Presynaptic effects of neuropeptide Y on [3H]noradrenaline and [3H]acetylcholine release

The electrically evoked release of radioactivity from mouse vas deferens and rat hypothalamic slices preloaded with [3H]noradrenaline was measured. In addition the release of [3H]acetylcholine from longitudinal muscle strip of guinea-pig ileum was also measured. Neurochemical evidence has been obtained that neuropeptide Y (NPY), although it co-exists and is released with (-)-noradrenaline (NA), it behaves differently as far as its effect on presynaptic modulation of chemical neurotransmission is concerned. It exerts a frequency-dependent presynaptic inhibitory effect on noradrenaline release from mouse vas deferens but has no effect on the electrically evoked release of NA from rat hypothalamus. Unlike NA, NPY does not influence the release of [3H]acetylcholine from the longitudinal muscle strip of guinea-pig ileum and does not potentiate the presynaptic effect of NA. It seems very likely, that the inhibitory effect of NPY is mediated via receptors. Its action is concentration dependent. While exogenous noradrenaline inhibited the release of noradrenaline by 91%, the maximum inhibition reached with NPY was not higher than 60%, indicating that either the intrinsic activity of NPY is lower or much less axon terminals are equipped with NPY receptors. Peptide YY (PYY) also reduced the release of NA from mouse vas deferens.     

Antagonism of presynaptic adenosine receptors by theophylline 9-beta-D-riboside and 8-phenyltheophylline

Theophylline 9-beta-D-riboside and 8-phenyltheophylline were evaluated as presynaptic adenosine receptor antagonists in the rat vas deferens in vitro. Stimulation of presynaptic adenosine receptors, which results in an inhibition of the twitch response to electrical field stimulation, was achieved with 2-chloroadenosine, an adenosine analogue that appears not to be a substrate for the adenosine transport system. The presynaptic inhibitory action of 2-chloroadenosine was antagonized by theophylline (10 and 100 microM) and by 8-phenyltheophylline (10 microM) but not by theophylline 9-beta-D-riboside (100 microM). It is concluded that the addition of a ribose moiety to theophylline does not enhance the antagonist potency of the molecule but actually renders the compound inactive. However, 8-phenyltheophylline is approximately three times more potent than theophylline at presynaptic adenosine receptors.     

Urodilatin attenuates sympathetic neurotransmission in the rabbit isolated vas deferens without activating guanylyl cyclase.

Natriuretic peptides are produced in cardiovascular, renal and neural tissues and are believed to reduce arterial blood pressure by augmenting sodium and water loss in the urine. Another potential antihypertensive action of these peptides involves a suppression of adrenergic neurotransmission. Atrial, brain and C-type natriuretic peptides suppress sympathetic neurotransmission but no data are available on neuromodulatory actions of urodilatin. This study investigates the hypothesis that urodilatin and brain natriuretic peptide inhibit sympathetic neurotransmission by elevating guanylyl cyclase activity. Both brain natriuretic peptide and urodilatin suppressed force generation in response to electrical stimulation of the vas deferens. Brain natriuretic peptide accelerated the production of cyclic guanosine monophosphate equipotently with its effects on neurotransmission. However, urodilatin failed to increase guanylyl cyclase activity, thus dissociating its effects on neurotransmission from guanylyl cyclase stimulation. None of the natriuretic peptides altered contractile effects of either adenosine triphosphate or norepinephrine, the two putative neurotransmitters secreted from adrenergic nerves in the vas deferens. These data are consistent with the following conclusions: 1) all of the known endogenous natriuretic peptides suppress adrenergic neurotransmission; 2) guanylyl cyclase activation is not required for the inhibition of sympathetic neurotransmission by natriuretic peptides; and 3) inhibitory effects of the natriuretic peptides on neurotransmission result from a suppression of neurotransmitter exocytosis. The novel findings of this study include both the suppression of sympathetic neurotransmission by urodilatin and its biological activity in the absence of guanylyl cyclase activation.     

Beta-endorphin. Biological activity of analogs containing dermorphin and dynorphin sequences: ileum and vas deferens assays

Biological activity of synthetic beta-endorphin (beta-EP) analogs containing dermorphin or dynorphin-A-(1-13) structure has been investigated using the guinea pig ileum and the vas deferens of the mouse, rat and rabbit. Replacement of NH2-terminal 1-7 segment of camel beta-EP [beta c-EP-(1-7)] with dermorphin caused a great increase in opiate potency of the analog. [Dermorphin (1-7)]-beta c-EP was 120 times more potent than beta c-EP in the guinea pig ileum assay, 49 times more potent in the mouse vas deferens assay; and only 4 times more potent in the rat vas deferens assay. Replacement of NH2-terminal 1-13 segment of human beta-EP [beta h-EP-(1-13)] with dynorphin-A-(1-13) caused an increase in opiate potency in both the guinea pig ileum and rabbit vas deferens assays, a complete loss of potency in the rat vas deferens assay, and no change in the mouse vas deferens assay. In comparison with dynorphin-A-(1-13), the hybrid peptide was less potent in the guinea pig ileum assay as well as in mouse and rabbit vas deferens assay. It is suggested that beta c-EP-(8-31) facilitates the dermorphin moiety to act on opiate mu and delta receptors but not on the epsilon receptor, while beta h-(14-31) reduces the action of dynorphin on mu, delta and kappa receptors.     

Pre- and postjunctional actions of neuropeptide Y and related peptides

The effects of neuropeptide Y (NPY) and related peptide fragments on blood pressure and vagal action at the heart were compared in the anaesthetized rat. A change in vagal action was taken as a measure of presynaptic activity and a change in blood pressure was taken as a measure of postsynaptic activity. NPY, NPY-(13-36), PYY-(13-36), des-Ser22-NPY-(13-36) and a stabilized 13-36 analogue of NPY (ANA NPY) all exerted pressor actions and attenuated vagal action at the heart. The maximum vagal inhibitory or presynaptic action in order of potency was NPY, ANA-NPY, PYY-(13-36) significantly greater than NPY-(13-36), des-Ser22-NPY-(13-36). The order of potency for the half time of this effect was NPY, ANA-NPY significantly longer than PYY-(13-36) and NPY-(13-36), which were significantly longer than des Ser22-NPY-(13-36). For the pressor or postsynaptic effects, NPY increased blood pressure significantly more and for a longer duration than all the 13-36 fragments, which were not demonstrably different in this respect. These results are consistent with the proposal that there are two populations of NPY receptors. The C-terminal flanking peptide of NPY (CPON) and desamido-NPY had no effect on either vagal action at the heart or on blood pressure.     

The effects of adenosine and adenine nucleotides on the guinea-pig vas deferens: evidence for existence of purinergic receptors.

The effect of adenosine 5′-triphosphate (ATP) and its congeners on the alpha-adrenergic neuroeffector transmission in the isolated vas deferens of the guinea pig was evaluated. Both intracellular activity and contractile response of the smooth muscle of the vas deferens were recorded by using the sucrose-gap method. Adenosine, adenosine diphosphate (ADP) and adenosine monophosphate (AMP) influenced alpha-adrenergic receptor-mediated excitatory responses by depolarizing the cell membrane potential. ATP, on the other hand, produced action potentials rather than sustained depolarization, and its activity was blocked by theophylline and 2, 2′-pyridylisatogen, an ATP antagonist, but not blocked by either phentolamine or phenoxybenzamine, which inhibit alpha-adrenoreceptor responsiveness caused by norepinephrine or phenylephrine. Furthermore, dipyridamole, an adenosine uptake blocker, potentiated both ATP and adenosine activities. These findings indicate that adenosine and adenine nucleotides may exert their action at an extracellular site. From these results, it may be speculated that alpha adrenoreceptors and purinergic receptors do indeed exist on the smooth muscle of the vas deferens.     

L-NAME pre-treatment partially inhibits the agmatine-evoked depression of the electrically induced twitch contraction of isolated rat vas deferens

The effect of the putative endogenous ligand for alpha(2)-adrenoceptors and imidazoline receptors agmatine was studied in sympathetic neurotransmission in the rat epididymal vas deferens. Tissues were obtained from N(varpi)-nitro-l-arginine methyl ester (l-NAME)-treated or normal animals and were contracted by electrical stimulation or by exogenous adenosine 5'-triphosphate (ATP). In the electrically stimulated epididymal end, agmatine produced an inhibitory effect on twitch contraction that was partially reversed in l-NAME-treated animals, whereas the inhibition produced by clonidine was not affected by l-NAME treatment. The nitric oxide (NO)-donor S-nitroso-N-acetyl-penicillamine (SNAP) also inhibited twitch contraction. Neither agmatine nor SNAP interfered with the responses induced by exogenous ATP in the epididymal end. Removal of the epithelium of the preparation did not modify the agmatine response. We conclude that a nitrergic pathway activated by agmatine plays a role in its inhibitory effect in rat vas deferens, but it remains to be investigated whether it results from a direct action on the enzyme NO-synthase or a receptor-mediated mechanism.     

Adrenergic receptors mediating prostaglandin production in the rabbit vas deferens

Contractile and prostaglandin E (PGE)-producing effects of adrenergic agonists were compared in the rabbit isolated vas deferens to determine which adrenergic receptor(s) potentially could mediate neural responses. Additionally, interactions among receptors were elucidated by comparing responses to norepinephrine, phenylephrine and isoproterenol to those in the presence of selective adrenergic agonists or antagonists. Norepinephrine increased the force of muscle contraction and the immunoassayable PGE concentrations in a concentration-dependent manner with EC50's of 55 +/- 8 and 112 +/- 39 microM, respectively. Propranolol (10 microM) enhanced the contractile effects of norepinephrine (p less than 0.01) whereas yohimbine (100 microM) or prazosin (1 microM) reduced norepinephrine-induced contractions and PGE production (p less than 0.01). Propranolol did not alter the PGE production induced by norepinephrine. Metoprolol (100 microM) also enhanced contractile effects of norepinephrine (p less than 0.05). The beta adrenergic agonist, isoproterenol (100 nM), decreased the contractile, but not the PGE-producing, effects of phenylephrine (p less than 0.001). Isoproterenol, given alone, increased PGE concentrations and inhibited electrically-induced force generation in a concentration-dependent manner. These results are consistent with the presence of alpha receptors on the vas deferens which mediate smooth muscle contraction and PGE generation. Beta receptors which mediate relaxation and PGE production also are present. Tentative identification of the beta receptor subtype revealed the presence of a beta 1 receptor.     

Somatostatin inhibits adrenergic and cholinergic neurotransmission in smooth muscle.

Somatostatin reduced the response to field stimulation in the guinea pig ileum and reduced the spontaneous contractions in the rabbit jejunum, an effect that was blocked by tetrodotoxin. Somatostatin also inhibited field stimulated alpha adrenergic contractions in the rat vas deferens and rabbit ear artery. However, the responses to direct application of either acetylcholine in the ileum or to norepinephrine in the ear artery or vas deferens were not affected by somatostatin. These results strongly suggest that somatostatin inhibits neuronal release of cholinergic and adrenergic transmitter substances in smooth muscle.     

Development of clonidine-tolerance in the rat vas deferens: Cross tolerance to other presynaptic inhibitory agents

Possibility of the development of clonidine-tolerance in the peripheral nervous tissue was examined using vas deferens isolated from rats chronically treated with clonidine. Rats were treated with clonidine for 10 days by adding the drug to drinking water (10 μg/ml). For the control rats, drug-free tap water was provided. Electrically evoked twitch response of vas deferens was suppressed by adenosine, β-endorphine and α₂-adrenergic agonists, such as clonidine and B-HT 933, both in control and clonidine-treated groups. Vas deferens isolated from clonidine-treated rats showed significantly lower responsiveness to the inhibitory effects of clonidine and B-HT 933 compared to those from control rats. Vas deferens from clonidine-treated rats also was less responsive to adenosine and β-endorphin, both of which interact with presynaptic inhibitory receptors other than α₂-adrenergic and muscarinic cholinergic stimulation responsiveness of the postsynaptic smooth muscle to both α-adrenergic and muscarinic cholinergic stimulation did not change after 10 days of treatment with clonidine. These results suggest that clonidinetolerance can be induced in the peripheral nervous system by chronic treatment of this drug and that the tolerance is not specific to α₂-adrenergic agonists. Some common pathway in the inhibitory mechanisms of various agents or possible interactions between the different types of presynaptic inhibitory receptors may be involved in this phenomenon.     

The effects of pancreatic polypeptides and neuropeptide Y on the rat vas deferens

In order to study the physiological significance of the coexistence of pancreatic polypeptide and norepinephrine (NE) in peripheral noradrenergic nerves, the effects of pancreatic polypeptides of several species were tested on the isolated rat vas deferens. Neuropeptide Y (NPY) was also studied because of its sequence homology to the pancreatic polypeptides. The contractile responses, which were mediated predominantly by activation of noradrenergic nerves following electrical stimulation, were inhibited by bovine pancreatic polypeptide (BPP), human pancreatic polypeptide (HPP), avian pancreatic polypeptide (APP) and NPY in a dose-dependent manner using a constant flow bath. The decreasing order of the inhibitory responses was as follows: BPP = HPP greater than NPY greater than APP. The inhibitory responses produced by BPP and HPP lasted more than 1 hr and displayed a marked tachyphylaxis. In contrast, the inhibitory effects induced by NPY and APP usually returned to the control level after 20-30 min and had minimal tachyphylaxis. The inhibitory action of NPY was still present during alpha-adrenergic blockade. Contractions produced by a single submaximal dose of exogenous NE or serotonin (5-HT) in unstimulated preparations were not affected by pretreatment with NPY. The amplitude of contractions was partially reduced 1 min after pretreatment with BPP or HPP; recovery occurred about 15 min after peptide pretreatment in a constant flow bath. These results suggest that an NPY receptor exists presynaptically in the rat vas deferens and that stimulation of the receptor by NPY inhibits the release of NE from noradrenergic nerves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine A2A receptor-mediated facilitation of noradrenaline release involves protein kinase C activation and attenuation of presynaptic inhibitory receptor-mediated effects in the rat vas deferens

In the epididymal portion of rat vas deferens, facilitation of noradrenaline release mediated by adenosine A2A receptors, but not that mediated by beta2-adrenoceptors or by direct activation of adenylyl cyclase, was attenuated by blockade of alpha2-adrenoceptors and abolished by simultaneous blockade of alpha2-adrenoceptors, adenosine A1 and P2Y receptors. The adenosine A2A receptor-mediated facilitation was not changed by inhibitors of protein kinase A, protein kinase G or calmodulin kinase II but was prevented by inhibition of protein kinase C with chelerythrine or bisindolylmaleimide XI. Activation of protein kinase C with phorbol 12-myristate 13-acetate caused a facilitation of noradrenaline release that was abolished by bisindolylmaleimide XI and reduced by antagonists of alpha2-adrenoceptors, adenosine A1 and P2Y receptors. Activation of adenosine A2A receptors attenuated the inhibition of noradrenaline release mediated by the presynaptic inhibitory receptors. This effect was mimicked by phorbol 12-myristate 13-acetate and prevented by bisindolylmaleimide XI. It is concluded that adenosine A2A receptors facilitate noradrenaline release by a mechanism that involves a protein kinase C-mediated attenuation of effects mediated by presynaptic inhibitory receptors, namely alpha2-adrenoceptors, adenosine A1 and P2Y receptors.     

Coexistence and cooperation between neuropeptide Y and norepinephrine in nerve fibers of guinea pig vas deferens and seminal vesicle

Fluorescence immunocytochemistry of guinea pig vas deferens and seminal vesicle revealed dense networks of nerve fibers containing both neuropeptide Y (NPY) and dopamine-beta-hydroxylase (DBH), a marker for adrenergic neurons. The effects of norepinephrine (NE) and NPY on the smooth musculature of these organs were studied in vitro. NE inhibited the response to electrical nerve stimulation and increased the basic tension in the vas deferens and contracted the smooth muscle of the seminal vesicle, but had no effect on the contractile response to transmural stimulation in the latter organ. NPY had similar effects on the vas and vesicula, i.e. it inhibited the electrically induced contractions and had no effect on the basic tension. The results suggest a role for NPY as a transmitter that acts before the site of the neuromuscular junction to modulate the release of other transmitters from motor nerve fibers in the smooth musculature.     

Changes of norepinephrine levels, tyrosine hydroxylase and dopamine-beta-hydroxylase activities after castration and testosterone treatment in vas deferens of adult rats

Norepinephrine levels and tyrosine hydroxylase and dopamine-beta-hydroxylase activities have been used to evaluate the effect of castration and testosterone treatment on the sympathetic innervation of the adult vas deferens. Castration was followed by a decrease in both norepinephrine content and tyrosine hydroxylase activity, even though the changes were not concomitant. Treatment of castrated animals with testosterone reversed the effect of castration on organ weight and norepinephrine content, but only a short-lasting increase in tyrosine hydroxylase activity occurred at the beginning of testosterone treatment. In contrast, the testosterone-induced recovery of norepinephrine content observed at this time was accompanied by a marked increase in dopamine-beta-hydroxylase activity. The results suggest that in rat vas deferens, norepinephrine levels are under androgenic control and that this regulation mainly involves changes in dopamine-beta-hydroxylase activity rather than a modulation of tyrosine hydroxylase.     

A novel cyclic analog of neuropeptide Y specific for the Y2 receptor.

The low-molecular-mass, cyclic analog of neuropeptide Y, [Ahx5-24, gamma-Glu2-epsilon-Lys30] NPY (YESK-Ahx-RHYINKITRQRY; Ahx, 6-aminohexanoic acid; NPY, neuropeptide Y), was synthesized and investigated for receptor binding, inhibition of forskolin-stimulated cAMP accumulation, inhibition of electrically stimulated rat vas deferens contractions and ability to increase blood pressure. Like the linear peptide [Ahx5-24] NPY (YPSK-Ahx-RHYINLITRQRY), the more rigid, cyclic analog showed good correlation between receptor binding to rabbit kidney membranes and biological activity in the vas deferens assay. Binding of this peptide to a new Y2-receptor-expressing cell line was slightly reduced, compared to the linear peptide [Ahx5-24] NPY, however inhibition of cAMP accumulation was even more efficient. Unlike the linear peptide [Ahx5-24] NPY, the cyclic analog did not induce a blood pressure increase in rats. Reduced binding to Y1 receptor-expressing SK-N-MC cells, as well as the loss of capability of signal transduction, suggest that only Y2-mediated activity is preserved after cyclization. The selectivity of the cyclic compound for Y2 subtypes of NPY receptors with respect to inhibition of cAMP accumulation is more than fortyfold increased, as compared to the linear NPY-(13-36) peptide, which has been used to determine Y2 selectivity so far.     

Long-term treatment with fluoxetine associates with peripheral effects on rat vas deferens contractility

The aim of this work was to study whether long-term treatment with fluoxetine could induce peripheral effects by modifying vas deferens contractile activity. For this purpose the contractile response to NE, and 5-HT of vas deferens isolated from male Wistar rats that received fluoxetine 10 mg/kg/day i.p., during 21 days, was studied using the isolated organ bath technique. Results show that vas deferens of treated rats presented spontaneous activity, an effect that was abolished by prazosin and isoproterenol and that was not affected by nitroprusside or indomethacin. In addition, fluoxetine did not modify the response to calcium suggesting that spontaneous activity was not a consequence of an abnormal calcium movement. Fluoxetine induced a significant increase in the response of vas deferens to 5-HT and to low NE concentrations while NE maximal effect was unaffected. Fluoxetine treatment did not modify the binding parameters of [3H]-prazosin to vas deferens. It is concluded that long-term treatment with fluoxetine modifies vas deferens contractile activity. This effect could be the result of an alteration of adrenergic neurotransmission and could account for some of the untoward effects observed during clinical course with fluoxetine.     

Beta-endorphin: peripheral opioid activity of homologues from six species

The peripheral opioid activity of six homologous beta-endorphins (beta-EPs) were assayed on the guinea pig ileum and the vas deferens of the mouse, the rat and the rabbit. In the guinea pig ileum assay, human beta-EP (beta h-EP) was less potent than camel, turkey, and ostrich beta-EPs, of the same potency as equine beta-EP and more active than des-acetyl salmon beta-EP. In the rat vas deferens, mammalian beta-EPs showed higher activity than those from the bird and the fish, whereas in the mouse vas deferens assay, beta h-EP is more active than those from other species. In the rabbit vas deferens, however, all homologous beta-EPs show very weak activity. The relative potency of beta-EP homologues obtained from rat vas deferens assay is in good correlation with the analgesic potency, while the receptor binding activity does not correlate with any of the four bioassays, but appears to be related to the charge properties of the peptides.     

Dissociation of the effects of neuropeptide Y on feeding and memory: evidence for pre- and postsynaptic mediation

In mice not deprived of food, centrally administered neuropeptide Y (NPY) increases feeding and improves retention. In this study, we examined the effect of C-terminal NPY fragments on feeding and on memory retention. Mice were trained to avoid footshock in a T-maze. After training NPY, NPY fragments (20-36 and 26-36) or saline were administered intracerebroventricularly. Food consumption was measured during the first hour after training and memory retention was measured one week after training. NPY elicited a 544% increase in feeding compared to the saline control. Neither NPY fragment significantly increased feeding. Both NPY and NPY(20-36) improved retention compared to the saline-treated group. NPY(26-36) did not improve retention. NPY administered to well-trained mice results in amnesia. As a further test of the differential effect of NPY on memory processing and eating, we determined in well-trained mice whether administration of NPY and NPY(20-36) resulted in amnesia. Both NPY and NPY(20-36) resulted in amnesia, but only NPY stimulated feeding. These results are compatible with NPY effects on feeding being mediated through postsynaptic (Y1)NPY receptors and effects on memory retention being mediated through presynaptic (Y2)NPY receptors.     

Supersensitivity of the isolated guinea-pig vas deferens induced by a toxic fraction of scorpion venom (Tityus serrulatus)

Tityustoxin (TsTx), a toxic fraction of Tityus serrulatus venom, was studied on the isolated guinea-pig vas deferens. It increased significantly the maximal response of the preparation to both norepinephrine and acetylcholine and decreased the effective median dose of norepinephrine. The effect of TsTx on norepinephrine median dose was unchanged when atropinized or pharmacologically "denervated" preparations were used but was abolished when both procedures were associated. Atropinization of pharmacologically denervated muscles almost never modify the TsTx-induced increase in the maximal response to norepinephrine. On denervated or phentolamine-treated muscles TsTx-induced increase in the maximal response to acetylcholine was abolished. It was concluded that toxin predominantly induces adrenergic postsynaptic supersensitivity. Of minor significance, it also induces presynaptic cholinergic and adrenergic supersensitivity. Comparison of these results with those of crude venom indicates that TsTx effects may result from the sum of the effects of subcomponents not demonstrated by the chemical procedures here utilized.     

Y2 receptors for neuropeptide Y in the nucleus of the solitary tract mediate depressor responses.

In anesthetized, spontaneously breathing rats, microinjections of selective agonists of neuropeptide Y (NPY) receptor subtypes were made into the medial region of the caudal nucleus of the solitary tract (NTS) at the level of the area postrema. This region of the rat NTS exhibits very high densities of NPY binding sites. Microinjections of the long C-terminal NPY fragment, NPY(13-36), a selective agonist at Y2 receptors, into the caudal NTS elicited pronounced, dose-related reductions in blood pressure and respiratory minute volume. Moreover, the specific pattern of cardiorespiratory responses elicited by NPY(13-36) was remarkably similar, over approximately the same dosage range, with the cardiorespiratory response pattern elicited by intact NPY. In contrast to the potent NTS-mediated responses evoked by NPY(13-36), similar microinjections conducted with either NPY(26-36), an inactive C-terminal NPY fragment, or [Leu31,Pro34]NPY, a NPY analog with specific agonist properties at Y1 receptors, into the same caudal NTS sites did not appreciably affect cardiorespiratory parameters even at 10-20-fold higher dosages. The present results with selective agonists for NPY receptor subtypes suggest that the depressor responses and reductions in minute volume elicited by microinjections of intact NPY and NPY(13-36) were mediated by Y2 receptors in the caudal NTS, likely distributed at presynaptic sites in the medial region of the subpostremal NTS.