Neuropeptide Y is a cotransmitter with norepinephrine in guinea pig inferior mesenteric vein

Peptides 21 (2000) 835– 843

Neuropeptide Y is a cotransmitter with norepinephrine in guinea pig inferior mesenteric vein夞 Lisa Smyth, Janette Bobalova, Sean M. Ward, Violeta N. Mutafova–Yambolieva* Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557-0046, USA Received 13 January 2000; accepted 13 March 2000

Abstract Neuropeptide Y (NPY) is a cotransmitter with noradrenaline in guinea pig inferior mesenteric vein. Tyrosine hydroxylase-like immunoreactivity and NPY-like immunoreactivity were colocalized in a dense network of fibers within the adventitial layer of guinea-pig inferior mesenteric vein. Vasoconstrictor responses to electrical field stimulation (0.2– 64 Hz, 0.1 ms, 12 V, for 10 s) appear to be mediated primarily by norepinephrine at 0.2 to 4 Hz and by NPY at 8 to 64 Hz. NPY Y1 receptors mediate the contractile responses to both endogenous and exogenous NPY. Norepinephrine and NPY are involved in neuromuscular transmission in guinea pig mesenteric vein suggesting that the sympathetic nervous system requires the coordinated action of norepinephrine and NPY to serve capacitance. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Neuropeptide Y; Sympathetic cotransmission; Guinea pig mesenteric vein

1. Introduction Intense activation of sympathetic nervous system as initiated by hemorrhage, intense exercise, or stress results in marked constriction of medium- to large-sized veins [32] especially veins in the splanchnic circulation. This redistributes venous blood and results in increases in venous return and central venous pressure, which are factors important in increasing cardiac efficiency and output. Sympathetic vasoconstrictor neurons typically utilize two or more cotransmitters. The most common cotransmitters are norepinephrine (NE), adenosine 5⬘-triphosphate (ATP), and neuropeptide Y (NPY) [5,21,36,38]. The roles of these cotransmitters vary considerably between different regions of vasculature and with different types of sympathetic stimulation [17,22,26]. The great majority of observations concerning cotransmission in the peripheral sympathetic nervous system in blood vessels originate from studies with arteries [5,8,27,29,38]. NE/NPY cotransmission has been suggested for some veins including the rabbit [7] and human 夞 This work was supported by US Public Health Service Grant HL 60031. * Corresponding author. Tel.: ⫹01-775-784-4302; fax: ⫹01-775-7846903. E-mail address: [email protected] (V.N. Mutafova–Yambolieva).

[31] saphenous vein and vena cava [28]. However, the functional significance of cotransmission specifically in mesenteric veins is less well understood. NPY is known to cause contraction of the vascular smooth muscles in some regions such as rat mesenteric arterial bed, human coronary artery, and cerebral circulation [1,23,39]. In some systems NPY can also play a role as a neuromodulator by prejunctional inhibition and postjunctional potentiation at the neuroeffector junction [20,21,39]. NPY is assumed to predominantly mediate responses to higher frequencies of stimulation and therefore play a role, specifically, at high level of sympathetic nerve activity [21]. Therefore, the role of this peptide in conjunction with NE in the control of venous tone might be particularly important. NPY directly constricts the rabbit saphenous vein [7]; human, rat, and guinea pig femoral vein [14]; and veins of the human forearm [30]. However, no contractions to NPY occur in canine saphenous and portal veins [16]. In view of the limited information of cotransmission in mesenteric veins, and in an attempt to study the role of NPY as a cotransmitter of NE, the present investigation utilizes guinea pig inferior mesenteric vein to pharmacologically examine the functional contributions of NE and NPY in the contractile responses to short stimulation of sympathetic nerves. NE/NPY cotransmission was further characterized morphologically by comparing the localization of tyrosine

0196-9781/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 1 9 6 - 9 7 8 1 ( 0 0 ) 0 0 2 1 7 - 5

836

L. Smyth et al. / Peptides 21 (2000) 835– 843

hydroxylase-like immunoreactivity (TH-LI) and NPY-like immunoreactivity (NPY-LI) in the vascular wall.

2. Methods 2.1. General Male guinea pigs (weighing 400 – 450 g) were euthanized by CO2 overdose followed by exsanguination in keeping with protocols approved by the University of Nevada’s Animal Care and Use Committee. The aorta, inferior mesenteric artery, vein and associated mesentery were immediately removed from the animal and placed in cold (10°C) oxygenated Krebs solution for further dissection of the mesenteric veins. Segments of first and second order branches of the inferior mesenteric vein (250 –500 ␮m in diameter) were dissected out and bathed in regular Krebs solution, whereas perfusing them with distilled water for 30 min to remove endothelium (for contractile experiments). The absence of endothelium was confirmed functionally by a lack of vasorelaxation in response to 0.1 ␮M bradykinin in vessels contracted with the histamine H1 receptor agonist 2-(2-aminoethyl)pyridine (100 ␮M). 2.2. Immunohistochemistry Mesenteric veins (250 –500 ␮m in diameter, 1.5–2 cm long) were removed and prepared for immunohistochemistry. For whole mount preparations, vessels were cut open longitudinally by using fine dissecting scissors and pinned flat to the Sylgard elasotmere (Dow Corning, Midland, MI, USA) base of a coated Petri dish. Vessels were subsequently fixed in paraformaldehyde (4% w/v made up in 0.1 M sodium phosphate buffer with 0.9% NaCl; phosphate-buffered saline [PBS], pH 7.4) at 4°C for 30 min. For cryostat sections, paraformaldehyde was injected into the lumen to distend the vessel so that its shape would be maintained during fixation and the vessel was subsequently immersed in fixative. After fixation, vessels were removed from the fixative and washed with 0.01 M PBS (pH 7.4) for 60 min (15 min ⫻ 4 washes). For cryostat sections tissues were dehydrated through graded sucrose solutions (5–20% in PBS, 1 h each). Sections (10 ␮m) were cut on a Leica (Deerfield, IL, USA) CM 3050 cryostat and mounted on Vectabond (Vector Laboratories, Burlingame, CA, USA) treated slides. Nonspecific antibody binding was reduced by incubation of the tissues in 1% bovine serum albumin (BSA) in 0.01 M PBS for 1 h at room temperature before addition of the primary antibodies. For double immunolabeling, primary antibody incubations, containing a mixture of two primary antisera raised in different species, were carried out concurrently for 48 h at 4°C in 0.01 M PBS containing 0.3% Triton X-100. The two combinations of antibodies were tyrosine hydroxylase (TH; Cat No. A-2027, Signal Transduction Products, San Clem-

ente, CA, at a dilution of 1:400) and NPY (Cat No. ApAB-7172R, Peninsula Laboratories, San Carlos, CA, USA; at a dilution of 1:400). The secondary antibody combination was fluorescein isothiocyanate (FITC)-coupled anti-sheep IgG for TH and Texas Red-conjugated anti-rabbit IgG for NPY. All secondary antibodies were purchased from Vector Laboratories and used at a dilution of 1:100 in 0.01 M PBS. Secondary incubations were performed for 1 h at room temperature in the dark. Control tissues were prepared by either omitting primary or secondary antibodies from the incubation solutions. All the antisera were diluted with 0.3% Triton X 100 in 0.01 M PBS (pH 7.4). Tissues were examined with a BioRad MRC 600 confocal microscope (Hercules, CA, USA) with an excitation wavelength appropriate for FITC (494 nm) and Texas Red (595 nm). Confocal micrographs are digital composites of Z-series scans of 10 –15 optical sections through a depth of 10 to 15 ␮m. Final images were constructed with BioRad Comos software and reconstructed by using Adobe Photoshop 4.0.1. 2.3. Contractile responses to electrical field stimulation or exogenous compounds Ring preparations (3 mm long) were mounted in 3-ml organ baths by inserting two stainless-steel triangle mounts into the lumen. The bottom triangle was attached to a stable hook while the top triangle was attached to a strain gauge (Grass FT03C). Electrical field stimulation (EFS, 0.2– 64 Hz, 0.1 ms duration, 15 V, for 10 s) was applied by means of two platinum wire electrodes that were positioned in parallel on both sides of the preparation and connected to a Grass S 88 electrical stimulator (Grass Instruments, Quincy, MA, USA). Changes in smooth-muscle tone were acquired and analyzed online by a Biopac system (MP100) and Acknowledge 2.3.4 software. The bath contained Krebs solution of following composition (mM): 118.5 NaCl; 4.2 KCl; 1.2 MgCl2; 23.8 NaHCO3; 1.2 KH2PO4; 11.0 dextrose; 1.8 CaCl2, aerated with 95% O2/5% CO2 and maintained at 37°C. The solution routinely contained indomethacin (1 ␮M) and N␻-nitro-L-arginine (l-NNA, 100 ␮M) to block any residual effects of endothelium that might have gone undetected. A resting force of 0.25 g was applied to the venous segment. This was found to stretch vessels to near the optimum length for tension development. In all experiments tissues were initially equilibrated for 1 h before at least three alternating 3-min exposures every 15 min to KCl (70 mM) and NE (1 ␮M) in order to establish viability and equilibrate the tissue. In some experiments the animals were treated intraperitoneally (i.p.) with reserpine (1 mg/ kg), 24 h before the experiment [18], in order to deplete noradrenaline. EFS was applied every 2 min with increasing frequencies of stimulation. When receptor antagonists were used the drugs were applied every time the tissue was washed between electrical stimulations. Contractile responses (area of the response during 90 s from the onset of the stimulation) elicited by EFS were

L. Smyth et al. / Peptides 21 (2000) 835– 843

expressed as a percentage of the contractile response produced with 70 mM KCl. Control experiments were performed to determine the consistency of the contractile response to repeated applications of KCl (70 mM) over the duration of time equivalent to the average duration of the protocols. At the end of the experiment the length and the weight (after blotting the tissue on a filter paper) of the tissue were measured and stress (mN/mm2) was calculated [11,15]. 2.4. Drugs Guanethidine, norepinephrine bitartrate (NE), prazosin, phentolamine, reserpine, tetrodotoxin, yohimbine (Sigma, St. Louis, MO, USA). ((R)-N2-(diphenylacetyl)-N-[(4-hydroxy-phenyl)methyl]-argininamide (BIBP3226), NPY, polypeptide Y (PYY), NPY13–36, [Leu31Pro34]NPY, and pancreatic peptide (PP; Peninsula Laboratories). All drugs were dissolved in redistilled water except for noradrenaline bitartrate and reserpine (1 mM ascorbic acid), and peptides (1% BSA in 0.09% NaCl).

837

provide innervation to the area of the adventitia within a different region of the vessel (Fig. 1C and D). 3.2. EFS-evoked contractions The contractile responses to EFS (0.2– 64 Hz for 10 s) were measured in vein and compared to the response obtained with 70 mM KCl (94.04 ⫾ 7.2 mN/min/mm2). These responses to 70 mM KCl are submaximal, i.e., they represent ⬃70% of the maximum contraction developed in response to combined application of the histamine H1 receptor agonist 2-(2-aminoethyl)pyridine (100 ␮M) and NE (10 ␮M) taken as 100%. All preparations tested responded with frequency-dependent contractions. The contractile responses typically consisted of a rapid initial phase (first 10 s) followed by a slower progressively declining phase that lasted for 90 to 150 s after the stimulation was terminated (Fig. 2). The contractile responses to EFS at all frequencies of stimulation (i.e., up to 64 Hz) were abolished by either tetrodotoxin (1 ␮M) or guanethidine (10 ␮M; n ⫽ 5, data not shown) suggesting that they were due to action-potential induced neurotransmitter release from sympathetic nerves.

2.5. Statistics Data are presented as means ⫾ SE mean. Means were compared by two-tailed paired or unpaired Student’s t-test. A probability value of less than 0.05 was considered significant. The concentration of exogenous peptides producing half-maximum contraction (EC50) was calculated by nonlinear regression analysis (sigmoidal dose-response equations) using GraphPad-Prizm 1.0 (Graph Pad Software, San Diego, CA, USA). The receptor antagonistic activity of BIBP3226 was assessed by Arlunlakshana–Schild plot [2] and pA2 was obtained.

3. Results

3.3. Influences of ␣-adrenoceptor antagonists Neural responses were further characterized by use of specific ␣-adrenoceptor antagonists. In the presence of either prazosin or phentolamine the initial rapid phase was affected more profoundly than the second, more sustained phase of contraction (Fig. 2). Phentolamine (1 ␮M), an ␣1/␣2-adrenoceptor antagonist, abolished responses to 0.5 to 2 Hz and significantly reduced the responses to greater frequencies (4 – 64 Hz) of stimulation (Fig. 3A). Prazosin (0.1 ␮M), an ␣1-adrenoceptor antagonist, abolished the responses to nerve stimulation at 0.5– 4 Hz and reduced the responses to 8 to 64 Hz (Fig. 3A). Yohimbine (0.1 ␮M), an ␣2-adrenoceptor antagonist, did not affect the responses to 0.5 to 1 Hz, and facilitated the responses to 2 to 16 Hz (Fig. 3A).

3.1. Localization of TH-LI and NPY-LI 3.4. Influence of reserpine pretreatment TH-LI in flat mounts of isolated mesenteric veins revealed a dense network of nerve fibers within the wall of the vessels (Fig. 1A). At high magnification some of these nerve fibers had varicose swellings along their processes. NPY-LI also revealed a dense network of nerve fibers within these veins. TH immunopositive nerves showed an identical distribution to nerve fibers that were immunopositive for NPY (Fig. 1A and B). Cryostat sections of mesenteric veins also revealed an identical distribution for both TH-LI (Fig. 1C) and NPY-LI (Fig. 1D). Both TH-LI and NPY-LI were observed within a dense layer of nerve fibers at the level of the adventitia. There were ⬃8 to 10 nerve fibers per 100 ␮m along the length of vein when cut in cross-section. Located at the outer extremities of the adventitia were TH-LI and NPY-LI nerves that may possibly

In another series of experiments, vein segments from reserpine-pretreated animals were examined. Nerve-evoked contractions in response to lower frequencies of stimulation (0.2– 4 Hz) were abolished, whereas the responses to 8 to 64 Hz were only reduced (Fig. 3B) suggesting that in this vessel a nonadrenergic component of nerve evoked contractions also occurs. Typically in these vessels the contractile response started with a delay, i.e., the contraction begins after the stimulation is terminated (Fig. 2). All these findings taken together suggest that the initial rapid phase of the contraction as well as a portion of the second phase of the contraction are mediated by NE that is released during nerve stimulation. These finding further suggest that a significant nonadrenergic component can be revealed.

838

L. Smyth et al. / Peptides 21 (2000) 835– 843

Fig. 1. Flat mount and cryostat sections of mesenteric vein labeled with TH-LI and NPY-LI. Panel A shows the distribution of TH-LI nerve fibers (arrows) in guinea pig mesenteric vein. Panel B shows the same area of vein labeled for NPY-LI (arrows). TH-LI and NPY-LI were colocalized in the same nerve network. Panels C and D show a cryostat section of a mesenteric vein labeled for TH-LI (arrows, Panel C) and NPY-LI (arrows, Panel D). The distribution of TH-LI and NPY-LI was also identical in cryostat sections suggesting colocalization of both neurotransmitters in the same nerve population. Scale bar in Panel D applies to all figures.

3.5. Influences of NPY Y1 receptor antagonist BIBP 3226 As shown in Fig. 4A the frequency-response curves to EFS were shifted to the right in the presence of BIBP 3226 (0.1–1 ␮M). BIBP 3226 (1 ␮M) when added to phentolamine (1 ␮M), abolished the EFS-evoked contractions at all frequencies of stimulation (Fig. 4B). BIBP 3226 (1 ␮M) also abolished the responses to nerve stimulation at all frequencies of stimulation in veins from reserpine-treated animals (Fig. 4C).

All these findings taken together suggest that a component of nerve evoked contractions is mediated by the NPY Y1 receptors. 3.6. Contractile responses to exogenous NPY and analogues and influence of BIBP3226 The receptors that mediate the responses to NPY in these vessels were studied by the use of different NPY analogues. NPY and analogues contracted the vessel segments but the

L. Smyth et al. / Peptides 21 (2000) 835– 843

839

Fig. 2. Electrical field stimulation (EFS, 0.1 ms, 12 V, for 10 s) evoked contractions in guinea pig mesenteric vein. Representative tracings showing the responses to 8 Hz in controls, after blockade of the ␣-adrenoceptors with prazosin (0.1 ␮M), depletion of NE stores with reserpine (1 mg/kg i.p., 24 h before experiment), and blockade of NPY Y1 receptors with BIBP 3226 (1 ␮M). Tracings are representative of those obtained from 4 –10 different preparations. The responses were compared with the response to 70 mM KCl.

potency of responses differed (see EC50 values in Table 1). The relative potency sequence of analogues was: NPY ⱖ [Leu31Pro34]NPY ⫽ PYY ⬎ PP ⬎ NPY13–36. There were also significant difference observed in the efficacy of some

Fig. 4. NPY-ergic component of the contractile responses to EFS (0.1 ms, 12 V, for 10 s) in guinea pig mesenteric vein. Panel A: Effects of the NPY Y1 receptor antagonist BIBP 3226 (0.3–1 ␮M) on EFS-evoked contractions. Panel B: Effects of BIBP 3226 (1 ␮M) on phentolamine-resistant component of EFS-evoked contractions. Panel C: Effect of BIBP 3226 (1 ␮M) on EFS evoked contractions in vessels from reserpine-treated (1 mg/kg i.p., 24 h before experiment) animals. Asterisks denote significant difference from controls (P ⬍ 0.05). Each point represents an n ⫽ 4 to 6 experiments; SE mean are presented by error bars.

peptides. For example, both [Leu31Pro34] NPY and PYY were less efficacious than NPY (Fig. 5). This potency order suggests that NPY Y1 receptors mediate the contractile responses to exogenous NPY in this tissue. Table 1 EC50 values (nM) of NPY-like peptides in inducing contractions in guinea-pig isolated mesenteric vein (means ⫾ s. e. mean, number of experiments in parenthesis). Fig. 3. Adrenergic component of the contractile responses to EFS (0.1 ms, 12 V, for 10 s) in guinea pig mesenteric vein. Panel A: Effects of ␣-adrenoceptor antagonists prazosin (1 ␮M), phentolamine (0.1 ␮M) and yohimbine (0.1 ␮M) on EFS-evoked contractions. Panel B: Effect of reserpine (1 mg/kg i.p. 24 h before experiment) on EFS-evoked contractions. Asterisks denote significant difference from controls (P ⬍ 0.05). Each point represents an n ⫽ 4 to 6 experiments; SE mean are presented by error bars.

EC50 NPY [Leu31,Pro34]NPY PYY PP NPY13-36 P ⬍ 0.05 between artery and vein groups.

*

1.00 ⫾ 0.3(12) 5.00 ⫾ 0.53(4) 6.00 ⫾ 0.49(5) 102 ⫾ 8.2(4) 300 ⫾ 22.2(4)

840

L. Smyth et al. / Peptides 21 (2000) 835– 843

[Leu31Pro34]NPY (0.3 nM), or NPY13–36 (0.3 ␮M). At these concentrations the peptides evoked either no contraction or very weak contractile response (see Fig. 5). Nerve evoked contractions were not changed in the presence of either peptide (Fig. 7). 3.8. Influences of NPY on the contractile responses to exogenous NE

Fig. 5. Concentration-response relationships for NPY and analogues ([Leu31Pro34]NPY, PYY, PP, and NPY13–36) in guinea pig mesenteric vein rings normalized to the response obtained with 70 mM KCl. Each point represents an n ⫽ 3 to 5; SE mean are presented by error bars.

Pretreatment with BIBP 3226 (0.1–3 ␮M) resulted in rightward shift of the concentration-response curve to NPY (Fig. 6A). Regression analysis of the Schild plot gave a pA2 value of 7.68 ⫾ 0.09, and slope of regression line 0.96. BIBP 3226 shifted to the right the concentration response curves of either PYY (Fig. 6B) or [Leu31Pro34]NPY (Fig. 6C) with pA2 values 7.40 ⫾ 0.1 and 7.53 ⫾ 0.13, respectively. BIBP 3226 shifted to the right the concentrationresponse curves for PP, as well (Fig. 6D). The contractile responses to NPY13–36 (0.3 and 1 ␮M) were also abolished in the presence of BIBP 3226 (1 ␮M; n ⫽ 3, data not shown). 3.7. Influences of NPY, [Leu31Pro34]NPY and NPY13–36 on EFS-evoked contractions The responses to EFS (0.2– 64 Hz) were tested in the presence of subthreshold concentrations of NPY (0.3 nM),

The contractile responses to NE (0.01–10 ␮M) were not changed in the presence of NPY (0.3 nM; n ⫽ 3; Fig. 8 ).

4. Discussion There is growing awareness that peripheral nerves release more than one transmitter, a process referred to as plurichemical neurotransmission or cotransmission [5]. NPY is considered to be a cotransmitter together with NE in the sympathetic nervous system [20]. The relative contribution of each of the neurotransmitters to contraction varies significantly between blood vessels. NPY is assumed to predominantly mediate responses to long-lasting and higher frequency stimulation and therefore plays a role specifically at high level of sympathetic nerve activity [21]. As mentioned in the introduction, intense activation of sympathetic nervous system appears to play a significant role for the capacitative function of veins [32]. Veins in splanchnic circulation in particular, are important for serving capacitance. The role of NE and NPY as excitatory cotransmitters in mesenteric veins is not clear. Our results suggest that NE is the primary excitatory neurotransmitter in vein of the guinea pig at low frequencies of stimulation and is also

Fig. 6. The effects of BIBP 3226 on the concentration-response relationships for NPY (A), [Leu31Pro34]NPY (B), PYY (C), and PP (D) in guinea pig mesenteric vein segments normalized to the response obtained with 70 mM KCl. Each point represents an n ⫽ 3 to 5; SE mean are presented by error bars.

L. Smyth et al. / Peptides 21 (2000) 835– 843

Fig. 7. Effects of NPY (0.3 nM), [Leu31Pro34]NPY (0.3 nM), and NPY13–36 (0.3 ␮M) on EFS (0.1 ms, 12 V, for 10 s)-evoked contractions in guinea pig mesenteric vein. Each point represents an n ⫽ 3 to 5 experiments; SE mean are presented by error bars.

responsible for the initiation of the contractile responses to high frequencies of stimulation. Evidence was obtained to suggest that NPY is involved in nerve-evoked contractions in vein even at brief stimulation and is primarily responsible for the maintenance of the contractile response. Our results further indicate that both NE and NPY may be released from the same sympathetic neurons. Immunohistochemical experiments in this study demonstrate, for the first time, the distribution of NPY-LI and TH-LI nerves in the guinea-pig mesenteric vein. Thus, our experiments revealed that both TH-LI and NPY-LI were distributed in a dense network of fibers within the adventitial layer. It is also apparent that TH-LI and NPY-LI were located within the same nerve population that innervates mesenteric veins. The chemical coding of inferior mesenteric ganglion neurons innervating mesenteric vessels of the guinea-pig has been recently studied [4]. Contrary to the present investigation, it was reported that NPY-LI was not found in the inferior mesenteric ganglia nerve cell bodies innervating the inferior mesenteric veins. Several differences exist between the present study and that of Browning [4]. We examined the distribution of nerves in the close proximity to their terminals. Examination of some neuropeptides in cell bodies has been reported to be difficult

Fig. 8. The effect of NPY (0.3 nM) on the contractile responses to exogenous NE in guinea pig mesenteric vein rings normalized to the response obtained with 70 mM KCl. Each point represents an n ⫽ 3 to 4 experiments; SE mean are presented by error bars.

841

without the use of an axonal transport inhibitor, such as colchicine [9,25,34,37]. Without the use of colchicine the number of nerve cell bodies that contain NPY-LI could have been underestimated in Browning’s study [4]. Secondly, we used mature male guinea pigs weighing 400 to 450 g in comparison to the female guinea pigs weighing 150 to 250 g in Browning’s study [4]. Further work will be necessary to resolve the differences between these two studies. The finding that TH-LI and NPY-LI were colocalized in a dense network of fibers within the adventitial layer in the guinea pig mesenteric vein presents a morphologic basis for NE/NPY cotransmission in this vessel. One should keep in mind, however, that recent results suggest that the colocalization of neurotransmitters does not always imply functional cotransmission [3,24]. In order to study the functional correlates of the immunohistochemical study we further examined the role of both NE and NPY in the contractile responses to stimulation of perivascular nerves. This study has demonstrated that repetitive transmural stimulation (10 s) of sympathetic neurons supplying the mesenteric vein usually produces contraction at frequencies as low as 0.5 Hz. A significant component is eliminated by either prazosin or phentolamine suggesting that ␣1-adrenoceptors mediate a large part of EFS-evoked contractions. A small nonadrenergic component was revealed in presence of these ␣-adrenoceptor antagonists when NE stores were intact. However, in vessels from reserpine-treated animals a larger nonadrenergic component was revealed. The reduction of this component under control conditions may be related to the well-known autoinhibition of transmitter release produced by NE acting at presynaptic ␣2 receptors [35]. This action could inhibit not only NE release but also the release of other potential cotransmitters including NPY. In agreement with this we observed that the ␣2-adrenoceptor antagonist yohimbine produced enhancement of neural responses at 2–16 Hz suggesting great autoinhibition by NE in this vessel. This reserpine-resistant (and, hence, nonadrenergic) component is abolished by the NPY Y1 selective receptor antagonist BIBP 3226 [33] suggesting that NPY mediates a component of nerve-evoked contractions. Even in preparations with intact NE stores BIBP3226 significantly reduced the contractions at frequency as low as 2 Hz. Finally, similar results were obtained in nonreserpinized vessels when the adrenergic component of the neural response was eliminated by the nonselective ␣-adrenoceptor antagonist phentolamine, i.e., BIBP 3226 abolished the phentolamine-resistant response. All these findings taken together provide strong evidence that NPY is a mediator of the nonadrenergic contractions of the guinea pig inferior mesenteric vein. The present study provided evidence that NE and NPY appear to be colocalized in the same neurons and that both adrenergic and BIBP3226-sensitive components of nerve stimulation are tetrodotoxin and guanethidine-sensitive. We conclude, therefore, that the NPY Y1 receptor-mediated component of the EFS-evoked contraction is mediated by NPY that is released from sympathetic nerves.

842

L. Smyth et al. / Peptides 21 (2000) 835– 843

The delay in onset of the sympathetic contractions in the presence of either ␣-adrenoceptor antagonists or reserpine implies that NE mediates primarily the initial phase of the contraction. The sustained phase of contraction, however, appears to be sensitive to BIBP3226 and, hence, is mediated by NPY. Thus, the two transmitters appear to serve interconnected functions when sympathetic nerves are activated, i.e., NE initiates the contractile response while NPY maintains it. Our results also imply that in guinea pig mesenteric vein NPY can serve this function even at brief (i.e., 10 s) sympathetic stimulation. It is of physiological significance that the sympathetic perivascular nerves costore and corelease NE and NPY, providing a physiological scenario for sympathetic cotransmission in this system. It is not clear whether the two transmitters are released simultaneously or in sequence. To our knowledge no studies on the time course of NE and NPY release from sympathetic neurons have been published. It is well known, however, that NPY is stored primarily in large dense-cored vesicles in the nerve terminal, whereas NE can be stored in both large densecored vesicles and small dense-cored vesicles [10]. Therefore, it can be speculated that the release of NE and NPY may be sequential or simultaneous depending on the frequency of stimulation and the type of the synaptic vesicles that are subjected to exocytosis at particular frequencies. NPY elicited prominent concentration-dependent contractions. There is increasing evidence that the vascular response to NPY is mediated by Y1 subtype of NPY receptors [12,19]. The pharmacological properties that we observed for contractions to NPY and analogues in the mesenteric vein are consistent with this conclusion. Thus, the potency order of NPY ⱖ [Leu31Pro34]NPY ⫽ PYY ⬎ PP ⬎ NPY13–36 suggested that NPY Y1 receptors primarily mediate the contraction to exogenous NPY and analogues. Likewise, the selective NPY Y1 receptor antagonist BIBP 3226 shifted to the right the concentration-response curves to all NPY analogues. Finally, BIBP 3226 abolished the nonadrenergic contraction to nerve stimulation as well. All these findings suggest that the postjunctional receptors that mediate the contractile responses to both endogenous and exogenous NPY are primarily of NPY Y1 subtype. In some systems NPY plays also a role of a neuromodulator by inhibiting the release of NE [39] or potentiating the vasoconstriction produced by other agonists, including cotransmitters [8]. The prejunctional inhibitory effect of NPY on sympathetic transmitter release is usually Y2-receptor mediated [19]. The NPY Y1 receptors can also be prejunctional and may inhibit sympathetic transmitter release, as in rabbit vas deferens [13]. In the present study however, neither NPY nor [Leu31, Pro34]NPY, a NPY analog with specific agonist properties at Y1 receptors, or NPY13–36, a selective agonist at Y2 receptors, affected EFS-evoked contractions suggesting that in this vessel NPY may not serve presynaptic neuromodulation at 0.2 to 64 Hz for 10 s. Further, the contractile responses to exogenous NE remained unchanged in the continuous presence of subthresh-

old concentration of NPY, suggesting that NPY may not serve postsynaptic modulation as well. In summary, three different lines of evidence all suggest that NPY serves as a neurotransmitter in vein, namely: 1) physical localization of NPY in sympathetic nerves of vein; 2) mimicry of the neurally induced contraction by exogenous NPY; 3) inhibition of the neural response with a NPY blocker. Further, NPY Y1 and NPY Y2 receptor agonists as well as NPY itself did not affect the nerve-evoked contractions suggesting lack of presynaptic neuromodulation. Likewise, contractile responses to exogenous NE were not affected by NPY suggesting lack of postjunctional neuromodulation. Given these observations the most likely conclusion is that NPY is released from sympathetic nerves in mesenteric vein along with NE and serves cotransmission rather than neuromodulation. In conclusion, both NE and NPY are involved in neuromuscular transmission in guinea pig mesenteric vein suggesting that the sympathetic nervous system requires the coordinated action of NE and NPY to serve capacitance.

References [1] Abounder R, Villemure JG, Hamiles E. Characterization of neuropeptide Y receptors in human cerebral arteries with selective agonists and the new Y1 antagonist BIBP 3226. Br J Pharmacol 1995;116:2245– 50. [2] Arunlakshana O, Schild HO. Some quantitative studies of drug antagonists. Br J Pharmacol Chemother 1959;14:48 –58. [3] Blitz DM, Nusbaum MP. Distinct functions for cotransmitters mediating motor pattern selection. J Neurosci 1999;19:6774 – 83. [4] Browning KN, Zheng Z, Kreulen DL, Travalgi RA. Two populations of sympathetic neurons project selectively to mesenteric artery or vein. Am J Phsyiol 1999;276:H1263–72. [5] Burnstock G. Co-transmission. The Fifth Heymens Memorial Lecture. Arch Int Pharmacodyn Ther 1990;304:7–33. [6] Burnstock G. Noradrenaline and ATP. Cotransmitters and neuromodulators. J Physiol Pharmacol 1995;46:365– 84. [7] Cadieux A, Pheng L, St. Pierre S, Fournier A, Taoudi M. The rabbit saphenous vein: a tissue preparation specifically enriched in NPY-Y1 receptor subtype. Regul Pept 1993;46:557–564. [8] Cortes V, Donoso MV, Brown N, Fanjul R, Lopez C, Fournier A, Huidobro–Toro JP. Synergism between neuropeptide Y and norepinephreine highlights sympathetic cotransmission: studies in rat arterial mesenteric bed with neuropeptide Y, analogs, and BIBP 3226. J Pharmacol Exper Ther 1999;289:1313–22. [9] Costa M, Brookes SJ, Steele PA, Gibbins I, Burcher E, Kandiah CJ. Neurochemical classification of myenteric neurons in the guineapig ileum. Neuroscience 1996;75:949 – 67. [10] De Potter WP, Kurzawa R, Miserez B, Coen EP. Evidence against differential release of noradrenaline, neuropeptide Y, and dopamine␤-hydroxylase from adrenergic nerves in the isolated perfused sheep spleen. Synapse 1995;19:67–76. [11] Dillon PF, Murphy RA. High force development and crossbridge attachment in smooth muscle from swine carotid arteries. Circ Res 1982;50:799 – 804. [12] Donoso MV, Brown N, Carrasco C, Cortes V, Fournier A, Huidobro– Toro JP. Stimulation of the sympathetic perimesenteric arterial nerves releases neuropeptide Y potentiating the vasomotor activity of noradrenaline: involvement of neuropeptide Y-Y1 receptors. J Neurochemistry 1997;69:1048 –59.

L. Smyth et al. / Peptides 21 (2000) 835– 843 [13] Doods HN, Krause J. Different neuropeptide Y receptor subtypes in rat and rabbit vas deferens. Eur J Pharmacol 1991;204:101–3. [14] Ekblad E, Edvinsson C, Wahlestedt C, Uddman R, Hakanson R, Sundler F. Neuropeptide Y co-exists and co-operates with noradrenaline in perivascular nerve fibers. Reg Peptides 1984;8:225–35. [15] Herlihy JT, Murphy RA. Length-tension relationship of smooth muscle of the hog carotid artery. Circ Res 1973;33:275– 83. [16] Hunter LW, Tyce GM, Rorie DK. Neuropeptide Y release and contractile properties: differences between canine veins and arteries. Eur J Pharmacol 1996;313:79 – 87. [17] Kennedy C, Saville VL, Burnstock G. The contributions of noradrenaline and ATP to the responses of the rabbit ear artery to sympathetic nerve stimulation depend on the parameters of stimulation. Eur J Pharmacol 1986;122:291–300. [18] Khoyi MA, Westfall DP, Buxton ILO, Akhtar–Khavari F, Rezaei E, Salaices M, Sanchez–Garcia P. Norepinephrine and potassium induced calcium translocation in rat vas deferens. J Pharmacol Exp Ther 1988;246:917–23. [19] Larhammar D, Soderberg C, Lundell I. Evolution of the neuropeptide Y family and its receptors. Ann NY Acad Sci 1998;839:35– 40. [20] Lundberg JM, Franco–Cereceda A, Lacroix JS, Pernow J. Neuropeptide Y and sympathetic neurotransmission. Ann NY Acad Sci 1990; 611:166 –74. [21] Lundberg JM. Pharmacology of cotransmission in the autonomic nervous system: integrative aspects on amines, neuropeptides, adenosine triphosphate, amino acids and nitric oxide. Pharmacol Reviews 1996;48:113–78. [22] Lundberg JM, Hokfelt T. Multiple co-existance of peptides and classsical transmitters in peripheral autonomic and sensory neurons –functional and pharmacological implications. Prog Brain Res 1986;68: 241– 62. [23] Macho P, Perez R, Huidobro–Toro JP, Domenech R. Neuropeptide Y: a coronary vasoconstrictor and potentiator of catecholamine-induced coronary vasoconstriction. Eur J Pharmacol 1987;167:67–74. [24] Marder E. Neural signalling: does colocalozation imply cotransmission? Curr Biol 1999;9:R809 –11. [25] Messenger JP, Furness JB. Projections of chemically-specified neurons in the guinea-pig colon. Arch Histol Cytol 1990;53:467–95. [26] Morris JL, Gibbins IL. Co-transmission and neuromodulation. In: Burnstock G, Hoyle CHV, editors. Autonomic neuroeffector mechanisms. Chur, Switzerland: Harwood, 1992. p. 33–119. [27] Morris JL. Cotransmission from sympathetic vasconstrictor neurons to small cutaneous arteries in vivo. Am J Physiol 1999;277:H58 – 64.

843

[28] Morris JL. Roles of neuropeptide Y and noradrenaline in symapthetic neurotransmission to the thoracic vena cava and aorta of gunea-pigs. Regul Pept 1991;32:297–310. [29] Mutafova–Yambolieva V, Westfall DP. Inhibitory and facilitatory presynaptic effects of endothelin on sympathetic cotransmission in the rat isolated tail artery. Br J Pharmacol 1998;123:136 – 42. [30] Pernow J, Lundberg JM, Kaijser L. Vasoconstrictor effects in vivo and plasma disappearance rate of neuropeptide Y in man. Life Sci 1987;40:47–54. [31] Racchi H, Irarrazabal MJ, Howard M, Moran S, Zalaquett R, Huidobro–Toro JP. Adenosine 5⬘-triphosphate and neuropeptide Y are cotransmitters in conjunction with noradrenaline in the human saphenous vein. Br J Pharmacol 1999;125:1175– 85. [32] Rothe CF. Reflex control of veins and vascular capacitance. Physiol Rev 1983;63:1281–347. [33] Rudolf K, Eberlein W, Engel W, Wieland HA, Willim KD, Entzeroth M. The first highly potent and selective non-peptide Y Y1 receptor antagonist: BIBP3226. Eur J Pharmacol 1994;271:R11–3. [34] Sang Q, Young HM. Chemical coding of neurons in the myenteric plexus and external muscle of the small and large intestine of the mouse. Cell Tiss Res 1996;284:39 –5. [35] Starke K. Presynaptic ␣-autoreceptors. Rev Physiol Biochem Pharmacol 1987;107:73–146. [36] Stja¨rne L, Åstrand P, Bao J-X, Gonon F, Msghina M, Stja¨rne E. Spatiotemporal pattern of quantal release of ATP and noradrenaline from sympathetic nerves: consequences for neuromuscular transmission. In: Stja¨rne L, Greengard P, Grillner S, Ho¨kfelt T, Ottson D, editors. Advances in second messenger and phosphoprotein research: molecular and cellular mechanisms of neurotransmitter release. New York: Raven Press, 1994. p. 461–96. [37] Stele PA, Costa M. Opoid-like immunoreactive neurons in secretomotor pathways of the guinea-pig ileum. Neuroscience 1990;38:771– 86. [38] Westfall DP, Dalziel HH, Forsyth, KM. ATP as a neurotransmitter, co-transmitter and neuromodulator. In: Phillis JW, editor. Adenosine and adenine nucleotides as regulators of cellular function. Boca Raton: CRC Press, 1991. p. 295–305. [39] Westfall TC, Carpentier S, Chen X, Beinfeld MC, Naes L, Meldrum MJ. Prejunctional and postjunctional effects of neuropeptide Y at the noradrenergic neuroeffector junction of the perfused mesenteric arterial bed of rat. J Cardiovasc Pharmacol 1987;10:716 –22.