Functional effects of neuropeptide Y receptors on blood flow and nitric oxide levels in the human nose.

The aim of this study was to examine dose-dependent effects of intranasal application of neuropeptide Y (NPY) on nasal mucosal blood flow, blood content, and intranasal nitric oxide (NO) concentration. Blood flow was measured by laser Doppler flowmetry (LDF) and blood content by rhinomanometry. Mucosal biopsies were taken for investigation of Y1 and Y2 receptor mRNA expression, using the reverse transcriptase-polymerase chain reaction (RT-PCR). Intranasal application of NPY evoked a dose-dependent reduction of nasal mucosal blood flow. Maximal vasoconstriction, seen at 12 nmol, was -37.5 +/- 6.2%, p < 0.05 (n = 9). The vasoconstrictive effect developed within 2 to 4 min and lasted > 17 min. NPY evoked a dose-dependent reduction of nasal airway resistance (NAR) on the ipsilateral side. Maximal decrease was -24.0 +/- 10.0% at 12 nmol, p < 0.05 (n = 9). There was a decrease in nasal NO production on the ipsilateral side after application of NPY 12 nmol (-7.4 +/- 1.2%, p < 0.05, n = 8). RT-PCR products corresponding to Y1 receptor but not Y2 receptor mRNA were obtained from biopsies of the nasal mucosa. In conclusion, NPY is a potent vasoconstrictor in the human nose reducing mucosal blood flow, as well as the blood content. The effect is probably mediated via Y1 receptors. NPY receptor agonists may prove beneficial in the treatment of the congested nose in allergic or vasomotor rhinitis.

The cloned guinea pig neuropeptide Y receptor Y1 conforms to other mammalian Y1 receptors.

We have cloned the guinea pig neuropeptide Y (NPY) Y1 receptor and found it to be 92-93% identical to other cloned mammalian Y1 receptors. Porcine NPY and peptide YY (PYY) displayed affinities of 43 pM and 48 pM, respectively. NPY2-36 and NPY3-36 had 6- and 46-fold lower affinity, respectively, than intact NPY. Functional coupling was measured by using a microphysiometer. Human NPY and PYY were equipotent in causing extracellular acidification with EC50 values of 0.59 nM and 0.69 nM, respectively, whereas NPY2-36 and NPY3-36 were about 15-fold and 500-fold less potent, respectively, than NPY. The present study shows that the cloned guinea pig Y1 receptor is very similar to its orthologues in other mammals, both with respect to sequence and pharmacology. Thus, results from previous studies on guinea pig NPY receptors might imply the existence of an additional Y1-like receptor sensitive to B1BP3226.

Cloning and functional expression of the guinea pig neuropeptide Y Y2 receptor.

Five neuropeptide Y (NPY) receptor subtypes have been cloned in mammals. The degree of sequence conservation differs considerably between subtypes as well as between evolutionary lineages. To shed further light on this, we have cloned the five NPY receptors in the guinea pig. Here, we report the cloning of the guinea pig Y2 receptor. The Y2 receptor is generally highly conserved, with 90-95% identity between different orders of mammals, including the guinea pig. The guinea pig receptor has a divergent cytoplasmic tail, indicating possible differences in regulation of signalling and/or down regulation. COS-7 cells transiently transfected with the gpY2 receptor show saturable 125I-PYY binding with a Kd = 6 pM. In displacement experiments, the gpY2 receptor was similar to the human and rat receptors with the following rank order of potencies: pNPY > pPYY > pNPY13-36 = pNPY22-36 >> [Leu31Pro34]NPY > BIBP3226. Thus, the guinea pig Y2 receptor is well conserved in comparison with human and rat with regard to both amino acid sequence and pharmacological profile.

Suppression of sensory C fiber-mediated contractions by neuropeptide Y Y1 receptors in the guinea pig bronchi.

The aim of the study was to examine which neuropeptide Y (NPY) receptor types that are coupled to inhibition of sensory C fiber-mediated contractions of the guinea pig bronchi. NPY and PYY evoked a concentration-dependent inhibition of the electrically stimulated contractions. The Y1 receptor-selective antagonist BIBP3226 (1 microM) evoked a rightward shift of the NPY-induced response. Also the Y1 (and Y4-Y6) receptor agonist [Leu31,Pro34]NPY suppressed the stimulated contractions with a potency similar to the parent molecule. BIBP3226 (1 microM) also attenuated the response induced by [Leu31,Pro34]NPY. The Y2 receptor agonist [Cys2, Aoc524, D-Cys27]NPY suppressed the stimulated contractions at 1 microM only. NPY 2-36 was much less potent than NPY itself and pretreatment with BIBP3226 did not affect the inhibitory response. Human pancreatic polypeptide (Y4-Y6 receptor agonist) was inactive (< or = 1 microM). In conclusion, NPY is capable of suppressing sensory nerve-mediated contractions in the guinea pig bronchi mainly via Y1 receptors.

Characterization of the receptor response for the neuropeptide Y-evoked suppression of parasympathetically-mediated contractions in the guinea pig trachea.

Neuropeptide Y (NPY) acts via several distinct receptor types. The aim of the present study was to examine which NPY receptors are coupled to inhibition of parasympathetically-mediated contractions of the isolated guinea pig trachea. Electrical field stimulation of tracheal rings evoked a rapid twitch, which was abolished by atropine (1 microM). NPY, the structurally related hormone peptide YY (PYY), the Y2 receptor agonist [Cys2, Aoc5-24, D-Cys27]NPY, as well as NPY 5-36 and NPY 13-36 evoked a concentration-dependent inhibition of the electrically-stimulated twitches. Pretreatment with the Y1 receptor-selective antagonist BIBP3226 (1 microM) failed to prevent the NPY-induced inhibition. Although less potent than NPY, the Y1 (and Y4-Y6) receptor agonist [Leu31, Pro34]NPY also inhibited the electrically-stimulated twitches. Another NPY-related peptide, pancreatic polypeptide, which recognizes Y4-Y6 receptors did not affect the stimulated twitches at concentrations up to 1 microM. However, pretreatment with the Y1 receptor-selective antagonist BIBP3226 (1 microM) virtually abolished the inhibition evoked by [Leu31, Pro34]NPY. None of the peptides affected the baseline tension and BIBP3226 (1 microM) per se did not affect the amplitude of the electrically-stimulated twitches. In conclusion, it seems that NPY and PYY are capable of suppressing parasympathetically mediated contractions in the guinea pig trachea mainly via Y2 receptors, but there is also a small contribution from Y1 receptors.

Pitfalls using metalloporphyrins in carbon monoxide research.

The proposal that endogenously produced carbon monoxide (CO) may act as a biological messenger has remained controversial. Carbon monoxide is generated by haem oxygenase isoenzymes in the degradation of haem-containing molecules. Certain metalloporphyrins, which are inhibitors of haem oxygenase, have been widely used as pharmacological tools in order to establish a messenger role for CO in the brain and periphery. However, increasing evidence shows that many metalloporphyrins are also associated with a large range of undesired effects, which make the interpretation of results using such compounds very uncertain. In this article, Lars Grundemar and Lars Ny evaluate the properties and describe the nonselective effect profile of such metalloporphyrins.

Electroconvulsive treatment evokes release of preprotachykinin-A mRNA into the cerebrospinal fluid and ocular aqueous humor of rabbits.

Following electroconvulsive treatment (ECT) of rabbits, preprotachykinin-A (PPT-A) mRNA was detected by Southern blot analysis of polymerase chain reaction (PCR)-amplified products in the cerebrospinal fluid (CSF) and aqueous humor of the eye. In contrast, no PPT-A mRNA could be detected in samples from untreated animals. In addition, several neuropeptides (substance P, neuropeptide Y, cholecystokinin, calcitonin gene-related peptide and pituitary adenylate cyclase activating peptide) were released into the CSF (and aqueous humor) following ECT. The results suggest that PPT-A mRNA was released together with neuropeptides into the CSF and aqueous humor in response to ECT. Indeed, previous studies have suggested that neurons can release neuropeptide mRNAs and that neurons are capable of taking up and expressing foreign mRNA. If neuropeptide mRNA can be taken up and utilized by another neuronal population, it might explain instances when neurons display 'phenotypic switch', i.e. the transient expression of novel neuropeptides.

Ocular inflammation induced by electroconvulsive treatment: contribution of nitric oxide and neuropeptides mobilized from C-fibres.

1. Electroconvulsive treatment (ECT) of rabbits produced ocular inflammation consisting of conjunctival hyperaemia, miosis and protein extravasation into the aqueous humour, reflected by the so-called aqueous flare response (AFR): the maximal reduction in pupil size was 3.8 +/- 0.1 mm (s.e. of mean, n = 16) while the maximal AFR was 28.1 +/- 2.8 (arbitrary units). 2. ECT also caused release of substance P (SP), pituitary adenylate cyclase-activating peptide (PACAP)-27, -38 and calcitonin gene-related peptide (CGRP). The concentrations of SP and CGRP in the aqueous humour of normal, untreated eyes were 10.6 +/- 1.4 and 117.4 +/- 12.4 pmol l-1, respectively, while the concentrations of PACAP-27 and -38 were below the detection limit. After ECT the concentrations of SP, PACAP-27, -38 and CGRP were 65.0 +/- 9.6, 46.9 +/- 8.4, 50.2 +/- 5.4 and 1109.9 +/- 133.1 pmol l-1, respectively (s.e. of mean, n = 12). Conceivably, ECT evoked an antidromic activation of sensory neurones in the trigeminal ganglion with the consequent release of neuropeptides from C-fibres in the uvea and the development of neurogenic inflammation. 3. Rabbits received the nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 200 mg kg 1, i.v.). This pretreatment inhibited the ECT-evoked conjunctival hyperaemia, miosis and AFR: under these circumstances the maximal reduction in pupil size was 1.9 +/- 0.1 mm while the maximal AFR was 2.7 +/- 0.9 (n = 16). L-NAME also inhibited the ECT-evoked release of SP, PACAP-27, -38 and CGRP into the aqueous humour; the concentrations of SP and CGRP were 13.2 +/- 1.5 and 204.8 +/- 33.5 pmol l-1, respectively, while PACAP-27 and -38 were below the detection limit (n = 12). 4. The ECT-evoked miosis was also inhibited by pretreatment with the tachykinin receptor antagonist D-Pal9 spantide 11 (90 nmol, intravitreal injection); under these circumstances the maximal reduction in pupil size was only 0.7 +/- 0.03 mm, indicating an important role for SP in the miotic response. Pretreatment of the eye with capsaicin, which is known to cause functional ablation of C-fibres, inhibited the conjunctival hyperaemia, miosis and AFR by 40-50%; the maximal reduction in pupil size being 2.2 +/- 0.2 mm and the maximal AFR 13.8 +/- 2.1 (arbitrary units) (n = 8). 5. The results suggest (1) that ECT evokes ocular inflammation through antidromic C-fibre activation; (2) that SP contributes to the ECT-evoked miosis; and (3) that NO contributes to the antidromic C-fibre activation and possibly to the vascular responses mediated by the C-fibre transmitters.

Characterization of Y3 receptor-mediated synaptic inhibition by chimeric neuropeptide Y-peptide YY peptides in the rat brainstem.

1. Neuropeptide Y (NPY) and peptide YY (PYY) act at receptors referred to as Y1 and Y2, while the Y3 receptor is specific to NPY and does not recognize PYY. The effects of NPY, its related peptides and a series of newly constructed chimeric NPY-PYY peptides were examined on excitatory and inhibitory postsynaptic currents (e.p.s.cs and i.p.s.cs, respectively) in rat dorsomedial nucleus tractus solitarius (NTS) neurones recorded in coronal brainstem slices. Monosynaptic activity was evoked by electrical stimulation in the region of the tractus solitarius. 2. NPY (5-500 nM) inhibited e.p.s.cs and i.p.s.cs in a concentration-dependent manner. In contrast, PYY (500 nM) failed to affect either e.p.s.cs or i.p.s.cs. The N- and C-terminal parts of a series of chimeric NPY-PYY peptides were joined at positions where NPY and PYY sequences differ. In binding experiments the chimeric peptides were all about equipotent with NPY and PYY in displacing [125I]-PYY from Y1 and Y2 binding sites on SK-N-MC cells and rat hippocampus respectively. 3. In the whole cell voltage clamp recordings of NTS neurones, NPY(1-23)-PYY(24-36) and NPY(1-14)-PYY(15-36) evoked a concentration-dependent inhibition of e.p.s.cs and i.p.s.cs, while NPY(1-7)-PYY(8-36) and NPY(1-3)-PYY(4-36) were inactive. The only differences in amino acid residues between NPY(1-14)-PYY(15-36) and NPY(1-7)-PYY(8-36) reside in positions 13 and 14. 4. Furthermore, [Pro34]NPY (500 nM) was equivalent in potency to NPY itself at inhibiting monosynaptic transmission in NTS, while [Leu31,Pro34]NPY and pancreatic polypeptide (both at 500 nM) failed to affect synaptic transmission. 5. The present study has shown that NPY acts at Y3 receptors to suppress both excitatory and inhibitory currents in the NTS. The different efficacy of the chimeric NPY-PYY peptides suggests that positions 13 and 14 are of great importance for Y3 receptor recognition. Finally, this receptor type readily recognizes [Pro34]NPY, but not [Leu31,Pro34]NPY.

Inhibition of stimulated cyclic AMP production by multiple neuropeptide Y receptors in the rat brainstem.

Neuropeptide Y (NPY) has been shown to modulate blood pressure, heart rate and to inhibit the baroreceptor reflex at the level of nucleus tractus solitarius (NTS). The aim of this study was to examine effects of NPY and its related peptides on forskolin (1 microM)-stimulated cyclic AMP production in slices of the rat NTS. Each peptide was present at 0.3 microM. Pretreatment with NPY inhibited the stimulated increase in cyclic AMP levels in slices of rat NTS. Also [Pro34]NPY, an analog, which activates Y1, Y3 (and Y5) receptors inhibited the stimulated increase in cyclic AMP levels. However, pretreatment with the Y1 receptor-selective antagonist BIBP3226 (3 microM) did not affect the [Pro34]NPY-evoked inhibition of cyclic AMP levels. In addition, [Leu31,Pro34]NPY, an Y1 (and PP1/Y4 and Y5) receptor agonist did not inhibit the stimulated increase in cyclic AMP production. Also the Y2 receptor-selective agonist C2-NPY inhibited the stimulated elevation of cyclic AMP levels, while peptide YY, which does not recognize Y3 receptors did not significantly affect the stimulated cyclic AMP production. In conclusion, it seems that Y2 and Y3 receptors are coupled to inhibition of adenylate cyclase activity in the rat NTS.

Effects of the neuropeptide Y (NPY)-receptor antagonist BIBP3226 on vascular NPY-receptors with different ligand requirements.

The aim was to examine effects of a newly developed neuropeptide Y (NPY)-receptor antagonist, BIBP3226 and to characterize NPY-receptors in the isolated guinea pig caval vein and human subcutaneous artery, respectively. BIBP3226 < or = 1 microM did not affect the basal tension. Pretreatment with increasing concentrations of BIBP3226 (10 nM-1 microM) resulted in a progressive rightward shift of the concentration-response curve to the Y1-receptor selective agonist [Pro34]NPY in the guinea pig caval vein. Regression analysis of the Schild plot gave a pA2-value of 7.58 (7.20-8.33, 95% confidence interval), slope of regression line 0.96 (0.52-1.39, 95% confidence interval) and a correlation coefficient of 0.78. NPY and the C-terminal NPY 2-36 evoked equipotent concentration-dependent contractions, both of which were sensitive to BIBP3226. Although less potent than NPY 2-36, also the contraction induced by NPY 5-36 was antagonized by BIBP3226. In the human subcutaneous artery [Pro34]NPY but not NPY 2-36 (< or = 0.3 microM) evoked a concentration-dependent contraction. Pretreatment with BIBP3226 (0.1 microM) resulted in a rightward shift of the concentration-response curve to [Pro34]NPY (from 7.38 +/- 0.10 to 6.95 +/- 0.16 (P < 0.05, n = 6). The present study has shown that the Y1-receptor-selective antagonist BIBP3226 potently antagonizes vascular NPY-receptors with different ligand requirements in the guinea pig caval vein and human subcutaneous artery, respectively. It appears that the guinea pig Y1-receptor is much less stringent in its demand on the N-terminal part of NPY than that of human Y1-receptors.

Localization and activity of haem oxygenase and functional effects of carbon monoxide in the feline lower oesophageal sphincter.

1. In the feline lower oesophageal sphincter (LOS), the distribution of the carbon monoxide (CO) producing enzymes haem oxygenase (HO)-1 and -2 was studied by immunohistochemistry and confocal microscopy, the HO activity was measured and the possible role for CO as a mediator of relaxation was investigated. 2. HO-2 immunoreactivity was abundant in nerve cell bodies of the submucosal and myenteric plexus. Approximately 50% of the HO-2-containing myenteric cell bodies were also nitric oxide synthase- and vasoactive intestinal peptide (VIP)-immunoreactive. In addition, HO-2 immunoreactivity was seen in nerve fibres, in non-neuronal cells dispersed in the smooth muscle and in arterial endothelium. HO-1 immunoreactivity was confined to non-neuronal cells in the smooth muscle, similar to those positive for HO-2. 3. Activity of HO, measured as CO production, was observed in LOS homogenates at a rate of 1.00 +/- 0.05 nmol mg-1 protein h-1. This production was inhibited by the HO inhibitor, zinc protoporphyrin-IX (ZnPP). 4. In isolated circular smooth muscle strips of LOS, developing spontaneous tone, exogenously administered CO evoked a concentration-dependent relaxation reaching a maximum of 93 +/- 3%. This relaxation was accompanied by an increase in cyclic GMP, but not cyclic AMP levels. The relaxant response was attenuated by methylene blue, but unaffected by tetrodotoxin. Repeated exposure to CO resulted in a progressive reduction of the relaxant response. 5. ZnPP caused a rightward-shift of the concentration-response curves for the relaxant responses to VIP, peptide histidine isoleucine, and pituitary adenylate cyclase activating peptide 27. 6. ZnPP and tin protoporphyrin-IX (another inhibitor of HO) did not affect nonadrenergic, noncholinergic relaxations induced by electrical field stimulation. Nor did ZnPP affect relaxations induced by 3-morpholino-sydnonimine or forskolin. 7. The present findings, showing localization of HO immunoreactivity to both neuronal and nonneuronal cells of the feline LOS, ability of LOS to produce CO and a relaxant effect of CO in circular LOS muscle, suggest a role for CO as a peripheral messenger.

Ligand binding and functional effects of systematic double D-amino acid residue substituted neuropeptide Y analogs on Y1 and Y2 receptor types.

In order to identify the signal epitopes of the neuropeptide Y (NPY) molecule, the conformation of the NPY molecule was pertubated by a systematic double D-amino acid replacement of neighbouring residues. These NPY-analogs were examined for receptor affinity and on biological activity. The rat cerebral cortex and hippocampus were used for binding characteristics on Y1 and Y2 binding sites, respectively, while the isolated guinea pig caval vein and rat vas deferens were used in functional characterization of Y1 and Y2 receptors, respectively. The NPY analogs were examined as ligands at [3H]NPY binding sites in homogenates of the rat brain. Pairwise D-substitutions of either of the first 6 amino acid residues in the N-terminal part of the molecule resulted in a 20-100-fold loss of affinity for Y1 binding sites compared with the native peptide. In comparison, the same analogs displayed affinities, which were about 8-40 times lower than NPY itself at Y2 binding sites. Especially [D-Ser3,D-Lys4]NPY had a low affinity to Y1 and Y2 binding sites. For many of the pairwise D-amino acid substituted NPY analogs, there were similar affinities for Y1 and Y2 binding sites in the cerebral cortex and hippocampus, respectively. D-Amino acid residue substitutions in positions 7 and 8 did essentially not affect the affinity to either type of binding site, while such replacements in positions 19 and 20 resulted in a drastic loss of affinity to both types of NPY binding site. In contrast, [D-Tyr21,D-Ser22]NPY was only slightly less potent than NPY itself on either type of binding site. Pairwise D-amino acid substitutions in the C-terminal (positions 27 to 36) decreased the affinity to Y1 and Y2 binding sites by 2 to 3 orders of magnitude. In the guinea pig vena cava the D-amino acid substituted NPY analogs evoked a concentration-dependent contraction with an rank order of potency similar to that of the respective analog at Y1 binding sites in the cerebral cortex. Similarly, in the rat vas deferens the D-amino acid substituted NPY analogs evoked a concentration-dependent inhibition of the electrically-stimulated twitches with a rank order of potency similar to that of the respective analog at Y2 binding sites in the hippocampus. However, D-amino acid replacements in positions 25 and 26 resulted in an analog which was virtually inactive in the vas deferens, but almost equipotent with NPY in the vena cava. In conclusion, the present study has shown that N-terminal double D-amino acid substitutions in the NPY molecule reduced the binding affinity to and activation more of the Y1 receptor, than of the Y2 receptor, while both receptors were quite sensitive to double D-amino acid changes in positions 19 and 20 and in the C-terminal end of the NPY molecule.

Modulation of carbon monoxide production and enhanced spatial learning by tin protoporphyrin.

Endogenous carbon monoxide (CO), produced by haem oxygenase (HO), may play a role in hippocampal long-term potentiation (LTP). Its role in learning and memory in intact animals is less well known. Tin protoporphyrin (Sn-PP; 25 mg kg-1, i.p.) effectively but transiently inhibited HO activity in brain homogenates, and improved acquisition in the Morris water maze. Locomotor activity was unaffected, indicating a behavioural specificity of the learning effect. The analogue zinc protoporphyrin (25 mg kg-1, i.p.), which does not pass the blood-brain barrier, did not affect learning. If the observed memory effect is related to inhibition of HO, the role of CO in spatial learning may be different from that suggested by LTP studies.

Carbon monoxide as a putative messenger molecule in the feline lower oesophageal sphincter of the cat.

The distribution of the carbon monoxide (CO) producing enzymes haem oxygenase (HO) type 1 and 2 were studied in the feline lower oesophageal sphincter (LOS), as were HO activity and functional effects of CO. HO-2 immunoreactivity was observed in nerve cell bodies in the submucosal and myenteric plexus, nerve fibres, non-neuronal cells surrounding smooth muscle bundles, and in arterial endothelium, HO-1 immunoreactivity was confined to non-neuronal cells in the smooth muscle layer. CO production, indicating HO activity, was demonstrated in tissue homogenates. CO relaxed the LOS, and activated the cyclic GMP system. These results show that HO is present in the LOS, and suggest that CO can be generated by neuronal and non-neuronal structures and may have a role as a peripheral messenger.

Inhibition by zinc protoporphyrin-IX of receptor-mediated relaxation of the rat aorta in a manner distinct from inhibition of haem oxygenase.

1. Carbon monoxide (CO), produced by haem oxygenase through degradation of haem, has been claimed to be a neuromessenger and a possible regulator of vascular tone. We examined whether the haem oxygenase inhibitor, zinc protoporphyrin-IX (ZnPP) and other porphyrins affect the relaxation evoked by various agents in the rat isolated aorta. 2. Pretreatment with ZnPP (0.1 mM) virtually abolished the relaxation evoked by vasoactive intestinal peptide (VIP) and atrial natriuretic peptide (ANP). ZnPP also evoked a rightward shift of the concentration-response curve for the relaxation induced by acetylcholine. 3. In contrast, ZnPP did not affect the relaxation evoked by forskolin and 3-morpholino-sydnonimine, agents which directly activate adenylate and guanylate cyclase, respectively. 4. Although, less effective than ZnPP, tin protoporphyrin-IX (SnPP; 0.1 mM) and protoporphyrin-IX (PP; 0.1 mM) also attenuated the VIP-evoked relaxation. 5. The elevation of cyclic AMP and cyclic GMP levels evoked by VIP and ANP, respectively, were abolished by pretreatment with ZnPP (0.1 mM). 6. ZnPP, SnPP and PP did not affect the contraction evoked by phenylephrine. 7. The results show that ZnPP inhibits relaxation induced by VIP, ANP and acetylcholine, probably by interfering with membrane receptor-coupled signal transduction pathways. This inhibition does not seem to be dependent upon inhibition of haem oxygenase. The lack of specificity of the haem oxygenase inhibiting metalloporphyrins makes them less suitable as pharmacological tools in the investigation of a messenger role for CO.