Neuropeptide y- and alpha-adrenergic receptors in pig spleen: Localization, binding characteristics, cyclic amp effects and functional responses in control and denervated animals

Neuroscience Vol. 24, No. 2, pp. 659412, 1988

0306-4522/88 $3.00 + 0.00

Printed in Great Britain

Pergamon Journals Ltd 0 1988IBRO

NEUROPEPTIDE Y- AND ALPHA-ADRENERGIC RECEPTORS IN PIG SPLEEN: LOCALIZATION, BINDING CHARACTERISTICS, CYCLIC AMP EFFECTS AND FUNCTIONAL RESPONSES IN CONTROL AND DENERVATED ANIMALS J. M. LUNDBERG,*? A. HEMS&N,* A. RUDEWLL,$ A. H.&RFSTRAND,§0. LARSSON,* A. SOLLEVI,~ A. SARIA,~/ T. H~~KFIZLT,$K. FUXE$$and B. B. FREDHOLM* Departments of *Pharmacology and §Histology, Karolinska Institutet fDepartment of Anesthesiology, Karolinska Hospital, Stockholm, Sweden IlDepartment of Experimental and Clinical Pharmacology, University of Graz, Graz, Austria

Ah&act-The localization of neuropeptide Y binding sites in the pig spleen, as revealed by ~‘zsI]Bolton-Hunter-la~U~ porcine neuropeptide Y and alpha,-adrener~c receptor binding sites, as revealed by [‘25I](2-beta/4-hydroxy-phenyl/-ethylaminomethyl)-tetralone as radioligand, was compared with the distribution of neuropeptide Y and noradrenaline nerves, the latter revealed by tyrosine hydroxylase and dopamine-~~-hydroxylase, using imm~ohist~hemist~. A large degree of codistribution was obtained between [‘25f]neuropeptide Y and alpha,-binding sites in the capsule, trabeculae, blood vessels and the red pulp of the spleen. Neuropeptide Y and tyrosine hydroxylase as well as dopamine-beta-hydroxylase-positive nerves were identical in the spleen and had a similar gross distribution pattern as the [‘2sI]neuro~ptide Y and alpha, binding sites. In functional studies using the isolated blood-perfused spleen from pentobarbital-anaesthetized pigs, neuropeptide Y, noradrenaline and the alpha,-selective agonist phenylephrine contracted the capsule and induced vasoconstriction in the spleen in r&o. However, the selective alphaZ-adrenoceptor agonists clonidine and azepexole bad no effects on blood flow or perfusion pressure, suggesting that postjunctional alpha-receptors were of the alpha, type. Neuropeptide Y inhibited the forskolin-evoked, cyclic adenosine monophosphate formation in vitro. The [12sI]neuropeptide Y binding, with an equilibrium-dissociation constant of 503 + 73 pM and a maximal number of specific binding sites of 23 f 3 fmol/mg protein, the neuro~ptide Y-induced perfusion-pressure increase in oivo and the inhibition of forskolin-evoked cyclic adenosine monophosphate formation in vitro were dependent on the amidation of the C-terminal portion of the peptide molecule. Furthermore, the effects of neuropeptide Y were not changed by alpha- and eta-adren~ptor blockade using prazosin and propranolol. Two weeks after postganglionic denervation the neuropeptide Y and the noradrenaline contents of the pig spleen were reduced by 97% and 99%, respectively. These changes were associated with a selective supersensitivity for the noradrenaline-induced perfusion-pressure increase in oiuo compared with the effect of neuropeptide Y. However, a similar potentiation of the noradrenaline effect was induced by the monoamine-uptake blocker desipramine in the absence of denervation, and there was no change in the functions response to phenyleph~ne after denervation. This indicated that the dene~ation supersensitivity for noradrenaline was of prejunctional rather than of postjunctional origin, although a significant increase in the maximal number of specific binding sites of the [‘Hlprazosin binding (75%) was observed in virro. No signiticant changes in the ~~lib~urn-diss~iation constant or in the maximal number of specific binding sites were observed after denervation for the [‘251]neuropeptide Y binding sites. Furthermore, the neuropeptide Y-induced inhibition of forskolin-induced cyclic adenosine monophosphate formation was not changed after denervation. It is concluded that alpha,-adren~ptor and neuropeptide Y binding sites are co-distributed and can be activated in parallel to induce muscle contraction in support of a co-transmitter role for noradrenaline and neuropeptide Y in the sympathetic nervous control of the pig spleen. Denervation was associated with a selective u~mgulation of the alphas-adren~ptor binding sites, but no changes in the binding characteristics were observed for neuropeptide Y, suggesting the absence of homeostatic responses in the regulation of neuropeptide Y receptors of the spleen.

tTo whom correspondence should be addressed at: Department of Pharmacology, Karolinska Institutet, Box 60400, S-104 01 Stockholm, Sweden. Abbreviations: CAMP, cyclic adenosine monophosphate; DBH, dopamine-beta-hydroxylase; DMI, desipramine; EDTA, ethylenediaminetetra-acetate; FITC, fluorescein iso~i~anate; HEAT, 2-eta-/~hydroxyphenyl/~thylaminomethyl-tetralone; HEPES, N-2-hydroxyethyl-

piperazine-W-2-ethanesulphonic acid. ICY, half maxima1 inhibition of specific binding; IR, immunoreactive; &, ~~~b~urn-diss~iation constant; -LI,-like immunoreactivity; NA, noradrenaline; NPY, neuropeptide tyrosine; pNPY, porcine NPY; PYY, peptide with Nand C-terminal tyrosine; RIA, radioimmunoassay; TH, tyrosine hydroxylase. 659

J. M.

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Increasing evidence suggests that the 36-aminoacid peptide neuropeptide tyrosine (NPY)‘l acts together with noradrenaline (NA) to regulate peripheral cardiovascular control.36 Thus, NPY is likely to co-exist with NA in sympathetic nerves around blood vessels and in the parenchyma of the heart and spleen.1’*29~30 NPY is co-released with NA from the spleen and heart upon sympathetic nerve stimulation3’.3’,45 and into the systemic circulation during reflexogenic, sympathetic activation (e.g. by physical exercise or in newborn infants upon vaginal Furthermore, NPY exerts both vasodelivery. 34.35,4’ constrictor actions per se in uivoq-28,31*43 and in vitro,” enhances the contractile effects of NA6.” as well as evoking the prejunctional inhibitory effects on NA via mechanisms independent of adrenorelease7.2q+32 ceptors. Therefore, NPY is one candidate to mediate the nerve stimulation-evoked ~rfusion-pressure increase, which remains in the cat” and pig37 spleen in the presence of adrenoceptor antagonists or after pretreatment with reserpine to deplete tissue NA. With regard to receptor mechanisms for NPY, specific, high-affinity binding sites for [‘251]NPY have been demonstrated in membranes of the rat brain,5.47,55suggesting the presence of NPY receptors. Furthermore, NPY has been shown to inhibit the forskolin-induced elevation of cyclic adenosine monophosphate (CAMP) in cat cerebral vesselsI and in brain tissue,20 indicating a link between NPY receptors and adenylate cyclase activity. In the present study we have compared the distribution and receptor characteristics for NPY and NA in the pig spleen with the functional effects in vivo in normal and chronically denervated animals.

EXPERIMENTAL

PROCEDURES

Functional experiments

The present studies were performed on spleens in ten control and five surgically denervated male pigs (25-35 kg). Denervation of the spleen was performed under aseptic conditions, using sodium pentobarbital (Nembutal, LEO, Sweden) anaesthesia by transecting and removing the postganglionic sympathetic nerves along the main splenic artery and vein for a distance of l-2 cm. The accessory vessels to the caudal pole of the spleen and the communicating vessels to the stomach from the hilus area were ligated and sectioned. Two weeks after the denervation procedure, the pigs were premeditated with ketamine (Ketalar, ParkeDavies, USA.; 20 mg/kg i.m.) and atropine (ACO, Sweden; 0.05 m&kg i.m.). Sodium nentobarbital (20 ma/kg i.v.) was used for the induction of anaesthesia. Pancuronium. (Organon, The Netherlands; 0.2 mg/kg i.v.) was then given to induce skeletal muscle paralysis, and artificial ventilation was performed. After heparinization (Kabi Vitrum, Sweden; 250 Il-ljkg i.v.), the splenic artery was catheterized and perfused with blood (25-3Oml/min) from one femoral artery, using a roller pump (Gilson, France) to obtain a mean perfusion pressure of 75-80 mmHg. The blood was collected from the splenic vein and directed into a femoral vein. The blood flow in the splenic venous eflluent was continuously monitored, using an electromagnetic flow meter (Nycotron, Norway) and changes were taken as an indication of capsule

contractions. Local intra-arterial infusions of the substances NA (nt-arterenol, Sigma, NO, U.S.A.), porcine NPY, NPY-free acid and peptide with N- and C-terminal tyrosine (PYY; Peninsula, CA, U.S.A.), BHT-933 (azepexole) and clonidine hydrochloride (Boehringer, Ingelheim, F.R.G.). L-phenylephrine hydrochloride (Sigma),- prazosin hydrochloride (Pfizer. U.K.) and uronranolol (ICI. U.K.) were performed via an inlet into the perfusion system close’to the spleen. Perfusion pressure in the system was recorded by a Statham pressure transducer (P23 DC; Statham Company, Puerto Rico), and was taken as an indication of local vascular resistance changes. About 10 g of the caudal portion of the spleen (representing about 10% of the total size) was removed, divided up and used for immunohist~hemistry, biochemical determinations of the content of NPY and NA, receptor-binding and second-messenger formation (see below). After blood stilling with a clamp at the incision site. the rest of the spleen was used for functional experiments. Desipramine hydrochloride (DMI; Sigma; 0.5 mg/kg i.v.) was given to five of the control animals 30 min prior to local infusions to inhibit the neuronal uptake of ~t~hoiamines.22 Receptor-binding autoradiography To localize ~pha,-ad~noceptor and NPY binding sites in splenic tissue [‘*31](2-beta/4-hydroxyphenyl/-ethylaminomethyl-tetralone (BE 2254; HEAT)‘2.‘s.46and [i2sI]porcine NPY @NPY), respectively, were used as radioligands in autoradiographic experiments based on a modified method.@ Small pieces of the splenic tissue were placed in NaCl (0.9%) at +4”C and 14pm cryostat sections were made. Six sets of duplicate (adjacent) sections were taken from the spleen for incubation. With the two ligands every duplicate set was incubated with a non-radioactive ligand (see below) to define the non-specific binding. All sections were then mounted on object glasses, which had previously been coated with a solution of 0.1% (v/v) polyethylenimine (Sigma). For the incubation procedure- with ]‘251]NPY (Amersham, Dagenham, U.K.), a 50 mM Tris-HCl buffer (PH 7.6) containing 5 mM MgCl,, aprotinin (0.065 TIU/ml; Sigma) and 0.1 M bacitracin was used. Based on the Ko value (0.503 nM. see Table 1). 0.5 nM NPY was used. aivine a high signal-to-noise ratio. The binding was perf&med under equilibrium conditions (60min 23°C). After incubation, the sections were rinsed three times (5 min each in 50 mM Tris-HCl, pH 7.6) and further rinsed in redistilled H,O. and then dried under a stream of cold dry air.19 The cytoarchitecture was verified by counterstaining sections with haematoxylin-eosin (red and white pulp, trabeculae and capsule etc.). To define non-specific binding, 10-6M pNPY was used. In a few experiments, blood cells from the splenic venous effluent were collected during nerve stimulation, frozen, sectioned and incubated, as described above. The incubation nrocedure for llZSIIHEAT (NEN. U.S.A.: sp. act. 22OOCi/mmol) was based on Sargent-Jones et aI.& A 160mM TrisHCl buffer (pH 7.4), containing 5 mM MgCl, and 1mM EDTA, was used. The sections were preincubated (2 x 5 min) in a 160mM Tris-HCl buffer (pH 7.4) and then incubated with 60 pM [‘251]HEAT(2 h at 23”C), which is close to the B_,. . value.‘2*‘5The sections were rinsed twice in the buffer and dried, as described above. To define non-specific binding, lO-.4M L-NA was added to the incubation solution. In all experiments (1’2511NPYand [‘ZSI]HEAT), tritium-sensitive film sheets .-(3~-Ultrofilm, LKB, Stockholm, Sweden) were used to visualize the bindExposure times were as follows. [‘251]NPY: 24-30 h; ln511HEAT: 40 h in X-rav cassettes at -20°C. In the analysis of the ‘H-Ultrofilm, an IBAS image analyser (Zeiss, Munich, F.R.G.) was used in combination with a Bosch video camera. ..l^

Receptor-binding biochemistry

[‘251]Bolton-Hunter-labelled

NPY was purchased from

Neuropeptide Y- and alpha-adrenergic Amersham, Dagenham, U.K. The specific activity of the tracer was determined by radioimmunoassay (RIA) for NPY and corrected for decay with time.48 Splenic tissue (about 2 g) was placed in 10 volumes of ice-cold (0.3 M) sucrose, containing 5mM HEPES at a pH of 7.4. After homogenization using a Polytron, the suspension was centrifuged at 1OOOg for 10 min after homogenization. The supernatant was collected and further centrifuged at 10,OOOgfor 30min. The pellet was resuspended in 0.9% NaCI, containing 5 mM HEPES, pH 7.4, to a tissue concentration of 1 g/ml and frozen in aliquots. The protein concentration was measured by the Lowry method, using bovine serum albumin as standard.27 For binding studies, 30~1 of membranes corresponding to 300-4OO~g protein was added to increasing concentrations of [‘2fI]pNPY (range 5-3000 PM) in Krebs buffer solution at pH 7.4 of the following composition in mM: NaCl 137, KC1 2.68, MgCl, 2.05, CaCl,, CaCl, 180 and HEPES 20. The non-specific binding was estimated by adding cold NPY to a final concentration of 0.1 /IM. After incubation at +23”C for 60min, the bound and free fractions were separated by centrifugation at 10,OOOgfor 2min. The supernatant was aspirated and the pellet was washed with ice-cold buffer as above and recentrifuged. The radioactivity in the supernatant and pellet was then counted in an LKB gamma counter. The equilibrium-dissociation constant (Ko) of the reeeptor_I’2sI]NPY complex and the maximal number of specific binding sites (B,,) were calculated using LIGAND, a program performing a computerized analysis of ligandbinding data.@ Half the maximal inhibition of the specific binding of the [iz51]NPY is given as the IC, value.’ The characteristics of alpha,-adrenoceptor binding in the same splenic membrane preparations were studied, using [rH]prazosin (NEN, U.S.A.; sp. act. 82Ci/mmol). The incubation medium consisted of 5OmM Tris-HCl buffer (PH 8.0). lO-6 M non-labeiled prazosin was used to determine the non-specific binding for [‘Hlprazosin. In addition to the membrane preparations from the whole spleen, the possible binding of [iz51]pNPY to blood elements in the spleen was studied using 50ml of blood collected during splenic nerve stimulation at 2 Hz, which caused the capsule to contract and to expel stored blood cells. Membrane preparations of these blood cells were then obtained, as described above. The occurrence and distribution of NPY-immunoreactive (-IR) nerves in relation to NA-containing neurons were studied with immun~hemist~, using the NPY antiserum 102D” and antisera raised aaainst tvrosine hvdroxvlase lTH and dopamine-beta-hydroxylase (DBH)].r6- Briehy, small tissue pieces from the coeliac ganglion, splenic nerves and splenic parenchyma were immersion-fixed for 4 h at 4°C in a mixture of 0.1 M phosphate buffer, containing 0.4% (w/v) picric acid, 2.5% (w/v) parabenzoquinone and 4% (w/v) paraforamaldehyde. After overnight rinsing at 4°C in a phosphate buffer containing 10% (w/v) sucrose, 4-pm-thick cryostat sections (Dittes, Heidelberg, F.R.G.) were incubated with primary rabbit antisera to NPY diluted I:400 or TH and DBH (diluted 1: 500 and 1: 100, respectively) for 24 h at 4°C. For detection of the tissue localization of the primary antisera, fluorescein isothiocyanate (FITC)-labelled donkey anti-rabbit IgG {Amersham, Dagenham, U.K.) was applied to the sections for 30min at 37°C. In addition, double-labelling experiments for NPY and TH were performed on the same sections, using NPY antibodies raised in sheep’ and the TH antiserum raised in rabbit. These antibodies were then detected by FITC-labelled antirabbit (Nordic, Tilburg, The Netherlands) and Texas Red-labelled secondary antibodies, respectively. After rinsing, the settions were mounted in paraphenylenediamine and examined in a Zeiss fluorescence microscope equipped with proper filters for FITC or Texas Red detection.

receptors

661

Cyclic adenosine monophosphate formation Splenic tissue was placed in ice-cold Krebs-Ringer solution (PH 7.4) of the following composition in mM: NaCl 118, KC1 4.85, MgSO, 1.1% CaCl, 2.5, KH,PG, 1.15, NaHCO, 25. alucose 1I. The material was minced into small pie&s (2-6 mm), using a McIlwain tissue chopper, and then washed three to five times in a buffer, which was continuously gassed with 6.5% CO, and 93.5% 02. After 60 min equilibration at 37°C the tissue pieces were distributed into plastic tubas containing 0.3 mM of the phosphodiesterase inhibitor isobutylmethylxanthine (Calbiochem, U.S.A.). After 5min of preincubation, 0.3pM forskolin (Sigma) was added alone or together with 1-1OOOnM of pNPY. The reaction was terminated after 10 min by addition of 0.25 ml ice-cold perchloric acid. The tissues were homogenized and centrifuged. The supematant was neutralized and assayed for CAMP content,r while the protein content of the pellet was measured by the method of Lowry et aL2’ Biochemical ~term~atio~ of ~ropeptide

Y and noradren-

aiine levels The splenic content of NPY-like immunoreactivity ( -LI) was determined by radioimmunoassay, using antiserum Nl (for details, see Ref. 53) after heating of specimens in 1 M acetic acid at 95°C for lOmin, homogenization and lyophilization of the supernatant. The tissue content of NA was determined, using high-~rfo~an~ liquid chromatography in combination with electrochemical detection).‘s*23*24 RESULTS

Receptor-binding

autoradiography

Specific [iZSI]NPY binding was demonst~ted in splenic sections, using autoradiography (Fig. la and b). A distinct pattern of [‘251]NPY binding sites was present with a dense labelling, especially in the red pulp and blood vessels, while the [‘*‘I]NPY binding was less dense in the capsule and trabeculae (Fig. la and b). In contrast, the white pulp proper was only weakly labelled by [1251]NPY, but relatively dense binding was associated with blood vessels, e.g. central arteries (Fig. la). No [‘2sI]NPY binding was detectable on blood cells from the spleen. A similar distribution pattern of [‘251]NPY binding was present both in control and denervated spleens (not shown). Autoradiographical labelling of alpha, binding sites, as revealed by [“‘I]HEAT binding, showed a distribution pattern, which was very similar to that of [12’I]NPY binding (cf. Fig. la and c). Thus, the [‘251]HEAT labelling was especially dense in the red pulp of the spleen (Fig. lc), while less binding was present in the trabeculae and capsule. In the white pulp, only blood vessels were labelled (Fig. 1~). When comparing sections from control and denervated spleens, no clear-cut change in the distribution of the [‘251]HEAT binding was detected (not shown). Immunohistochemistry and neuropeptide adrenaI~e determinations

Y and nor-

The localization of [‘251]NPY and [I””I]HEAT binding in the spleen, as revealed by receptor autoradiography, was closely reiated to the occurrence of NPY-, TH- and DBH-IR nerve fibres (Fig. 2). Thus, NPY-IR nerves were abundant in the capsule and

f-j.7 ‘, Visualization of [“‘IINPY (0.5 nM) and [‘251]HF~\T 160pM) binding as revealed t eptor .ecific du%xddiography in adjacent sections (14 am) from the ~KIII-(1:rervated pig spleen. The low r hi~~thng of [‘2’I]NPY (b) and [‘rSI]HEAT (d) was revealed b) xddition of cold ligands (1 JIM ‘1i and white 100 {JM tN.4, respectivei! I. Note the high labcihng in the re 1 pulp (red colour) compared it’ pulp (blue colour) with a moderate iabelling (yellow colour) of capsule, trabeculae and centr,li :teries m the white pulp (black arrow). White arrows point to cap! ule with capsular trabecula. Bar ~n&cates I mm. All micrographs have the same magnification. The col mr scale for binding in (a) is the ~tne for all micrographs (a-d). The figures (ad) are photographs of displays on the screen of the IBAS image analyser after colour cc ding. __-“‘--i- ~~-

Fig. 2. Immunofluorescence micrograph (montage) of the pq spleen after incubation with anti$erum to NPY. A moderately dense to very dense fibre network can b: seen m the capsule, along the trabeculae (tr) as well as in the red pulp. Note NPY-positive fibres aro md central arteries (curved arrows) m the white pulp (w). Open arrow points to an intensely fluorescent small nerve bundle at the outer surface of the capsule (caps). Bar = 50 pm.

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J. M. LUNDBERGet al.

Fig. 3. Immunofluorescence micrographs of the pig spleen of control (a, b) and denervated (c, d) animals after incubation with antiserum to NPY (a, c) and tyrosine hydroxylase (TH) (b, d). NPY- and TH-positive fibres have a similar distribution in the spleen, as seen in the adjacent sections (a, b). Arrows point to some positive fibres. Two weeks after denervation, the neuronai immunoreactivities disappear completely (cf. a, b with c, d). Asterisks indicate venous sinusoids. tr, trabecula. (a) Bar = 50pm. AI1 micrographs have the same magnification.

Neuropeptide Y- and alpha-adrenergic receptors capsular trabeculae as well as around blood vessels in both the white and red pulp. In addition, nerve fibres were often seen without any clear-cut association to the vasculature in the red but not in the white pulp (Figs 2, 3a, 4a and c). NPY- and TH-IR nerve fibres in the spleen (Fig. 3a and b) as well as DBH-positive nerves (not shown) had identical distribution patterns. Double-labelling analysis on the same section revealed an apparent total overlap between NPYand TH-IR nerves in the spleen (Fig. 4a-d). Two weeks after denervation, there was a marked loss

Fig. 4. Immunofluorescence double-staining technique secondary antiserum (a, secondary antiserum (b, identity of nerve fibres orientation,

665

of both NPY- and TH-IR nerve fibres in the spleen (Fig. 3c and d). This was paralleled by a reduction of 97% of the content of NPY-LI, as determined by RIA (Table I), which in turn was closely correlated to the loss of NA (99%, Table 1). Most cells in the coeliac ganglion were both NPYand TH-IR as well as DBH-positive (Fig. 5a and b). The NPY-IR was granular in appearance and associated with the Golgi apparatus, while the TH and DBH stains were more homogenously distributed in the cytoplasm (Fig. 5a-d). Furthermore, double-

micrographs of two sections from the pig spleen processed according to the by incubation with antiserum to NPY raised in sheep with FITC-labelled c) and tyrosine hydroxylase (TH) raised in rabbit with Texas Red-labelled d). Note that both in the capsule (a, b) and in the red pulp (c. d), there is an containing NPY- and TH-like immunoreactivities. Arrows are given for (a) Bar = 50 pm. All micrographs have the same magnification.

666

J. M. LUNDBERG et al.

Fig. 5. hnmunofluorescence micrographs of adjacent sections of the pig coeliac ganglion after incubation with antiserum to NPY (a, c) and tyrosine hydroxylase (TH) (b, d). Most cell bodies are NPY-immunoreactive, but single cells (arrows) seem to lack this peptide (a). In the adjacent section it can be seen that virtually all cell bodies are TH-positive (b). (c), (d) In the double-seining experiments it is shown that many cells (l-5) are both NPY- (as revealed by FITC) and TH-positive (Texas Red labelhng), demonstrating co-existence of the two immunoreactivities. Asterisks point to some TH-positive nerve cells, apparently lacking NPY-like immunoreactivity. Bars = SOpm. (a, b) and (c, d) have the same magnification, respectively.

Neuropeptide Y- and alpha-adrener~c

receptors

667

Table 1. The effects of splenic nerve transection on noradrenaline and neuropeptide Y mechanisms in pig spleen [3Hjprazosin binding

Control Two weeks’ denervation Change (%)

P

NA content (nmolig) 7.80 _+2.60 0.04 If:0.02 -99 0.01

[‘251]NPY binding

CC-G)

Elmai (fmol/mg protein)

NPY-LI content (Pmol/g)

tiKG)

224 f 49

36 + 6

18.6 + 1.4

503 k 73

23 + 3

388 f 57 +73 NS

6326 i-75 <0.05

0.5 * 0.1 -97 CO.01

448&40 -11 NS

25*2 i-9 NS

B,, (fmol/mg protein)

Splenic contents of NA (nmol/g) and NPY-LI (pmol/g) are shown as well as the binding of [3H]prazosin and [iZsI]NPY in splenic membrane preparations in five control pigs and in five pigs subjected to surgical transection of the splenic nerves two weeks prior to being killed. KDand B,, values are given for both types of binding. Data are expressed as means + S.E.M., and a possible statistical significance between control and denervated preparations was evaluated using Mann-jitney U-test.

staining experiments revealed that the NPY-IR ganglion cells were also TH-positive (Fig. 5c and d), but occasional ceils were TH-IR with an apparent lack of NPY-IR (Fig. 5a-d). Receptor-binding biochemistry [1251]NPYbound to spienic membranes in a specific, saturable and time-dependent manner (Fig. 6A). Equilib~um binding was obtained after 40 min of incubation at 23°C. Scatchard plots of the data had a best fit to a straight line, indicating a single population of binding sites (Fig. 6C). The IC,, values for the displacement of [‘251]NPY binding for NPY and the structurally related peptide, PYY, were 250 and I50 nM, respectively (Fig. 6B). The nonamidated form of NPY was much less potent (IC&, around 1 PM; Fig. 6B). The calculated Kn value for the [1251]NPYbinding in the spleen from control pigs was 503 + 73 pM with a B,,, value of 23 + 3 fmol/mg protein (mean It S.E.M., n = 5; Table 1). The tcso value of NPY binding was not influenced by the presence of a combination of 10e6M prazosin and IOU6M propranolol. No specific [I” I]NPY binding could be demonstrate on membrane preparations from splenic blood cells at similar protein concentrations (not shown). Two weeks after postganghonic denervation (Fig. 6D), the Kn or B,,, values for the [‘251]NPY binding were similar to those obtained in membrane preparations from control spleens (Table 1). Biochemical dete~ination of alpha,-adrenoceptors, using [3H]prazosin, revealed a specific and saturable binding. The Scatchard analysis of the [3H]prazosin binding showed a best fit to a straight line and a Kb value of 250 5 11 pM and a B_ value of 36 f 6 fmol/mg protein (Table 1). Two weeks after denervation, a significant increase (by 75%) in the Bm, value was observed for the [3H]prazosin binding, while the increase in the Kn value was not significant. Cyclic adenosine monophosph~te sorption Addition of 0.3 /IM forskolin increased the CAMP

levels three- to four-fold in both control and denervated splenic tissue. NPY by itself did not change the basal levels of CAMP (not shown). However, the forskolin-induced increase in tissue CAMP content was inhibited by NPY in a concentration-de~ndent manner (Fig. 7). A threshold effect was observed at 10-s M NPY with an inhibition of the forskolin effect by about 75% at 10m6M NPY (Fig. 7). The NPY

effect on CAMP formation was not influenced by pretreatment with prazosin and propranolol. The non-amidated form of NPY did not influence the forskolin-induced CAMP formation. The NPYinduced inhibition of the forskolin-evoked CAMP

Fig. 6. Binding of [1251]NPYto membrane preparations from the spleen of control pigs. (A) Time-course for total (O----O), unspecific (O-0) and specific (a---@) binding. (B) Displacement of specific [‘r’I]NPY binding by increasing concentrations of NPY (@--a), PYY (m-_II) and the non-amidat~ form of NPY (o-r]). Data are expressed as percentage of [iz51jNPY binding in the absence of added peptide. Each point represents mean values from four to six separate experiments performed in duplicates (variability ~5%). Examples of equilibration binding for [‘*‘IJNPY in a control spleen (C) has been compared with preparations obtained two weeks after denervation (D).

J. M. LUNDBERGet al.

perfusion pressure and the blood flow from the spleen in vivo (Figs 8 and 9). The non-amidated form of NPY had no effect up to an estimated local plasma concentration of 10V6M (Fig. 8). Since the perfusionpressure response was reproducible, while the increase in the venous outflow of blood, which reflects capsular contraction, tended to decline upon repeated infusions, data are presented only for perfusionpressure changes. NPY and PYY were about fivefold more potent than NA on a molar basis to increase the ) ~rfusion pressure in control spleens (Fig. 9). NA t i was in turn slightly more active than phenylephrine 9 8 7 6 (Fig. 9A and B). The NA effect, but not the phenyl-log M NW ephrine and NPY responses, was enhanced by pretreatment with DMI (Fig. 9A-C). After denervatlon, Fig. 7. Inhibition of forskolin (0.3 PM) stimulated CAMP formation in slices from the pig spleen by increasing concenthe NPY and phenylephrine effects were similar to trations of NPY. Each value represents mean + S.E.M. of those observed in the control spleens, while the five separate experiments. The effects in control animals dose-response curve to NA was significantly shifted (e-0) are compared with the response obtained two weeks after denervation (O---O). *P < 0.05, **P-c0.01, to the left (about an eightfold reduction in the q0 value), reaching a rather similar position as after compared to the effects of forskolin in the absence of NPY, using Kruskal-Wallis analysis of variance with multiple DMI treatment (Fig. 9A). The maximal response to comparisons. NA, phenylephrine (10-5M) or NPY (5 x t0-‘M) was not changed by DMI pretreatment or after formation was similar in control and denervated chronic denervation. The NA response (10e6M) on spleens (Fig. 7). perfusion pressure was abolished by a combination of locally infused prazosin ( 10e7 M) and propranolol Functional experiments ( IOm5M), while the NPY effect of a similar magniLocal intra-arterial infusion of NPY, PYY, NA tude (5 x lo-“’ M) was uninfluenced by pretreatment and phenylephrine dose-dependently increased the with these adrenoreceptor antagonists (not shown).

I

Blood FIOW m!/mm

I

8. 40 y+----“-

1-P

anion...

-/‘-

-

-

NA

NPY

__/----

-__NPY free acld

PYY

Fig. 8. Increase in perfusion pressure (mmHg) and venous blood flow (ml/min) from the pig spleen induced by local in&a-arterial infusions for 2 min of noradrenaline (NA) estimated local plasma concentrations C10m6M), NPY (5 x 10-sM), PYY (5 x lo-* M) and the free-acid form of NPY (10S6M). (Al Noradrenaline

(B) phenyiepephrine

CC) NPY

wtusion Pressure Increase mm Hg

98765

9

8

7

6

5

9

8

7

6

5

-logM

Fig. 9. Perfusion pressure increased in the blot-~rfus~ pig spleen upon local intra-arterial infusion of increasing amounts of (A) noradrenaline, (B) phenylephrine and (C) NPY. Data are given as means f S.E.M. (n = 5). The estimated local plasma concentrations obtained are given in -log M. The effects in the control animals (0-O) are compared with the responses after pret~atment with DMI (0.5 mg/kg Lv.; m--m) and two weeks after preganglionic denervation (O---O).

~euro~pt~de Y- and alpha-ad~ner~c receptors Infusion of the selective alphas-a~eno~ptor agonists clonidine and azepexole did not influence the basal perfusion pressure in control or denervated spleens at estimated local plasma concentrations from lo-’ to IO-‘M (n = 5) (data not shown). DISCUSSION Neuro~eptide Y binding sites in the pig spleen The present data show that specific, high-a~nity binding sites for [rz51]NPY can be demonstrated both by receptor autoradiography and biochemical techniques in the pig spleen. Judging from the binding characteristics for [12sIJNPY with regard to timecourse, saturability and the KD and B,,,, values, the receptors in the spleen resemble those in the rat brain.5.47~55Furthermore, Scatchard analysis suggested that the [lz5IJNPY binding consisted of a single population of sites, as reported earlier. The occurrence of [**‘I]NPY binding, as seen by autoradiography, was closely in parallel with the distribution of NPY-, TH- and DBH-IR nerve fibres in the spleen. It seems clear from the immunohistochemi~l analysis of the coeliac ganglion, the origin of the postganglionic sympathetic fibres in the spleen and of the splenic nerve-terminal areas that NPY-IR was COlocalized with TH-IR in postganglionic, presumably NA~ontaining, sympathetic nerves. With regard to the more detailed distribution of [1251]NPYbinding sites, labelling was observed in the capsule and in blood vessels, which was in accordance with a constrictor effect of NPY both on the smooth muscle of the capsule (as revealed by increase in splenic venous blood flow) and blood vessels (as revealed by a perfusion-pressure increase). A striking difference was obtained between the red and white pulp both with regard to autoradiographic labelling with [‘**IJNPY and [‘2sI]HEAT as well as the occurrence of NPY-, TH- and DBH-IR nerves, which were all associated mainly with the red pulp. The flzsI]NPY binding sites in the red pulp could be associated with microvessels or possibly blood-cell elements. Both the binding analysis of membranes from splenic bloodcells and receptor autoradiography of splenic blood, however, did not reveal any specific [‘*‘I]NPY binding. This suggests that the main portion of the presently observed NPY binding was associated with the splenic stroma rather than blood-cell elements. Neuropeptide Y binding and cyclic udenosine mono~hosphQfe inhibition after denervation With regard to the pre- and/or postjunctional localization of [‘251]NPY-binding sites, no significant change of the binding characteristics was obtained two weeks after denervation. Furthermore, no clearcut evidence for an increased NPY-receptor sensitivity occurred after denervation with regard to a change in the postjunctional functional response to NPY in vivo or regarding inhibition of the forskolininduced CAMP formation in vitro. Taken together,

669

these results suggest that the majority of [rz51]NPYbinding sites in the pig spleen have a postjunctional rather than prejunctional localization. Furthermore, the inhibition of the CAMP formation after denervation is likely to be a postjunctional event. In in vitro studies from isolated blood vesseis,7*42vas deferens2v.32and the heart,r3 NPY seems to exert a prejunctional, inhibitory action on NA release. This suggests that there are prejunctional NPY receptors on sympathetic nerve terminals, although they must be much fewer than the postjunctional receptors, as indicated from the present denervation experiments. The [‘251]NPY binding to splenic membranes in vitro, the perfusion-pressure increase in NPY in vivo and the inhibition of CAMP formation in vitro were dependent on the amidation of the C-terminal portion of the peptide in accordance both with binding data on rat brain and with the demonstrated, prejunctionai, functional effects on the rat vas deferens.s*s6 Furthermore, the present correlation between functional effects and binding characteristics indicate that the [‘25I]NPY binding represented functionally active recognition sites and not association with, for instance, degradative enzymes. Neither were the abovementioned effects of NPY influenced by adrenoceptor antagonists, suggesting that the NPY response can occur independently of NA mechanisms. Furthermore, the lack of detectable changes in the NPY response in vivo after denervation suggests that, in contrast to NA (see below), the effect of NPY is not influenced by the presence of sympathetic nerves.” This observation favours the view that peptides, in contrast to monoamines, are not removed from the vicinity of the nerve-ending after release by local re-uptake or other inactivation mechanisms located on the nerve endings. The large difference (several orders of magnitude) in IC, values for NPY, regarding the displacement of the {1251]NPYbinding from membrane preparations and vasoconstrictor effects, are similar to the corresponding values for calcitonin gene-related peptide in the rat spleen.50 These differences may reflect a variety of factors including degradation, diffusion barriers and the long incubation period (60min) used for the binding studies compared to the 2 min infusions in vivo. The absence of clear-cut, postjunctional supersensitivity to NPY in vivo (perfusion-pressure increase) or in vitro (CAMP inhibition), as well as the lack of changes in binding characteristics of [12SI]NPY after denervation in the spleen, may depend on NPY release occurring only intermittently, which may not be sufficient to regulate receptor sensitivity. Intracellular second-messengers for alpha,receptor activation seem to involve an increased turnover of phosphatidyl inositol, ultimately leading to a mobilization of intracellular calcium and the activation of protein kinase C.‘* In accordance with the present findings on the pig spleen, the NPY response in cerebral blood-vessels seems to be associ-

670

J.

M. LUNDBERGet

ated with an inhibition of adenylate cyclase.i4 Whether this change is directly coupled to the vasoconstrictor effect remains to be established, especially since the basal CAMP levels were uninfluenced by NPY. However, it should be pointed out that this biochemical measure of the receptor activation did not change after denervation. This indicates that the denervation had neither altered the number of receptors nor the way in which these receptors are coupled to the effector, the adenylate cyclase. Noradrenaline receptors in the pig spleen

The NA-evoked contractions of the splenic capsule and vasculature are classically mediated via alphareceptors (see, for instance, Ref. 8). The present study suggests that alpha-receptors in the pig spleen on smooth muscle cells in the blood vessels, trabeculae and capsule are of the alpha, type. Thus, the functional, in vivo contractile effects of NA were antagonized by the alpha,-selective antagonist prazosin (in addition to beta-adrenoceptor blockade to inhibit the simultaneous vasodilatation). Furthermore, the alpha,-selective agent phenylephrine, but not the preferential alpha*-agonists clonidine or azepexole,“,” induced vasoconstriction and contraction of the capsule. Finally, [rZSI]HEAT binding sites, as demonstrated by autoradiography, were abundant in the pig spleen. It is known from receptor-binding studies that alpha2-adrenoceptors are also present in the spleen of rabbit and man.39 A large proportion of these alpha,-adrenoceptors are probably present on blood-cell elements, such as plateIetss9 In addition, prejunctional alpha,-adrenoceptors are likely to be present on the sympathetic nerve endings, where they modulate the release of NA and NPY.37,38Functional studies suggest that some alpha,-receptors may also be present on smooth muscle cells in the cat spleenZ6 Denervafion-induced, prejunctional supersensitivity for nora~enaIine

After chronic denervation, the perfusion-pressure response to NA in vivo was significantly enhanced in accordance with data from the cat spleen.*,” Furthermore, there was an increase in the B,,, value of the [3H]prazosin binding, suggesting an increase in the number of postjunctional alpha,-receptors. Pretreatment with the monoamine-uptake blocker DMI in intact pigs, however, enhanced the NA response to a degree similar to that in denervated animals. The perfusion-pressure response to phenylephrine (which is most likely not taken up into sympathetic nerves)4 in vivo was uninfluenced by denervation, suggesting that this increase in the binding sites for [3H]prazosin binding was not related to physiologically active receptor sites on splenic smooth muscle. Alternatively, this increase in the alpha, binding sites may have been compensated by the decrease in affinity (increased B,,,,,). Taken together, this suggests that the increased sensitivity to exogenous NA found after denervation is related mainly to prejunctional

al

changes rather than to a regulation of postjunctional alphas-r~eptor characteristics. Thus, the postjunctional response to locally infused NA may increase after denervation due to reduced uptake into nerve endings close to receptor sites. It is known that the number of adrenergic nerve terminals and the distance between the varicosities and the smooth muscle cells are of importance for the ability of NA-uptake inhibitors like DMI to cause a leftward shift of the NA concentration-response curve. Thus, in a richly innervated tissue like the rat vas deferens, the blockade of neuronal uptake by cocaine was found to cause an l&fold supersensitivity to NA.” In the more sparsely innervated smooth muscle, however, such as the aorta or the m~ente~c artery, cocaine produced ony a two- to fourfold increase in NA sensit~vity.2’,s7The ~MI-indu~d su~r~nsitivity to NA in the pig spleen was in the present experiments found to be intermediate between these two categories, and the histological analysis revealed a relatively dense adrenergic nerve supply. The present findings on the pig spleen are in accordance with conclusions based on studies regarding NA supersensitivity after denervation from experiments of the cat spleen2*r7 Thus, the situation in the pig spleen seems to be different from that in another sympathetically innervated tissue, the nictitating membrane of the cat, where a marked postjunctional supersensitivity to amines and other agents is present two weeks after dene~ation.~ An incomplete denervation does not seem to be responsible for this tissue difference in the present experiments, since an almost total depletion of both NA (99%) and NPY (97%) was obtained in the pig spleen, and denervation supersensitivity develops in the nictitating membrane at a much less complete NA depletion.x It remains to be established whether the selective prejunctional type of supersensitivity to NA obtained upon denervation of the cat spleen is due to an inte~ittent activation of the sympathetic nerves in this organ?,“’

CONCLUSION

The present data show an identity between NPYand NA-containing sympathetic nerves, a codistribution of NPY and.a1pha,-adrenore~ptor binding sites as well as similar contractile smooth-muscle effects of NPY and NA in the pig spleen. The NPY and NA effects can occur independently. Furthermore, chronic postganglio~~ dene~ation leads to a selective supersensitivity for the classical transmitter, mainly of a prejunctional origin due to a lack of the re-uptake mechanisms for NA, apparently without inthtencing the ~stjunction~ effects of the coexisting peptide NPY. Acknowledgements-The present study was supported by

grants from the Swedish Medical Research Council (t4X-6554, 04X-715, 07X-I I5), the American Council for

Neuropeptide Y- and alpha-adrenergic Tobacco Research, the Swedish Tobacco Company, Petrus och Augusta Hedlunds Stiftelse, the Laerdai’s Foundation, the Society for Medical Sciences and funds from the Karo-

receptors

671

linska Institute. For expert technical assistance we are grateful to Miss Margareta Stensdotter and Miss Kerstin Lundgren and for secretarial help to Mm Hilka Lindberg.

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