European Journal of Pharmacology, 177 (1990) 75-80

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Elsevier EJP 51179

Pertussis toxin.modifies the effect of central morphine on rat intestinal motility D a n i e l a P a r o l a r o 1, G a b r i e l a P a t r i n i 1, P a o l a M a s s i 1 M a r c o P a r e n t i 2, T i z i a n a R u b i n o Gabriella Giagnoni 1 and Enzo God t

1,

1 Institute of Pharmacology, Faculty of Sciences, University of Milan, Milan, Italy, and e Department of Pharmacology, School of Medicine, University of Milan, Milan, Italy

Received 20 July 1989, revised MS received 28 September 1989, accepted 28 November 1989

To find whether the antipropulsive effect of morphine administered intracerebroventricularly (i.c.v.) depends on a G-protein-mediated mechanism, we studied the effect of i.c.v, pertussis toxin (PTX) pretreatment on morphine-induced inhibition of intestinal motility. The influence of PTX was evaluated on intestinal transit (charcoal meal test) and by monitoring of intestinal myoelectrical activity. The antitransit effect of morphine (10/~g/rat) was antagonized by about 70% 3, 6, 9 and 12 days after PTX pretreatment (1/tg/rat) and it was partially restored after 25 days. I.c.v. morphine abolished the regular appearance of the myoelectric migrating complex (MMC) recorded in the rat jejunum and this effect was completely antagonized by PTX pretreatment. When morphine was injected 25 days after PTX, it significantly reduced MMC frequency, confirming the partial recovery seen in the transit experiments. The pertussis toxin-catalyzed ADP ribosylation of a 39-41 kDa substrate in membranes prepared from hypothalamus and midbrain of rats injected with toxin 6 days before was strongly reduced as compared to the controls. On the contrary, after 25 days, ADP ribosylation was the same in treated and control rats. Thus the antipropulsive effect of central morphine could be initiated at receptor sites which interact with G-protein substrates of pertussis toxin. Pertussis toxin; Morphine; Intestinal transit; Myoelectrical activity; ADP ribosylation

1. Introduction The molecular events subsequent to opiate receptor stimulation involve inhibition of adenylate cyclase (Sharma et al., 1977; Costa et al., 1983; Kurose et al., 1983), interaction with potassium channels or inhibition of voltage-dependent calcium channels (North, 1986). All these events require a signal transduction mechanism operated by G-proteins (Holz et al., 1986; Dolphin, 1987; Hescheler et al., 1987; Yatani et al., 1987).

Correspondence to: D. Parolaro, Institute of Pharmacology, Faculty of Sciences, University of Milan, Via Vanvitelli 32/A, 20129 Milan, Italy.

Recent studies suggest that pertussis toxin (PTX)-sensitive G-proteins, independently of the type of opioid receptors or effector systems, are a necessary transduction step for initiation of the molecular events that lead to an antinociceptive opioid response in the central nervous system (Parenti et al., 1986; Przewlocki et al., 1987). It now appears that the CNS could also be a target site for opiate effects on gut. In fact, at very small doses, centrally administered opioids block gastrointestinal propulsion in rats, mice and guinea pigs (Manara and Bianchetti, 1985). To verify whether the antipropulsive effect of i n t r a c e r e b r o v e n t r i c u l a r l y (i.c.v.) administered morphine (M) can be attributed, like analgesia, to an interaction between receptor and G-proteins,

0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

76 we studied this effect in rats after i.c.v, pretreatment with PTX by investigating intestinal transit (in a charcoal meal test) and monitoring intestinal myoelectrical activity.

2. Materials and methods

2.1. Animals Female Sprague-Dawley rats weighing 180-200 or 280-300 g were used depending on the experiment. They were fed a pellet diet with water ad libitum and the environmental conditions were standardized ( 2 2 + 2°C, 60% humidity and 12 h artificial lighting per day). Before treatment the animals were randomized according to a complete block design and were fasted for 15-18 h.

2.2. Lc.v. microinjection The rats were anesthetized with tribromoethanol (200 m g / k g i.p.) and prepared for i.c.v. microinjections according to Altaffer's procedure (Altaffer et al., 1970). Briefly, anesthetized rats were fixed in a stereotaxic apparatus and the right lateral ventricle was located using a stereotaxic atlas (Paxinos and Watson, 1982). A permanent polyethylene cannula (Ulrich and Co., type PE 10) was implanted so as to penetrate the ventricle 4.5 mm from the top of the skull, to which it was fixed with dental cement (Hottinger Baldwin Messtechnik, type X 60). After the operation the animals were placed in individual cages and allowed to recover for five days. Conscious rats were injected with drug solutions in a constant volume of 5 / t l by inserting a Hamilton microsyringe into the cut cannula tip. The control rats were injected i.c.v, with 5 /~1 of saline. At the end of each experiment 5 #1 Evans blue dye (0.5%) was microinjected through the cannulas, the brains were removed after 5 min and placed in 10% formalin. The brains were frozen 24 h later, cut to a thickness of 80 # m and examined microscopically to verify cannula placements.

2.3. Intestinal transit Intestinal transit was assessed in fasted rats on the basis of the progression of a charcoal meal through the small intestine (Parolaro et al., 1977). The animals were killed by cervical dislocation 20 min after the charcoal meal. The entire gastrointestinal tract was quickly and carefully removed and the whole length of the small intestine was gently stretched out. The distance the meal had traveled from the pylorus to the caecum was measured and expressed as percentage inhibition versus control transit (To) as follows: (Tc - T t ) / T c • 100 where T t is the transit in treated animals. A preliminary test of covariance showed that the total intestinal length was independent of the weight of the animal and did not differ significantly from rat to rat.

2.4. Motility recording Motility was recorded in conscious adult female rats weighing 280-300 g. Anesthetized rats were prepared for chronic treatment with pairs of insulated N i / C r electrodes (100 # m in diameter) implanted along the jejunum (40-45 cm from pylorus). The free ends of the electrodes were carried s.c. to the back of the neck, exteriorized on top of the skull and connected to the socket (Souriau, Boulogne, France). Soldered connections to the socket were encapsulated in dental acrylic cement (Hottinger Baldwin Messtechnik, type X 60). The animals were also fitted for i.c.v, microinjections. Measurements of intestinal myoelectric activity began 7-10 days after the operations, when the animal had completely recovered. Each animal was housed in a wire-bottom cage and was fasted 18 h before recordings were made. Water was allowed ad libitum. Bipolar recordings were made from conscious, unrestrained animals by making appropriate connections between the electrode socket and a direct writing polygraph (Battaglia Rangoni, Bologna, Italy), at a constant time of 0.1 s. Myoelectrical activity was separated into slow waves and spikes using low band-pass and high band-pass filters. Spike electrical activity and slow waves were summed every 20 s by an integrator circuit connected to a potentiometric

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recorder. This integrated record permitted a clear determination of the pattern of intestinal activity.

3. Results

3.1. Intestinal transit 2.5. ADP ribosylation The midbrain and hypothalamus, dissected from rats treated with PTX or their controls, were frozen at - 7 0 ° C. Crude membrane preparations were obtained by homogenization of the various tissues in Tris-HC1 buffer (50 mM, pH 7.4 at 0 ° C, aprotinin 0.3 TIU/ml, phenyl methylsulfonil fluoride 0.2 raM) using a Polytron, followed by centrifugation at 17000 x g for 20 min at 0°C. The pellets were resuspended in 50 #1 Tris-HC1 buffer (75 mM, pH 7.4); ribosylation with PTX (1 /~g/sample) using [32p]NAD (N.E.N. 10-50 Ci/mmol) was done, in a total volume of 60/~1, as described by Ribeiro-Neto et al. (1985). The reaction was stopped with 20% trichloroacetic acid and the product was centrifuged at 11000 x g. The supernatants were aspirated off, washed once with 1.5 ml ethyl ether and collected by centrifugation at the same speed. The ether was discarded and the pellets were resuspended in Laemmli sample buffer (Laemmli, 1970), boiled for 3 rnin and loaded on SDS-PAGE (10% gel strength). The gels were stained with Coomassie blue, dried and the PTX substrates were visualized by autoradiography. G-protein bands were excised from dried gels and counted in a liquid scintillation counter.

Figure 1 shows the effect of PTX pretreatment (1 # g / r a t i.c.v.) at various intervals on the intestinal inhibition elicited by i.c.v. M (10 /~g/rat). PTX alone did not affect intestinal transit but strongly reduced the antipropulsive effect of M by about 70% on days 3, 6, 9 and 12; the antitransit effect of M was partially restored 25 days after PTX although the inhibition was still less than in rats treated with vehicle + M.

3.2. Myoelectrical activity The myoelectrical activity of jejunum before and after i.c.v. M is shown in fig. 2. The typical recording from small intestine after an 18 h fast comprises a basal rhythm (slow waves) and spiking activity organized in migrated myoelectric complexes (MMC) at regular intervals of 13.8 + 1.39 rain. The i.c.v, microinjection of 15/zg/rat of M (a dose causing about 70% intestinal inhibition) completely abolished the regular MMC for about 150 min. The MMC were restored at the end of this period. The basal rhythm was unaffected by M (fig. 2). Figure 3 shows the effect of PTX on the inhibition of MMC by central M. PTX per se did not affect the MMC; in contrast, 6 and 12 days

Vehicle + M B I PT× +I~ PTX+Soline

2.6 D m ~ The following drugs were used: pertussis toxin (List Biological Labs., Campbell, CA, USA) dissolved in 0.1 M sodium phosphate buffer pH 7 with 0.5 M NaCI; morphine hydrochloride (S.I.F.A.C., Milan, Italy) dissolved in saline.

2. 7. Statistical analysis

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One-way analysis of variance (ANOVA) was done by collating data across all groups. This analysis was followed by individual group comparison using Student's t-test (Steel and Torrie, 1960).

3rd

6 th

9 th

ii 12 th

25t h

DAYS AF'I'ER PRIEIRF_.,ATMENT

Fig. 1. Time course of the effect of pertussis toxin (PTX) pretreatment (1 ~ g / r a t i.c.v.) on intestinal inhibition elicited by m o r p h i n e ( 1 0 / L g / r a t i.c.v.); * * P < 0.01 vs. vehicle + mor phine.

78 HV

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MORPHINE (15 p g ] r a t i,c.v.)

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Fig. 2. Effect of i.c.v, morphine (15/tg/rat) on the myoelectrical activity of the small intestine in fasted rats (integrated records).

after PTX, M no longer abolished spiking activity, the MMC being maintained at the same frequency as before M treatment. However, M again modified the MMC pattern at 25 days after PTX. The frequency was markedly reduced (28.2 + 2.4 vs.

il L r M (15pg//rat

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Fig. 4. In vitro pertussis toxin (PTX)-catalyzed ADP ribosylation of membranes from control (C) and PTX-treated rats 6 days (A) or 25 days (B) before. MB = midbrain; HY = hypothalamus.

basal frequency 13.6 + 0.67) and this effect lasted about 150 min. The basal frequency was restored subsequently.

3.3. A D P ribosylation

M

M

M

Fig. 3. Effect of pertussis toxin (PTX) pretreatment (1 /.tg/rat i.c.v.) on morphine-induced inhibition of migrating myoelectrical complexes (MMC). (A) Morphine alone; (B) morphine 6 days after PTX; (C) morphine 12 days after PTX; (D) morphine 25 days after PTX.

To confirm the effect of in vivo PTX treatment on ADP ribosylate G-proteins, we used [32p]NAD to examine the degree of PTX-catalyzed ADP ribosylation of a 39-41 kDa substrate in membranes prepared from midbrain and hypothalamus of control and treated rats killed 6 and 25 days after the drug testing procedure. There was less labeling of the 39-41 kDa band in all the cerebral membranes from rats treated with PTX 6 days before than in the controls (fig. 4A). The ADP ribosylation of PTX-sensitive Gproteins determined by counting the excised bands was decreased by exposure to the toxin in vivo, by 50% in the midbrain (17.2 + 0.3 x 10 3 cpm / m g protein in controls vs. 8.4 _+0.2 x 103 cpm / m g protein in PTX-treated rats) and by 64% in the hypothalamus (18.2 +_ 0.4 × 103 cpm / m g protein

79 in the controls vs. 6.6 _+ 0.2 × 10 3 c p m / m g protein in PTX-treated rats). Residual [32p]ADP ribose incorporation was still detectable in treated animals, suggesting that the toxin does not completely penetrate all cells in the brain areas under these experimental conditions. By 25 days after PTX treatment the level of in vitro ADP ribosylation was the same in the controls and in treated rats (fig. 4B), indicating that the integrity of the G-proteins system had been restored.

4. Discussion The main finding of this study was that pretreatment of rats with PTX greatly reduces the antitransit effect of centrally administered M. This reduction was detectable for at least 25 days. M no longer slowed gastrointestinal transit or prevented regular MMC during this period. As M blocks transit in fasted unanesthetized rats by abolishing the MMC (Weisbrodt et al., 1980; Primi and Bueno, 1987), our findings suggest that these events are closely related to PTX-sensitive G-proteins. The effect of PTX on M-induced inhibition of both intestinal transit and MMC pattern was clearly related. As also observed by Parenti et al. (1986) for analgesia, the effect took time to develop (3 days), indicating a slow diffusion of the toxin molecule into brain tissue and the need for a lag period for its activation and appearance of a biochemical effect on G-proteins. The M responses had partially recovered 25 days after PTX when it caused about 50% reduction of intestinal transit and a decrease in MMC frequency, thus slowing the velocity of intestinal transit. These responses were not the same as those after acute M but indicate that this lag period is apparently necessary for partial restoration of the integrity of the transduction system underlying the intestinal effect. On the other hand, by catalyzing the transfer of an ADP ribose group from N A D to a cysteine residue, PTX induces a covalent modification of G-proteins (Dolphin, 1987). De novo synthesis of G-proteins is thus probably necessary to restore the transduction system linked to activation of the

opioid receptor and 25 days may be the interval required for protein synthesis. The validity of this hypothesis is borne out by the results we obtained with ADP ribosylation studies. Six days after toxin exposure in vivo, there was clearly less ADP ribosylation of a 40 K D a substrate in the midbrain and hypothalamus than in the controls. By 25 days after PTX the degree of ADP ribosylation was the same in treated and control areas. These data seem to suggest that the effect of the toxin had been overcome and the integrity of transmembrane signalling was restored. Experiments on in situ hybridization in the brain areas considered are now in progress to better clarify whether this recovery could be attributed to new G-protein synthesis. Finally, the apparent discrepancy between the biochemical data (showing a complete recovery of G-proteins) and the results of in vivo experiments (where the effects of M were not completely restored) can be explained by the possibility that, in spite of the presence of G-proteins, the efficiency of the transduction system underlying the M-receptor interaction could still be impaired. In conclusion, the findings from this study indicate that G-proteins play an important role in mediating the intestinal effect of centrally administered M. The neuronal G-proteins involved in the effect of opioids on the gut are not known. Various findings (Sharma et al., 1975; Costa et al., 1983; Kurose et al., 1983; Tucker, 1984; Crain et al., 1986; Lux and Schulz, 1986) from experiments with tissue systems and isolated cells indicate that changes in the activity of the cAMP-generating system coupled to G i may be a fundamental step in the modulation of some acute and chronic effects of opiates. However, as PTX also interferes with other G-proteins coupled to different effector systems (e.g. ion channels) (Hescheler et al., 1987), our data cannot exclude the possibility that the toxin may be active on another second messenger or on ionic channel-linked G-proteins (Pfaffinger et al., 1985; Holz et al., 1986; Dolphin, 1987; Hescheler et al., 1987; Yatani et al., 1987). Moreover, since the intestinal effect induced by central opiates is extremely complex because of the involvement of different neurotransmitter systems,

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PTX could be affecting opiate- and/or nonopiate-linked-G-proteins or any G-protein associated with other modulators.

Acknowledgement We are grateful to Judy Baggott for style revision.

References Altaffer, F.B., V. De Balbian, S. Hall, C.J. Long and P. D'Encarnacao, 1970, A simple and inexpensive cannula technique for chemical stimulation of the brain, Physiol. Behav. 5, 119. Costa, T., K. Aktories, G. Schultz and M. Wuster, 1983, Pertussis toxin decreases opiate-receptor binding and adenylate inhibition in a neuroblastomaxglioma hybrid cell line, Life Sci. 33, 219. Crain, S.M., B. Crain and E.R. Peterson, 1986, Cyclic AMP or forskolin rapidly attenuate the depressant effects of opioids on sensory-evoked dorsal-horn responses in mouse spinal cord-ganglion explants, Brain Res. 370, 61. Dolphin, A.C., 1987, Nucleotide binding protein in signal transduction and disease, Trends Neurosci. 10, 53. Hescheler, J., W. Rosenthal, W. Trautwein and G. Schultz, 1987, The GTP-binding protein, Go, regulates neuronal calcium channels, Nature 325, 445. Holz, G.G., S.G. Rane and K. Dunlap, 1986, GTP-binding proteins mediated transmitter inhibition of voltage-dependent calcium channels, Nature 319, 670. Kurose, H., T. Katada, T. Amano and M. Ui, 1983, Specific uncoupling by islet-activating protein, pertussis toxin, of negative signal transduction via a-adrenergic cholinergic and opiate receptors in neuroblastomaxglioma hybrid ceils, J. Biol. Chem. 258, 4870. Laemmli, U.K., 1970, Cleavage of structural proteins during the assembly of the head of bacteriophage T 4, Nature 227, 680. Manara, L. and A. Bianchetti, 1985, The central and peripheral influences of opioids on gastrointestinal propulsion, Ann. Rev. Pharmacol. Toxicol. 25, 249.

North, R.A., 1986, Opioid receptor types and membrane ion channels, Trends Neurosci. 9, 114. Parenti, M., F. Tirone, G. Giagnoni, N. Pecora and D. Parolaro, 1986, Pertussis toxin inhibits the antinociceptive action of morphine in the rat, European J. Pharmacol. 124, 357. Parolaro, D., M. Sala and E. Gori, 1977, Effect of intracerebroventricular administration of morphine upon intestinal motihty in rat and its antagonism with naloxone, European J. Pharmacol. 46, 329. Paxinos, G. and C. Watson, 1982, The Rat Brain in Stereotaxic Coordinates (Academic, Inc., New York). Pfaffinger, P.J., J.M. Martin, D.D. Hunter, N.M. Nathanson and B. Hille, 1985, GTP-binding proteins couple cardiac muscarinic receptors to a K channel, Nature 317, 536. Primi, M.P. and L. Bueno, 1986, Effects of centrally administered naloxone on gastrointestinal myoelectrical activity in morphine-dependent rats, J. Pharmacol. Exp. Ther. 240, 320. Przewlocki, R., T. Costa, J. Lang and A. Herz, 1987, Pertussis toxin abolishes the antinociception mediated by opioid receptors in rat spinal cord, European J. Pharmacol. 144, 91. Ribeiro-Neto, F.A.P., R. Mattera, J.I. Hildebrandt, J. Codina, J.B. Field, L. Birnbaumer and R.D. Sekura, 1985, ADPribosylation of membrane components by pertussis and cholera toxin, Meth. Enzymol. 109, 566. Sharma, S.K., W.A. Klee and M. Nirenberg, 1975, Dual regulation of adenylate cyclase accounts for narcotic dependence and tolerance, Proc. Natl. Acad. Sci. 72, 3092. Sharma, S.K., W.A. Klee and M. Nirenberg, 1977, Opiate-dependent modulation of adenylate cyclase, Proc. Natl. Acad. Sci. 74, 3365. Steel, F.G. and J.H. Torrie, 1960, Principles and Procedures of Statistics (McGraw-Hill, New York). Tucker, J.F., 1984, Effect of pertussis toxin on normorphinedependence and on acute inhibitory effects of normorphine and clonidine in guinea pig isolated ileum, Br. J. Pharmacol. 83, 326. Weisbrodt, N.W., S.E. Sussman, J.J. Stewart and T.F. Burks, 1980, Effect of morphine sulphate on intestinal transit and myoelectric activity of the small intestine of the rat, J. Pharmacol. Exp. Ther. 214, 333. Yatani, A., J. Codina, A.M. Brown and L. Birnbaumer, 1987, Direct activation of mammalian atrial muscarinic potassium channels by GTP regulatory Gk, Science 235, 207.

Pertussis toxin modifies the effect of central morphine on rat intestinal motility.

To find whether the antipropulsive effect of morphine administered intracerebroventricularly (i.c.v.) depends on a G-protein-mediated mechanism, we st...
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