G-protein coupling of vagal CCK binding sites and comparisons of transport rates

0031-9384/93 $6.00 + .00 Copyright© 1993 PergamonPressLtd.

Physiology& Behavior.Vol. 53, pp. 1061-1065, 1993 Printed in the USA.

G-Protein Coupling of Vagal CCK Binding Sites and Comparisons of Transport Rates J U L I A N G. M E R C E R , 1 C. B R U C E L A W R E N C E A N D P E T E R J. M O R G A N

Rowett Research Institute, Bucksburn, Aberdeen A B 2 9SB, U K Received 9 S e p t e m b e r 1992 MERCER, J. G., C. B. LAWRENCE AND P. J. MORGAN. G-protein coupling ofvagal CCK binding sites and comparisons of transport rates. PHYSIOL BEHAV 53(6) 1061-1065, 1993.--The ability of nanomolar concentrations of guanine, but not adenine, nucleotides to inhibit specific 12~l-Bolton-HunterCCK binding to ligated rat vagus nerve demonstrated that vagal CCK binding sites were linked to G-proteins during axonal transport. The GTP analogue, GTP[S], reduced specific binding to both anterogradely and retrogradely transported binding sites by more than 90% at 1 t~M. Transport of these putative receptor-Gprotein complexes was examined under conditions of food deprivation or physiological hyperphagia induced by either lactation or genetic obesity. None of the physiological or imposed manipulations of food intake had any effect on the axonal transport of CCK binding sites. Transection of the cervical vagus resulted in an accumulation of binding sites at the lesion site that was indistinguishable from that seen following ligation for the same period. Vagus

Axonal transport

CCK receptor

G-protein

EXOGENOUS cholecystokinin (CCK) reduces food intake in various mammalian species, and evidence is accumulating that this effect may mimic a physiological role of meal-released hormone (13). The satiety signals induced by injection of CCK are transmitted via the vagus nerve (14), and the demonstration of transport of CCK binding sites in the cervical and subdiaphragmatic vagus of rats (6,9,10,16) identifies sites at which CCK may directly modulate vagal activity. These binding sites thus represent a candidate receptor population for the mediation of the satiety effects of exogenous and endogenous CCK (9,10). However, as CCK binding site transport cannot as yet be traced below the major subdiaphragmatic branches of the vagus, it is not known how these putative receptors regulate cellular functioning at neuronal termini in the abdominal cavity. A logical first step in resolving this sequence of events is to establish whether membrane-bound receptors are coupled to guanine nucleotide-dependent regulatory proteins, or G-proteins. Functional coupling of vagal CCK binding sites and G-proteins during axonal transport was assessed by characterising guanine nucleotide-induced inhibition of specific binding. In addition to investigating mechanisms of signal transduction, we have examined the possibility that rates of transport of CCK binding sites might be modulated under different food intake regimes, where plasma CCK titre would also be altered. The effects of hyperphagia in the obese Zucker rat, lactational hyperphagia, and 48-h food deprivation have been investigated. Cholecystokinin binding sites are only present in low densities in intact vagus nerve, and the study of binding site pharmacology and transport in the rat vagus nerve requires the establishment

Requests for reprints should be addressed to Julian G. Mercer.

1061

Food intake

of a false axonal terminus by nerve ligation, thereby interrupting axonal flow. We now report that quantitatively similar binding site accumulation can be obtained through the more rapid technique of nerve transection. Some of the data in this report were presented in a preliminary form to the Society for Endocrinology (8). METHOD Unless otherwise specified, all investigations employed Hooded Lister (HL) rats fed a pelleted diet (Labsure) ad lib. Animals were prepared for axonal transport studies by unilateral ligation or transection of the cervical vagus under brief halothane anaesthesia as described previously (6). After 24 h, animals were euthanised, and excised nerve segments were mounted in TissueTek (Miles Inc.) and stored at - 7 0 ° C prior to cryostat sectioning. Longitudinal sections (10 #m) from individual nerves were collected onto a set of five slides, with adjacent sections on consecutively numbered slides. Slides were incubated with 100 p M 125I-Bolton-Hunter CCK-8 (2000 Ci/mmol; Amersham) and processed as described previously (6). Dried slides were placed in an X-ray cassette and apposed to Kodak AR OMAT X-ray film for 2 weeks at 4°C. Optical density measurements of images produced on X-ray film were obtained from a Torch Quad X work station. Inclusion of brain paste and polymer standards (Amersham) with autoradiographs permitted conversion of data to fmol/mg protein (11). Specific binding was determined as the difference between total binding (T) to sections incubated with 100 p M ~25I-Bolton-Hunter CCK-8, and nonspecific binding

1062

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corroboration ofgenotype; the daily lood intake ol obese animals was approximately double that of leans. When right cervical vagi were ligated, profiles of specific binding to nerves from obese individuals were indistinguishable from their lean counterparts (Fig. 2). Analysis of binding site density revealed no significant interaction between genotype and position relative to the ligature, and no overall effect ofgenotype either proximal [ANOVA, F( I, 12) 0.01, p > 0.05] or distal to the ligature [ANOVA, 1:( 1, 12) - 4.08, p > 0.05]. Similarly, no effect ofgenotypc on binding site density was observed between two groups of three male Zucker rats (results not shown).

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FIG. 1. Inhibition of specific lZSI-Bolton-Hunter CCK-8 binding to 0. l 7mm proximal segments of ligated fight cervical vagus by guanine or adenine nucleotides. Data are mean _+SE from three nerves of percentage specific binding relative to controls. For each nerve, specific binding in the presence of a selected concentration of nucleotide, relative to controls, was determined for two or three sections, and the results averaged.

(NSB) to adjacent sections on slides incubated in the additional presence of 10 -6 M sCCK-8 (Bachem). The inhibition of specific binding by guanine or adenine nucleotides was assessed as follows: two slides from each set of five carrying the sections of a single nerve were incubated as above to measure T and NSB, and the remaining three slides were incubated with different concentrations of a selected nucleotide. Specific binding in the presence of nucleotides was expressed relative to control specific binding to adjacent sections, and data from sections of a single nerve were averaged. The ICso values were calculated using the log-logit transformation. For comparative studies of axonal transport, specific binding was averaged for two sections from each nerve. Data were analysed for significance by ANOVA. RESULTS

Effect of Guanine or Adenine Nucleotides on Specific t251-Bolton-Hunter CCK-8 Binding to Sections of Ligated Rat Vagus Four guanine and two adenine nucleotides were tested for the ability to inhibit specific binding to ligated right cervical vagus. The nucleotides could be divided into two groups on the basis of their ability to inhibit specific binding proximal to the ligature (Fig. 1); GTP, its nonhydrolysable analogue, GTP[S], and G D P had IC50 values of 10 7 M o r less (GTP[S], 5 × 10 -8 M; GTP, 8 × 10 s M; GDP, 10 7 M), while GMP, ATP, and ADP had IC50 values of 7 × 10 -5 M or greater (GMP, 7 × 10 -5 M; ATP, 8 × 10 -4 M; ADP, 6 × 10 -4 M). Similar data were obtained from the much lower densities of C C K binding sites that accumulated distal to the site of ligation, and were thus undergoing retrograde axonal transport; for example, GTP[S] had an IC5o of 6 × 10 s M a t distal binding sites. Limited studies indicated that CCK binding sites accumulating at ligatures on the subdiaphragmatic vagus were also guanine nucleotide sensitive.

Axonal Transport of CCK Binding Sites in Lean (Fa/?) and Obese (/alia) Zucker Rats Seven female Zucker rats of each genotype were ligated at 10.5 weeks of age. Body weights and food intake provided clear

Prior to weaning, the food intake of lactating rats exceeded 60 g per day. Immediately following weaning, 10 lactating animals had ligatures placed on either the right (n - 7) or left (n = 3) cervical vagus. A second group was ligated 2 weeks later (six right cervical vagus, three left cervical vagus). The latter group remained hyperphagic during the 24-h period immediately following the removal of litters (mean intake/female during 24 h _+ SE: 42.2 _+ 1.5 g), but stabilised at an intake of approximately 20 g per day thereafter (24-48 h after removal of litter: 20.5 _+ 1.2 g). The duration of the interval between weaning and ligation had no significant effect on binding site density in the right cervical vagus either proximal [ANOVA, F(I, i 1) = 2.67, p > 0.05] or distal to the ligature [ANOVA, F( 1, 11) - 0.18, p > 0.05], or in the left cervical vagus [proximal, F(I, 4) - 0.01, p > 0.05; distal, F(1, 4) = 0.60, p > 0.05] (Table 1). There were no significant interactions between animal group and position relative to the ligature. Using the same methodology, animals ligated 1 week after weaning also demonstrated similar binding profiles to hyperphagic individuals ligated at weaning.

Axonal Transport oJCCK Binding Sites in 48-h Food-Deprived and Ad Lib-I:ed HL Rats The body weight of male HL rats fell by an average of 13% from a starting weight of 250 g during 48-h food deprivation. Ligation of the right cervical vagus gave rise to similar profiles

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FIG. 2. Effect ofZucker genotype and attendant hyperphagia on specific ~25I-Bolton-Hunter CCK binding to longitudinal sections of fight cervical vagus ligated for 24 h. Densities were measured on 0.33-mm segments of nerve, six proximal (negative values on the abscissa) and four distal (positive values) to the site of ligation (0 on abscissa). Data are mean specific binding (+_SE) to nerves from seven obese (open circles) and seven lean animals (filled circles).

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Segment Number 3

Segment Number 4

Values are m e a n _+ SEM. N u m b e r o f subjects in brackets. * 0 . 3 3 - m m s e g m e n t s o f nerve; s e g m e n t 1 is i m m e d i a t e l y a d j a c e n t to the ligature.

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Specific Binding (fmol/mg protein)

Distal to Ligature*

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Proximal to Ligature*

EFFECT OF 48-h FOOD DEPRIVATION ON SPECIFIC J:~I-BOLTON-HUNTER C C K BINDING TO LONGITUDINAL SECTIONS OF RIGHT CERVICAL VAGUS LIGATED FOR 24 h

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Segment Number

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Distal to Ligature*

Proximal to Ligature*

Values are m e a n +__SEM. N u m b e r of subjects in brackets. * 0 . 3 3 - m m s e g m e n t s o f nerve; s e g m e n t 1 is i m m e d i a t e l y a d j a c e n t to the ligature.

R i g h t cervical vagus Ligated at w e a n i n g [7] Ligated 2 weeks after w e a n i n g [6] Left cervical vagus Ligated at w e a n i n g [3] Ligated 2 weeks after w e a n i n g [3]

Specific Binding (fmol/mg protein)

TABLE EFFECT OF LACTATIONAL HYPERPHAGIA ON SPECIFIC t251-BOLTON-HUNTER CCK BINDING TO LONGITUDINAL SECTIONS OF RIGHT OR LEFT CERVICAL VAGUS LIGATED FOR 24 h

0.15 + 0.04 0.15 _+ 0.02

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1064

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FIG. 3. Effect of different lesions of 24-h duration on specific L251-Bolton-HunterCCK binding to longitudinal sections of right cervical vagus. Densities were measured on 0.25-mm segments of nerve, six proximal (negative values on the abscissa) and four distal (positive values) to the site of ligation (0 on abscissa). Data are mean specific binding (_+SE)to nerves from four transected (open circles) and three ligated animals (filled circles).

of binding sites in the deprived and ad lib groups, each of which contained five animals. There were no significant interactions between animal group and position relative to the ligature, and no overall effect of 48-h deprivation on binding site density either proximal [ANOVA, F(1, 8) = 0.33, p > 0.05] or distal to the ligature [ANOVA, F(I, 8) = 0.01, p > 0.05] (Table 2).

Axonal Transport of CCK Binding Sites in Transected Cervical Vagus Nerve Unilateral vagal ligations or transections of the right cervical vagus were performed and animals were euthanised after 24 h. Both surgical interventions resulted in a characteristic profile of binding site density proximal and distal to the site of the lesion (Fig. 3). Analysis revealed no significant interaction between lesion type and position relative to the ligature, and no overall effect of lesion type on binding site density either proximal [ANOVA, F( 1, 5) = 0.24, p > 0.05] or distal to the ligature [ANOVA, F(I, 5) = 0.71, p > 0.05]. DISCUSSION

The specificity with which guanine nucleotides inhibited specific binding to vagal CCK binding sites (Fig. 1) demonstrated that binding sites were functionally coupled to guanine nucleotide-dependent regulatory proteins, or G-proteins, during axonal transport towards the abdominal cavity. G-protein coupling is consistent with vagal CCK binding sites functioning as physiological receptors at their transport termini. Vagal CCK binding sites are located on afferent fibres (9), and consequently, the binding site-G-protein complex must be assembled within the cell bodies of the nodose ganglion prior to transport. Although G-protein regulated during axonal transport, vagal CCK binding

sites are assumed to become functional only once inserted into the terminal plasma membrane. However, the effector systems with which the binding site-signal transduction complex is associated within the terminal membrane are unknown, and, as a consequence, a functional role for these putative receptors has yet to be established. Although CCK binding sites accumulating distal to the ligature represent only a small proportion of the population going towards the periphery, binding here is also guanine nucleotide sensitive. Studies of other neurotransmitter receptors in peripheral nerves have also indicated the transport of receptor-G-protein complexes, although a loss of guanine nucleotide sensitivity has been reported in retrogradely transported receptors (12). The degree to which GTP[S] inhibited specific binding (by 90% at micromolar concentrations) suggested that the majority, if not all, the CCK binding sites observed in sections of ligated vagus nerve were coupled to G-proteins. As the population of CCK binding sites undergoing anterograde axonal transport in the rat vagus comprises similar numbers of type A and type B receptors (2,7), both these receptor subtypes must be G-protein coupled in the vagus nerve. Type B CCK binding sites in rat vagus may thus exhibit differences in their transducer-effector systems from the same receptor subtype in the central nervous system; micromolar concentrations of the GTP analogue, GppNp, have only a limited effect on specific CCK binding to rodent cortical membranes (3,15). Transport of CCK binding sites in the rat vagus is not significantly altered by either acute food deprivation or the extended physiological hyperphagia induced by either lactation or genetic obesity (Fig. 2, Tables 1 and 2). It should be emphasised, however, that axonal transport measured in the present study is the sum of transport over a 24-h ligation period. Ligation periods

G-PROTEINS AND VAGAL CCK RECEPTORS

1065

of several hours are the minimum required for quantitative autoradiographic assessment of transport, and the effects of the consumption of individual meals would thus be difficult to quantify. Consequently, we have concentrated on more chronic paradigms where 24-h food intake was substantially modulated. The rationale behind an examination of binding site transport under different food intake regimes was that some resetting of peripheral satiety signals might be expected in different appetite states, where meal size and frequency are varied. For example, a satiety agent whose titres reflect the size and frequency of meals would act against the maintenance of hyperphagia in the absence of any modulation of the satiety signal. An adaptation of this type is indicated by observations that hyperphagic lactating female rats, which have elevated plasma CCK titres (5), are less sensitive to the satiety effects of exogenous CCK than are the same rats 2 weeks after the weaning of their litters (1). Genetically obese, hyperphagic, rodents also exhibit reduced sensitivity to the satiety effect ofCCK (1). Besides demonstrating the consistency of axonal transport rates at different levels of food intake, the data show no effect of Zucker genotype on axonal transport of CCK binding sites. Additional studies have shown that the pharmacology of vagal CCK binding sites in the Zucker rat is similar to that recorded in other strains of rat; both MK-329 and L-365,260 inhibited specific binding, indicating that a mixed population of CCK-A and CCK-B binding

sites undergo axonal transport towards the periphery (Mercer and Lawrence, unpublished). Agonist-receptor interactions frequently induce changes in receptor activity through desensitisation or down-regulation (4). The latter, a reduction in receptor density at the cell surface, may involve reduced de novo receptor synthesis. The absence of any observable effect of food intake on the accumulation of CCK binding sites at vagal ligatures indicates that any regulation of vagal CCK binding sites by meal-released CCK does not involve changes in de novo synthesis. Should signals induced by the interaction of endogenous CCK and vagal binding sites contribute to physiological satiety, some modulation of the satiety signal could be effected through signal transduction and turnover events at the terminal membrane. However, as already discussed, the locations of these transport termini within the abdominal cavity have yet to be determined. In addition to nerve transection being a more rapid method of studying axonal transport, which involves less physical manipulation of the nerve trunk, the demonstration that CCK binding sites undergo axonal transport at similar rates in transected and ligated nerves indicates that the type of lesion does not affect rates of binding site synthesis in the short term (Fig. 3). It may be possible to examine the cellular functions of CCK binding sites in newly established, and anatomically localised, terminal membranes by allowing severed vagal trunks to commence regeneration.

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9. Moran, T. H.; Norgren, R.; Crosby, R. J.; McHugh, P. R. Central and peripheral vagaltransport of cholecystokininbinding sitesoccurs in afferent fibers. Brain Res. 526:95-102; 1990. 10. Moran, T. H.; Smith, G. P.; Hostetler, A. M.; McHugh, P. R. Transport of cholecystokinin (CCK) binding sites in subdiaphragmatic vagal branches. Brain Res. 415:149-152; 1987. 11. Nazarali, A. J.; Gutkind, J. S.; Saavedra, J. M. Calibration of ~251polymer standards with ~251-brainpaste standards for use in quantitative receptor autoradiography.J. Neurosci. Methods 30:247-253; 1989. 12. Palacios, J. M.; Pazos, A. Axonal transport of neurotransmitter receptors studied by quantitative autoradiography. In: Boast, C. A.; Snowhill, E. W.; Altar, C. A., eds. Quantitative receptor autoradiography. New York: Alan R. Liss; 1986:173-197. 13. Silver,A. J.; Morley,J. E. Role of CCK in regulation of food intake. Prog. Neurobiol. 36:23-34; 1991. 14. Smith, G. P.; Jerome, C.; Norgren, R. Afferentaxons in abdominal vagusmediate satietyaffect of cholecystokininin rats. Am. J. Physiol. 249:R638-R641; 1986. 15. Wennogle, L.; Wysowskyj, H.; Steel, D. J.; Petrack, B. Regulation of central cholecystokinin recognition sites by guanyl nucleotides. J. Neurochem. 50:954-959; 1988. 16. Zarbin, M. A.; Wamsley, J. K.; Innis, R. B.; Kuhar, M. J. Cholecystokininreceptors: Presenceand axonal flowin the rat vagusnerve. Life Sci. 29:697-705; 1981.