Development and characterization of two novel 68 Ga-labelled neuropeptide Y short analogues with potential application in breast cancer imaging.

In vivo receptor targeting with radiolabelled peptide-based probes is an attractive approach for the development of novel radiotracers for molecular imaging. This work presents the development and characterization of two novel neuropeptide Y analogues labelled with a positron emitter 68 Ga, for potential use in breast cancer imaging. Both analogues share the same amino acid sequence and were derivatized with NOTA through either a lysine linker (L1) or an acetylated lysine (L2). In both cases, a single product with radiochemical purity higher than 95% was obtained. The two complexes were hydrophilic, showed remarkable in vitro stability, good cellular uptake, binding affinity in the nanomolar range and high cellular internalization rate. Biodistribution studies revealed low blood uptake and elimination through the urinary tract. The addition of an acetyl group in the spacer increased the lipophilicity of C2 and modified the reactivity of the ε-amino group of the lysine which resulted in lower protein binding and lower percentage of injected dose in bladder and urine. The tumour versus muscle ratio was (3.8 ± 0.4) for 68 Ga-L1 and (4.7 ± 0.4) for 68 Ga-L2. These results encourage performing further studies in order to complete the evaluation of both tracers as potential radiopharmaceutical for breast cancer imaging.

A Randomized Controlled Trial of Intranasal Neuropeptide Y in Patients With Major Depressive Disorder.

BACKGROUND: Since about one-third of patients with major depressive disorder (MDD) do not respond adequately to available antidepressants, there is a need for treatments based on novel mechanisms of action. Neuropeptide Y (NPY), a normal brain constituent, is reduced in cerebrospinal fluid of patients with MDD and post-traumatic stress disorder and in corresponding rodent models. Moreover, NPY administered centrally or intranasally rescues pathophysiology in these models. Consequently, we conducted the first, to our knowledge, controlled trial of NPY as a treatment for MDD. METHODS: Thirty MDD patients on a stable dose of a conventional antidepressant insufflated 6.8 mg NPY (n = 12) or placebo (n = 18) in a double blind randomized fashion. Effects were assessed at baseline, +1 hour, +5 hours, +24 hours, and +48 hours. The primary outcome was change in depression severity measured with the Montgomery-Åsberg Depression Rating Scale (MADRS). RESULTS: NPY was superior to placebo at +24 hours (change -10.3 [95% CI: -13.8; -6.8]) vs -5.6 (95% CI: -8.4; -2.7); group*time F = 3.26, DF = (1,28), P = .04; Cohen's d = 0.67). At +5 hours MADRS decreased -7.1 ([95% CI: -10.0; -4.2] vs -3.5 [95% CI: -5.8; -1.2]; group*time F = 2.69, DF = (1,28), P = .05; Cohen's d = 0.61). MADRS reduction at +48 hours was not significant. CONCLUSIONS: Since no results regarding the trajectory of NPY effects existed prior to this study we extrapolated from the known NPY biology and predicted the effects will occur 5-48 hours post insufflation. We chose +48 hours as the primary endpoint and +1, +5, and +24 hours as secondary endpoints. The results, the first of their kind, indicate that insufflated NPY is antidepressant, despite not meeting the primary outcome, and call for dose ranging and repeated NPY insufflation trials. CLINICAL TRIAL REGISTRATION: EudraCT Number: 2014-000129-19.

Novel chemically modified analogues of neuropeptide Y for tumor targeting.

The successful use of peptides as potential radiopharmaceuticals essentially requires the modification of the bioactive peptide hormones to introduce chelators for radiolabeling. In this study, four Y 1/Y 2 receptor-selective NPY analogues with different receptor subtype specificities have been investigated. For in vitro studies, the cold metal surrogate was used. Gallium and indium complexes were introduced by using 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid as bifunctional chelator. The peptides were synthesized by solid-phase peptide synthesis (SPPS), the chelator was coupled either at the N-terminus or at the N(epsilon) side chain of Lys(4) of the resin-bound peptide, and the labeling was performed in solution after cleavage. Competitive binding assays showed high binding affinity of the receptor-selective analogues at NPY receptor expressing cells. To test internalization of the novel peptide analogues and the metabolic stability in human blood plasma, the corresponding 5(6)-carboxyfluorescein (CF) analogues were prepared and investigated. One of the most promising analogues, the Y 1-receptor selective [Lys(DOTA)(4), Phe(7), Pro(34)]NPY was labeled with (111)In and injected into nude mice that bear MCF-7 breast cancer xenografts, and biodistribution studies were performed. In vitro and in vivo studies suggest that receptor-selective analogues of NPY have promising characteristics for future applications in nuclear medicine for breast tumor diagnosis and therapy.

Internalization of neuropeptide Y Y1 and Y5 and of pancreatic polypeptide Y4 receptors is inhibited by lithium in preference to sodium and potassium ions.

The receptor-linked internalization of [125I] human neuropeptide Y (NPY) in Chinese hamster ovary (CHO) cells expressing the guinea-pig Y1 receptors or in human endometrial carcinoma-1B (Hec-1B) cells expressing the human Y5 receptor, as well as the receptor-linked internalization of human pancreatic polypeptide (hPP) receptor expressed in CHO cells, is selectively inhibited by low molarities of the Li+ cation. The Na+ and K+ cations decreased the receptor-linked internalization of agonist peptides only at high molar inputs, and largely in proportion to the reduction of cell surface binding of Y ligand peptides, dependent on ion concentration and the type of Y receptor examined. With particulates isolated from disrupted cells, there was no preferential inhibition by Li+ relative to Na+ in the binding of type-specific ligand peptides to Y receptors of any type. The observed difference could be connected to the known ability of Li+ to modify active conformations of signal transducers, which may also directly or indirectly affect the internalization motors. The decrease in the rate of Y receptor internalization by Li+ also points to a possible alteration of Y receptor signaling in vivo by lithium at acute therapeutically employed dose levels.

Neuropeptide Y selectively potentiates alpha1-adrenoceptor-mediated contraction through Y1 receptor subtype in rat femoral artery.

The aim of this study was to investigate the synergism between neuropeptide Y and other vasoconstrictors (phenylephrine and serotonin) and which neuropeptide Y receptor subtype is responsible for the neuropeptide Y-induced potentiation. Exogenous neuropeptide Y (10 nM) potentiated alpha1-adrenoceptor-mediated (PE-induced) contraction in rat femoral artery permissively without its direct action, but not in the thoracic aorta. In contrast, neuropeptide Y produced no change in serotonin-induced contraction in both arteries. Increasing concentrations of neuropeptide Y caused dose-dependent potentiation of the phenylephrine-induced contraction in the femoral artery. This potentiation was blocked by a selective neuropeptide Y-Y1 receptor antagonist, BIBP3226 [(R)-N2-(diphenylacetyl)-N-[4-hydroxyphenyl)methyl]-argininamide] (1 microM). Semiquantitative reverse transcriptase polymerase chain reaction showed the selective expression of neuropeptide Y-Y1 receptor mRNA in the femoral artery. These findings indicated that the neuropeptide Y-induced selective potentiation of alpha1-adrenoceptor-mediated contraction is mediated through neuropeptide Y-Y1 receptor in rat femoral artery.

Localization and characterization of NPY/PYY receptors in rat frontoparietal cortex during development.

Neuropeptide Y (NPY) is present in most cerebrocortical areas during fetal and postnatal development. In the rat frontal cortex, a dense radial fiber network containing NPY immunoreactivity is observed transiently as early as embryonic day 17 (E17) and disappears at the end of the first postnatal week. We have investigated the distribution of NPY receptors in the frontoparietal cortex at 13 stages of development, from E15 fetuses to adults, by in vitro autoradiography, using (125)I-pPYY as a radioligand. Quantitative receptor density was measured through all cortical layers at each developmental stage. Pharmacological identification of (125)I-pPPY binding sites was made by competition experiments using pNPY or [Leu(31),Pro(34)]pNPY and pNPY(13-36), as selective competitors for Y1 and Y2 receptors, respectively. NPY receptors were first detected in the cerebral cortex at low densities at E19 in a thin layer of tissue corresponding to the inner half of the intermediate zone (IZ) and the upper ventricular zone (VZ). The neuroepithelium did not contain binding sites. High densities of sites were observed by E21 onward to P10 in the deep cortical layers corresponding to the IZ and layers V-VI. A decreasing gradient of receptor density was observed from layer VI to the marginal zone (layer I). The distribution of NPY receptors does not match with the perikarya of transient NPY-immunoreactive neurons located in the cortical plate but does coincide with their axonal extension. The receptor density decreased abruptly between P10 and P12 in deep layers, whereas a moderate expression of binding sites is detected from P10 to P12 in layers I-III. By P14, the binding level was the lowest observed in the postnatal period. From P21 onward, receptors were observed in superficial layers I-III, and their density rose by two- to threefold up to adulthood. Competition studies indicated that the NPY receptors located in the deep cortical layers of the E21 or P1 rat cortex exhibit Y2 receptor type characteristics. The binding sites detected in the superficial layers from P10 to P12 rats also show Y2 receptors characteristics, unlike the NPY receptors in layers II-III of the adult, which behave like Y1 receptors. These data show that different NPY receptor types are successively expressed in specific layers during late gestation and early postnatal life in the rat frontoparietal cortex.

99mTc-labeled neuropeptide Y analogues as potential tumor imaging agents.

The possible use of neuropeptide Y (NPY) as a novel radiopeptide has been investigated. NPY is a 36-amino acid peptide of the pancreatic polypeptide family, which is expressed in the peripheral and central nervous system, and is one of the most abundant neuropeptides in the brain. Its receptors are produced in a number of neuroblastoma and the thereof derived cell lines. As structure-activity relationships of NPY are well-known, we could assume where a radionuclide might be introduced without affecting receptor affinity. We applied the novel [99mTc(OH2)3(CO)3]+ aqua complex and PADA (2-picolylamine-N,N-diacetic acid) as bifunctional chelating agent. The peptides were synthesized by solid-phase peptide synthesis, and PADA was coupled to the side chain of Lys4 of the resin-bound peptide. Upon postlabeling of [K4(PADA)]-NPY, 99mTc(CO)3 did not only bind to the desired PADA, but presumably as well to the His in position 26. Since the replacement of His26 by Ala only slightly decreased binding affinity, [K4(PADA),A26]-NPY was specifically postlabeled, and the 185Re surrogate maintained high binding affinity. Furthermore, the prelabeling approach has been applied for the centrally truncated analogue [Ahx5-24]-NPY, which is highly selective for the Y2 receptor. The resulting Ac-[Ahx5-24,K4(99mTc(CO)3-PADA)]-NPY was produced with a yield of only 16%. Therefore, postlabeling was applied for the short analogue as well, again substituting His26 by Ala. Competitive binding assays using (185)Re as a surrogate for 99mTc showed high binding affinity of Ac-[Ahx5-24,K4(185Re(CO)3-PADA),A26]-NPY. Internalization studies with the corresponding 99mTc-labeled analogue revealed receptor-mediated internalization. Furthermore, biodistribution studies were performed in mice, and stability was tested in human plasma. Our centrally truncated analogue revealed a 6-fold increased stability compared to the natural peptide NPY. We conclude that Ac-[Ahx5-24,K4(99mTc(CO)3-PADA),A26]-NPY has promising characteristics for future applications in nuclear medicine.

Structure-activity relationships of neuropeptide Y Y1 receptor antagonists related to BIBP 3226.

Analogues of BIBP 3226, (R)-N(alpha)-diphenylacetyl-N-(4-hydroxybenzyl)argininamide, were synthesized and investigated for Y1 antagonism (Ca2+-assay, HEL cells) and binding on Y1, Y2 and Y5 receptors. Replacing the benzylamino by a tetrahydrobenzazepinyl group preserves most of the Y1 activity. Combination with a N(G)-phenylpropyl arginine and a N(alpha)-p-biphenylylacetyl moiety shifted the NPY receptor selectivity towards Y5.

Aspectos morfo-funcionales de arterias y venas mamarias usadas en revascularización cardíaca / Morphologic and functional aspects of mammary arteries and veins used for myocardial revascularization surgery

Los vasos sanguíneos están inervados por el sistema nervioso simpático autonómico. La fisiología y neuroquímica de los nervios perivasculares humanos ha sido poco estudiada. Con el propósito de contribuir a las investigaciones sobre la fisiología de la co-transmisión simpática humana, esta investigación se concentró en: i) estudiar el contenido de los neurotransmisores simpáticos, noradrenalina (NA) y neuropéptido y (NPY) en vasos de arteria y vena mamaria interna humana; ii) detectar mediante técnicas inmunohistoquímicas la presencia de los nervios simpáticos perivasculares de estos vasos; iii) caracterizar la reactividad vascular de la arteria mamaria interna, como un modelo usado en implantes de revascularización cardíaca. Se estudió además, la vena mamaria derivada de la misma biopsia. La arteria y vena mamaria contienen 50 veces más NA que NPY, el contenido de NA y NPY en la arteria y en la vena es muy similar. La detección inmunohistoquímica de los nervios simpáticos demuestra que éstos se localizan entre las capas musculares de los vasos. La estimulación de los filetes nerviosos perivasculares produce respuestas vasomotoras sensibles a tetrodotoxina y guanetidina, lo que es consistente con la naturaleza simpática de la respuesta, confirmando que parte de los nervios perivasculares son simpáticos. Los músculos lisos se estimulan por NA y por ATP, que sólo no contrae, facilita las respuestas vasomotoras de la NA. Estos resultados permiten concluir que en la arteria y la vena mamaria interna humana NA, ATP y NPY cooperan en la respuesta vasomotora, evidenciando la co-transmisión simpática en humanos

Nonsaturable entry of neuropeptide Y into brain.

Neuropeptide Y (NPY) is found and is active both in the periphery and brain, but its crossing of the blood-brain barrier (BBB) in either direction has not been measured. We used multiple time-regression analysis to determine that radioactively labeled NPY injected intravenously entered the brain much faster than albumin, with an influx constant of 2.0 x 10(-4) ml. g. -1. min-1. However, this rate of entry was not significantly changed by injection of 10 microgram/mouse of excess NPY, by leptin, or by food deprivation. HPLC showed that most of the NPY entering the brain was intact, and capillary depletion with and without washout showed that the NPY did not remain bound to endothelial cells or associated with vascular elements. Perfusion in a blood-free solution eliminated binding to serum proteins as an explanation for the lack of saturation. Efflux of labeled NPY from the brain occurred at the same rate as albumin, reflecting the normal rate of reabsorption of cerebrospinal fluid. Thus NPY can readily enter the brain from blood by diffusion across the BBB.

Labelling of peptides with 1.4-nm gold particles to demonstrate their binding sites in the rat spinal cord.

Recently we presented a method to label the neuropeptide substance P with a 1.4-nm gold particle covalently bound at the N-terminus that can be used for demonstrating its binding sites in histological sections. In this study we examined whether the peptides neuropeptide Y, somatostatin, calcitonin gene-related peptide and bradykinin can be labelled in the same way. Polyacrylamide gel electrophoresis revealed a reduction in mobility for peptide-gold conjugates over gold particles alone consistent with peptide binding. In cryostat sections of the rat lumbar spinal cord, the peptides showed a distinct binding pattern in the grey matter corresponding to data of studies using autoradiographic methods. Therefore, we conclude that this simple and fast method can be used for labelling peptides in general to demonstrate their binding sites in histological sections, provided the peptide binds by its C-terminus.

The perifornical area: the major focus of (a) patchily distributed hypothalamic neuropeptide Y-sensitive feeding system(s).

Neuropeptide Y (NPY), a neurochemical found in high concentrations within hypothalamic neurons, is believed to participate in the control of eating behavior and body energy balance and elicits a powerful eating response when injected into the hypothalamus. To delineate precisely the locus of this effect, NPY (78 pmol) or its artificial cerebrospinal fluid vehicle was injected in the extremely small volume of 10 nl through chronic guide cannulae into an array of 47 different hypothalamic areas in satiated rats and the elicited food intake was measured. To determine the anatomical resolution of this technique, the spread and recovery of [125I]NPY injected in 10 nl was also assessed. Results indicate that as much as 95% of the injected label was recovered within the brain tissue and guide cannulae and that 100% of the tissue label was localized to within 0.8 mm of the injection site. Behavioral results show that the perifornical hypothalamus (PFH), at the level of the caudal paraventricular nucleus, is the most sensitive hypothalamic site for NPY-induced eating. NPY there elicited mean increases in food intake of 12.5 g over baseline at 1 h and 20.0 g at 4 h postinjection. Injections bracketing the PFH in all directions were substantially less effective. Additionally, significant effects were also observed in at least seven other sites that were distributed throughout the hypothalamus. These findings suggest both that the PFH may be the primary hypothalamic site containing feeding-related NPY-sensitive receptors and that other sites distributed within the hypothalamus also can mediate NPY's effects.

Splanchnic and renal vasoconstrictor and metabolic responses to neuropeptide Y in resting and exercising man.

The local clearance of neuropeptide Y (NPY) and whether NPY influences splanchnic and renal metabolism in man have not been investigated previously. The influence of NPY on splanchnic and renal blood flows at physiologically elevated levels has also not been investigated. The effects of a 40-min constant NPY infusion (3 pmol kg-1 min-1) at rest and during 130 min of exercise (50% of VO2max) were studied in six healthy subjects and compared with resting and exercising subjects receiving no NPY. Blood samples were drawn from arterial, hepatic and renal vein catheters for the determination of blood flows (indicators: cardiogreen and para-aminohippuric acid [PAH]), NPY, catecholamines, glucose, lactate and glycerol. NPY infusion was accompanied by: (1) significant fractional extraction of NPY-like immunoreactivity (NPY-Li) by splanchnic tissues at rest (58 +/- 5%) and during exercise (53 +/- 6%), while no arterial-venous differences could be detected across the kidney; (2) a reduction in splanchnic and renal blood flows of up to 18 and 13% respectively (P less than 0.01-0.001) at rest without any additional changes during exercise; and (3) metabolic changes as reflected in: (a) a more marked fall in arterial glucose during exercise compared to the reference group (P less than 0.05); (b) a 35% lower splanchnic glucose release (P less than 0.01) during exercise due to diminished glycogenolysis (P less than 0.01); and (c) a lower arterial lactate level (18% P less than 0.05) together with unchanged splanchnic lactate uptake during exercise, suggesting reduced lactate production by extrahepatic tissues. The disappearance of plasma NPY-Li after the infusions was biphasic with two similar half-lives at rest (4 and 39 min) and during exercise (3 and 43 min).

Regional distribution of binding sites for neuropeptide Y in cat and monkey visual cortex determined by in vitro receptor autoradiography.

The goal of this study was to elucidate the precise regional and laminar distribution of neuropeptide Y (NPY) binding sites in feline and primate visual cortex. By means of in vitro receptor autoradiography, NPY binding sites in primate and feline visual cortex were specifically labeled with 3H-NPY. In cat area 17, the highest density of NPY-binding sites was present in lamina I and the upper half of lamina II. The density then gradually decreased towards lamina VI. Areas 18 and 19 exhibited a similar binding site-density profile. The decrease in density from superficial to deep layers was more gradual in area 18 than in areas 17 and 19. In monkey primary visual cortex (V1), layer IVc presented a high concentration of NPY binding sites, in addition to a dense zone of binding sites in layer I. Monkey secondary visual cortex (V2) displays a similar dense zone in layer I, but lacks such high density of NPY binding sites in layer IV. Therefore, the border between primary and secondary visual cortex coincides with the abrupt disappearance of this latter high density in layer IV. In cat as well as in monkey visual cortex, no significant differences were found between regions representing central vision and those representing the peripheral parts of the visual field. Comparison of our results for NPY binding sites with the distribution of alpha 1-adrenergic receptors, as recently described by Rakic et al. (J. Neurosci. 8(10):3670-3690, 1988) for primate and Parkinson et al. (Brain Res. 457:70-78, 1988) for feline visual cortex, revealed that those two patterns are very similar.

Co-release and functional interactions of neuropeptide Y and noradrenaline in peripheral sympathetic vascular control.

1. The immunohistochemical results suggest that NPY-LI co-exists with NA in sympathetic periarterial nerves of skeletal muscle. The corresponding veins are associated with few or no sympathetic nerves. 2. SNS evoked a detectable overflow of NPY-LI from the dog gracilis muscle in vivo during high-frequency but not low-frequency stimulation. After the administration of alpha-adrenoceptor antagonists, the overflow of NPY-LI was enhanced, indicating prejunctional inhibition of release via a mechanism similar to that for NA. Reserpine pretreatment depleted both NA and NPY-LI from skeletal muscle. The reserpine-induced depletion of NPY-LI, but not of NA, was highly dependent on intact nerve activity, since it was prevented by decentralization. 3. Slowly developing and long-lasting vasoconstriction was evoked by exogenous NPY and by high-frequency SNS in the gracilis muscle in the presence of adrenoceptor antagonists that completely blocked the vasoconstrictor response to exogenous NA. After reserpine treatment, considerably larger vasoconstrictor responses to SNS were observed in the decentralized muscle than in the intact one, in parallel with the overflow of NPY-LI. These results suggest that the released NPY may be the mediator of the adrenoceptor antagonist- and reserpine-resistant functional responses to SNS in the gracilis muscle. ATP mechanisms seem to be of less importance for the sympathetic control of gracilis muscle vasculature. 4. The plasma levels of NPY-LI and NA in man increased during physical exercise, suggesting a co-release from sympathetic nerves. This release appears to be attenuated by activation of alpha-adrenoceptors since phentolamine and clonidine, respectively, enhanced and suppressed the exercise-induced increase in the plasma levels of both NPY-LI and NA. The plasma levels of NPY-LI were increased during control conditions, and altered by clonidine and phentolamine, mainly at high work loads, which may indicate preferential release of the peptide at high levels of sympathetic activity. 5. Local i.a. infusion of NPY in man reduced forearm blood flow with a threshold effect at a plasma concentration in the low nmolar range. The reduction in forearm blood flow induced by NPY was considerably more long-lasting than that evoked by NA, which may partly be related to the long metabolic half-life of NPY. 6. NPY induced contractile effects in vitro that varied between different arteries and veins. The most pronounced effects were evoked in small human skeletal muscle arteries, where NPY caused a maximal contraction similar to that of NA.(ABSTRACT TRUNCATED AT 400 WORDS)

Neuropeptide Y and noradrenaline mechanisms in relation to reserpine induced impairment of sympathetic neurotransmission in the cat spleen.

The mechanisms underlying the reserpine-induced impairment of the functional responses to sympathetic nerve stimulation and output of noradrenaline (NA) and neuropeptide Y (NPY)-like immunoreactivity (-LI) were studied using the isolated blood-perfused cat spleen. Splenic nerve stimulation (10 Hz for 2 min) during control conditions caused perfusion-pressure increase, volume reduction and an increased output of NA and NPY-LI. After administration of phenoxybenzamine, the nerve stimulation-induced perfusion-pressure increase was almost abolished, the volume reduction inhibited and the output of NPY-LI enhanced. After subsequent addition of propranolol, a clear-cut increase in perfusion pressure upon nerve stimulation reappeared. Local infusion of NPY caused a potent, long-lasting, adrenoceptor-resistant increase in perfusion pressure and a relatively smaller volume reduction of the spleen. Twenty-four hours after reserpine pretreatment (1 mg kg-1 i.v.), which depleted the splenic content of NA greater than 95% and NPY-LI by about 50%, the functional responses upon nerve stimulation were markedly reduced. Preganglionic denervation or pretreatment with the ganglionic-blocking agent chlorisondamine did not influence the NA depletion after reserpine treatment. A considerable, adrenoceptor antagonist-resistant, long-lasting functional response as well as a markedly enhanced output of NPY-LI then occurred upon nerve stimulation. In conclusion, reserpine treatment combined with interruption of preganglionic impulse flow reveals non-adrenergic, nerve stimulation evoked splenic functional responses which could be mediated by release of a cotransmitter peptide like NPY.

Central catecholamine-neuropeptide Y interactions at the pre- and postsynaptic level in cardiovascular centers.

Central catecholamine (CA)-neuropeptide Y (NPY) interactions and their regulation by glucocorticoids have been analyzed in vivo and in vitro, especially in the dorsal cardiovascular center of the medulla oblongata, including the nucleus tractus solitarius (nTS), using immunocytochemical, receptor autoradiographical, biochemical, and physiological techniques. Intraventricular (i.v.t.) injections of NPY in a low (7.5 pmol) or a high (1.25 nmol) dose increased adrenaline levels 4 h later in the caudal part of the dorsomedial medulla. Furthermore, NPY immunoreactivity (IR) tended to decrease in the rostral part of the dorsomedial medulla 5 min after injection of clonidine (1 microgram) in the alpha-chloralose anaesthetized rat. Thus, presynaptic interaction between NPY and adrenaline (A) mechanisms may exist in the dorsal cardiovascular center taking place at the network local circuit level or the membrane level of the NPY/A costoring synapses of the dorsomedial medulla. In vitro NPY (10 nM) reduced the affinity of the alpha 2-adrenergic agonist binding sites in the nTS, and clonidine (10 nM) reduced the 125I-NPY binding in the dorsomedial medulla. These results indicate the existence of postsynaptic receptor-receptor interactions between alpha 2-adrenergic and NPY receptors in the dorsal cardiovascular center. This interaction may in part take place at the level of the Ni protein, since NPY (300 nM) inhibited cyclic AMP (cAMP) accumulation in slices of the dorsomedial medulla. However, the interactions also probably take place at the proteins carrying the recognition sites, since NPY and adrenaline together given i.v.t. significantly antagonized the hypotensive effects of one another. Thus, the reduced affinity of the alpha 2-adrenergic receptor induced by NPY may reflect a reduced efficiency of this receptor and not an increased coupling of Ni protein to the adenylate cyclase. Thus, the postsynaptic interaction between the two receptors represents inter alia a sensitivity regulation of the two receptors. Evidence is also presented for the existence of a glucocorticoid regulation of NPY IR neurons, especially of those innervating the locus coeruleus, since after 2 weeks adrenalectomy reduced NPY IR in this area. Furthermore, glucocorticoid receptor IR was demonstrated in the nuclei of NPY nerve cell bodies of the nTS. Thus, glucocorticoids exert direct actions on cardiovascular NPY/CA costoring neurons, actions that may contribute to their hypertensive effects in humans.(ABSTRACT TRUNCATED AT 400 WORDS)