Pertussis toxin inhibits neuropeptide Y-induced feeding in rats

Peptides,Vol. 10, pp. 1283-1286. ©PergamonPress plc, 1989. Printedin the U.S.A.

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Pertussis Toxin Inhibits Neuropeptide Y-Induced Feeding in Rats W. T. C H A N C E , * ? S. S H E R I F F , * T. F O L E Y - N E L S O N , * J. E. F I S C H E R * A N D A. B A L A S U B R A M A N I A M *l

*Division of G.I. Hormones, Department of Surgery, University of Cincinnati Medical Center and #Veterans Affairs Medical Center, Cincinnati, OH 45267 Received 10 July 1989

CHANCE, W. T., S. SHERIFF, T. FOLEY-NELSON, J. E. FISCHER AND A. BALASUBRAMANIAM. Pertussistoxin inhibits neuropeptide Y-inducedfeeding in rats. PEPTIDES 10(6) 1283-1286, 1989.--Neuropeptide Y (NPY) is the most powerful peptide drug stimulating feeding in rats. Rats with paraventricular hypothalamic (PVH) cannulae were used to investigate the mechanisms involved in NPY-induced feeding. Consistent with previous reports, injection of 2 p.g of NPY into the PVH significantly increased the cumulative food intake over 1-, 2- and 4-hr periods. Ad lib feeding decreased significantly two days after pertussis toxin (PT) administration, but recovered to nearly normal levels on the fourth day. PT had no immediate effect on NPY-induced feeding; however, four days after PT was injected NPY (2 ~g) did not increase the food intake compared to control. In vitro investigations showed that isoproterenol-stimulated adenylate cyclase activity in the hypothalamus of control rats was inhibited by NPY. In PT-treated rats, however, no inhibition of cAMP production was observed. These results suggest that cAMP may mediate NPY-induced feeding and that a PT-sensitive G protein may be involved in this signal transduction. Cyclic AMP

Feeding

Neuropeptide Y

Pertussis toxin

SINCE the initial isolation of neuropeptide Y (NPY) (23), the effects of this hormone upon the central nervous system have spawned numerous investigations. These studies have demonstrated that the administration of neuropeptide Y (NPY) into third (6) or lateral ventricle (16) or directly into the hypothalamus (17) induced a robust feeding response in rats. This peptide is now regarded as the most powerful stimulator of feeding yet observed in experimental studies (6, 16, 17). Although NPY is colocalized with norepinephrine (NE) in the brain (8), feeding induced by NPY is not antagonized by phentolamine (17,22), an alphaadrenergic antagonist that readily blocks NE-induced feeding (15). This observation suggests that NPY may stimulate feeding directly without involving NE receptors and by a mechanism independent of the alpha-adrenoreceptor. The specific alphw2-adrenergic antagonist, yohimbine, has been reported recently to reduce NPYinduced feeding, suggesting that this effect may be mediated partly by the alphaz-adrenoreceptor (5). Recent research concerning the effects of NPY upon cardiovascular tissue suggests an involvement of cAMP and a pertussis toxin (PT)-sensitive G protein in the actions of NPY on the heart (14). Since NPY was also found to inhibit NE-stimulated adenylate cyclase in cerebral cortex (25), we investigated the effects of

NPY on feeding and hypothalamic adenylate cyclase. In addition we determined whether pretreatment with PT would alter NPYinduced feeding or hypothalamic adenylate cyclase activity. METHOD The effects of NPY and PT upon feeding were investigated using 11 adult (350--450 G) male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) as subjects. The rats were housed individually in a temperature- and humidity-controlled vivarium under a 12-hr light/dark cycle, and were maintained ad lib on Purina rat chow and water throughout the experiment. To permit the intrahypothalamic injection of NPY and PT, 24-ga cannulae were surgically implanted into the paraventricular hypothalamus of anesthetized rats at the following stereotaxic coordinates (18): anterior 6.5 mm, lateral 1.0 mm, ventral - 2 . 0 mm. All rats were allowed at least 2 weeks to recover from these operations prior to any experimental manipulations. The experimental period was initiated by assessing the feeding response 1, 2, 3 and 5 hours after the injection of 1 I~1 of artificial cerebral spinal fluid (CSF) (4). The injection procedure was identical to that reported previously (4) employing a 31-ga

1Requests for reprints should be addressed to A. Balasubramaniam, Department of Surgery, University of Cincinnati Medical Center, Cincinnati, OH 45267-0558.

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hypodermic needle that terminated flush with the implanted cannulae. Two days later 1 txl of CSF was again injected and was followed 60 min later by the injection of 2 ixg of synthetic NPY (2) contained in 1 ixl of CSF. This dose of NPY was chosen since preliminary studies indicated it to elicit a robust feeding effect when applied to this brain area. Food intake was assessed for the one-hour period following CSF and for 1, 2 and 4 hours following the injection of NPY. Six days later this procedure was repeated but 2 txg of PT was injected instead of NPY. This dose of PT was selected so as to be comparable to that of NPY. Four days after the PT treatment the orexigenic effect of NPY was again assessed. One day after this final NPY treatment all rats were sacrificed to reveal cannulae placement and to provide hypothalamic tissue for adenylate cyclase investigations. Two brains were fixed in 10% formalin solution and the placement of cannulae was estimated using a dissecting microscope. In the remaining NPY-PT-treated ( n = 4 ) and CSF-treated ( n = 5 ) rats the hypothalamus was dissected free for the determination of adenylate cyclase activity. Membranes were prepared from the hypothalamus according to the published procedures (7). Protein was estimated using BSA standard (19). Adenylate cyclase activity was measured by the modified method of Rosselen and Freychet (1973) (20). The incubation solution contained in a 250 p,1 final volume: Tris-HC1 30 mM pH 7.4, NaC1 150 mM, MgC12 8.25 mM, EGTA 0.75 mM, theophylline 1.5 mM, aprotinin 20 Ixg/ml, bacitracin 100 p,g/ml, BSA 1 mg/ml, 1 mM ATP, 20 mM creatine phosphate, 1 mg/ml phosphocreatine kinase, isoproterenol 10 p,M, GTP 10 IxM, and various concentrations of NPY (0-10 p,M). Reaction was initiated by the addition of 50 p,g (50 p,1) of membrane protein. After incubating at 35°C for 10 min in a shaking water bath the reaction was terminated by the addition of 100 txM EDTA and boiling for 3 minutes, cAMP was extracted and quantitated by radioimmunoassay using a kit obtained from DuPont (cat. No. NEK 033). The recovery of cAMP during the extraction procedure was approximately 85 to 90% as determined by 3H-cAMP marker. RESULTS

Injection of 2 ~g of NPY significantly (p<0.01; by repeated measures ANOVA) stimulated cumulative food intake as compared to the CSF-treated rats over 1-, 2- and 4-hr periods (Fig. 1). This dose of NPY has been previously shown to produce the maximum response (6). Administration of 2 p~g of PT had no immediate effect on the ad lib (Fig. 2) or NPY-induced feeding. Two days after injection of PT, however, food intake was significantly (p<0.01) reduced by about 40%. Ad lib feeding was no longer reduced significantly four days after PT treatment. When NPY was injected on this date, however, food intake at each measurement period was reduced significantly (p<0.01) as compared to its previously obtained values (Fig. 1). This response to NPY was not significantly different from that observed following the injection of CSF alone. Body weight also decreased (p<0.01) following the injection of PT from a mean preinjection weight of 506--- 16 to 467 --- 18 g on the day of sacrifice. Histological examination revealed the m e a n ± SEM cannula placement to be A = 7 . 2 - 0 . 2 , L = l . 3 ± 0 . 1 and V = 0 . 9 ± 0 . 2 mm above internal line, indicating that the cannulae were centered somewhat lateral and ventral of the PVH nuclei. In vitro experiments revealed that NPY inhibited isoproterenolstimulated cAMP production in a dose-dependent manner in the hypothalamic membranes prepared from the control rats; however, no inhibition of cAMP production was observed in the hypothalamus of PT-treated rats (Fig. 3). DISCUSSION

The results presented in this paper agree with previous obser-

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NPY BB PERTUSSB TOXIN (PT)

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FIG. 1. Cumulative food intake by rats over 1, 2 and 4 hr after paraventricular hypothalamic administration of 1 p.l artificial cerebral spinal fluid, 2 I~g NPY, 2 p,g pertussis toxin or 2 I~g NPY four days after the injection of pertussis toxin. *=p<0.01 compared to control, a= p<0.01 compared to 2 p,g of NPY before PT treatment (ANOVA). n = 5 for CSF and n = 6 NPY and PT experiments.

vations (6, 16, 17) that central administration of NPY causes a robust feeding response in rats. This effect of NPY was greater and longer lasting than an equivalent dose of NE (10). Ad hqa feeding, which decreased significantly after PT injection, returned to nearly normal levels four days later. This observation suggests that even if animals had undergone nonspecific sickness associated with PT injection, they have recovered from it on the fourth day. However, animals showed no obvious signs of discomfort or disease during this experimental period. The stimulatory effect of NPY on the food intake was completely blocked on the fourth day after PT treatment. PT, an exotoxin produced by Bordetella pertussis, is known to block the inhibitory messages from the hormone receptor to

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FIG. 2. Food intake following the injection of 2 p,g of PT into the rat hypothalamus (n = 6). * =p<0.01 compared to the ad lib feeding on day one.

PERTUSSIS INHIBITS NPY-INDUCED FEEDING / 100 1

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FIG. 3. Inhibition of isoproterenol-stimulated cAMP production by rat hypothalamic membranes prepared from CSF- and pertussis toxin-treated rats. cAMP was estimated as described in the Method section.

adenylate cyclase by ADP-ribosylation of the inhibitory guanine nucleotide binding protein (Gi) (9). PT has been therefore widely used to explore the role of adenylate cyclase in a number of biological processes. Peripheral administration of PT has been associated with a number of biological effects including lymphocytosis, hypoglycemia, altered cardiovascular hemodynamics, and disturbances in renin secretion and fat metabolism (1). Similarly, central administration of PT has been shown to block the autore-

ceptor-mediated dopamine synthesis (3), 5HT]A and GABA B receptor-mediated inhibition of serotonergic neurons (13), and locus coeruleus activity (21). These central and peripheral effects associated with PT treatment have been therefore described as due, at least in part, to the blocking of the cAMP-dependent pathway (1, 3, 13, 21). However, recent research has revealed that PT also blocks phosphotidylinositol (PI) hydrolysis by acting on a GTPbinding protein required for stimulation of PI pathway (24). These observations suggest that the PT inhibition of NPYinduced feeding could have been caused by the interruption of either cAMP or PI pathway. Furthermore, the presence of two types of NPY receptors in brain, one linked to PI hydrolysis (Y]) and the other coupled to adenylate cyclase inhibition (Y:), has been proposed based on the experiments with NPY, PYY and their fragments (11). However, NPY is known to stimulate the hydrolysis of PI in frontal cortex, hippocampus and striatum of rat brain, but not in the hypothalamus (12). It appears, therefore, that the abolishment of NPY-induced feeding by PT might have been caused by the interruption of cAMP pathway. Consistent with this suggestion NPY inhibited the cAMP production in the hypothalamus of control rats and had no effect on the cAMP production in the hypothalamus obtained from PT-treated rats. It has also been shown that NPY binding to rat brain is sensitive to GTP, suggesting that the NPY receptors are coupled to adenylate cyclase system (25). Based on these observations, it may be suggested that cAMP mediates NPY-induced feeding and that a PT-sensitive G protein may be involved in this signal transduction. ACKNOWLEDGEMENTS

This work was supported in part by NIH Grants GM 38601 (A.B.) and CA48057 (W.T.C.), and a VA Grant (W.T.C.). We thank L. Carovillano and Arlene Orth for secretarial services. This paper was presented at the 1989 meeting of the Society for Neuroscience.

REFERENCES

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