Introduction

Top Abstract Introduction Experimental Procedures Results Discussion References

Histamine (HA) is one of the aminergic neurotransmitters playing an important role in the regulation of several physiological and pathological processes. In the mammalian brain, histamine is synthesized by a population of neurons whose cell bodies are restricted to the tuberomamillary nuclei of the posterior hypothalamus (Schwartz et al., 1991). These histaminergic neurons have been implicated in several brain functions (e.g., sleep/wakefulness, hormonal secretion, cardiovascular control, thermoregulation, food intake, and memory formation). In peripheral tissues, histamine is stored in mast cells, basophils, and enterochromaffin-like cells. In these tissues, histamine is considered one of the most important mediators of allergy and inflammatory responses acting through H₁ receptors (Black et al., 1972) as well as in gastric acid secretion acting through H₂ receptors (Ash and Schild, 1966).

Histamine H₃ receptors were first identified as presynaptic autoreceptors on brain histamine neurons, although they are also present in many other cell types (Schwartz et al., 1991). H₃ autoreceptors control the stimulated release of histamine (Arrang et al., 1983) as well as histamine synthesis (Arrang et al., 1987b). Histamine formation from its precursor histidine is catalyzed by the enzyme L-histidine decarboxylase (HDC; EC 4.1.1.22). The cDNA of rat HDC (Joseph et al., 1990) encodes a protein with a molecular mass of 74 kDa (655 amino acid residues) that is post-translationally processed to obtain an active enzyme of 54 kDa (469 amino acid residues). The HDC sequence has two regions fitting consensus phosphorylation sites for PKA. This suggests the possibility that PKA may participate in HDC modulation, although very little is known about the mechanisms involved in the regulation of the enzyme. The cloning and functional characterization of human and rat H₃ receptor cDNA (Lovenberg et al., 1999, 2000) show that the receptor belongs to the family of G-protein-coupled receptors. In these studies, H₃ receptor agonists decreased cAMP accumulation elicited by forskolin stimulation of adenylate cyclase in receptor-transfected cells. Also, it had been observed previously that stimulation of [³⁵S]GTP[S] binding through H₃ receptor agonists in cerebral cortical membranes was prevented by pertussis toxin (Clark and Hill, 1996). All these results demonstrate that histamine H₃ receptors coupled negatively to adenylate cyclase through G_i/o proteins. Nevertheless, is not known whether H₃ autoreceptors in histaminergic neurons regulate histamine synthesis through cAMP formation. If this hypothesis is true, high cAMP levels should stimulate histamine synthesis. However, some evidence against that was presented by Huszti et al. (1991), who observed that HDC activity could be decreased in phosphorylating conditions [including ATP, Mg₂Cl, cAMP, and cAMP-dependent protein kinase (PKA)].

To investigate the role of adenylate cyclase (AC)-PKA pathway in H₃ receptor-regulated histamine synthesis, we prepared rat brain miniprisms as described originally by Arrang et al. (1983). In our preparations, we confirmed that [³H]histamine synthesis is stimulated by depolarization and modulated by H₃ receptor ligands as reported previously by Arrang et al. (1987a). Subsequently, we studied the effects of stimulators and inhibitors of AC-PKA pathway, such as forskolin, dibutyryl-cAMP (db-cAMP), 3-isobutyl-1-methylxanthine (IBMX), or Rp-adenosine 3',5'-cyclic monophosphothioate triethylamine (Rp-cAMPS) on histamine synthesis in nondepolarizing conditions. Finally, we tested the effect of H₃ receptor ligands on cAMP-stimulated histamine synthesis.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Materials. Ring-labeled [2,5-³H]L-histidine stocks (1 mCi; 50 Ci/mmol) were obtained from Amersham Biosciences (Little Chalfont, Buckinghamshire, UK) and were purified by high performance liquid chromatography (HPLC) before their use (Ortiz et al., 2000). Nonradiolabeled histidine, histamine, thioperamide maleate, imetit, clobenpropit dihydrobromide, Rp-cAMPs triethylamine salt, octanesulfonic acid, and trichloroacetic acid were purchased from Sigma/RBI (Steinheim, Germany). PKI_14-22, db-cAMP, and IBMX were obtained from Calbiochem/Merck KGaA (Darmstadt, Germany). Forskolin was supplied from Alomone-Labs (Jerusalem, Israel). IBMX and forskolin stocks were prepared in dimethyl sulfoxide. All other reagents were of the maximum purity available. A reversed-phase C18 HPLC column, 25 × 0.46 cm (Tracer Extrasil ODS-2, 5-µm particle size) equipped with a 2 × 20 mm guard column (Upchurch) was purchased from Teknokroma (Barcelona, Spain). Amberlite IRA-900 (mesh 16-50) strong anion exchange resin (Supelco) was also from Teknokroma. Microspin filter Ultrafree-MC tubes with low-binding Durapore membrane of 0.45-µm pore size were obtained from Millipore GmbH (Eschborn, Germany). OptiPhase "HiSafe"-3 liquid scintillation cocktail was purchased from PerkinElmer Wallac (Turku, Finland).

Preparation and Incubation of Brain Slices. Male Sprague-Dawley rats of 200 to 250 g (Servei d'Estabulari, Universitat Autonoma de Barcelona, Spain) were killed by decapitation between 9 and 10 AM. Brains were immediately placed into ice-cold modified Krebs-Ringer-bicarbonate medium (KRM) of the composition 120 mM NaCl, 0.8 mM KCl, 2.6 mM CaCl₂, 0.67 mM MgSO₄, 1.2 mM KH₂PO₄, 27.5 mM NaHCO₃, and 10 mM glucose, pH 7.4. Working in a cold environment (4°C), meninges were removed and cortical lobes were dissected without white matter. A McIlwain tissue chopper (Mickle Laboratory Engineering Co, Gomshall, Surrey, UK) was used to obtain cerebral cortex miniprisms of 0.3 × 0.3 mm. The miniprisms were suspended in KRM and washed three times with ice-cold KRM to remove debris of damaged cells. Then, the miniprisms were allowed to settle and the excess KRM was removed. Aliquots of 100 µl from the settled slice suspension (usually containing 2-3 mg of protein) were distributed (using a yellow pipette tip cut at 1 cm from the tip) into 2-ml polypropylene tubes. The tubes were preincubated for 25 min at 37°C in a shaking water bath under 95% O₂/5% CO₂ atmosphere. After preincubation, prepurified [³H]histidine (6.25 µCi; final concentration, 0.5 µM) was added to all samples and the tubes were incubated for 5 min further to allow for [³H]histidine uptake. Then, miniprisms were incubated for 30 min to synthesize [³H]histamine in a final volume of 250 µl. If depolarizing conditions were required, the buffer added to bring volume to 250 µl contained concentrated KCl to make a final concentration of 30 or 60 mM potassium (K⁺) and NaCl concentration was decreased proportionally to maintain the isotonicity.

Protein Quantification. To take into account the variability of miniprism amounts pipetted into each tube, protein was determined in an aliquot of each homogenate after sample sonication. Protein content was measured by the method of Lowry et al. (1951), using bovine serum albumin as standard.

Purification of [³H]Histidine. Aliquots of the commercial [³H]histidine standard were purified before their use to check specific activity and avoid excessive radiolysis (Ortiz et al., 2000). Briefly, the HPLC system (Kontron 325 pump) was set to perform a linear gradient from 1 to 6 mM sodium phosphate buffer (pH 3) in 12.5 min at a flow rate of 1 ml/min. A constant percentage of methanol (2%) and 0.1 mM octanesulfonic acid were present throughout. Under these conditions, histidine was eluted at 9 to 10 min and it was detected at 225 nm on a UV 432 detector (Kontron Instruments, Watford, Herts, UK). To calculate the quantity of the purified [³H]histidine, a linear regression of nonradiolabeled histidine (0.2- to 20-nmol standards) versus their corresponding areas was performed. Finally, the specific activity of purified [³H]histidine was determined by dividing total dpm obtained in the histidine fraction by the amount of histidine detected.

Histamine Purification by HPLC. [³H]histamine levels in each sample were determined by HPLC separation after elimination of excess [³H]histidine by ion-exchange on Amberlite IRA 900 resin (Ortiz et al., 2000). Briefly, 100 µl of resin, previously equilibrated with NaOH, was added to deproteinized supernatants into the top half of Ultrafree Microspin tubes. The tubes were vortexed for 10 min in a multitube shaker at room temperature. During this step, the resin bound approximately 85% of the [³H]histidine. Finally, the tubes were centrifuged at 4,000g for 5 min and the filtrates recovered in the bottom half of tubes were injected into the HPLC system.

Statistical Analysis. Concentration-response curves obtained with H₃ receptor ligands (imetit or thioperamide) in depolarizing conditions were analyzed by nonlinear regression using Prism (GraphPad Software, San Diego, CA). Each concentration-response curve was fitted to a logistic function of the form: E = E_max[A]^n_H / ([A]^n_H + (EC₅₀)^n_H), where [A] is the ligand concentration, E is the agonist effect, E_max is the maximal response, n_H is the Hill coefficient, and EC₅₀ is the ligand concentration giving half-maximal response.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Potassium Depolarization Stimulates Histamine Synthesis. To confirm data reported previously (Arrang et al., 1987b), we studied the effect of depolarization with potassium on histamine synthesis in rat brain cortical miniprisms. Potassium produced a dose-dependent increase of histamine synthesis, reaching the maximal effect at 60 mM K⁺ (+192 ± 19.5% versus 2 mM K⁺). Histamine synthesis in samples with 2, 30, and 60 mM K⁺ was 20 ± 0.5, 41 ± 0.9 and 59 ± 1.8 fmol of HA/mg of protein/h, respectively. All groups were statistically different from 2 mM K⁺ (P < 0.01).

Imetit and Thioperamide Modulate Potassium-Stimulated Histamine Synthesis. To assess that presynaptic H₃ receptors were pharmacologically functional in our preparations, we studied the effects of an agonist or an inverse agonist on histamine synthesis in potassium-stimulated conditions (30 mM K⁺). Imetit, a potent and selective H₃ receptor agonist (Garbarg et al., 1992), inhibited the K⁺-stimulated histamine synthesis in a dose-dependent manner. The maximal inhibition (75%) was obtained at of 100 nM and the half-maximal inhibitory concentration was 5.8 nM (Fig. 1A). Conversely, the H₃ antagonist thioperamide (Arrang et al., 1987a), recently classified as H₃ inverse agonist (Morisset et al., 2000), increased the K⁺-stimulated histamine synthesis in a dose-dependent way. Thioperamide maximal stimulation (50%) was reached at 10 nM and the half-maximal stimulatory concentration was 0.92 nM (Fig. 1B). Similar values were obtained in at least two or three additional experiments (not shown).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Effects of H3 receptor ligands on K+-stimulated histamine synthesis. A, effect of imetit (agonist) on [3H]histamine synthesis in 30 mM K+ depolarized cerebral cortex miniprisms. B, effect of thioperamide (inverse agonist) on [3H]histamine synthesis in depolarizing conditions. Results are means ± S.E.M. of two to four replicates. In both figures, 100% [3H]histamine synthesis corresponds to the difference between 30 and 2 mM K+ and results are expressed as a percentage of this value. [3H]histamine synthesis was 24 ± 0.2 and 48 ± 0.3 fmol of HA/mg of protein/h in the presence of 2 and 30 mM K+, respectively. Similar results were obtained in at least two or three additional experiments (not shown).

Forskolin, db-cAMP, and IBMX Stimulate Histamine Synthesis. Because HDC has two PKA consensus phosphorylation sites (Joseph et al., 1990), we studied the effect of several drugs increasing cAMP levels on [³H]histamine synthesis. Forskolin (100 µM), a potent activator of AC that synthesizes cAMP from ATP (Daly et al., 1982; Seamon et al., 1983), elicited an increase of 34 ± 4.3% in the [³H]histamine synthesis in nondepolarized conditions (2 mM K⁺) (Fig. 2). db-cAMP (1 mM), a cell-permeable cAMP analog that directly activates PKA (Posternak and Weiman, 1974), also produced an increase of 29 ± 6.2% in the [³H]histamine synthesis (Fig. 2). Finally, IBMX (1 mM), a nonspecific inhibitor of cAMP and cGMP phosphodiesterases (Beavo and Reifsnyder, 1990; Morgan et al., 1993), elicited an [³H]histamine increase of 47 ± 5.6% (Fig. 2). In addition, we studied the effect of forskolin (100 µM) or db-cAMP (1 mM) on histamine synthesis in the presence of IBMX (1 mM). IBMX potentiated both forskolin- and db-cAMP-stimulation, producing increases of 69.6 ± 1.9% and 89.6 ± 8.1%, respectively.

View larger version (41K): [in this window] [in a new window]   Fig. 2.   Stimulators of AC-PKA pathway increase histamine synthesis. Forskolin, db-cAMP or IBMX were added to the medium 15 min previously to the incubation period. Results are means ± S.E.M. Number of replicates is indicated in brackets above the columns. **, P < 0.01 compared with controls (2 mM K+). 100% [3H]histamine synthesis corresponds to 20 ± 0.5 fmol of HA/mg of protein/h.

Rp-cAMPS Prevents Histamine Synthesis Stimulated by Forskolin and db-cAMP plus IBMX. To confirm that cAMP-stimulated [³H]histamine synthesis was mediated through PKA activation, we examined whether the PKA inhibitor (Rp-cAMPS) could prevent the stimulatory effects produced by forskolin (100 µM) or db-cAMP (1 mM) plus IBMX (1 mM). Rp-cAMPS (Botelho et al., 1988; Wang et al., 1991) decreased forskolin-stimulated [³H]histamine synthesis in a dose-dependent manner, reaching its maximal inhibition (45%) at a concentration of 500 µM (Fig. 3). Similarly, the stimulatory effect produced by db-cAMP plus IBMX was also blocked dose dependently by the PKA inhibitor. In this case, Rp-cAMPS (500 µM) blocked (80%) the effects of cotreatment with db-cAMP plus IBMX. Rp-cAMPS alone did not statistically modify histamine synthesis in basal conditions.

View larger version (48K): [in this window] [in a new window]   Fig. 3.   The PKA inhibitor Rp-cAMPS prevents forskolin- or db-cAMP plus IBMX-stimulated histamine synthesis. Rp-cAMPS was added to the medium 5 min before the stimulatory drugs. Results represent means ± S.E.M. Number of replicates is indicated in brackets above the columns. a, P < 0.01 compared with controls (2 mM K+). *, P < 0.05; **, P < 0.01 compared with forskolin stimulation. #, P < 0.05; ##, P < 0.01 compared with db-cAMP plus IBMX cotreatment. 100% [3H]histamine synthesis in 2 mM K+ was 20 ± 0.5 fmol of HA/mg of protein/h.

Imetit Prevents Forskolin- and IBMX-Stimulated Histamine Synthesis. Because activation of recombinant H₃ receptors decreases intracellular cAMP levels (Lovenberg et al., 1999), we studied whether imetit (H₃ agonist) could prevent forskolin- or IBMX-stimulated [³H]histamine synthesis. Imetit strongly blocked forskolin-stimulated synthesis in a dose-dependent manner. In the presence of 100 nM imetit, forskolin did not produce a statistically significant increase of [³H]histamine synthesis compared with controls without forskolin (Fig. 4). Similarly, the IBMX-stimulation was also dose dependently prevented by imetit, which at 100 nM absolutely blocked IBMX effect. Imetit alone (at concentrations studied) did not statistically modify histamine synthesis under basal conditions (2 mM K⁺).

View larger version (46K): [in this window] [in a new window]   Fig. 4.   Imetit prevents forskolin- and IBMX-stimulated histamine synthesis. Imetit was added to the medium 5 min before the addition of forskolin or IBMX to rat cerebral cortex miniprisms. Results are means ± S.E.M. Number of replicates is indicated in brackets above the columns. a, P < 0.01 compared with controls (2 mM K+). **, P < 0.01 compared with forskolin stimulation. ##, P < 0.01 compared with IBMX-stimulated [3H]histamine synthesis samples. 100% [3H]histamine synthesis in the presence of 2 mM K+ and without any drug represents 21 ± 0.8 fmol of HA/mg of protein/h.

Thioperamide Increases IBMX- and K⁺-Stimulated Histamine Synthesis. Thioperamide alone (H₃ inverse agonist) did not increase histamine synthesis in nondepolarizing conditions (2 mM K⁺). Thus, we studied whether this drug could show effects in IBMX- or K⁺-stimulated conditions. In the presence of IBMX (1 mM) and 2 mM K⁺, thioperamide produced a dose-dependent increase of [³H]histamine synthesis. The maximum potentiation by thioperamide of IBMX effects was about 47% compared with IBMX stimulation (Fig. 5). Similarly, thioperamide produced a dose-dependent increase of histamine synthesis in depolarizing conditions (30 mM K⁺ and no IBMX). The maximal increase was of 45% at a concentration of 100 nM thioperamide, compared with K⁺ stimulation (Fig. 5).

View larger version (45K): [in this window] [in a new window]   Fig. 5.   The stimulatory effect of thioperamide on IBMX- or K+-enhanced histamine synthesis. IBMX and thioperamide were added to the incubation of rat brain cortical miniprisms 15 min before the incubation period. Results represent means ± S.E.M. Number of replicates is indicated in brackets above the columns. a, P < 0.01 compared with 2 mM K+, no drugs. *, P < 0.05, **, P < 0.01 compared with IBMX stimulation. ##, P < 0.01 compared with stimulation produced by 30 mM K+. 100% [3H]histamine synthesis in the presence of 2 mM K+ and without drugs was 23 ± 0.6 fmol of HA/mg of protein/h.

Rp-cAMPS Prevents Thioperamide Effects in IBMX- and K⁺-Stimulated Histamine Synthesis. To confirm whether the effect of thioperamide in stimulated conditions (presence of IBMX or K⁺) was mediated through AC-PKA pathway, we studied whether the PKA inhibitor Rp-cAMPS could prevent the effect of the inverse agonist. Pretreatment with Rp-cAMPS (10 µM) completely blocked the stimulatory effect of thioperamide (100 nM) on IBMX-stimulated samples (Fig. 6). Higher concentrations of the PKA inhibitor (100 and 500 µM) also decreased the stimulatory effect produced by IBMX. In depolarizing conditions (30 mM K⁺), the effect of thioperamide (100 nM) was also prevented by 10 µM Rp-cAMPS (Fig. 6). In contrast to IBMX, higher concentrations of the PKA inhibitor (100 and 500 µM) were unable to decrease the effect produced by K⁺.

View larger version (45K): [in this window] [in a new window]   Fig. 6.   The PKA inhibitor Rp-cAMPS prevents thioperamide stimulated [3H]histamine synthesis in the presence of IBMX or K+. Rp-cAMPS was added to the medium 5 min before thioperamide and IBMX addition to cerebral cortical miniprisms. The other drugs were added to the medium as indicated in Fig. 5. Results represent means ± S.E.M. Number of replicates is indicated in brackets above the columns. a, P < 0.01 compared with IBMX stimulation. **, P < 0.01 compared with IBMX plus thioperamide cotreatment. b, P < 0.01 compared with K+ stimulation. ##, P < 0.01 compared with K+ plus thioperamide cotreatment. 100% [3H]histamine synthesis was 23 ± 0.5 fmol of HA/mg of protein/h in 2 mM K+ with no drugs addition. Note that at 500 µM, Rp-cAMPS also decreases IBMX effects, but not K+ effects.

PKI_14-22 Prevents Clobenpropit-Stimulated Histamine Synthesis in K⁺-Depolarized Samples. To further confirm the specificity of the effects observed in Fig. 6, we looked at the effects on histamine synthesis of a different histamine H₃ antagonist (clobenpropit; Kathmann et al., 1993) and a different cAMP-dependent protein kinase inhibitor (PKI_14-22; Glass et al., 1989). As expected, clobenpropit (10 nM) stimulated histamine synthesis in the presence of 30 mM K⁺ (+59% compared with 30 mM K⁺ stimulation) (Fig. 7). Similarly to thioperamide, clobenpropit had no effects in the absence of depolarization by K⁺. Finally, PKI_14-22 (10 µM) prevented clobenpropit potentiation of histamine synthesis in K⁺-depolarized samples (72% compared with clobenpropit stimulation) (Fig. 7).

View larger version (35K): [in this window] [in a new window]   Fig. 7.   Histamine synthesis stimulation by clobenpropit in the presence of 30 mM K+ is impaired by PKI14-22. PKI14-22 was added to the medium 5 min before clobenpropit addition to cerebral cortical miniprisms. Results represent means ± S.E.M. Number of replicates is indicated in brackets above the columns. a, P < 0.01 compared with 2 mM K+, no drugs. **, P < 0.01 comparison with 30 mM K+. *, P < 0.05 compared with clobenpropit in 30 mM K+. 100% of histamine synthesis was 24.2 ± 0.2 fmol of HA/mg of protein/h in 2 mM K+ without drugs addition.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

This is the first study of the signal transduction pathway used by H₃ autoreceptors to regulate histamine synthesis in histaminergic neurons. The recent cloning of histamine H₃ receptors by Lovenberg et al. (1999) showed that H₃ receptor agonists decreased cAMP accumulation elicited by forskolin stimulation of adenylate cyclase in recombinant receptor-transfected cells. Taking into account that H₃ receptor agonists inhibit histamine synthesis (Arrang et al., 1983) and that the histamine-synthesizing enzyme (histidine decarboxylase) has PKA phosphorylation sites (Joseph et al., 1990), it would be easy to hypothesize a connection among H₃ receptors, the AC-PKA pathway and histamine synthesis. Nevertheless, this fact had never been demonstrated. For these reason, the goals of this study were 1) to study the involvement of AC-PKA pathway on the histamine synthesis and 2) to asses whether this is the signal transduction mechanism used by H₃ autoreceptors to regulate histamine synthesis.

The present study clearly demonstrates that the adenylate cyclase-PKA pathway modulates histamine synthesis. This conclusion is based on results obtained with different kinds of drugs: [³H]histamine synthesis was increased by forskolin (directly activating AC), db-cAMP (directly activating PKA), or IBMX (increasing intracellular cAMP levels through inhibition of phosphodiesterases) treatments. On the other hand, the PKA inhibitor Rp-cAMPS prevented forskolin- and db-cAMP plus IBMX-stimulated histamine synthesis. Because Rp-cAMPS is a highly specific PKA inhibitor (Botelho et al., 1988; Wang et al., 1991), all these results demonstrate that the increment of cAMP levels and the subsequent activation of PKA stimulates histamine synthesis. Nevertheless, further studies are necessary to clarify whether PKA-stimulated histamine synthesis is mediated through a direct phosphorylation of histidine decarboxylase into the PKA phosphorylation consensus sites described by Joseph et al. (1990). Alternatively, an indirect effect of PKA through modulation of other protein kinase cascades could also be possible.

In apparent disagreement with our data, results published previously by Huszti et al. (1991) suggested that PKA should negatively modulate histidine decarboxylase. In their work, the activity of crude histidine decarboxylase obtained from hypothalamus and lung was decreased by phosphorylating conditions. However, their results are difficult to interpret because of the low selectivity of the conditions used. In addition, their results might not be mediated by PKA, because the addition or omission of exogenous PKA into the phosphorylation medium had no significant effects.

Once we demonstrated that the AC-PKA pathway modulates histamine synthesis, we wanted to clarify whether native H₃ autoreceptors regulate histamine synthesis through this cascade. As hypothesized, the H₃ agonist imetit prevented both forskolin- and IBMX-stimulated [³H]histamine synthesis. Conversely, the H₃ inverse agonist thioperamide potentiated the effects of IBMX. Furthermore, in depolarizing conditions, the effects of two H₃ antagonists/inverse agonists (clobenpropit and thioperamide) were blocked by different PKA inhibitors. This clearly confirms that histamine H₃ receptors use the AC-PKA pathway to modulate histamine synthesis. A similar mechanism has been described for the regulation of the tyrosine hydroxylase activity by dopamine D₂ autoreceptors (El Mestikawy et al., 1985). In this case, D₂ autoreceptors are negatively coupled to AC modulating intracellular cAMP levels and PKA activity, which subsequently regulates tyrosine hydroxylase through direct phosphorylation (Arita and Kimura, 1986; Salah et al., 1989).

High forskolin concentrations may also have non-AC mediated effects (Laurenza et al., 1989), such as the modulation of several ion channels. Part of the forskolin effects we observed may not be related to AC. This would explain why Rp-cAMPS and imetit prevented forskolin stimulation only partially. In contrast, Rp-cAMPS and imetit completely impaired db-cAMP and IBMX effects. Thus, our results are strengthened by the fact that different drugs stimulated histamine synthesis acting at different levels of the AC-PKA pathway (H₃ receptors, AC, PKA, and cAMP degradation). Furthermore, different compounds inactivating the pathway at two levels (acting on H₃ receptors or blocking PKA) reversed these effects.

H₃ ligands did not modify basal (2 mM K⁺) histamine synthesis. However, they had effects in the presence of AC-PKA stimulators (forskolin, IBMX) or in depolarized conditions (30 mM K⁺). This suggests that histamine synthesis needs to be stimulated to be regulated by H₃ receptors. At present, we do not know what mechanisms are involved in K⁺-stimulation of histamine synthesis. Thus, further work will be necessary to assess whether K⁺-stimulation and AC-PKA activation converge at some point on histamine synthesis regulation.

The observation that H₃ receptors use the AC-PKA pathway to exert their function on neurotransmitter synthesis in histaminergic neurons does not exclude the possibility that additional signal transduction mechanisms are involved in this or other H₃ receptor-mediated events. Several reports describe an interaction of H₃ receptors with calcium related processes. For instance, Takeshita et al. (1998) reported that H₃ receptor could modulate voltage-sensitive calcium channels in neurons dissociated from the rat tuberomamillary nucleus. Schlicker et al. (1994) indicated that H₃ receptor-mediated inhibition of noradrenaline release was dependent of Ca²⁺ influx. Also, Blandizzi et al. (2001) suggested that H₃ receptor-inhibition of intestinal acetylcholine release could be mediated by modulation of N-type calcium channels. A direct coupling of G protein complexes to Ca²⁺ channels might produce these effects of H₃ receptors on voltage-dependent calcium channels (Diverse-Pierluissi et al., 1997 and 2000). It has also been described that H₃ receptors can modulate the effects of a calcium ionophore on arachidonic acid release (Morisset et al., 2000). On the other hand, other reports of signal transduction mechanisms related to H₃ receptors have been published. Cherify et al. (1992) described a negative coupling of histamine H₃ receptor to phospholipase C in human intestinal HGT-1 cells. Finally, Drutel et al. (2001) reported that activation of several rat H₃ receptor isoforms led to activation of the MAP kinase signaling cascade via pertussis toxin-sensitive G proteins. Several H₃ receptor isoforms varying in the length of their third intracellular loops have been identified (Tardivel-Lacombe et al., 2000; Drutel et al., 2001). Because this is the molecular domain thought to be responsible for coupling to G proteins, it is possible that different H₃ isoforms have differences in their signaling pathways. It should be mentioned that we have obtained results on the involvement of AC-PKA pathway in histamine synthesis in the absence of depolarization. This fact gives more consistency to our conclusions, because the effects observed in depolarizing conditions involve general neurotransmitters release, which could complicate the interpretation of the results observed.

In conclusion, we have demonstrated that brain histamine synthesis can be stimulated by activators of cAMP-dependent protein kinase. Subsequently, we have shown that this protein kinase participates in the H₃ receptor transduction mechanism modulating histamine synthesis. However, the involvement of other signaling pathways can not be excluded.