL to lactating, nonsuckled rats increased basal plasma concentrations of OT. rGH was ineffective, but administration of oGH did produce some stimulation of OT release. Both oPRL and rPRL significantly enhanced the electrical stimulation-induced release of OT from isolated stalk-neurointermediate lobes, in vitro, without affecting the basal release of the peptide. oGH was ineffective on basal or stimulated in vitro OT release, and neither hormone altered basal or stimulation-induced release of vasopressin from these tissues. Both rPRL and oPRL reversed the inhibitory effect of the D-2 dopamine agonist bromocriptine. Immunoneutralization of circulating PRL with a highly specific antiserum abolished the increases in OT in response to either suckling or to administration of the D-2 dopamine antagonist domperidone.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of feeding-associated peptides and receptors by nicotine.

Although numerous epidemiological studies have provided convincing evidence for the inverse association between tobacco smoking and body weight, the molecular mechanisms underlying this relationship are not well-understood. Nicotine, as a potent secretagogue, could be expected to influence the levels and expression of many classes of neurotransmitters, as well as of cell-membrane constituents linked to neurotransmission, including signal transducers and related effectors. A potentially major group of candidate molecules that could be involved in feeding-related actions of nicotine are the numerous neuropeptides and peptide hormones shown in the past two decades to regulate food intake and energy expenditure. These could include neuropeptide Y (NPY), orexins, leptins, and uncoupling proteins (UCPs). Some of these peptides were already shown to respond to nicotine treatment in terms of regulation of levels and of activity at the level of cell-membrane receptors. The primary objective of this review is to summarize our current understanding of the regulatory effects of nicotine on the food intake and energy expenditure as related to the expression levels of leptin, NPY, orexin, uncoupling proteins, and of NPY and orexin receptors.

Agonist internalization by cloned Y1 neuropeptide Y (NPY) receptor in Chinese hamster ovary cells shows strong preference for NPY, endosome-linked entry and fast receptor recycling.

In Chinese hamster ovary (CHO) cells expressing the cloned guinea-pig Y1 receptor, the saturable, receptor-linked internalization of NPY (NPY)-related peptides showed the rank order of human/rat neuropeptide Y (hNPY)>pig/rat peptide YY (pPYY)>=(Pro(34))human PYY>(Leu(31),Pro(34))hNPY>(Leu(31),Pro(34))hPYY>>BVD-11 (a selective Y1 antagonist). All agonists accessed similar numbers of Y1 sites in particulates from disrupted cells, with relatively small affinity variation. The rate of internalization could significantly depend on the overall interactivity of the agonist peptide (reflected in sensitivity to chaotropic agents, as well as in the level of non-saturable binding and internalization). Concentration-dependent inhibition of the agonist-driven CHO-Y1 internalization was found with filipin III (a cholesterol-complexing macrolide), and confirmed with inhibitors of clathrin lattice formation, phenylarsine oxide (PAO) and sucrose. In the concentration range affecting Y1 internalization, none of the above treatments or agents significantly alter agonist affinity for Y1 cell surface or particulate receptors. Largely similar responses to the above inhibitors were observed in CHO-Y1 cells for internalization of human transferrin. Internalization of CHO-Y1 receptor apparently is driven by NPY in strong preference to other naturally encountered agonists. At 37 degrees C, most of the internalized receptor is rapidly recycled through endosome-like membrane elements, detectable in Percoll gradients.

Activation of Central D-1 Dopamine Receptors Stimulates Oxytocin Release in the Lactating Rat: Evidence for Involvement of the Hypothalamic Paraventricular and Supraoptic Nuclei

The stimulatory effect of dopamine (DA) on the release of oxytocin (OT) in lactating rats is exerted at the D-1 DA receptor subtype. Because the neural loci mediating this effect have not been identified, the objective of the present studies was to test whether OT release in the lactating rat would be elevated after central administration of a D-1 DA receptor agonist into the third ventricle (3V) or directly into either the rostral paraventricular/anterior commissural nucleus area (PVN/ACN), the central paraventricular nucleus area, or the supraoptic nucleus (SON), all of which contain OT neurosecretory cells. Lactating rats were implanted with a stainless steel cannula directed into one of the above areas or into the arcuate-ventromedial region of the medial basal hypothalamus (MBH), or sites dorsal to the PVN/ACN or SON, which served as anatomical controls. After 6-7 days of recovery, each animal received an intra-atrial cannula for sequential blood sampling, and was used in experiments 24 h later. Animals were separated from their litters, and following a period of basal blood sampling, received central microinjections of either vehicle, the D-1 DA receptor agonist SKF-38393, or the D-2 DA receptor agonist quinpirole, and blood samples were removed periodically for 60 min. An injection of angiotensin II (Ang II, 100 ng) was made into each site as a positive control for OT release.(ABSTRACT TRUNCATED AT 250 WORDS)

Excitatory and Inhibitory Dopaminergic Regulation of Oxytocin Secretion in the Lactating Rat: Evidence for Respective Mediation by D-1 and D-2 Dopamine Receptor Subtypes

The present experiments were designed to test whether the previously reported excitatory and inhibitory effects of dopamine (DA) on the secretion of oxytocin (OT) in lactating rats are exerted at different DA receptor subtypes, and to examine whether one or both of these effects might occur at the level of the posterior pituitary. The basal release of OT in nonsuckled, lactating rats was increased after intravenous administration of the D-1 DA agonist SKF 38393, and this effect, as well as the suckling-induced release of OT, was prevented by treatment with the D-1 DA antagonist SCH 23390, suggesting that DA may exert an important stimulatory influence over OT secretion through an action at the D-1 DA receptor subtype. A small stimulation of basal PRL release was also produced by SKF 38393, but blockade of the D-1 DA receptor did not prevent the suckling-induced release of this hormone. Stimulation of the D-2 DA receptor with PPHT had no effect on basal OT release in nonsuckled rats, but this agent, as well as another D-2 DA agonist, bromocriptine, prevented the suckling-induced release of both OT and PRL. The inhibitory effect of D-2 DA receptor stimulation was blocked by the D-2 DA antagonist domperidone, which increased the basal release of both hormones when given alone. These observations confirm previous findings that inhibitory effects of DA on suckling-induced OT release are mediated through activation of the D-2 DA receptor. To test whether either dopaminergic effect occurs at the level of neurosecretory endings in the neurointermediate lobe (NIL), the stalk-NIL was isolated from lactating rats and perifused in vitro. The stalk-NIL junction was electrically stimulated for 4 s, and the effects of selective D-1 DA and D-2 DA agonists and antagonists on the basal and electrically evoked release of OT and vasopressin (VP) was assessed using the two stimulation (S2/S1) paradigm. Electrical stimulation produced marked increases in release of both neural lobe peptides in a Ca(2+)-dependent manner, and the electrically evoked release of OT, but not VP, was enhanced by the opiate antagonist naltrexone (10 microM). Consistent with the in vivo results, SKF-38393 (20 microM) produced a small, but statistically significant, increase in electrically induced OT release, while SCH 23390 (20 microM) was without significant effect. Neither drug affected the basal release of OT or the basal or electrically stimulated release of VP.(ABSTRACT TRUNCATED AT 400 WORDS)

Ligand internalization by cloned neuropeptide Y Y5 receptors excludes Y2 and Y4 receptor-selective peptides

In human embryonic kidney-293 (HEK-293) cells, the cloned human neuropeptide Y Y5 receptor saturably internalized agonists, with the rank order of neuropeptide Y-(19-23)-[Gly1,Ser3,Gln4,Thr6,Ala31,Aib32,Gln34]human pancreatic polypeptide (neuropeptide Y-Aib-pancreatic polypeptide)>human neuropeptide Y>porcine peptide YY>[Pro34]human peptide YY>[Leu31,Pro34]human peptide YY>>human peptide YY-(3-36). Human pancreatic polypeptide competed [125I]neuropeptide Y binding and internalization in neuropeptide Y Y5 receptor-expressing cells, but itself showed no internalization. The internalization was strongly dependent on temperature. The surface binding, and especially the internalization, of human neuropeptide Y were highly sensitive to the clathrin network inhibitor phenylarsine oxide, and to the cholesterol-complexing antibiotic filipin III. The internalized ligands were present in particles corresponding to secondary endosomes in Percoll gradients, but especially in particles banding with the acid hexosaminidase lysosomal marker. At any temperature tested, internalization of the neuropeptide Y Y5 receptor driven by human neuropeptide Y in HEK-293 cells was much slower than the internalization of the neuropeptide Y Y1 receptor reported in the same cells, or in Chinese hamster ovary (CHO) cells. The neuropeptide Y Y5 receptor subtype could be the metabotropic receptor responding to protracted challenges by neuropeptide Y-like peptides, and its density could be little sensitive to concentration of extracellular agonists.

Central Stimulation of Oxytocin Release in the Lactating Rat by N-Methyl-D-Aspartate: Requirement for Coactivation through Non-NMDA Glutamate Receptors or the Glycine Coagonist Site

Excitatory amino acid (EAA) neurotransmitters participate in the regulation of secretion of several neuropeptides, including oxytocin (OT), via actions at different receptors. In earlier studies, release of OT could be achieved reliably by injection into the supraoptic nucleus (SON) of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptor agonists, but not by treatment with N-methyl-D-aspartate (NMDA) alone. This prompted further examination of the possible role of NMDA receptors in OT release following central coapplication of NMDA and AMPA-site agonists, or of NMDA and agonists active at the glycine coagonist site. The agonists were injected into the right SON, the right paraventricular nucleus (PVN), or into the third ventricle (3V) of nonsuckled lactating rats. Cotreatment with NMDA and AMPA (using doses that alone did not include OT release) elicited a strong OT release in all animals by either the SON or the PVN route, and this was attenuated by pretreatment/cotreatment with specific antagonists of either the NMDA or the AMPA receptor. The SON area or 3V coinjection of NMDA and the NMDA/glycine site agonists glycine or D-serine also induced OT discharges in all animals, while cotreatment in the PVN did not result in uniform OT discharges. This release was potently reduced by cotreatment with the specific NMDA/glycine site antagonist 5, 7-dichlorokynurenate (DCK). L-Serine somewhat increased the frequency of discharge-type response to NMDA, while intra-SON coinjection of L-leucine did not stimulate OT release. D-Serine alone stimulated the release of OT much less than in combination with NMDA, and with no obvious dose dependence. The suckling-induced release of OT was attenuated, but not abolished, by DCK, while PRL release was briefly stimulated by this agent. A physiological role for the NMDA receptor in OT release is clearly supported by these studies. NMDA receptor activation in the lactating rat may result from either an allosteric stimulation by glycine site agonists, or a synergistic interaction with the AMPA/kainate group of excitatory amino acid receptors.