Introduction

Top Abstract Introduction Experimental Procedures Results Discussion References

Neuropeptide Y (NPY) is a 36-amino-acid peptide (Tatemoto et al., 1982) that is widely distributed in the central and peripheral nervous systems (Dumont et al., 1991; Morris and Gibbins, 1992). It is released from postganglionic sympathetic nerve terminals in combination with norepinephrine (Lundberg et al., 1990). Because NPY produces little or no contractile responses in smooth muscle preparations but can enhance contractile responses elicited by agonists or nerve stimulation (Edvinsson et al., 1984; Lopez et al., 1989) and can inhibit catecholamine release (Pernow et al., 1986; Grundemar et al., 1992), it is regarded as a modulator of sympathetic transmission and could play this role when coreleased with norepinephrine (Burnstock and Ralevic, 1996). However, there is a lack of conclusive evidence of cotransmission (Lundberg, 1996).

Rat anococcygeus muscle is a smooth muscle preparation with a dense intramural plexus of sympathetic nerve terminals that produces -adrenergic-mediated contraction (Gillespie, 1980) and a nonadrenergic, noncholinergic (NANC), putatively nitrergic, innervation (Gillespie et al., 1989; Liu et al., 1991), whose activation produces relaxation if the sympathetic nerves are blocked. Vila et al. (1992) reported that in rat anococcygeus muscle NPY could increase nerve-induced contraction but not norepinephrine-induced contraction, without itself producing contraction or altering transmitter release. This paradoxical preferential potentiation of nerve-mediated rather than norepinephrine-mediated contraction by NPY was explained, at least in part, by inhibition of the NANC nerve-induced relaxation. This provided no support for a cotransmitter role for NPY in this preparation.

Since then, two developments encouraged us to revisit the role of NPY in the rat anococcygeus muscle: 1) the observation by Iravani and Zar (1997) that NPY can increase contraction to norepinephrine in rat anococcygeus muscle when, in contrast to the study of Vila et al. (1992), blockers of the neuronal and extraneuronal uptake of catecholamines were absent and 2) the characterization of NPY receptor subtypes and the availability of pharmacological tools for their study.

At present, six NPY receptors (Y1-Y6) have been described. Y1 and Y2 receptors have been best characterized (Michel, 1991; Gehlert, 1994; Grundemar and Hakanson, 1994; Michel et al., 1998). Both Y1 and Y2 receptors have been implicated in postjunctionally procontractile effects and inhibition of transmitter release (Wahlestedt et al., 1986; Grundemar et al., 1992; McAuley and Westfall, 1992). The physiological effects of NPY on Y1 and Y2 receptors can be distinguished by using truncated neuropeptide agonists (Wahlestedt et al., 1990; Michel, 1991). The Y1 receptor possesses a high affinity for full-length NPY (Fuhlendorff et al., 1990), and the Y2 receptor exhibits a high affinity for short [e.g., NPY (13-36)] fragments (Wieland et al., 1995a). Furthermore, a nonpeptide ligand BIBP 3226 [(R)-N²-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]argininamide] has been developed (Rudolf et al., 1994) and described as a selective and potent antagonist of Y1 receptors (Doods et al., 1996).

The aim of this study was to clarify whether endogenous NPY can increase adrenergic nerve responses, to characterize the subtype of NPY receptor involved, and to establish whether endogenous NPY released with norepinephrine plays a role in excitatory transmission.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Male Sprague-Dawley rats (300-350 g) were sacrificed by decapitation. The anococcygeus muscle was dissected as described by Gillespie (1972) and set up in 20-ml organ baths containing physiological salt solution composed of 112.0 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl₂, 1.1 mM KH₂PO₄, 1.2 mM MgSO₄, 25.0 mM NaHCO₃, and 11.1 mM glucose maintained at 37°C and continuously gassed with 95% O₂, 5% CO₂. A resting tension of 4.90 mN was placed on the tissue, and changes in tension were recorded with a PIODEN (UF-1; Pioden Controls, Kent, UK) isometric transducer attached to an Omniscribe pen recorder (Allen Datagraph, Salem, NH). The preparations were left to equilibrate for 45 min with frequent washes. Tension was readjusted if necessary. After equilibration, the preparation was contracted three or four times with KCl (60 mM) every 5 min until the contractile responses were similar. The tissue was then washed, and when the initial tension was achieved, it was left to equilibrate for an additional 30-min period before starting the experiments.

Responses to Norepinephrine. The effects of NPY alone and in the presence of BIBP 3226 were assessed on norepinephrine-induced contraction in the absence of neuronal and extraneuronal uptake blockers. In preliminary experiments, we found that the maximum contraction in the second concentration-response curve to norepinephrine was slightly smaller than in the first. Therefore, second curves were compared throughout. Two cumulative concentration-response curves to norepinephrine (0.001-30 µM) were constructed in each of three sets of preparations. In the first set, two control concentration-response curves were constructed. In the other two sets, before the second concentration-response curve, in either the absence or the presence of 1 µM BIBP 3226 (30 min), NPY (0.01 or 0.1 mM) was applied for 7 min. The effects of NPY in the presence or absence of BIBP 3226 were then assessed against the time control tissue. Contractions were calculated as a percentage of the maximum response obtained in the first concentration-response curve (control) to the agonist.

Nerve-Mediated Responses to Electrical Field Stimulation. For electrical field stimulation, the anococcygeus muscle was suspended between a pair of platinum ring electrodes. Square wave pulses of 0.1 ms and supramaximal voltage at 0.25 to 5 Hz for 10 s every 3 min using a Grass stimulator causes frequency-related contractions. Three frequency-response curves, separated by a 30-min period, were generated in each tissue. Under these conditions, contractile responses were abolished by 1 µM tetrodotoxin (TTX) as previously reported (Gillespie, 1972).

Effects of Agonists and Antagonists on Contractile Responses to Electrical Field Stimulation. Three sets of experiments were performed. In a first set, three control frequency-response curves in the presence of 0.1% BSA (the vehicle used to dissolve NPY) were carried out as time controls. In the second and third sets, after a control frequency-response curve, paired anococcygeus muscles were incubated with 0.01 and 0.1 µM NPY or NPY (13-36) for 7 min before a further frequency-response curve with or without a 30-min preincubation with BIBP 3226 (1 µM).

Effects of NPY and BIBP 3226 on NANC Nerve-Induced Responses to Electrical Field Stimulation. We assessed whether BIBP 3226 could influence the previously observed inhibition of NANC responses by NPY (Vila et al., 1992). NANC-induced relaxation to field stimulation was obtained after raising muscle tone and eliminating the sympathetic nerve-induced contraction. The sympatholytic drugs phentolamine (1 µM) and guanethidine (30 µM) were added for 40 and 30 min, respectively; then, tone was raised to a submaximal plateau by carbachol (50 µM). Two frequency-response curves separated by a 30-min period were then constructed. In control experiments, inhibitory responses were greater in the second frequency-response curve compared with the first curve. Thus, comparisons were made between second curves. NPY (0.1 µM, 7 min) was added before the second frequency-response curve in either the absence or the presence of BIBP 3226 (1 µM, 30 min). Results obtained in the second frequency-response curve in the presence of the vehicle, NPY, or NPY in the presence of BIBP 3226 were compared. Field stimulation was applied at supramaximal voltage, 1-ms duration at 0.25 to 5 Hz applied for 10 s every 3 min, according to the protocol established by Gillespie (1972). Under these conditions, the inhibitory responses were abolished by TTX (1 µM) as previously reported (Gillespie, 1972).

Effects of BIBP 3226 on Norepinephrine and Adrenergic and NANC Nerve-Induced Responses to Electrical Field Stimulation. After a control concentration-response curve, increasing concentrations of BIBP 3226 (1-10 µM) were applied for 30 min before a second concentration-response or frequency-response curve, all in the absence of neuronal and extraneuronal uptake blockers.

Analysis and Statistics. Experimental points are expressed as mean ± S.E. The number of animals (n) is indicated in the legend of the figures. The forces developed in contractile responses were calculated either as percentage of the control response elicited by the highest frequency of stimulation (5 Hz) or as percentage of the maximum () agonist response of the first curve. NANC relaxations were calculated as the percentage reduction from the tone existing at the onset of nerve stimulation.

Materials. Porcine NPY and BIBP 3226 were a gift from Dr. Karl Thomae GmbH (Biberach, Germany). NPY (13-36) was purchased from Calbiochem (England). ()-Norepinephrine bitartrate, phenylephrine, desipramine HCl, normetanephrine HCl, and BSA were purchased from Sigma Chemical Co. (St. Louis, MO). Phenoxybenzamine HCl was obtained from Research Biochemicals International (Natick, MA). Stock solutions of 0.1 mM NPY and NPY (13-36) were dissolved in BSA (0.1%) and 0.4 mM BIBP 3226 in distilled water, aliquoted, and stored at 70°C in silicon-coated tubes. This procedure prevents the adsorption of the peptide to the plastic and glass tubes. Stock phenoxybenzamine solution (1 mM) was dissolved in 2 mM tartaric acid and kept at 20°C. Further dilutions of phenoxybenzamine were prepared in physiological salt solution. Norepinephrine was prepared daily in 23 µM Na₂EDTA. All chemicals used were of analytical grade.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

In the absence of uptake blockers, norepinephrine contracted the anococcygeus muscle in a concentration-related manner (Fig. 1). NPY (0.01 or 0.1 µM) caused no contraction. However, NPY (0.01 µM) significantly (P < .05) increased the responses to norepinephrine in the range of 0.1 to 3 µM (Fig. 1a). This effect was fully reversed by 1 µM BIBP 3226. A higher concentration of NPY (0.1 µM; Fig. 1b) clearly enhanced (P < .05-P < .001) responses to all submaximal concentrations of norepinephrine. BIBP 3226 prevented this increase of norepinephrine-induced contractions. In the presence of neuronal and extraneuronal uptake blockers and after exposure to phenoxybenzamine, the norepinephrine concentration-response curve (pEC₅₀ = 5.25 ± 0.01, n = 6) was shifted to the left (pEC₅₀ = 5.64 ± 0.08, n = 6; P < .001) by NPY (0.1 µM) (Fig. 2a). This effect was prevented by BIBP 3226 (1 µM; Fig. 2b).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   The antagonism by BIBP 3226 of NPY-induced increase of contractions to norepinephrine in the absence of blockers of norepinephrine uptake. Each curve is the second curve obtained from a different set of tissues: time control, NPY, and NPY in the presence of BIBP 3226 (1 µM). a, 0.01 µM NPY. b, 0.1 µM NPY. Contraction is expressed as percentage of the maximum response obtained in the control concentration-response curve. Lines represent pragmatic logistic curve fitting (see text). Results are the mean ± S.E. (n = 5-10) by two-way ANOVA (treatment and concentration) with repeated measures in both factors. Vertical pairwise contrast: *P < .05, **P < .01, ***P < .001.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   The antagonism by BIBP 3226 of NPY-induced increase of contractions to norepinephrine after incubation with 0.1 µM phenoxybenzamine and in the presence of desipramine (0.1 µM) and normetanephrine (1 µM). a, control followed by 0.1 µM NPY. b, control followed by 0.1 µM NPY in the presence of 1 µM BIBP 3226. Contraction is expressed as percentage of the maximum response obtained in the first concentration-response curve for each experiment. Lines represent pragmatic logistic curve fitting (see text). Results are the mean ± S.E. (n = 5-10) by two-way (treatment and concentration) ANOVA with repeated measures in both factors. Vertical pairwise contrast: *P < .05, ***P < .001.

Electrical field stimulation induced reproducible, frequency-dependent contraction of rat anococcygeus muscle (Fig. 3a). NPY (0.01 or 0.1 µM) significantly increased, in a concentration-dependent manner, the responses induced at all frequencies of electrical field stimulation (Fig. 3b). BIBP 3226 (1 µM) partly reversed the increase by NPY of electrical field stimulation-induced responses at all frequencies used (Fig. 3c). To evaluate whether Y2 receptors could contribute to the enhancement by NPY of responses induced by electrical field stimulation, the effect of the Y2 receptor agonist NPY (13-36) was studied. NPY (13-36) (0.01 µM) did not modify the frequency-response curve (Fig. 4a). However, a higher concentration (0.1 µM) produced small but significant increases (Fig. 4a) at all frequencies except 0.25 Hz. BIBP 3226 (1 µM) completely blocked this increase (Fig. 4b).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   The antagonism by BIBP 3226 of NPY-induced increase of contractions to electrical field stimulation. a, three consecutive time-control curves. b, effects of NPY alone: control followed by 0.01 µM NPY, followed by 0.1 µM NPY. c, as in b but in the presence of 1 µM BIBP 3226 after the first curve. Results are the mean ± S.E. (n = 14-20) by two-way (treatment and frequency) ANOVA with repeated measures in both factors. Vertical pairwise contrast: *P < .05, **P < .01, ***P < .0001.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   The antagonism by BIBP 3226 of NPY (13-36)-induced increase of contractions to electrical field stimulation. a, effects of NPY (13-36) alone: control followed by 0.01 µM NPY (13-36), followed by 0.1 µM NPY (13-36). b, as in a but in the presence of 1 µM BIBP 3226 after the first curve. Results are the mean ± S.E. (n = 5-8) by two-way (treatment and frequency) ANOVA with repeated measures in both factors. Vertical pairwise contrast: *P < .05, **P < .01.

Electrical field stimulation in the presence of sympathetic nerve blockade elicited the known frequency-related NANC relaxation (Fig. 5). NPY (0.1 µM) significantly reduced the NANC responses at 0.25 and 0.5 Hz. BIBP 3226 (1 µM) had no effect on the inhibition by NPY of NANC-induced relaxation.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   BIBP 3226 (1 µM) shows no antagonism of the inhibitory effect of 0.1 µM NPY on relaxation of anococcygeus muscle to NANC nerves. Responses are expressed as a percentage reduction from the tone existing at the onset of nerve stimulation. Results are the mean ± S.E. (n = 5-8) by two-way (frequency and treatment) ANOVA with repeated measures in both factors. Vertical pairwise contrast: *P < .05.

The effects of increasing concentrations of BIBP 3226 on electrical field stimulation-induced (Fig. 6a) and norepinephrine-induced (Fig. 6b) contractions were studied to evaluate the participation of endogenous NPY in the nerve-induced responses. BIBP 3226 (1 µM) decreased responses at 1 and 5 Hz. Higher concentrations diminished responses at all frequencies of stimulation except at the lowest frequency, which was reduced only by the highest concentration of BIBP 3226 (Fig. 6a). BIBP 3226 had no effect on norepinephrine-induced contraction at 1 or 3 µM (results not shown) or at 10 µM (Fig. 6b). BIBP 3226 at 1 to 10 µM had no effect on NANC-induced relaxation (results not shown).

View larger version (22K): [in this window] [in a new window]   Fig. 6.   .. Effect of BIBP 3226 on the frequency-response curve for contraction to electrical field stimulation (a) and concentration-response curve for contraction to norepinephrine (b) in the absence of neuronal and extraneuronal uptake blockers. Results are the mean ± S.E. (n = 8) by two-way (treatment and concentration/frequency) ANOVA with repeated measures in both factors. Vertical pairwise contrast: *P < .05, **P < .01, P < .001.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

This study shows that exogenous NPY increases contractile responses to stimulation of sympathetic, adrenergic nerves via postjunctional Y1 receptors. The participation of endogenous NPY via this mechanism, as a facilitatory, excitatory cotransmitter, is demonstrated.

NPY produced a concentration-dependent increase of the contractile responses to norepinephrine. This was clear cut at 0.1 µM NPY, and the threshold occurred at around 0.01 µM NPY, where there was a significant increase in individual norepinephrine concentrations. On its own, this suggests a postjunctional synergistic action that facilitates the contractile effect of norepinephrine. We tested and excluded an indirect origin for this effect through blockade of cellular uptake of norepinephrine by showing that NPY could still cause an increase in the presence of neuronal and extraneuronal norepinephrine uptake blockers. This confirms an action of NPY to enhance the postjunctional effect of norepinephrine. This facilitatory action of NPY was completely blocked by 1 µM BIBP 3226. This was a selective action against NPY because the contraction to norepinephrine per se was unaffected by BIBP 3226. The affinity of BIBP 3226 for Y2 receptors (K_i > 10 µM) is 1000 times less than that for Y1 receptors (K_i = 6.8 ± 0.7 nM) in rat (Wieland et al., 1995b). Thus, blockade by 1 µM BIBP 3226 of facilitation by NPY of the action of norepinephrine indicates an interaction at Y1 receptors.

NPY increased the electrical field stimulation-induced contraction, which arises from activation of postganglionic sympathetic nerves, whose major transmitter is norepinephrine (Gillespie, 1972; Gillespie and McGrath, 1973). Analysis of the basis for this enhancement is complicated because inhibitory NANC nerves are activated simultaneously by field stimulation and attenuate the contractile effect of sympathetic nerve stimulation. These NANC nerves are believed to be nitrergic (Gillespie et al., 1989, Liu et al., 1991). They can be pharmacologically blocked by nitric oxide synthase inhibitors to isolate the adrenergic response. This allowed the original demonstration that NPY has a potentiating effect on sympathetic nerve responses in rat anococcygeus muscle (Vila et al., 1992). Conversely, in this study, the inhibitory effect of NPY on the NANC response could be illustrated when the NANC response was isolated pharmacologically (guanethidine and phentolamine). This inhibition was totally unaffected by BIBP 3226 at the concentration that eliminated the facilitatory effect on norepinephrine-mediated contraction. This indicates that NPY blocks the action of inhibitory nerves by an action that is independent of Y1 receptors and thus separate from the Y1-mediated enhancement of noradrenergic contractile responses.

The interpretation of the action of NPY on the contractile responses to nerve stimulation is now straightforward. The adrenergic component of the response to sympathetic nerve activation is enhanced by a Y1 receptor-mediated action, such as potentiation of the responses to norepinephrine. In addition, the inhibition of the NANC nerve-mediated relaxation response by a non-Y1 receptor-mediated mechanism contributes to NPY enhancement of the contractile response. This component of the NPY effect explains why BIBP 3226 can completely block NPY enhancement of norepinephrine but incompletely blocks its enhancement of contraction to nerve stimulation.

A further question is whether the non-Y1 receptor-mediated effect of NPY on nerve stimulation might be mediated by the Y2 receptors. Evidence against this was that NPY (13-36) produced no BIBP 3226-resistant facilitatory action against the nerve responses. Given the greater affinity and efficacy of this peptide for Y2 than for Y1 receptors (Wieland et al., 1995b), this can be taken as clear evidence for the lack of a Y2 receptor capable of facilitatory action in this tissue. It follows from this interpretation that in contrast to some other tissues, there is no evidence for a prejunctional Y2 receptor (Wahlestedt et al., 1986) or Y1 receptor (McAuley and Westfall, 1992).

Taken together, these results provide positive evidence for a functional postjunctional Y1 receptor that facilitates norepinephrine responses or adrenergic transmission and for a non-Y1 action of NPY that is inhibitory to NANC transmission. Its non-Y1 site of action (prejunctional or postjunctional) cannot be conclusively identified. However, because relaxation to sodium nitroprusside is attenuated by NPY (Vila et al., 1992), at least part of this action seems to be postjunctional. These data require us to modify the conclusions from our earlier study of NPY in rat anococcygeus muscle. We missed the facilitatory effect of NPY on norepinephrine contraction, which was subsequently shown by Iravani and Zar, (1997), leading us to argue against a postjunctional modulatory effect of NPY (Vila et al., 1992). The present results show that NPY does increase contraction to exogenous norepinephrine but that this effect is not seen if the norepinephrine neuronal and extraneuronal uptake mechanisms are blocked. This raised the question of whether the absence of the effect of NPY in the presence of uptake blockers is because it blocks norepinephrine uptake. This possibility was eliminated by demonstrating that after phenoxybenzamine, NPY still caused enhancement of norepinephrine-induced contractions when uptake blockers were present. This allows us to conclude that NPY can enhance the response to exogenous norepinephrine by a direct postjunctional action on smooth muscle.

The final important question raised by our observations is whether NPY is a functional excitatory cotransmitter. NPY-immunoreactive nerve fibers are present in rat anococcygeus muscle (Iravani and Zar, 1997). Thus, if NPY is present in the same nerves as norepinephrine, as in other tissues, and is coreleased with norepinephrine, NPY would be expected to facilitate transmission by the Y1 receptor-mediated action that we have demonstrated.

BIBP 3226 produced a concentration-related inhibition of electrical field stimulation-induced contraction. Even the highest concentration of BIBP 3226 had no effect on exogenous norepinephrine. This suggests that BIBP 3226 was acting via blockade of the effects of endogenous NPY coreleased with norepinephrine. Excitatory transmission in rat anococcygeus muscle can be entirely blocked by -adrenoceptor antagonists (Gillespie and McGrath, 1974; Docherty and Starke, 1981) in contrast to adrenergic/purinergic cotransmission, where blockade of responses to either norepinephrine or ATP leaves the response to the other transmitter intact (Blakeley et al., 1981; Sneddon and Westfall, 1984; Bulloch and McGrath, 1988). NPY may, therefore, be more accurately described as a modulatory cotransmitter.

In conclusion, in rat anococcygeus muscle, exogenous NPY can act through Y1 receptors on smooth muscle to increase the contractile effect of norepinephrine. Endogenous NPY coreleased with norepinephrine enhances -adrenergic transmission through this Y1 receptor. This tissue provides a model system for the further study of the cotransmitter role of NPY, and the approach that was necessary to illustrate this may prove fruitful in other tissues.