Differential modulation of NMDA-stimulated [3H]dopamine release from rat striatum by neuropeptide Y and sigma receptor ligands

Although the identity of the endogenous ligands for sigma (sigma) receptors is unknown, neuropeptide Y (NPY) has been named as a possible candidate for a natural transmitter at these receptors. Using a superfusion system, we compared the effect of NPY on NMDA-stimulated [3H]dopamine release in rat striatum to that of the sigma agonists (+)-pentazocine and BD737. In contrast to (+)-pentazocine- or BD737-mediated inhibition of release, NPY enhanced release. However, the same sigma antagonists (BD1008, DuP734, haloperidol and DTG) that reverse (+)-pentazocine- or BD737-mediated inhibition, as well as a Y receptor antagonist, PYX-1, all reversed the enhancement. PYX-1 also reversed the (+)-pentazocine- and BD737-mediated inhibition of release. Peptide YY (PYY) and [Leu31,Pro34]NPY did not mimic the effect of NPY. NPY13-36 enhanced release to the same extent as NPY but the effect was not reversed by sigma antagonists. Our findings are consistent with the potential role of NPY as an endogenous ligand for a subtype of sigma receptor with characteristics different from Y1, Y2 and Y3 receptors but sensitive to PYX-1.     

3H]-BIBP3226 and [3H]-NPY binding to intact SK-N-MC cells and CHO cells expressing the human Y1 receptor

We have studied the binding of [3H]-NPY and the newly developed non-peptide Y1 receptor antagonist [3H]-BIBP3226 to intact SK-N-MC cells and CHO-K1 cells transfected with the human NPY Y1 receptor gene i.e. CHO-Y1 cells. Whereas the association and dissociation of the specific [3H]-NPY binding was slow, the binding kinetics of [3H]-BIBP3226 binding was very rapid. Saturation binding of both radioligands reveal the presence of an apparently homogeneous population of high affinity binding sites in both cell lines. The corresponding equilibrium dissociation constants are similar for the two cell lines and are close to those obtained from previous competition binding experiments. The specific binding of both radioligands was completely and with high affinity displaced by BIBP3226 and its inactive (S)-enantiomer BIBP3435 was much less potent. Whilst the NPY Y1 agonists NPY, PYY and [Leu31-Pro34]-NPY completely and potently displaced [3H]-NPY binding, they could only displace 70 to 80% of the [3H]-BIBP3226 binding sites in CHO-Y1 and SK-N-MC cells. A possible explanation can be that only part of the receptors are G-protein coupled. In agreement pertussis toxin was found to reduce high affinity [3H]-NPY binding sites in CHO-Y1 cells whereas [3H]-BIBP3226 binding parameters remained unchanged.     

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.     

ORL1 receptor-mediated inhibition by nociceptin of noradrenaline release from perivascular sympathetic nerve endings of the rat tail artery

In the present study the effect of the opioid heptadecapeptide nociceptin, also termed orphanin FQ, an endogenous ligand for the orphan receptor named ORL1 (opioid receptor-like 1) receptor, was investigated on [3H]noradrenaline release induced by electrical field stimulation (24 pulses at 0.4 Hz, 200 mA, 0.3 ms duration) in the rat tail artery in the absence and presence of an alpha2-adrenoceptor antagonist, rauwolscine 3 microM. Nociceptin inhibited the electrically-evoked tritiated noradrenaline release in a concentration-dependent manner from rat tail arteries. This inhibitory effect of nociceptin was enhanced in the presence of the alpha2-adrenoceptor antagonist rauwolscine (maximum inhibition by 25% and 50% in the absence and presence of rauwolscine, respectively). At a supramaximal concentration (10 microM), the inhibitory action of DAGO, a selective micro-opioid receptor agonist, was less pronounced than that of nociceptin. The inhibitory effect of nociceptin was counteracted by naloxone benzoylhydrazone (3 microM) which by itself did not change the stimulation-evoked noradrenaline overflow. Naloxone (10 microM), a non-selective opioid receptor antagonist, did not affect the inhibitory effect of nociceptin whereas it abolished that of DAGO. In conclusion, these results suggest that nociceptin modulates noradrenergic neurotransmission by acting on prejunctional ORL1 receptors located on nerve terminals innervating the rat tail artery. They also demonstrate that prejunctional ORL1 receptors interact with prejunctional alpha2-adrenoceptors. The physiological significance of this phenomenon remains to be determined.     

Exogenous NPY modulation of cardiac autonomic reflexes and its pressor effect in the conscious rabbit

1. Neuropeptide Y (NPY) may inhibit sympathetic and vagal transmission via presynaptic Y2 receptors and cause vasoconstriction via postsynaptic Y1 receptors. We examined the effects of NPY and related peptides on cardiovascular parameters and autonomic reflexes in the conscious rabbit. Further, the postjunctional effects of NPY and related peptides were assessed on acetylcholine (ACh) and isoprenaline agonist dose-chronotropic response curves. 2. In conscious rabbits the cardiac baroreceptor-heart rate reflex (baroreflex), Bezold-Jarisch like and nasopharyngeal reflexes were assessed in control, propranolol-treated or methscopolamine-treated (baroreflex only) groups, before and 30 min after i.v. administration of NPY (10 microg kg[-1] + 5 microg kg[-1] min[-1]) or vehicle (saline, 10 ml h[-1]). The effects of equivalent pressor doses of [Leu31, Pro34]NPY or methoxamine on the baroreflex were also examined. In separate animals, dose-heart rate (HR) response curves to isoprenaline or ACh were constructed before and 15 min after administration of NPY, [Leu31,Pro34]NPY (ACh only) or [Leu31,Pro34]NpY + sodium nitroprusside (ACh only). 3. Administration of NPY-receptor agonists caused sustained bradycardia (in the absence of methscopolamine) and rightward shifts of the barocurves in all 3 groups. The range of sympathetically-mediated tachycardia was significantly decreased by NPY or [Leu31,Pro34]NPY in the methscopolamine-treated group. However, these changes in the baroreflex were no different from those elicted by equipressor doses of methoxamine. There was no vagal inhibition by any NPY-receptor agonist in all three autonomic reflexes examined. ACh or isoprenaline dose-HR response curves were not affected by NPY peptide administration. 4. We conclude that in the conscious rabbit, at a single dose that elicits a significant pressor response, exogenous NPY has no direct effect on modulation of cardiac and autonomic reflexes. Non-specific effects of exogenous NPY on the baroreflex may be fully explained by its pressor action. There was no effect of NPY on postjunctional ACh or isoprenaline agonist dose-response curves. Therefore, it is unlikely that endogenous NPY has a functional role in directly modulating cardiac autonomic neurotransmission in the rabbit.     

Inhibition of vagal vasodilatation by a selective neuropeptide Y Y2 receptor agonist in the bronchial circulation of anaesthetised dogs

Neuropeptide Y (NPY) is both co-stored and co-released with noradrenaline from sympathetic nerve terminals. In the cardiovascular system, NPY acts on two main receptor subtypes. At postjunctional, or Y1 receptors, NPY can cause both direct vasoconstriction and the potentiation of various constrictor agents. NPY acting at the presynaptic, or Y2 receptor, inhibits the release of neurotransmitter from autonomic nerves. In the present paper, we have used both sympathetic stimulation and the selective NPY Y2 receptor agonist, N-acetyl [Leu28,Leu31] NPY24-36, to examine the role of NPY in the inhibition of vagally mediated vasodilatation in the bronchial circulation of the anaesthetised dog. Stimulation of the cardiac end of the cervical vagus nerve at 1 Hz for 15 s (1 ms, 70 V) increased bronchial vascular conductance by 45%. This increase in flow was abolished by atropine. Sympathetic stimulation for 2.5 min at 16 Hz (1 ms, 20 V) produced a significant (P < 0.05) and prolonged (9 min) inhibition of the subsequent parasympathetically evoked vasodilatation. Similarly, the NPY Y2 receptor agonist, N-acetyl [Leu28,Leu31] NPY24-36, produced a significant (P < 0.05) and prolonged (15 min) inhibition of parasympathetically evoked vasodilatation. When vagus was stimulated at 2.5 Hz for 30 s (1 ms, 70 V), an atropine-resistant, but capsaicin-sensitive vasodilatation was observed. Neither sympathetic stimulation nor the NPY Y2 receptor agonist could be demonstrated to inhibit this vasodilatation. These results suggest that NPY can inhibit cholinergic parasympathetic vasodilatation in the bronchial circulation by an action on NPY Y2 receptors.     

Co-release of endogenous ATP and [3H]noradrenaline from rat hypothalamic slices: origin and modulation by alpha2-adrenoceptors

The release of endogenous ATP, measured by the luciferin-luciferase assay, and of [3H]noradrenaline from the in vitro superfused rat hypothalamic slices were studied. ATP and [3H]noradrenaline were released simultaneously during resting conditions and in response to low and high frequency field electrical stimulation; the release of both substances were frequency dependent between 2 Hz and 16 Hz. The stimulation-induced release of ATP and [3H]noradrenaline was diminished by more than 80% under Ca2+-free conditions. Tetrodotoxin inhibited the majority of the evoked release of both ATP and [3H]noradrenaline, however, it was less effective in reducing the release of [3H]noradrenaline, than that of ATP. Bilateral stereotaxic injection of 6-hydroxydopamine (4 microg/side) to the ventral part of the ventral noradrenergic bundle, originating from the A1 cell group in the brainstem, resulted in a 55% reduction of endogenous noradrenaline content of the hypothalamic slices, and the tritium uptake and the stimulation-evoked release of [3H]noradrenaline was also markedly reduced. While the basal release of ATP was not affected, the evoked release was diminished by 72% by this treatment. Perfusion of the slices with noradrenaline (100 microM) initiated rapid and continuous tritium release; on the other hand, it did not release any ATP. In contrast, 6 min perfusion of (-)nicotine and 1,1-dimethyl-4-phenyl-piperazinium iodide evoked parallel release of ATP and [3H]noradrenaline which was inhibited by the nicotinic receptor antagonist mecamylamine; 6-hydroxydopamine lesion of the ventral part of the ventral noradrenergic bundle did not affect the nicotine-evoked ATP and [3H]noradrenaline release. While CH 38083, a non subtype-selective alpha2-antagonist and BRL44408, the subtype-selective alpha2AD antagonist augmented the evoked release of [3H]noradrenaline, ARC239, a selective alpha2BC antagonist was without effect. In contrast, neither of the alpha2-antagonists significantly affected the evoked-release of ATP. In summary, we report here that endogenous ATP and [3H]noradrenaline are co-released stimulation-dependently from superfused rat hypothalamic slices. A significant part of the release of both compounds is derived from the nerve terminals, originating from the A1 catecholaminergic cell group of brainstem nuclei. Unlike that from the peripheral sympathetic transmission, noradrenaline and alpha1-adrenoceptor agonists were unable to promote the release of ATP. Conversely, parallel ATP and noradrenaline release could be induced by nicotine receptor activation, but this release does not originate from the same nerve endings. The evoked-release of [3H]noradrenaline is inhibited by endogenous noradrenaline via alpha2AD subtype of adrenoreceptors, while the release of ATP is not subject to this autoinhibitory modulation. In conclusion, our results support the view that ATP is involved in the neurotransmission in the hypothalamus, but the sources of the released ATP and noradrenaline seem to be not identical under different stimulatory and modulatory conditions.     

Modulation of [3H]acetylcholine release from cultured amacrine-like neurons by adenosine A1 receptors

We have investigated the effect of endogenous adenosine on the release of [3H]acetylcholine ([3H]ACh) in cultured chick amacrine-like neurons. The release of [3H]ACh evoked by 50 mM KCl was mostly Ca2+ dependent, and it was increased in the presence of adenosine deaminase and in the presence of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), an adenosine A1 receptor antagonist. The effect of adenosine on [3H]ACh release was sensitive to pertussis toxin (PTX) and was due to a selective inhibition of N-type Ca2+ channels. Ligand binding studies using [3H]DPCPX confirmed the presence of adenosine A1 receptors in the preparation. Using specific inhibitors of the plasma membrane adenosine carriers and of the ectonucleotidases, we found that the extracellular accumulation of adenosine in response to KCl depolarization was due to the release of endogenous adenosine per se and to the extracellular conversion of released nucleotides into adenosine. Activation of adenosine A1 receptors was without effect on the intracellular levels of cyclic AMP under depolarizing conditions, but it inhibited the accumulation of inositol phosphates. Our results indicate that in cultured amacrine-like neurons, the Ca2+-dependent release of [3H]ACh evoked by KCl is under tonic inhibition by adenosine, which activates A1 receptors. The effect of adenosine on the [3H]ACh release may be due to a direct inhibition of N-type Ca2+ channels and/or secondary to the inhibition of phospholipase C and involves the activation of PTX-sensitive G proteins.     

Prostaglandin E2 suppression of acetylcholine release from parasympathetic nerves innervating guinea-pig trachea by interacting with prostanoid receptors of the EP3-subtype

1. We have demonstrated recently that exogenous prostaglandin E2 (PGE2) inhibits electrical field stimulation (EFS)-induced acetylcholine (ACh) release from parasympathetic nerve terminals innervating guinea-pig trachea. In the present study, we have attempted to characterize the pre-junctional prostanoid receptor(s) responsible for the inhibitory action of PGE2 and to assess whether other prostanoids modulate, at a prejunctional level, cholinergic neurotransmission in guinea-pig trachea. To this end, we have investigated the effect of a range of both natural and synthetic prostanoid agonists and antagonists on EFS-evoked [3H]-ACh release. 2. In epithelium-denuded tracheal strips pretreated with indomethacin (10 microM), PGE2 (0.1 nM-1 microM) inhibited EFS-evoked [3H]-ACh release in a concentration-dependent manner with an EC50 and maximal effect of 7.62 nM and 74% inhibition, respectively. Cicaprost, an IP-receptor agonist, PGF2alpha and the stable thromboxane mimetic, U46619 (each at 1 microM), also inhibited [3H]-ACh release by 48%, 41% and 35%, respectively. PGD2 (1 microM) had no significant effect on [3H]-ACh release. 3. The selective TP-receptor antagonist, ICI 192,605 (0.1 microM), completely reversed the inhibition of cholinergic neurotransmission induced by U-46619, but had no significant effect on similar responses effected by PGE2 and PGF2alpha. 4. A number of EP-receptor agonists mimicked the ability of PGE2 to inhibit [3H]-ACh release with a rank order of potency: GR63799X (EP3-selective) > PGE2 > M&B 28,767 (EP3 selective) > 17-phenyl-omega-trinor PGE2 (EP1-selective). The EP2-selective agonist, AH 13205 (1 microM), did not affect EFS-induced [3H]-ACh release. 5. AH6809 (10 microM), at a concentration 10 to 100 times greater than its pA2 at DP-, EP1- and EP2-receptors, failed to reverse the inhibitory effect of PGE2 or 17-phenyl-omega-trinor PGE2 on [3H]-ACh release. 6. These results suggest that PGE2 inhibits [3H]-ACh release from parasympathetic nerves supplying guinea-pig trachea via an interaction with prejunctional prostanoid receptors of the EP3-receptor subtype. Evidence for inhibitory prejunctional TP- and, possibly, IP-receptors was also obtained although these receptors may play only a minor role in suppressing [3H]-ACh release when compared to receptors of the EP3-subtype. However, the relative importance of the different receptors will depend not only on the sensitivity of guinea-pig trachea to prostanoids but on the nature of the endogenous ligands released locally that have activity on parasympathetic nerves.     

The human 5-ht5A receptor couples to Gi/Go proteins and inhibits adenylate cyclase in HEK 293 cells

The G protein coupling of human 5-hydroxytryptamine5A (h5-ht5A) receptors was investigated in stably transfected human embryonic kidney (HEK) 293 cells, using radioligand and guanosine-5'[gamma-35S]thiotriphosphate binding to membranes and cyclic adenosine monophosphate measurements in cells. 5-Carboxamido[3H]tryptamine bound to high- and low-affinity sites on h5-ht5A-HEK 293 cell membranes. Guanylyl-imidodiphosphate addition and pertussis toxin pre-treatment abolished high-affinity binding, indicating coupling to G proteins of the Gi/Go family. [N-methyl-3H]Lysergic acid diethylamide bound to a single site; guanylyl-imidodiphosphate and pertussis toxin did not alter lysergic acid diethylamide affinity. 5-Hydroxytryptamine stimulated guanosine-5'[gamma-35S]thiotriphosphate binding to 130% over basal and this effect was completely abolished by pertussis toxin. Various 5-hydroxytryptamine receptor ligands were tested for inhibition of 5-carboxamido[3H]tryptamine binding and in guanosine-5'[gamma-35S]thiotriphosphate binding assays. 5-Hydroxytryptamine consistently inhibited forskolin-induced cyclic adenosine monophosphate formation by 25% in h5-ht5A-HEK 293 cells; no effect was detected on basal cyclic adenosine monophosphate levels, on intracellular Ca2+ concentration or arachidonic acid release. Our studies demonstrate functional coupling of the h5-ht5A receptor to pertussis toxin-sensitive G proteins and to inhibition of adenylate cyclase activity.     

Inhibition of glucose stimulated insulin secretion by neuropeptide Y is mediated via the Y1 receptor and inhibition of adenylyl cyclase in RIN 5AH rat insulinoma cells

Neuropeptide Y (NPY) has been shown to inhibit insulin secretion from the islets of Langerhans. We show that insulin secretion in the insulinoma cell line RIN 5AH is inhibited by NPY. 125I-Peptide YY (PYY) saturation and competition-binding studies using NPY fragments and analogues on membranes prepared from this cell line show the presence of a single class of NPY receptor with a Y1 receptor subtype-like profile. Inhibition of insulin secretion in this cell line by NPY fragments and analogues also shows a Y1 receptor-like profile. Both receptor binding and inhibition of insulin secretion showed the same orders of potency with NPY > [Pro34]-NPY > NPY 3-36 > NPY 13-36. The Y1 receptor antagonist, BIBP 3226, blocks NPY inhibition of insulin secretion from, and inhibits 125I-PYY binding to, RIN 5AH cells. Northern blot analysis using a Y1-receptor specific probe shows that NPY Y1 receptors are expressed by RIN 5AH cells. Y5 receptors are not expressed in this cell line. Neuropeptide Y inhibition of insulin secretion is blocked by incubation with pertussis toxin, implying that the effect is via a G-protein (Gi or Go) coupled receptor. Neuropeptide Y inhibits the activation of adenylyl cyclase by isoprenaline in RIN 5AH cell lysates, and the stimulation of cAMP by glucagon-like peptide-1 (7-36) amide (GLP-1). It also blocks insulin secretion stimulated by GLP-1, but not by dibutyryl cyclic AMP. Hence, we suggest that NPY inhibits insulin secretion from RIN 5AH cells via a Y1 receptor linked through Gi to the inhibition of adenylyl cyclase.     

Nonpeptide peptidomimetic antagonists of the neuropeptide Y receptor: benextramine analogs with selectivity for the peripheral Y2 receptor

We synthesized a new series of benextramine analogs as neuropeptide Y (NPY) functional group mimetics and tested them for N-[propionyl-3H]NPY ([3]NPY) displacement activity in rat brain membrane homogenates and for NPY receptor antagonist activity in the rat femoral artery. The tetraamine, carbon analog N,N'-bis[6-[N-(2-naphthylmethyl)amino]hexyl]-1,6-hexanediamine (15) was equipotent with benextramine (based on comparison of the relevant IC50's) in a rat brain [3H]NPY displacement assay, suggesting that the disulfide is not a necessary feature of benextramine's [3H]NPY displacement activity, although this analog maintained selectivity for the benextramine-sensitive binding site population. The bis(N,N-dialkylguanyl) disulfide and carbon analogs 14a-c were 3-4 times more potent than their respective controls in displacing [3H]NPY from rat brain membrane homogenates with IC50's ranging from 15 to 18 microM and maintained selectivity for the benextramine-sensitive, Y1 binding site population. However, the activity of the carbon analog N,N'-bis[6-[N-(2-naphthylmethyl)amino]hexyl]-N,N'-(1,6- hexanediyl)diguanidine tetrahydrochloride (14b) showed a different profile in a femoral artery vasoconstriction assay; at 1.0 nM, this analog shifted the concentration-effect curve of the Y2-selective agonist NPY13-36 to the right (pA2 = 9.2; Kd = 0.63 nM) without a significant change in the maximum effect, while even at 1.0 mM it had no effect on the vasoconstrictive activity of the Y1-selective agonist [Leu31,Pro34]NPY. Thus, the bis(N,N-dialkylguanidine) analogs of benextramine are selective, competitive antagonists of the postsynaptic NPY receptor in the femoral artery.     

P2-receptor modulation of noradrenergic neurotransmission in rat kidney

1. ATP has previously been shown to act as a sympathetic cotransmitter in the rat kidney. The present study analyses the question of whether postganglionic sympathetic nerve endings in the kidney possess P2-receptors which modulate noradrenaline release. Rat kidneys were perfused with Krebs-Henseleit solution containing the noradrenaline uptake blockers cocaine and corticosterone and the alpha2-adrenoceptor antagonist rauwolscine. The renal nerves were electrically stimulated, in most experiments by 30 pulses applied at 1 Hz. The outflow of endogenous noradrenaline (or, in some experiments, of ATP and lactate dehydrogenase) as well as the perfusion pressure were measured simultaneously. 2. The P2-receptor agonist adenosine-5'-O-(3-thiotriphosphate) (ATPgammaS, 3-30 microM) reduced the renal nerve stimulation (RNS)-induced outflow of noradrenaline (estimated EC50 =8 microM). The P2-receptor antagonist cibacron blue 3GA (30 microM) shifted the concentration-inhibition curve for ATPgammaS to the right (apparent pKB value 4.7). 3. Cibacron blue 3GA (3-30 microM) and its isomer reactive blue 2 (3-30 microM) significantly increased RNS-induced outflow of noradrenaline in the presence of the P1-receptor antagonist 8-(p-sulphophenyl)theophylline (8-SPT, 100 microM) by about 70% and 90%, respectively. The P2-receptor antagonist suramin (30-300 microM) only tended to enhance RNS-induced outflow of noradrenaline. When the nerves were stimulated by short pulse trains consisting of 6 pulses applied at 100 Hz (conditions under which autoinhibition is inoperative), reactive blue 2 did not affect the RNS-induced outflow of noradrenaline. 4. RNS (120 pulses applied at 4 Hz) induced the outflow of ATP but not of the cytoplasmatic enzyme lactate dehydrogenase. 5. ATPgammaS (3-30 microM) concentration-dependently reduced pressor responses to RNS at 1 Hz. Cibacron blue 3GA, reactive blue 2 as well as suramin also reduced pressor responses to RNS (maximally by 50 to 70%). 6. This study in rat isolated kidney, in which the release of endogenous noradrenaline was measured, demonstrates that renal sympathetic nerves possess prejunctional P2-receptors that mediate inhibition of transmitter release. These prejunctional P2-receptors are activated by endogenous ligands, most likely ATP, released upon nerve activity. Both, P2-receptor agonists and P2-receptor antagonists reduced pressor responses to RNS either by inhibiting transmitter release or by blocking postjunctional vasoconstrictor P2-receptors.     

Role of adenosine in the hypoxic induction of vascular endothelial growth factor in porcine brain derived microvascular endothelial cells

The distribution of neuropeptide Y (NPY)-immunoreactive (IR) nerves, as well as the functional effects of NPY and the Y1- and Y2-receptor agonists, [Leu31,Pro34]NPY and NPY(13-36), respectively, have been investigated in vitro in both visceral and arterial smooth muscle of the horse intravesical ureter. NPY-IR nerve fibres were widely distributed along the entire length of the ureter, although the intravesical part was the most richly innervated region, and the only one where NPY-IR ganglion cells were found. NPY (10(-7) M) did not affect either basal tone or spontaneous rhythmic contractions of the isolated intravesical ureter, but significantly enhanced the increases in both tone and frequency of phasic activity elicited by noradrenaline (10(-6) and 10(-5) M). The Y1-receptor agonist, [Leu31,Pro34]NPY (10(-7) and 10(-6) M) did not significantly alter either ureteral basal tone or the contractile activity induced by noradrenaline, whereas the Y2-receptor agonist, NPY(13-36) (10(-7) M), mimicked the potentiating effect of NPY on noradrenaline responses. In ureteral resistance arteries (effective lumen diameters of 130-300 microm), NPY (10(-10) to 10(-7) M) elicited concentration-dependent contractions, which were inversely correlated with the arterial lumen diameter. Submaximal concentrations of NPY (10(-8) M) significantly increased the sensitivity of ureteral arteries to noradrenaline. [Leu31,Pro34]NPY (10(-10) to 10(-7) M), but not NPY(13-36), induced a contractile effect of similar magnitude and potency as those of NPY, and also potentiated noradrenaline responses. The present results demonstrate a rich NPY-innervation in the intravesical ureter and reveal functional effects of the peptide enhancing motor activity in both ureteral and arterial smooth muscles, although the receptors mediating such effects seem to be different. Thus, NPY potentiates the phasic contractions and tone elicited by noradrenaline through Y2-receptors, whereas it both contracts and potentiates noradrenaline vasoconstriction in ureteral arteries via Y1-receptors.     

The non-peptide neuropeptide Y Y1 receptor antagonist BIBP3226 blocks the [Leu31,Pro34]neuropeptide Y-induced modulation of alpha 2-adrenoceptors in the nucleus tractus solitarii of the rat

The potential blockade of the neuropeptide Y (NPY) Y1 receptor agonist [Leu31,Pro34]NPY-induced modulation of the characteristics of alpha 2-adrenoceptor agonist [3H]p-aminoclonidine binding sites by a selective non-peptide NPY Y1 receptor antagonist BIBP3226, was studied in the nucleus tractus solitarii of the rat by means of quantitative receptor autoradiography. [Leu31,Pro34]NPY at a concentration of 10 nM significantly increased the Kd value of [3H]p-aminoclonidine binding sites in the nucleus tractus solitarii without influencing the Bmax, suggesting the existence of an antagonistic modulation by NPY Y1 receptors of alpha 2-adrenoceptors in the nucleus tractus solitarii. BIBP3226 at 100 nM fully blocked the [Leu31,Pro34]NPY-induced increase in Kd of the [3H]p-aminoclonidine binding sites. The present results therefore provide evidence, by use of a NPY Y1 receptor antagonist, for the existence of a NPY Y1/alpha 2 receptor interaction in the nucleus tractus solitarii.     

Pathohistological study of adriamycin-induced tracheal agenesis in the fetal rat

Mr 2266 [(-)-(1R,5R,9R)-5,9-diethyl-2-(3-furylmethyl)-2'-hydroxy-6,7-benzomorpha n] is an antagonist at kappa-opioid receptors and at ORL1 receptors as well. The aim of our study was to examine whether the known stereoselective antagonism of Mr 2266 at kappa-opioid receptors also extends to ORL1 receptors. In mouse brain cortex membranes, the binding of the ORL1 receptor agonist [3H]nociceptin was equipotently inhibited by Mr 2266 and its enantiomer Mr 2267 (pK(i) 4.82 and 5.14, respectively), whereas the binding of the kappa-opioid receptor agonist [3H]U-69,593 was inhibited by Mr 2266 more potently (pK(i) 9.11) than by its enantiomer Mr 2267 (pK(i)7.15). In mouse brain cortex slices preincubated with [3H]noradrenaline, the concentration-response curve of nociceptin for inhibition of the electrically evoked overflow of tritium was equipotently shifted to the right by Mr 2266 and Mr 2267 (pA2 5.77 and 5.64, respectively). On the other hand, the inhibitory effect of U-69,593 on the electrically evoked overflow of tritium in guinea-pig brain cortex slices preincubated with [3H]noradrenaline was more potently antagonized by Mr 2266 (pA2 8.81) than by Mr 2267 (pA2 7.15). These data show that the stereoselective antagonism of Mr 2266 at kappa-opioid receptors does not extend to ORL1 receptors.     

The ligand binding site of NPY at the rat Y1 receptor investigated by site-directed mutagenesis and molecular modeling

The ligand binding site of neuropeptide Y (NPY) at the rat Y1 (rY1,) receptor was investigated by construction of mutant receptors and [3H]NPY binding studies. Expression levels of mutant receptors that did not bind [3H]NPY were examined by an immunological method. The single mutations Asp85Asn, Asp85Ala, Asp85Glu and Asp103Ala completely abolished [3H]NPY binding without impairing the membrane expression. The single mutation Asp286Ala completely abolished [3H]NPY binding. Similarly, the double mutation Leu34Arg/Asp199Ala totally abrogated the binding of [3H]NPY, whereas the single mutations Leu34Arg and Asp199Ala decreased the binding of [3H]NPY 2.7- and 5.2-fold, respectively. The mutants Leu34Glu, Pro35His as well as Asp193Ala only slightly affected [3H]NPY binding. A receptor with a deletion of the segment Asn2-Glu20 or with simultaneous mutations of the three putative N-terminal glycosylation sites, displayed no detectable [3H]NPY binding, due to abolished expression of the receptor at the cell surface. Taken together, these results suggest that amino acids in the N-terminal part as well as in the first and second extracellular loops are important for binding of NPY, and that Asp85 in transmembrane helix 2 is pivotal to a proper functioning of the receptor. Moreover, these studies suggest that the putative glycosylation sites in the N-terminal part are crucial for correct expression of the rY1 receptor at the cell surface.     

Peptide YY receptors in the proximal tubule PKSV-PCT cell line derived from transgenic mice. Relation with cell growth

Receptors for peptide YY (PYY) were identified in the PKSV-PCT renal proximal tubule cell line, derived from transgenic mice (SV40 large T antigen under the control of the rat L-type pyruvate kinase 5'-regulatory sequence). Binding of [125I-Tyr36]monoiodo-PYY ([125I] PYY to cell was specific, saturable, and reversible. The order of potency for peptides for inhibiting [125I]PYY binding was: PYY > neuropeptide Y (NPY) = PYY (13-36) > pancreatic polypeptide. A single class of receptors was observed with a Kd of 0.37 +/- 0.05 nM and a Bmax of 103 +/- 10 fmol/mg protein. After cross-linking, electrophoresis of covalent [125I]PYY-receptor complexes revealed a single band of M(r) 50,000. PYY receptors were exclusively present at the basolateral membrane surface of polarized cells and were coupled negatively to adenylylcyclase by a pertussis toxin-sensitive G protein. PKSV-PCT cell growth and T antigen expression could be modulated by D-glucose in the medium. PYY receptors were exclusively expressed in proliferative cells cultured in the presence of D-glucose. PYY receptors disappeared in the absence of D-glucose and were expressed again when proliferation was activated by reintroduction of D-glucose. PYY stimulated cell growth (17-26% increase) and promoted [methyl-3H]thymidine incorporation into DNA (64% increase; ED50 = 5 nM PYY) of cells grown in D-glucose-enriched medium. This latter effect of PYY was largely reversed by pretreatment of cells with pertussis toxin. These findings suggest that PYY receptors play a role in epithelial cell growth.     

Age-related alterations in pre-synaptic and receptor-mediated cholinergic functions in rat brain

Fractional [3H]acetylcholine (ACh) release and regulation of release process by muscarinic receptors were studied in corpus striatum of young and aged rat brains. [3H] Quinuclidinyl benzilate (QNB) binding and carbachol stimulated phosphoinositide turnover, on the other hand, were compared in striatal, hippocampal and cortical tissues. High potassium (10 mM)-induced fractional [3H]ACh release from striatal slices was reduced by aging. Although inhibition of acetylcholinesterase with eserine (20 microM) significantly decreased stimulation-induced fractional [3H]ACh release in two groups of rats, this inhibition slightly lessened with aging. Incubation of striatal slices with muscarinic antagonists reversed eserine-induced inhibition in fractional [3H]ACh release with a similar order of potency (atropine = 4-DAMP > AF-DX 116 > pirenzepine) in young and aged rat striatum, but age-induced difference in stimulated ACh release was not abolish by muscarinic antagonists. These results suggested that fractional [3H]ACh release from striatum of both age groups is modulated mainly by M3 muscarinic receptor subtype. Although both muscarinic receptor density and labeling of inositol lipids with [myo-3H]inositol decreased with aging, carbachol-stimulated [3H]myo inositol-1-fosfat (IP1) accumulation was found similar in striatal, cortical and hippocampal slices.