Peptides,Vol. 12, pp. 549-553. ©PergamonPress plc, 1991. Printedin the U.S.A.
0196-9781/91 $3.00 + .00
Treatment With Pertussis Toxin Does Not Prevent Central Effects of Eel Calcitonin F. G U I D O B O N O , P. B E T I ' I C A , I. V I L L A , F. P A G A N I , C. N E T T I , V. S I B I L I A A N D A. P E C I L E
Department of Pharmacology, Chemotherapy and Medical Toxicology University of Milan, Via Vanvitelli 32, 20129 Milan, Italy Received 20 June 1990
GUIDOBONO, F., P. BETTICA, I. VILLA, F. PAGANI, C. NETTI, V. SIBILIA AND A. PECILE. Treatment with pertussis toxin does not prevent central effects of eel calcitonin. PEPTIDES 12(3) 549-553, 1991.--To determine whether or not the CNS inhibitory activity of eel calcitonin (eCT) on adenylyl cyclase is the endocellular mechanism underlying the antinociceptive effect of the peptide, as shown for morphine analgesia, we administered Bordetella pertussis toxin (PTX) by intracerebroventricular (ICV) injection (0.5 ~g/rat) to block the receptor-mediated inhibition of adenylyl cyclase. In PTX-treated rats there was no change in eCT (2.5 ~g/rat, ICV)-induced antinociceptive activity (hot-plate test) nor in eCT (100 ng/rat, ICV) inhibition of gastric acid secretion (Shay test) whereas morphine (5 l~g/rat, ICV) analgesia was significantly reduced. In vitro studies showed no reduction of eCT binding in the CNS of rats treated with PTX in vivo. Moreover, PTX treatment did not change the inhibitory effect of eCT on adenylyl cyclase in isolated membranes from rat striatum in contrast with opiates (DAME and morphine) whose effects were lost. As PTX is known to inactivate the guanidine binding inhibitory protein Gi, these data suggest that a G protein, distinct from the Gi protein involved in the coupling of opiate receptors into a functional response, could be responsible for regulating the intracellular pathways resulting in eCT-induced antinociceptive effect and inhibition of gastric acid secretion. Eel calcitonin
Morphine
Antinociceptive activity
Gastric acid secretion
THE established primary action of calcitonin (CT) is to inhibit osteoclastic bone resorption through a direct action on osteoclasts (11). This is the reason why CT is a useful drug in certain bone diseases. However, another reason for its therapeutic use is the antinociceptive effect exerted by the peptide, shown both in animals and in humans under a variety of conditions (6, 13, 20). CT antinociceptive activity appears to involve peripheral and central actions. An interaction of the peptide with binding sites identified in the CNS appears to be selective for areas known to participate in pain perception control systems (9). Different classes of receptors for CT have been identified both in peripheral tissues and in the CNS (2,10). The transductional mechanism activated by CT interaction with its receptors seems to be linked to an increase of adenylyl cyclase activity in peripheral tissues (bone, kidney and some human cancer cells) and to an inhibition of adenylyl cyclase activity in the brain (17, 21, 23, 25). It is still unclear what other biochemical events are required for CT receptor binding to be transduced into an activity on bone and into an analgesic response. Since the control of adenylyl cyclase is achieved by transfer of the information from receptor-agonist complex to catalytic subunits of the adenylyl cyclase by means of guanine nucleotide binding proteins (G proteins), which could be inhibitory (Gi) or stimulatory (Gs) (16), we have examined the possibility that G i proteins may represent the link between CT receptors and CT-induced antinociceptive activity. In view of the evidence that treatment with pertussis toxin (PTX) by ribosylation of a Gi protein blocks the receptor-mediated inhibition of adenylyl cyclase (1), we studied whether or not
Adenylyl cyclase
Pertussis toxin
PTX pretreatment into rat brain ventricles (ICV) had any effect on eel CT (eCT)-induced antinociceptive activity as reported for morphine by Parenti et al. (18). PTX pretreatment was also tested on another central activity induced by eCT, the inhibition of gastric acid secretion. As it has been shown that the inactivation of G i protein by PTX, and the consequent uncoupling of Gi and adenylyl cyclase, reduces the binding of several ligands to their receptors (7), we evaluated the possible changes induced by PTX on eCT binding site densities in the rat CNS. We have also examined the effect of PTX treatment in vivo on eCT inhibition of adenylyl cyclase in vitro in isolated membranes from rat striatum. In the same membrane preparations the effect of morphine and of an opiate peptide agonist, DAME, was tested as a control for the effectiveness of PTX treatment.
METHOD
Animals Male Sprague-Dawley rats weighing 150-200 g (Charles River, Italy) were used. Rats were kept in standardized environmental conditions, artificial light on 12 h per day.
Drugs The following drugs were used: eCT (Sclavo, Italy); PTX (Sigma, USA); morphine hydrochloride (Farmitalia-Carlo Erba,
549
550
GUIDOBONO ET AL.
i
.o
.Tx
•
2O . . . . . . . . . . . . . . . . . . . . . /,,
.
,,/ ..'" ...'"
eCT
• p'rx+ eCT
rrZT1 ~x..c,
0...................................................~.........................................................& PTx _ 2o
~ I
~-"-"T
~
sa,,~
FIG. 1. Time-course of the antinociceptive effect of eCT (2.5 ixg/rat, ICV) in the hot-plate test in PTX-pretreated rats (0.5 Ixg/rat, ICV, 6 days before). Hot-plate latencies are expressed in MPE% (see text for explanation). Each point is the mean---SE, n=12-16. **p
0196-9781/91 $3.00 + .00
Treatment With Pertussis Toxin Does Not Prevent Central Effects of Eel Calcitonin F. G U I D O B O N O , P. B E T I ' I C A , I. V I L L A , F. P A G A N I , C. N E T T I , V. S I B I L I A A N D A. P E C I L E
Department of Pharmacology, Chemotherapy and Medical Toxicology University of Milan, Via Vanvitelli 32, 20129 Milan, Italy Received 20 June 1990
GUIDOBONO, F., P. BETTICA, I. VILLA, F. PAGANI, C. NETTI, V. SIBILIA AND A. PECILE. Treatment with pertussis toxin does not prevent central effects of eel calcitonin. PEPTIDES 12(3) 549-553, 1991.--To determine whether or not the CNS inhibitory activity of eel calcitonin (eCT) on adenylyl cyclase is the endocellular mechanism underlying the antinociceptive effect of the peptide, as shown for morphine analgesia, we administered Bordetella pertussis toxin (PTX) by intracerebroventricular (ICV) injection (0.5 ~g/rat) to block the receptor-mediated inhibition of adenylyl cyclase. In PTX-treated rats there was no change in eCT (2.5 ~g/rat, ICV)-induced antinociceptive activity (hot-plate test) nor in eCT (100 ng/rat, ICV) inhibition of gastric acid secretion (Shay test) whereas morphine (5 l~g/rat, ICV) analgesia was significantly reduced. In vitro studies showed no reduction of eCT binding in the CNS of rats treated with PTX in vivo. Moreover, PTX treatment did not change the inhibitory effect of eCT on adenylyl cyclase in isolated membranes from rat striatum in contrast with opiates (DAME and morphine) whose effects were lost. As PTX is known to inactivate the guanidine binding inhibitory protein Gi, these data suggest that a G protein, distinct from the Gi protein involved in the coupling of opiate receptors into a functional response, could be responsible for regulating the intracellular pathways resulting in eCT-induced antinociceptive effect and inhibition of gastric acid secretion. Eel calcitonin
Morphine
Antinociceptive activity
Gastric acid secretion
THE established primary action of calcitonin (CT) is to inhibit osteoclastic bone resorption through a direct action on osteoclasts (11). This is the reason why CT is a useful drug in certain bone diseases. However, another reason for its therapeutic use is the antinociceptive effect exerted by the peptide, shown both in animals and in humans under a variety of conditions (6, 13, 20). CT antinociceptive activity appears to involve peripheral and central actions. An interaction of the peptide with binding sites identified in the CNS appears to be selective for areas known to participate in pain perception control systems (9). Different classes of receptors for CT have been identified both in peripheral tissues and in the CNS (2,10). The transductional mechanism activated by CT interaction with its receptors seems to be linked to an increase of adenylyl cyclase activity in peripheral tissues (bone, kidney and some human cancer cells) and to an inhibition of adenylyl cyclase activity in the brain (17, 21, 23, 25). It is still unclear what other biochemical events are required for CT receptor binding to be transduced into an activity on bone and into an analgesic response. Since the control of adenylyl cyclase is achieved by transfer of the information from receptor-agonist complex to catalytic subunits of the adenylyl cyclase by means of guanine nucleotide binding proteins (G proteins), which could be inhibitory (Gi) or stimulatory (Gs) (16), we have examined the possibility that G i proteins may represent the link between CT receptors and CT-induced antinociceptive activity. In view of the evidence that treatment with pertussis toxin (PTX) by ribosylation of a Gi protein blocks the receptor-mediated inhibition of adenylyl cyclase (1), we studied whether or not
Adenylyl cyclase
Pertussis toxin
PTX pretreatment into rat brain ventricles (ICV) had any effect on eel CT (eCT)-induced antinociceptive activity as reported for morphine by Parenti et al. (18). PTX pretreatment was also tested on another central activity induced by eCT, the inhibition of gastric acid secretion. As it has been shown that the inactivation of G i protein by PTX, and the consequent uncoupling of Gi and adenylyl cyclase, reduces the binding of several ligands to their receptors (7), we evaluated the possible changes induced by PTX on eCT binding site densities in the rat CNS. We have also examined the effect of PTX treatment in vivo on eCT inhibition of adenylyl cyclase in vitro in isolated membranes from rat striatum. In the same membrane preparations the effect of morphine and of an opiate peptide agonist, DAME, was tested as a control for the effectiveness of PTX treatment.
METHOD
Animals Male Sprague-Dawley rats weighing 150-200 g (Charles River, Italy) were used. Rats were kept in standardized environmental conditions, artificial light on 12 h per day.
Drugs The following drugs were used: eCT (Sclavo, Italy); PTX (Sigma, USA); morphine hydrochloride (Farmitalia-Carlo Erba,
549
550
GUIDOBONO ET AL.
i
.o
.Tx
•
2O . . . . . . . . . . . . . . . . . . . . . /,,
.
,,/ ..'" ...'"
eCT
• p'rx+ eCT
rrZT1 ~x..c,
0...................................................~.........................................................& PTx _ 2o
~ I
~-"-"T
~
sa,,~
FIG. 1. Time-course of the antinociceptive effect of eCT (2.5 ixg/rat, ICV) in the hot-plate test in PTX-pretreated rats (0.5 Ixg/rat, ICV, 6 days before). Hot-plate latencies are expressed in MPE% (see text for explanation). Each point is the mean---SE, n=12-16. **p
