Direct stimulatory effect of calcitonin on [3H]5-hydroxytryptamine release from the rat spinal cord

European Journal of Pharmacology, 156 (1988) 13-23 Elsevier

13

EJP 50490

Direct stimulatory effect of calcitonin on [3H]5-hydroxytryptamine release from the rat spinal cord S. B o u r g o i n *, M. Pohl, M. Hirsch, A. M a u b o r g n e , F. Cesselin a n d M. H a m o n I N S E R M U.288, Neurobiologie Cellulaire et Fonctionnelle, Facult~ de Mddecine Pitid-Salp~trikre, 91, Boulevard de l'H3pital, 75634 Paris Cedex 13, France Received 19 May 1988, revised MS received 19 July 1988, accepted 2 August 1988

The in vitro effects of porcine, salmon and human calcitonin on the K+-evoked overflow of [MetS]enkephalin, substance P and [3H]5-HT (previously taken up) were investigated in superfusion experiments with spinal cord slices. Porcine and salmon calcitonin did not affect the release of [MetS]enkephalin and substance P but enhanced that of [3H]5-HT. In contrast, human calcitonin was inactive. The stimulatory effect of porcine and salmon calcitonin on K+-evoked [3H]5-HT overflow was found with slices from the dorsal or the ventral half of the lumbar enlargement but not with hippocampal or hypothalamic slices. The calcitonin effect on [3H]5-HT outflow persisted in the absence of extracellular Ca 2+ but was totally suppressed by 5-HT uptake inhibitors such as citalopram and chlorimipramine and by the 5-HT-releasing agent, p-chloroamphetamine. Direct investigation of the possible action of porcine calcitonin on [3H]5-HT uptake and release demonstrated that the enhanced [3H]5-HT overflow resulted from a p-chloroamphetamine-like 5-HT-releasing effect of the hormone at the spinal level. This action might be involved in the potent analgesic effect of intrathecal calcitonin. Calcitonins; Spinal cord; [3H]5-HT; [MetS]enkephalin; Substance P; (In vitro release, Salmon, Human, Porcine)

1. Introduction

Calcitonin is a 32-amino acid peptide hormone which is secreted by the 'C' cells of the thyroid gland and which protects the skeleton from calcium loss (Munson, 1976). In addition to this major physiological function, calcitonin has been shown to exert various pharmacological effects, notably an antinociceptive action in both animals (Bates et al., 1981; Bobalik et al., 1974; Clementi et al., 1984; 1985) and humans (Allan, 1983; Chrubasik, 1987; Fraioli et al., 1982; Gennari et al., 1985; Kessel and Wtirz, 1987). Analgesia can be induced by the peripheral (Allan, 1983), intracerebroventricular (Pecile et al., 1975; Welch et

* To whom all correspondence should be addressed.

al., 1986) or intrathecal (Candeletti et al., 1984; Fiore et al., 1983; Spampinato et al., 1984) administration of calcitonin. The latter route seems to require the lowest doses for reducing pain in patients suffering from chronic, intractable oncological pain (Fraioli et al., 1982; Fiore et al., 1983). An effect of calcitonin at the spinal level is indeed likely since specific binding sites for calcitonin are particularly abundant within the superficial layers of the dorsal horn in various mammalian species (Guidobono et al., 1986b; 1987). Thus, it can be hypothesized that calcitonin produces analgesia by acting at these specific sites to block the relay of nociceptive signals from primary afferent fibres to postsynaptic neurones projecting to supraspinal structures (see Besson and Chaouch, 1987). Several neurotransmitters are involved in the transfer and control of nociceptive signals within

0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

14 the dorsal horn, notably substance P (SP) contained in some primary afferent fibres, enkephalins mainly in local interneurones, and serotonin (5-HT) in descending bulbo-spinal pathways (Besson and Chaouch, 1987; Hamon et al., 1988). Previous investigations of the possible involvement of these neurotransmitters in the analgesic effect of calcitonin have yielded contradictory data. For instance, some authors claim that the opioid antagonist, naloxone, prevents calcitonininduced analgesia (Bates et al., 1981; Welch et al., 1986) whereas others have denied the involvement of opioids by showing that this drug and other opioid antagonists do not affect the antinociceprive activity of calcitonin (Braga et a1.,1978; Candeletti et al., 1984; Yamamoto et al., 1979). Similarly the participation of serotoninergic neurones in the analgesic effect of calcitonin is a matter of controversy since data in favour of (Clementi et al., 1984; 1985; Nakhla and Majumdar, 1978) and against (Guidobono et al., 1986a) such a hypothesis have been reported. In an attempt to at least partly solve the problem, we have re-examined the involvement of 5-HT and/or neuropeptides (SP, [Met 5]enkephalin: ME) in the spinal action of calcitonin by exploring possible calcitonin-induced changes in the release of these neurotransmitters from the rat spinal cord in vitro. For this purpose calcitonin from three different species, salmon, pig and human, was used. The data are further support for the possibility that calcitonin-induced analgesia might result from some stimulatory action of this hormone on 5-HT release at the spinal level.

2. Materials and methods

[3H]5-Hydroxytryptamine ([3H]5-HT, generally labelled, 17-20 Ci/mmol, Amersham, U.K.) was purified by ion exchange chromatography just prior to each experiment (Hamon et al., 1974). Salmon, porcine and human calcitonin was from Laboratoires Armour-Montagu (Levallois-Perret, France). Other drugs were: pargyline (Abbott), citalopram (Lundbeck), chlorimipramine (CibaGeigy) and p-chloroamphetamine (Regis). The

other compounds were the purest commercially available (Merck, Prolabo). All experiments were performed with tissues from adult male Sprague-Dawley (Charles River strain) rats weighing 250-300 g. The animals were killed by decapitation and their brains and spinal cords were rapidly removed in the cold (4 ° C). Brain structures (hippocampus, hypothalamus) and the dorsal and ventral halves of the lumbar enlargement of the spinal cord were dissected (Cesselin et al., 1984; Glowinski and Iversen, 1966) and immediately used for the superfusion experiments.

2.1. Superfusion experiments Dissected tissues from 8-12 rats were sliced (thickness: 0.3 mm) with a Mcllwain tissue chopper and were suspended in an artificial cerebrospinal fluid (CSF, composition in mM: NaC1 136; KC1 5.6; MgC12 1.2; NaH2PO 4 1.2; CaC12 2.2; NaHCO 3 16.2; glucose 5) maintained at pH 7.4 by continuous bubbling with O2:CO2 (95:5%). For the measurement of [3H]5-HT outflow (see below), the tissue suspension was first incubated for 15 min at 37°C in the presence of 25-35 nM [3H]5-HT and 10 /~M pargyline. The labelled tissues were then collected by filtration through Whatman No. 3 filters and resuspended in the same CSF as above (without drugs). Aliquots of the tissue suspensions (usually 0.50 ml corresponding to 35-40 mg of fresh tissue) were finely dispersed in thermostated chambers (Cesselin et al., 1984) for continuous superfusion with the same CSF (at 37 ° C) at a flow rate of 0.25 ml/min. After washing for 20-25 min, 1 ml superfusate fractions were collected for 80 min in ice-cold tubes then frozen at - 3 0 ° C until the time of radioimmunological measurements of neuropeptide (SP, ME)-like materials (LM), or were immediately processed for radioactivity counting (experiments with [3H]5-HT). The tissues were depolarized twice in the course of superfusion by changing the superfusing fluid to a K+-enriched medium (KC1 = 30 mM, NaC1 = 111.6 mM; other salts as above) for 8 min during the collection of fractions 7-8 (Ka) and 16-17 (K2) (see fig. 1). The K+-induced overflow of [3H]5-HT

15

[K + ]

"

30

mM

m o ~ 4 x E

[K + ]

-

30

mM

porcine Calcijtonin II~M)

':~I

,

3 -r I I

~2

DORSAL ZONE LUNBAR ENLARC~NENT 0

I

l

I

I

I

| 5

7

8

~0

~2

t5

~s

17

20

Fraction number

Fig. 1. Time course of [3H]5-HT outflow from slices of the dorsal zone of the lumbar enlargement superfused in the absence or the presence of porcine calcitonin. The slices were labelled with [3H]5-HT, collected by filtration then dispersed in thermostated chambers for continuous superfusion with artificial CSF at a flow rate of 1 ml/4 min. Normal CSF (K ÷= 5.6 mM) was used throughout, except for the collection of fractions 7 and 8, and fractions 16 and 17 (each of 1 ml), where the K ÷ concentration was raised to 30 mM (grey areas). The effect of porcine calcitonin (1 /~M) was assessed by exposing the slices to this substance on and after collection of fraction 12 (hatched area). The K÷-evoked overflow (K1, K2) of [3H]5-HT for both K + pulses corresponds to the radioactivity above that estimated from basal spontaneous outflow (dotted line on the figure) in the three fractions (7, 8, 9 and 16, 17, 18) collected from the beginning of superfusion with K+-enriched medium. The data are the means+ S.E.M. of 12 and 6 separate experiments in the absence (continuous line) and the presence (dotted line) of 1/~M porcine calcitonin, respectively. * P < 0.05 when compared to [3H]5-HT in corresponding fractions (No, 16, 17) from tissues superfused without calcitonin.

or n e u r o p e p t i d e - L M was estimated for each dep o l a r i z a t i o n a n d the ratio of the overflow due to the 2 n d K ÷ pulse to the overflow due to the first one, K 2 / K ~ , was calculated. This ratio is rem a r k a b l y c o n s t a n t u n d e r control c o n d i t i o n s (see Cesselin et al., 1984; M a u b o r g n e et al., 1987) so that a n y change i n its value w h e n a drug is a d d e d to the superfusing fluid before the second K ÷ pulse can be ascribed to the effect of this particular drug o n the release process. I n the present study, drugs (e.g. calcitonins) were added to the superfusing fluid from the b e g i n n i n g of the collection of fraction No. 12 up to the e n d of the experiment (see fig. 1). Other t r e a t m e n t s a r e d e scribed in the Results section. I o n exchange c h r o m a t o g r a p h y with A m b e r l i t e C G 5 0 a n d Dowex A G 5 0 W X 4 c o l u m n s ( H a m o n

et al., 1974; 1976) revealed that [3H]5-HT regularly a c c o u n t e d for 85-90% of total radioactivity in the fractions collected whether superfusion was achieved with n o r m a l or depolarizing ( K + = 30 m M ) C S F in the absence or the presence of 0.1-10 /~M c a l c i t o n i n from a n y of the species.

2.2. [3H]5-HT uptake Tissue slices p r e p a r e d as above were i n c u b a t e d for 15 m i n at 3 7 ° C in the presence of 24 n M [3H]5-HT, 10/~M p a r g y l i n e a n d various drugs (see Results). T h e labelled tissues were then collected b y centrifugation, washed with ice-cold C S F a n d their radioactivity was c o u n t e d (see H a m o n et al., 1976, for details). T h e b l a n k s were similar samples i n c u b a t e d at 0 ° C i n s t e a d of 37 o C.

16 [3H]5-HT uptake in crude synaptosomal (P2) fractions was measured by means of 60 nM of the radiolabelled indoleamine as described in detail elsewhere (Hamon et al., 1981). A 4-min incubation at 37 ° C was followed by collection of labelled synaptosomes by filtration through G F / B filters which were then washed with ice-cold CSF, dried and finally immersed in Aquasol ® (New England Nuclear) for radioactivity counting. The blanks for these experiments were made by incubating similar samples supplemented with the potent and selective 5-HT uptake inhibitor citalopram (10 /~M) (Hyttel and Larsen, 1985).

2.3. [3H]5-HT release Spinal cord slices were first incubated for 15 min in CSF supplemented with 10 /~M pargyline and 24 nM [3H]5-HT. The tissue was then collected by filtration through Whatman No. 3 filters, rapidly washed on the filters with 5 ml of CSF, and immediately reincubated for 15 min at 37 ° C in the same medium containing various concentrations of drugs. At the end, the samples were centrifuged (4000 × g, 3 rain) and the radioactivity in tissues and medium was then counted (see Hamon et al., 1976). In agreement with previous observations (Hamon et al., 1976), authentic [3H]5-HT was found to represent more than 95% of the total radioactivity accumulated in the tissues ([3H]5-HT uptake) or released in the medium ([3H]5-HT release) under such conditions.

2.4. Radioimmunoassays 2.4.1. Substance P The SP-LM content of superfusate fractions was estimated by radioimmunoassay as described elsewhere (Mauborgne et al., 1987). Previous in vitro release studies with spinal cord slices have shown that the SP-LM recognized by the specific antiserum we used is at least 90% authentic SP (Mauborgne et al., 1987). 2.4.2. [Met 5]enkephafin The procedure of Cesselin et al. (1984) was applied since it allows the measurement of as little

as 0.25 pg of ME-LM in 0.25 ml of spinal cord superfusate. Chromatographic analyses have demonstrated that ME-LM released from slices of the dorsal half of the rat lumbar enlargement under similar in vitro conditions corresponds to authentic ME (Cesselin et al., 1984). The neuropeptide-LM contents for both SP and ME in superfusate fractions are expressed in pg of neuropeptide equivalents producing the same displacement of the radiolabelled tracer bound to specific antibodies. Standard curves with authentic neuropeptides were obtained from radioimmunoassays performed under the same conditions as for superfusate samples, i.e. in the presence of drug(s) at the same concentration(s) as in these samples. Statistical analyses were made according to Snedecor and Cochran (1967). When a P value (Student's t-test) was higher than 0.05, the difference was considered as being non-significant.

3. Results

3.1, Effects of porcine, salmon or human calcitonin on K +-evoked overflow of SP-LM, ME-LM, and [3H]5-HT from the dorsal half of the lumbar enlargement Slices of the dorsal half of the lumbar enlargement of the spinal cord released 4.5 + 0.3 pg equivalents ME, and 2.4 _+ 0.2 pg equivalents SP per 1 ml fraction (corresponding to 4 min of superfusion) (means + S.E.M., n = 12, for each neuropeptide) under basal resting conditions. These levels increased markedly when [K +] was raised from 5.6 to 30 m M in the superfusing fluid, particularly for the first depolarizing pulse (K1): the M E L M and SPLM contents in fractions No. 7 and 8 were thus - 7 and - 5-fold those in fractions No. 5 and 6, respectively (not shown). The second K + pulse was less efficient in this regard, so that the K z / K 1 ratio was less than 1.0 for both SP-LM and ME-LM (table 1). As shown in table 1, the addition of porcine calcitonin (0.1-10 #M) to the superfusing fluid for the second part of the experiments (see Materials and methods) did not significantly alter the K 2 / K a values for both

17 TABLE 1 K+-evoked overflow of M E - L M and SP-LM from slices of the dorsal zone of the lumbar enlargement exposed to porcine calcitonin. The slices were depolarized twice (K1, K2) by 30 m M K + in the course of superfusion with artificial CSF (see Materials and methods) and the K+-evoked overflow of neuropeptide-like material was measured in specific radioimmunoassays. Porcine calcitonin was added to the superfusing fluid only for the second half of the experiment (including K2). The K 2 / K 1 (i.e. neuropeptide-LM overflow for K 2 / n e u r o p e p t i d e LM overflow for K 1) is the mean 4- S.E.M. of 6-10 independent determinations. NS non-significant Addition

K 2/K1 ME-LM

SP-LM

None 0.464 + 0.055 Porcine calcitonin 0.1/~M 0.419 + 0.063 NS 1.0 ~ M 0.539+0.058 NS 10/~M 0.453+0.062 NS

0.540 + 0.085 _ 0.635 +0.051 NS 0.5034-0.052 NS

As illustrated in fig. 1, K + depolarization also enhanced the [3H]5-HT outflow from prelabelled slices and, as noted for neuropeptides, the second K + pulse released less [3H]amine than did the first ( K z / K 1 < 1.0). However, in contrast to the case of neuropeptides, the addition of 1 btM porcine calcitonin to the superfusing fluid significantly enhanced the [3H]5-HT overflow (fig. 1), so that the K 2 / K 1 ratio for release of the amine was higher in the presence than in the absence of the hormone (fig. 2). Similarly, salmon calcitonin (1 /~M) enhanced the K z / K 1 ratio but human calcitonin was inactive (fig. 2). The relative potencies of the three calcitonins between 0.1 and 10 /LM revealed that human calcitonin was inactive throughout this range whereas porcine and salmon calcitonins were approximately equipotent: no significant effect was noted at 0.1 /~M, and the maximal increase in

peptides. In addition, the spontaneous outflow of SP-LM and ME-LM remained unchanged in the presence of the porcine hormone (not shown). TABLE 2

0.

Effects of salmon or porcine calcitonin on K+-evoked [3 H]5-HT overflow from various spinal and brain regions. Slices from various regions were labelled by [3H]5-HT then superfused with artificial CSF for 80 min (allowing the collection of 20 fractions, each 1 m l / 4 rain). Salmon or porcine calcitonin (1 # M ) was added to the superfusing fluid from the 44th min up to the end of the experiment. [3H]5-HT release was evoked twice by superfusion with K÷-enriched CSF (30 m M K ÷ ) for 8 rain, before (K1) and during (K2) superfusion with calcitonin and the K 2 / K 1 ratio was calculated for each condition. Each value is the m e a n + S.E.M. of at least 9 independent determinations.

~0.

0.

K2/K1 Control L u m b a r enlargement dorsal half 0.556 + 0.017 ventral half 0.515 + 0.041 Hippocampus 0.471 + 0.048 Hypothalamus 0.488 -t- 0.044

Salmon calcitonin

Porcine calcitonin

0.715 a + 0.035 0.819 a -t-0.081 0.517 + 0.043 0.501 + 0.040

0.725 a + 0.033 0.748 a 4- 0.102 0.523 4- 0.065 0.572 4- 0.071

a p < 0.05 when compared to respective control values (K l and K 2 in the absence of calcitonin).

C

10 - ?

t0 - s ICalciton~n]

tO -m P4

Fig. 2. Effects of various concentrations of salmon, porcine and h u m a n calcitonin on K+-evoked [3H]5-HT overflow from the dorsal zone of the lumbar enlargement. The experiments were as described in the legend to fig. 1, except that 0.1, 1 or 10 /zM of each calcitonin was used instead of only 1 ~ M porcine calcitonin. The ratio of [3H]5-HT overflow due to the 2nd K ÷ pulse (in the absence: C on abscissa, or the presence of 0.1-10 /~M calcitonin) to that evoked by the first pulse (in the absence of drugs), K 2 / K 1 , was calculated for each experiment. The bars are the m e a n s + S . E . M , of at least 5 independent determinations. * P < 0.05 when compared to the K 2 / K 1 value in the absence of calcitonin (C on the abscissa).

18

K 2 / K 1 was noted with 1o0-10 /~M of either hormone (fig. 2).

3.2. Effects of porcine and salmon calcitonin on K +-evoked [3H]5-HT overflow from various spinal and brain regions The stimulatory effect of porcine or salmon calcitonin on the [3H]5-HT overflow was observed not only in the dorsal half but also in the ventral half of the lumbar enlargement of the spinal cord (table 2). In contrast, neither porcine calcitonin nor salmon calcitonin significantly affected the K 2 / K 1 ratio for [3H]5-HT release in the hippocampus and the hypothalamus. This ratio tended to increase ( + 17%), however, when hypothalamic slices were exposed to 1 /~M porcine calcitonin (table 2).

3.3. Effects of Ca 2+ removal from, or addition of 5-HT uptake inhibitors to, the superfusing fluid on the stimulation by porcine calcitonin of [3H]5-HT overflow from the rat spinal cord When Ca 2+ was removed and 0.1 mM E G T A added to the superfusing fluid from the beginning of the collection of fraction No. 12 up to the end of the experiment, the second K ÷ pulse produced

NO A D D I T I O N

CITALOPRAM

CHLORIMIPRAMINE

(2 ~M)

(2 uM)

0.

~o.6

0,

~!

Fig. 3. Blockade by citalopram and cNorimipramine of the stimulatory effect of salmon and porcine calcitonin on K +evoked [3H]5-HT overflow from the dorsal zone of the lumbar enlargement. The slices were labelled with [3H]5-HT and dispersed in thermostated chambers for continuous superfusion with artificial CSF (flow rate: 1 m l / 4 min) containing either no drug, 2 # M citalopram or 2 # M chlorimipramine. The tissue was depolarized twice by 30 m M K + (during the collection of fractions 7-8, and 16-17) and exposure to 1/~M salmon calcitonin or 1 # M porcine calcitonin began on the collection of fraction No. 12, as described in the legend to fig. 1. Each bar is the m e a n + S.E.M. of at least 5 independent determinations of K 2 / K 1 (see legend to fig. 2) for each condition. * P < 0.05 when compared to respective control value. [] Control; [] salmon calcitonin (1 #M); [] porcine calcitonin (1 t~M).

TABLE 3 Ca2+-independence of the effect of porcine calcitonin on K ÷evoked [3H]5-HT overflow from the dorsal half of the lumbar enlargement. The slices were labelled by [3H]5-HT then superfused with artificial CSF containing or not 1 /tM porcine calcitonin a n d / o r no Ca 2÷ but 0.1 m M EGTA for the second part of the superfusion experiment (from the 44th min up to the end). The [3H]5-HT overflow evoked by 30 m M K ÷ was then measured under normal conditions (K1, first part of the superfusion experiment) and after the change in CSF composition (K2, second part). The values of the ratio K 2 / K ~ are the means + S.E.M. of at least 6 independent determinations. Addition

2.2 m M Ca 2+ 0 Ca 2÷, 0.1 m M E G T A

K 2/K1 None

Porcine calcitonin

0.580+0.052 0.048 +0.031

0.738-t-0.048 a 0.173+0.024 a

a p < 0.05 when compared to respective values in the absence of porcine calcitonin.

no [3H]5-HT overflow, which resulted in a K 2 / K 1 ratio not different from 0 (table 3). However porcine calcitonin (1 /~M) still stimulated the [3H]5-HT release in the absence of Ca 2+ (table 3). Interestingly, the absolute increase in the K z / K 1 ratio due to porcine calcitonin was similar in the presence (0.738-0.580 = 0.158) and in the absence (0.173-0.048 = 0.125) of Ca 2+ in the superfusing fluid (table 3). Superfusion of slices of the dorsal half of the lumbar enlargement with a CSF containing 2 # M citalopram or 2 /~M chlorimipramine throughout the experiment altered neither the spontaneous outflow of [3H]5-HT nor its K+-evoked overflow as shown by the unchanged value of the K 2 / K ~ ratio (fig. 3). However in the presence of either

19 TABLE 4 Effects of porcine calcitonin on [3H]5-HT release evoked by p-chloroamphetamine from the dorsal half of the lumbar enlargement. The slices are labelled with [3H]5-HT and superfused as described in Materials and methods except that the [3H]5-HT overflow was evoked by 10 /xM p-chloroamphetamine instead of 30 mM K ÷. The first application of p-chloroamphetamine, C1, occurred before the addition of I t~M porcine calcitonin to the superfusing fluid while the second application of the drug, C2, was during superfusion with the hormone. Each value is the mean + S.E.M. of 7 independent determinations of the C 2 / C 1 ratio of the [3H]5-HT overflow due to the second p-chloroamphetamine application over that due to the first application• Ns Non-significantly different from C 2 / C 1 in the absence of porcine calcitonin.

3.5. Effects of porcine calcitonin in comparison with citalopram and p-chloroamphetamine on [3H] 5-HT uptake and release at the spinal level As expected from its selective inhibitory action on 5-HT uptake (Hyttel and Larsen, 1985), citalopram was highly potent (ICs0 = 45 nM) to prevent [3H]5-HT accumulation in slices of the dorsal half of the lumbar enlargement (fig. 4). The 5HT-releasing drug, p-chloroamphetamine, also inhibited [3H]5-HT accumulation but did so at much I

C2/C1 Control Porcine calcitonin

I

|

I

I

lOO

0.491 _+0.025 0.499 _+0.037 NS

_

_

5-HT uptake inhibitor, porcine or salmon calcitonin was no longer able to significantly enhance the K+-induced [3H]5-HT overflow and did not alter the K 2 / K 1 ratio (fig. 3). The same results were obtained in superfusion experiments with slices of the ventral half of the lumbar enlargement (data not shown).

5O

.0 .,-t

fi

) ;/i

~t

3.4. Effects of porcine calcitonin on p-chloroamphetamine-induced [;H]5-HT overflow from the dorsal half of the lumbar enlargement Two successive applications of the drug (Ca = fractions No. 7-8; C 2 = fractions No. 16-17) were also inequally effective when the [3H]5-HT overflow was induced by 10 #M p-chloroamphetamine instead of 30 mM K +. Thus the absolute increase in [3H]5-HT outflow due to the first application ( × 3.5-4.0 over basal value) was about twice that evoked by the second one, leading to a C2/C 1 ratio close to 0.5 (table 4). However in contrast to the result when [3H]5-HT overflow was induced by K ÷ depolarization (fig. 1), the addition of 1 /~M porcine calcitonin to the superfusing fluid for the second part of the experiment did not significantly alter the releasing effect of p-chloroamphetamine, as shown by the unchanged value of C 2 / C 1 (table 4).

::::::::z

0 -8

-7

-6

-5

-4

log [MI

Fig. 4. Effects of porcine calcitonin, citalopram or p-chioroamphetamine on the accumulation of [3H]5-HT in slices of the dorsal zone of the lumbar enlargement. The slices were incubated for 15 min at 3 7 ° C in the presence of 24 nM [3H]5-HT, 10 /xM pargyline, and 10 nM-0.1 m M of drugs as indicated on the abscissa. The tissues were collected by centrifugation, washed and [3H]5-HT accumulation was calculated as the difference between the radioactivity entrapped at 37 ° C minus that bound at 0 o C. The data were calculated as % inhibition of [3H]5-HT uptake according to the formula: % inhibition = 100 × ([3H]5-HT in control tissues-[3H]5-HT in tissues incubated with the drug)/([3H]5-HT in control tissues). Each point is the mean+S.E.M, of 5 independent determinations. At the end of the incubation period the control slices had accumulated 2.39-t-0.10 laCi of [3H]5-HT per g of fresh tissue.

20

higher concentrations (IC50 -- 4.3 #M), and porcine calcitonin was inactive up to 30 /~M, the highest concentration which could be obtained on account of the limited solubility of the peptide hormone (fig. 4). Complementary experiments with P2 fractions confirmed that porcine calcitonin as well as salmon calcitonin between 10 nM and 10 /~M exerted no significant effect on the high-affinity [3H]5-HT uptake in synaptosomes from spinal cord, hypothalamus or hippocampus (data not shown). i

t50

-

I

As shown in fig. 5, the outflow of [3H]5-HT previously taken up by slices of the dorsal half of the lumbar enlargement was markedly increased by p-chloroamphetamine and also by citalopram but to a lesser extent. Under the same experimental conditions, porcine calcitonin significantly enhanced the [3H]5-HT outflow, and at the lowest effective concentration, 0.1 /~M, the peptide hormone was as potent as p-chloroamphetamine (fig. 5). However, porcine calcitonin was clearly less active at higher concentrations than p-chloroamphetamine (fig. 5).

I

• porcine

calcitontn

4. Discussion

0 cttalopram 0 p-chloroamphetamlne

x

100

--

L (.1 C

50

0

~

I

I

1

I

-8

-7

-6

-5

-4

log

IN]

Fig. 5. Effects of porcine calcitonin, citalopram or p-chloroamphetamine on [3H]5-HT outflow from slices of the dorsal zone of the lumbar enlargement. The slices were incubated for 15 min at 37 ° C with 24 nM [3H]5-HT and 10/~M pargyline, then were collected by filtration and washed before resuspension in medium containing various concentrations of porcine calcitonin, citalopram or p-chloroamphetamine (abscissa). After incubation for 15 min at 37°C, tissue and medium were separated by centrifugation and their radioactivity was counted. In the absence of drugs, the ratio M / T of 3H in medium over that in tissues was equal to 0.319-t-0.012 (n = 16). Each point is the mean_+S.E.M, of 5 independent determinations of the percent increase in the M / T ratio due to each drug concentration. * P < 0.05 when compared to M / T in the absence of drugs (in the case of citalopram and p-chloroamphetamine, M / T was significantly higher than the control value for 0.1 #M-0.1 m M of either drug).

Of the three neurotransmitters now investigated, only 5-HT appeared to be directly affected by calcitonin at the spinal level. Porcine calcitonin altered the release of [3H]5-HT but not that of SP(LM) and ME(LM) from the dorsal half of the lumbar enlargement. These negative results were not unexpected in the case of ME since several groups (Braga et al., 1978; Yamamoto et al., 1979; Candeletti et al., 1984; Spampinato et al., 1984) have reported the failure of opioid antagonists to prevent calcitonin-induced analgesia. However, it must be emphasized that the present results were obtained in vitro with slices in which the neuronal circuits were partly destroyed, and further in vivo studies are necessary before one can infer that no opioid link is involved in the antinociceptive action of calcitonin. Several features of the stimulatory effect of calcitonin on the [3H]5-HT outflow at the spinal level strongly suggest a hormonal action through the 5-HT carrier in the presynaptic membrane instead of some modulation of the physiological exocytotic releasing process. Thus, the [3H]5-HT release due to Ca2+-dependent exocytosis triggered by depolarization was totally suppressed in the absence of Ca 2+ in the extracellular fluid, but the release evoked by porcine calcitonin was still present. In contrast, blockade of the 5-HT carrier by citalopram or chlorimipramine did not alter the K÷-evoked overflow of [3H]5-HT, but inhibited the releasing effect of the hormone. However,

21 porcine calcitonin did not act directly at the 5-HT carrier level since neither [3H]5-HT uptake in synaptosomal fractions nor [3H]5-HT accumulation in spinal cord slices were significantly altered in the presence of hormone concentrations as high as 10-30/~M. One could suspect instead an action similar to that of the 5-HT-releasing agent, p-chioroamphetamine, since: (1) like this drug, porcine calcitonin stimulated the outflow of [3H]5-HT previously taken up in spinal cord slices; (2) the effects of the two agents were not additive, and the releasing action of calcitonin could have been masked by that of p-chloroamphetamine; (3) 5-HT uptake inhibitors such as citalopram and chlorimipramine completely suppressed [3H]5-HT overflow due to porcine calcitonin, as previously shown for the amine release triggered by p-chloroamphetamine (Fuller, 1980; Maura et al., 1982). Therefore, as previously shown for the latter drug (Maura et al., 1982), porcine calcitonin probably acts through an increased inside--* out activity of the 5-HT carrier at presynaptic serotoninergic terminals. The molecular events triggering 5-HT carrier activation are however completely unknown and will require further investigation before any working hypothesis can be proposed. The first step involved in the stimulatory action of calcitonin on [3H]5-HT outflow might well be hormone interaction with specific binding sites since there is a clearcut relation between the hormone affinity for these sites and its effect on [3H]5-HT release. Thus both porcine and salmon calcitonin bind in the nanomolar range to specific sites (Nakamata et al., 1981; Guidobono et al., 1986b; 1987) and stimulated [3H]5-HT release, whereas human calcitonin, which exhibits a much lower affinity for these sites (Nakamata et al., 1981; Henke et al., 1985; Guidobono et al., 1986b), exerted no significant influence on amine release. In addition, the interaction of hormone with specific sites might account at least partly for the regional specificity of calcitonin action. Thus, calcitonin-induced [3H]5-HT overflow was found with slices from the dorsal and ventral halves of the spinal cord, where specific binding sites are abundant (Guidobono et al., 1986b; 1987), but not with hippocampal tissues which contain only a low density of these sites (Henke et al., 1983;

Olgiati et al., 1983). Nevertheless, (still unknown) factor(s) other than the local density of high affinity binding sites probably accounted for the regional specificity of the hormone action since [3H]5-HT release was only slightly (and non-significantly) increased by porcine calcitonin from the hypothalamus, in spite of the abundance of calcitonin binding sites in this structure (Guidobono et al., 1986b; 1987). The functional significance of calcitonin-induced [3H]5-HT release within the ventral zone of the spinal cord is still unclear but might be related to motor disturbances previously described in rats treated either intracerebroventricularly (Wiesenfeld-Hallin and Persson, 1984) or intrathecally (Twery et al., 1986) with the hormone. At the level of the dorsal zone of the spinal cord, the stimulatory effect of calcitonin on [3H]5-HT release might be functionally relevant to the analgesic action of the hormone, since several lines of evidence clearly demonstrate that 5-HT exerts a negative influence on the transfer of nociceptive signals in the dorsal horn (see Yaksh and Wilson, 1979; Besson and Chaouch, 1987). In agreement with a calcitonin action mediated via 5-HT release, Clementi et al. (1984; 1985) already noted that 5-HT depletion by reserpine, blockade of 5-HT receptors by specific antagonists and selective lesioning of serotoninergic neurones all suppress the analgesic effect of the hormone. Furthermore, the specific receptors mediating the analgesic action of calcitonin might well be identical with those involved in the hormone effect on [3H]5-HT release at the spinal level. Indeed the pharmacological characteristics of these receptors are closely correlated since salmon or porcine calcitonin but not the human hormone was particularly efficient both to release spinal [3H]5-HT (this paper) and to induce analgesia in rodents (Satoh et al., 1979; Fabbri et al., 1985). Recently Guidobono et al. (1986a) proposed that spinal noradrenergic projections might be even more critically involved than the serotoninergic system in calcitonin-induced antinociception. Studies similar to those on [ 3H]5-HT release should show whether calcitonin also affects [3H]noradrenaline release within the dorsal horn of the spinal cord.

22

Acknowledgements This research was supported by grants from INSERM, DRET (contract No. 87/132), and the Laboratoires ArmourMontagu. We are grateful to the pharmaceutical companies for generous gifts of drugs.

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