CAPÍTULO DE ANÁLISIS

There is an abundance of evidence that autoregulation greatly contributes to the modulation of noradrenaline release. Moreover, in vivo studies (detailed below) have confirmed that autoreceptors, activated by released transmitter, reduce the firing rate of noradrenergic neurones, indicating a physiological role for the az-autoreceptor.

In 1973, Starke and Montel reported that the tt2-agonist, clonidine, decreased the

stimulation-induced release of noradrenaline from neurones of the rat cerebral cortex. Later, in 1975, using single-unit recording in vivo, Svensson et a l reported that intravenous and microiontophoretic administration of clonidine inhibited the spontaneous firing rate of noradrenaline-containing neurones in the locus coeruleus. Since then, modulation of neuronal firing-rate and noradrenaline release by somatodendritic a2-autoreceptors, in the locus coeruleus (Cedarbaum and Aghajanian 1976, 1977; Dresse

and Scuvée-Mongeau 1986) and lateral tegmental system (Andrade and Aghajanian, 1982 [A5 cell group], Moore and Guyenet 1983 [A2 cell group]), has been intensively investigated. It is noteworthy that, in all these studies, the animals were anaesthestised. Yet, interactions between a2-adrenoceptors and anaesthetic agents have been well

documented (Wikberg et a l 1987; Saunier et al. 1993; Shimokawa et al. 1998) and challenge the validity of experiments carried out on anaesthetised rats.

More recent dual-probe microdialysis studies in freely-moving animals confirmed the existence of auto-regulation by somatodendritic (%2-autoreceptors. Mateo et al. (1998)

infused the noradrenaline reuptake blocker, desipramine, into the locus coeruleus. They found that noradrenaline efflux increased in the locus coeruleus, but decreased in the cingulate cortex. Local administration of the (%2-antagonist, RX 821002, into the locus

coeruleus, abolished the decrease seen in the cingulate cortex. These results suggested that desipramine inhibited the firing of locus coeruleus neurones by indirect activation of somatodentritic a2-adrenoceptors. Similarly, Mateo and Meana (1999) reported that local

administration of the a2-agonists, clonidine or bromoxidine, in the locus coeruleus,

decreased noradrenaline efflux in the cingulate cortex, and that the selective ct2AÆ>-adrenoceptor antagonist, BRL 44408, abolished this effect. When administered

Chapter 1: General Introduction

s p e c i f i c a z B /c -a n ta g o n is t s d id n o t a n t a g o n is e t h e in c r e a s e in d u c e d b y c l o n i d i n e o r

b r o m o x i d i n e , a n d d i d n o t i n d u c e c h a n g e s in n o r a d r e n a lin e e f f l u x , a lo n e . T o g e t h e r , t h e s e

r e s u l t s s u g g e s t t h a t th e s o m a t o d e n d r it ic a 2 - a d r e n o c e p t o r s m o d u la t in g n o r a d r e n e r g ic

a c t i v i t y in th e l o c u s c o e r u le u s a r e t o n i c a l l y a c t iv a t e d a n d o f t h e a2 A /o s u b ty p e .

Somatodendritic a2-autoreceptors are not the only a2-adrenoceptors which

modulate noradrenaline release (see Figure 1.2). Presynaptic (%2-autoreceptors, situated

on noradrenergic terminals, and a2-adrenoceptors located postsynaptically

(heteroceptors), can also regulate noradrenaline efflux, directly or via feedback loops. Most (%2-adrenoceptor antagonists do not discriminate between pre- and post-junctional

(%2-adrenoceptors but Ruffolo et al. (1987) reported the existence of a potent antagonist at

postjunctional sites only (SK&F 104078), confirming the existence of the two subtypes. However, SK&F 104078 had higher affinity towards 5-HTia and 5-HT2 receptors than

towards (%2-adrenoceptors in the rat brain (Kilpatrick et al. 1989) and this could explain

why this drug has not been extensively used since its discovery.

The existence of presynaptic (%2-adrenoceptors in the cental nervous system has

also been demonstrated in vivo. In a microdialysis study in freely-moving animals, Dennis et al. (1987) showed that the (%2-antagonist, idazoxan (see more details in 1.2.3.1),

infused via the dialysis probe into the frontal cortex, in the aim of eliminating the contribution of somatodendritic a2-adrenoceptors, increased noradrenaline efflux.

Radioligand binding has been used to quantify presynaptic (%2-adrenoceptors in

certain brain areas. Comparing [^H]-idazoxan binding in rat brain membranes taken from control and DSP-4 pretreated animals, 3 days after treatment. Heal et al. (1993) estimated that presynaptic a2-adrenoceptors represent 2 0 % of the total (%2-adrenoceptor population

in the cortex, hippocampus and cerebellum, and 40 % of the total population in the hypothalamus. The authors considered that proliferation of remaining (%2-adrenoceptors

was négligeable 3 days after treatment with DSP-4. Moreover, the 5-HT reuptake inhibitor, zimeldine, was administered prior to DSP-4 to ensure that DSP-4 would selectively lesioned noradrenergic neurones.

som atodentritic

a2-ad re n o c ep to rs in the

p resynap tic a2-a d re n o c e p to rs

on n erve term in als cell body regions

postsynaptic a2-a d re n o c e p to rs NA local polysynaptic circuit 5HT n eg ative fe e d b a c k by long m ultisynaptic p athw ays projecting to n o rad ren alin e cell bodies

0 a2-ad re n o c ep to rs

Figure 1.2 Location o f a2-adrenoceptors modulating noradrenaline release.

Adapted from Dennis et al. (1987). Details o f the subtypes involved as yet unknown.

1.2.3 Atipamezole, a selective a2-antagonist

1.2.3.1 Pharmacology of atipamezole

The a2-antagonist atipamezole (4-(2-ethyl-2,3-dihydro-lH-inden-2-yl)-l H-

imidazole) is a highly specific antagonist at az-adrenoceptors. Early binding studies indicated that it was a potent ai-adrenoceptor antagonist with a afcLx selectivity ratio greater than that o f idazoxan or yohimbine (Virtanen et al. 1989). However, these displacement studies were using clonidine, an imidazoline / a2-adrenoceptor agonist

ligand, to define antagonist binding at a2-adrenoceptor sites. More recently, the

selectivity o f atipamezole for a2-adrenoceptors compared with ai-adrenoceptors was

confirmed using selective a2-antagonists (Table 1.1). It was also established that

Chapter 1: General Introduction

(Table 1.1; Haapalina et al. 1997). Moreover, in contrast with the imidazoline idazoxan, atipamezole, an imidazole, shows négligeable binding to imidazoline h sites (Table 1.1; Savontaus er a/. 1997).

Table 1.1 K ifo r atipamezole and various a-adrenoceptor subtypes.

Ki(nM) Selectivity ratio ( a / a x )

r ai 4786 1 h tt2A 0.891 5 371 h tt2B 1.175 4 073 h a 2 c 1.950 2 454 ra2D 0.323 14 817 5754.39

Results of experiments of displacement by atipamezole of -prazosin in rat (r) cerebral cortex tissue, and ^ ^ [^H]-RX-821002 in cultured SI 15 cell lines expressing one of the human (h) 0C2A.B.c-&drenoceptor subtypes, [^H]-RX 821002 in rat submandibular gland, and [^H]-idazoxan in rat liver tissue. (pAl obtained from Savontaus et al. 1997 and Haapalinna et al.

1997).

Table 1.2 Selectivity o f atipamezole and other a2-antagonists for the human

a2A-odrenoceptor compared with the human 5-HT]a receptor.

Ki (nM) h 02A h 5-HTia Selectivity ratio h 0 2A/ h 5-HTia

Atipamezole 3.16 4 074 1 288

Idazoxan 25.7 93.3 3.6

Yohimbine 5.7 54.9 9.5

Results of experiments of displacement of [ H]-RX 821002 in CHO cells expressing h tt2A receptor (non-specific binding defined with phentolamine) and [^H]-8-OH-DPAT in

CHO cells expressing h 5-H T ia receptor (nonspecific binding defined with 5-HT) b y test

compounds. (pATi obtained from Newman-Tancredi et al. 1998).

Finally, unlike other az-adrenoceptor ligands that are widely used in the laboratory (RX 821002; yohimbine; rauwolscine; idazoxan), atipamezole shows negligible binding to 5-HTia receptors (Table 1.2 and Table 1.3; Meana et al. 1996; Newman-Tancredi et al. 1998). This makes it an ideal tool to assess the role of a2-adrenoceptors in modulation of central systems.

Table 1.3 Selectivity o f atipamezole and other a2-antagonists fo r the rat

a2A-adrenoceptor compared with the rat 5-HTia receptor.

K\ (nM) r a z o r 5 - H T i a S e l e c t i v i t y ratio r «2 0/ r 5-HTu

Atipamezole 0.12 7 413 63 358

Idazoxan 1.91 97.7 51.3

Yohimbine 28.8 120.2 4.17

Results of experiments of displacement of [^H]-RX 821002 in rat cerebral cortex tissue (non specific binding defined with phentolamine) and [^H]-8-OH-DPAT in rat hippocampus tissue expressing h 5 - H T i a receptor (nonspecific binding defined with 5-HT) by test compounds. (pA"i

obtained from Newman-Tancredi et al. 1998).

1.2.3.2 M icrodialysis s tu d ies

Several in vivo microdialysis studies have indicated that administration of atipamezole increases extracellular noradrenaline in the medial hypothalamus of anaesthetized rats (Laitinen et a l 1995). Local administration (50 pM, applied for 1 h) and systemic administration (1 mg/kg s.c.) of atipamezole induced a two-fold increase in

noradrenaline efflux (mean basal release of 53 ± 7 fmol/30 min; probes perfused at 2 pl/min; membrane length exposed 2 mm) (Laitinen et al. 1995). However, the increase was transient and started to decline after 2 h. This increase in noradrenaline efflux supports earlier reports (Mateo and Meana 1999; refer to section 1.2.2 for more details) that ct2-adrenoceptors are tonically activated.

Kask et al. (1997) also reported a 2-fold increase in fronto-cortical noradrenaline dialysate concentration after injection of 3 mg/kg atipamezole (i.p.) in anaesthetised rats (mean basal release of 52 ± 6 fmol/20 min; probes perfused at 2 pl/min; membrane length

exposed 4 m m). However, they had included the noradrenaline reuptake inhibitor nomifensine (10 pM) in the perfusate '"because previous attempts to measure NA from dialysate in the frontal cortex had been unsuccessful. ..”

Finally, local infusion of atipamezole increases noradrenaline efflux. When 0.5 pM atipamezole was infused into the frontal cortex of halothane-anaesthetized rats, Dailey and Stanford (1995) observed a progressive 2-fold increase in noradrenaline extracellular concentration which stabilised after 1 h.

Chapter 1: General Introduction