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(The FASEB Journal. 2001;15:164-170.)
© 2001 FASEB

Melatonin protects against 6-OHDA-induced neurotoxicity in rats: a role for mitochondrial complex I activity

FEDERICA DABBENI-SALA, STEFANIA DI SANTO, DAVIDE FRANCESCHINI, STEPHEN D. SKAPER* and PIETRO GIUSTI1

Department of Pharmacology, University of Padova, 35131 Padova, Italy; and
* Department of Neuroscience Research, SmithKline Beecham Pharmaceuticals, Harlow, Essex CM19 5AW, U.K.

1Correspondence: Department of Pharmacology, L.go Meneghetti, 2, 35131 Padova, Italy. E-mail: giusti{at}ux1.unipd.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Unilateral injection into the right substantia nigra of the catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA) produces extensive loss of dopaminergic cells (‘hemi-parkinsonian rat’). The pineal hormone melatonin, which is a potent antioxidant against different reactive oxygen species and has been reported to be neuroprotective in vivo and in vitro, was evaluated for potential anti-Parkinson effects in this model. Imbalance in dopaminergic innervation between the striata produced by intranigral administration of 6-OHDA results in a postural asymmetry causing rotation away from the nonlesioned side. Melatonin given systemically prevented apomorphine-induced circling behavior in 6-OHDA-lesioned rats. Reduced activity of mitochondrial oxidative phosphorylation enzymes has been suggested in some neurodegenerative diseases; in particular, selective decrease in complex I activity is observed in the substantia nigra of Parkinson’s disease patients. Analysis of mitochondrial oxidative phosphorylation enzyme activities in nigral tissue from 6-OHDA-lesioned rats by a novel BN-PAGE histochemical procedure revealed a clear loss of complex I activity, which was protected against in melatonin-treated animals. A good correlation between behavioral parameters and enzymatic (complex I) analysis was observed independent of melatonin administration. A deficit in mitochondrial complex I could conceivably contribute to cell death in parkinsonism via free radical mechanisms, both directly via reactive oxygen species production and by decreased ATP synthesis and energy failure. Melatonin may have potential utility in the treatment of neurodegenerative disorders where oxidative stress is a participant.—Dabbeni-Sala, F., Di Santo, S., Franceschini, D., Skaper, S. D., Giusti, P. Melatonin protects against 6-OHDA-induced neurotoxicity in rats: a role for mitochondrial complex I activity.


Key Words: Parkinson’s disease • neurodegeneration • pineal hormone • neuroprotection • oxidative phosphorylation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Parkinson’s disease (PD) is an age-related disorder characterized by a progressive degeneration of dopaminergic neurons of the substantia nigra (SN) pars compacta. The underlying cause of this selective cell death is not understood, although several hypotheses have been advanced (1) . Generation of reactive oxygen species (ROS) caused by oxidative stress, together with a relative paucity of antioxidant defenses in the SN and nigrostriatal dopaminergic pathway, is widely considered as the final cause of neuronal death (2 , 3) .

A similar loss in nigro-striatal dopaminergic neurons is produced on intracerebral administration of the catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA). In this animal model of PD, termed ‘the hemi-parkinsonian rat’ (4 , 5) , unilateral injection of 6-OHDA into the nigro-striatal pathway results in extensive loss of dopaminergic cells in the ipsilateral SN (6 7 8) . The resulting imbalance in dopaminergic innervation between the striata produces a postural asymmetry that manifests itself in the animal rotating away from the nonlesioned side (6) . The 6-OHDA lesion model has been used to evaluate potential anti-Parkinson agents (6) . The clinical relevance of 6-OHDA toxicity has been strengthened by the detection of 6-OHDA in urine of parkinsonian patients treated with L-DOPA (9) .

Damage induced by 6-OHDA in vivo and in cultured neuronal cells is similar to that induced by ROS, i.e., increased lipid peroxidation, a decrease in reduced glutathione, and an increased iron content (10) . In fact, 6-OHDA-induced release iron from ferritin (11) may enhance oxidative stress through induction of hydroxyl radical formation via the Fenton reaction (reviewed in ref 12 ). Increasing evidence points to a correlation between neurodegenerative diseases and reduced activity of mitochondrial oxidative phosphorylation (OXPHOS) enzymes (13 , 14) . For instance, a selective 30–40% decrease in complex I activity has been found in the SN of PD patients (15) . Such a defect could be due to an inherited mutation, although disease-related mitochondrial DNA mutations are not known; more probably, toxic insult secondary to oxidant stress is responsible. A mitochondrial complex I deficit could contribute to cell degeneration in PD via a direct generation of ROS together with a decrease in ATP synthesis leading to energy failure. Catecholamines themselves are potent inhibitors of mitochondrial respiration (16) . Moreover, 6-OHDA toxicity can be attenuated by free radical scavengers (16 17 18) .

We have shown that the pineal hormone melatonin prevents the neurotoxic effects of ROS triggered by kainic acid receptor activation (19 20 21 22) . Melatonin antioxidant effects directed to different ROS species (23 , 24) have been documented in various in vitro and in vivo models (25 26 27 28) . Moreover, this pineal product increased mRNA levels and activity of several antioxidant enzymes and inhibited apoptosis caused by 6-OHDA in PC12 cells (29) .

In the present study we have used the hemi-Parkinson rat model of intranigral 6-OHDA administration to analyze the effects of melatonin on apomorphine-induced circling behavior and mitochondrial OXPHOS enzyme activities. Furthermore, a blue native-polyacrylamide gel electrophoresis (BN-PAGE) histochemical method was used for the first time to evaluate damage and repair in a central nervous system degenerative pathology.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Adult male Sprague-Dawley rats (body weight 250±20 g) were obtained from Harlan Italy (S. Pietro al Natisone, UD). Animals were housed under conditions of controlled temperature (23±2°C) and illumination (12 h light; 12 h darkness; darkness: 19:00–07:00) with free access to standard diet and water. Experiments were performed between 10:00 and 17:00 in compliance with the European Communities Council directive of 24 November 1986 (86/609/IIC) concerning the experimental use of animals.

6-OHDA lesions
Animals (seven per group) were anesthetized intramuscular with a solution of ketamine (87 mg/kg) plus xylazine (13 mg/kg) and secured in a Kopf stereotaxic instrument with the tooth bar set +5 mm above the interaural line. Lesions were made by the unilateral injection of 6-OHDA (8 µg in 2 µl) into the right SN at the following coordinates: AP, -4.8; L, -2.2; DV, -8.7 mm from Bregma (30) . The sham-operated animals received vehicle only (0.1% ascorbate in 0.9% saline) at the same coordinates. 6-OHDA administration was performed using a 27 gauge Hamilton syringe connected to an infusion minipump (Harvard Compact Infusion Pump, Holliston, Mont.) at a rate of 0.5 µl/min. The syringe was left in place for 5 min before slowly retracting it to allow for toxin infusion and prevent reflux.

Melatonin administration and measurement
Melatonin was administered immediately after intranigral 6-OHDA injection. Osmotic minipumps filled with a solution of melatonin (50 mg/ml in saline plus 50% of DMSO; 220 µl) were placed in the subcutaneous (s.c.) tissue between the scapulae. The delivery rate was constant at 1 ± 0.15 µl/h (50±7.5 µg melatonin/h). This produced a plasma concentration of 1660 ± 240 pg melatonin/ml for at least 7 days. Other groups of seven animals received a bolus injection of melatonin s.c. (5.0 mg/kg) immediately after 6-OHDA; controls were administered vehicle.

Melatonin was measured as described by Chanut et al. (31) . Plasma samples of 1 ml were added to 5 ml of dichloromethane after alkalinization with 100 µl 1N KOH; 6-fluorotryptamine (10 µl of a 0.05 µM solution) was added as internal standard. Samples were shaken for 10 min and then centrifuged (10 min, 1500 g, 4°C). The aqueous phase was removed and the lower (organic) phase taken to dryness under a nitrogen stream. The residue was dissolved of 100 µl mobile phase buffer and 50 µl subjected to high-performance liquid chromatography using a Beckman System Gold PSM 125 pump equipped with a Rheodyne injection valve and a reversed-phase C18 (150 mm x 4.6 mm) Ultrasphere column. The mobile phase consisted of water-acetonitrile (80:20, v/v) containing 0.01 mM EDTA, 0.1 mM KH2PO4, and 0.5 mM octan-sulfonic acid (pH 4.7): flow rate 1.4 ml/min. Electrochemical detection was performed with an amperometric detector (BAS, LC-4B) at a working potential of +0.9 V.

Behavioral testing
Two weeks after right intranigral stereotaxic injection of 6-OHDA, animals were subjected to rotational behavior testing (6 7 8) . Rats were injected s.c. with R-(-)-apomorphine hydrochloride, melatonin, or vehicle, placed in a cylindrical cage (240 mm in diameter, 300 mm high), and the number of rotations over a 1 h period, both ipsilateral and contralateral, were recorded using an automatic rotometer (Rota-Count 8; Columbus Instruments, Columbus, Ohio).

Electrophoretic analysis
Five rats each from groups receiving 6-OHDA plus melatonin (minipump) or 6-OHDA plus vehicle were used. Animals were killed after behavioral testing; the right and left SN were excised according to Heffner et al. (32) and immediately frozen in liquid nitrogen. On the day of analysis, 5 mg of frozen SN were homogenized in 1 ml of 0.44 M sucrose, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, and 20 mM 3-(N-morpholino)propanesulfonic acid, pH 7.2, using a tight-fitting Teflon pestle and Beckman polycarbonate centrifuge tube. The homogenate was centrifuged at 20,000 g for 20 min; the supernatant was discarded and the pellet resuspended in 1 ml buffer and centrifuged as above. The pellet was then suspended in 40 µl 1M aminocaproic acid, 50 mM Bis-Tris/HCl pH 7.0 (4°C) containing protease inhibitors (20 µM phenylmethylsulfonyl fluoride, 20 µM N-tosyl-L-phenylalanine chloromethyl ketone, 1 µg/ml pepstatin and 1 µg/ml leupeptin) and membranes solubilized by adding 15 µl 10% freshly prepared dodecylmaltoside. After centrifugation for 25 min at 100,000 g, the supernatants were collected and the pellets were re-extracted with the same volume of buffered detergent and centrifuged for 25 min at 100,000 g. The first and second extracts were pooled, the final volume being 100–120 µl for each tissue sample. The extracts were used immediately for BN-PAGE or stored in small aliquots at -80°C for several months. BN-PAGE was performed using a minigel apparatus (miniprotean, Bio-Rad, 1x70x82 mm). A 5–11% polyacrylamide gradient was used; 14 µl of supernatant plus 1 µl 5% Serva Blue G in 1M aminohexanoic acid was applied to each lane. BN-PAGE was performed as described (33) , with the blue cathode buffer being replaced by a clear buffer immediately after the sample passed the stacking gel. Gels were sliced into individual lanes for histochemical and Coomassie staining.

Histochemical evaluation of BN-PAGE gels
Histochemical staining of BN-PAGE was performed as described (34) . Complex I (NADH, NADH dehydrogenase) activity was determined by incubating gel slices with 2 mM Tris-HCl pH 7.4, 0.1 mg/ml NADH, and 2.5 mg/ml NTB (Nitro Blue Tetrazolium) at room temperature. Complex II (succinate dehydrogenase) activity was evaluated by incubating gel slices at room temperature with 4.5 mM EDTA, 10 mM KCN, 0.2 mM phenazine methosulfate, 84 mM succinic acid, and 50 mM NTB in 1.5 mM phosphate buffer (pH 7.4). Complex IV (cytochrome oxidase) activity was assessed by incubating gel slices with 5 mg 3,3'-diaminobenzidine tetrahydrochloride dissolved in 9 ml phosphate buffer (0.05 M, pH 7.4), 1 ml catalase (20 µg/ml), 10 mg cytochrome c, and 750 mg sucrose. Maximum color was achieved with a 3 h incubation. Color development for complex I, II, and IV reacting bands was preserved by fixing gels in 50% methanol/10% acetic acid for 15 min; the fixed gel was stored in 10% acetic acid. Complex V (ATPase) activity was measured by incubating gel slices overnight in 35 mM Tris, 270 mM glycine, 14 mM MgSO4, 0.2% Pb(NO3)2, and 8 mM ATP, pH 7.8, at room temperature. The gel was then washed and stored in distilled water. The remaining gel slice from each sample group was fixed and stained with Coomassie blue G (33) . Color intensity of the stained bands was assessed by scanning the still wet gel with a HP3 Scanjet; areas were integrated using Sigma-plot Jandel (Sigma Chemical Co., St. Louis, Mo.). Band area was expressed either in arbitrary units or relative to the area for Coomassie blue-stained complex V. This allowed for BN-PAGE results to be given quantitatively (34) .

Materials
6-OHDA, melatonin, and xylazine HCl were purchased from Sigma. 6-OHDA HBr was dissolved in sterile saline containing 0.1% ascorbic acid; melatonin in 0.9% saline/50% DMSO. R-(-)-apomorphine HCl was obtained from ICN Biomedical Inc. (Costa Mesa, Calif.) and dissolved in sterile saline. Ketamine HCl was obtained from Virbac (Milan, Italy). ALZET mini-osmotic pumps (model 2001) were purchased from Alza Pharmaceuticals (Palo Alto, Calif.). Serva Blue G (Coomassie blue) was from by Serva (Heidelberg, Germany), dodecylmaltoside from Boehringer (Mannheim, Germany), digitonin from Merck (Darmstad, Germany), 6-aminocaproic acid from Fluka (Milan, Italy), acrylamide and bis-acrylamide from Bio-Rad (Milan, Italy), and nitrocellulose filters for Western blotting from Pharmacia (Uppsala, Sweden). All other chemicals were purchased from Sigma-Aldrich (Milan, Italy).

Statistics
Behavioral test results were subjected to Kruskall-Wallis nonparametric analysis of variance, followed by a two-tailed Mann-Whitney U test. Data were expressed as the mean ± SE despite the probable non-normality of the distribution of scores. OXPHOS respiratory chain enzyme activities on right and left sides and the effects of 6-OHDA and 6-OHDA plus melatonin were analyzed by a t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Apomorphine induces circling behavior after 6-OHDA lesioning
Rats with unilateral (right SN) 6-OH-DA lesions exhibited apomorphine-induced contralateral rotations: 220 ± 50 turns/h at 0.15 mg/kg apomorphine increasing to 856 ± 99 turns/h at 1.6 mg/kg apomorphine (Table 1 ) (see also ref 35 ). Lesioned rats showed an increased level of spontaneous ipsilateral rotation after vehicle treatment, reflecting basal dopamine activity in the nonlesioned side. Apomorphine dose-dependently decreased ipsilateral turning in this group of animals (Table 1) . Subsequent studies used a dose of 1 mg/kg s.c. apomorphine. Sham-operated animals failed to exhibit increased contralateral and ipsilateral rotations upon apomorphine administration (Table 1) .


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  		Table 1. Effect of apomorphine on circling behavior in rats with unilateral 6-OHDA stereotaxic injectionsa

Melatonin effect on circling behavior in 6-OHDA lesioned rats
Rats subjected to sham stereotaxic injection 2 wk before and receiving melatonin (5 mg/kg, s.c.) had the same number of contralateral and ipsilateral rotations as vehicle. Likewise, melatonin did not modify the turning behavior of sham-lesioned animals when given either as a bolus or by minipump immediately after surgery (Table 2 ). When rats were stereotaxically injected with 6-OHDA and immediately given vehicle, melatonin (5 mg/kg, s.c.) or minipumps containing melatonin (50±7.5 µg/h x 7 days), challenge with vehicle on day 14 produced the same number of contralateral rotations in all groups. Ipsilateral turning increased significantly in the latter case, and melatonin (5 mg/kg, s.c.) did not affect this behavior. In contrast, 6-OHDA-infused animals receiving melatonin by minipump exhibited a significant decrease (P<0.01) in ipsilateral rotations (6.3±3.5 vs. 23±4.0) when challenged with vehicle (Table 2) . Minipumps were not implanted prior to 6-OHDA administration, as damage caused by the neurotoxin is fully evident after 2 wk and still present 10 months later (36) . The first week, and not the initial hours after 6-OHDA treatment, appears critical.


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  		Table 2. Effect of melatonin on circling behavior in rats with unilateral 6-OHDA stereotaxic injectionsa

Effects of melatonin on apomorphine-induced circling
A dose of 1 mg/kg s.c. apomorphine was used as standard challenge for interaction studies, as noted above. This dose induced in lesioned plus vehicle animals a consistent and pronounced contralateral rotation behavior, i.e., 652 ± 46 turns/h (Fig. 1 ); ipsilateral turns were 1.9 ± 0.74/h (data not shown). Melatonin (5 mg/kg, s.c.) given immediately after stereotaxic 6-OHDA injection failed to modify these parameters. However, when melatonin was administered via osmotic pump immediately after 6-OHDA injection, a striking decrease (P<0.0001) in apomorphine-induced contralateral turning behavior was observed (from 652±46 to 37±10 turns/h) (Fig. 1) .



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  		Figure 1. Effect of melatonin on apomorphine-induced contralateral rotation behavior in rats with unilateral stereotaxic injection of 6-OHDA. A group of 7 animals received a stereotaxic injection of 6-OHDA in the right SN. Melatonin (5 mg/kg s.c. or osmotic pump, 50 ± 7.5 µg/h x 7 days) immediately after 6-OHDA (8 µg in 2 µl). Two weeks later, the number of rotations was recorded during a 1 h period immediately after apomorphine (1 mg/kg, s.c.) administration. Results are means ± SE *P<0.0001 compared to the vehicle group.

Effect of 6-OHDA on mitochondrial OXPHOS enzymes: rescue by melatonin
Figure 2 shows standard BN-PAGE patterns of detergent-extracted SN tissue samples after Coomassie blue (lanes 1 and 2) or enzymatic histochemical staining for mitochondrial enzymes (lanes 3–10). Even and odd numbers indicate right (6-OHDA infused) and left SN, respectively. Quantification of OXPHOS enzyme analysis after 6-OHDA plus or minus melatonin is summarized in Table 3 . In comparison to left SN, right SN showed a significant (P=0.0079) inhibition (-20±4.5%) of NADH dehydrogenase activity. Melatonin given by osmotic minipump completely prevented the inhibitory effect of 6-OHDA on complex I. Application of 6-OHDA produced a nonsignificant reduction (P=0.070) in complex IV activity that was reversed by melatonin administration via osmotic minipump.



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  		Figure 2. Histochemical enzymatic staining of rat nigral tissue on BN-PAGE: effect of 6-OHDA lesion. A) Lanes 1 and 2: Coomassie blue staining; lanes 3, 4: NADH dehydrogenase activity staining; lanes 5, 6: cytochrome oxidase activity staining; lanes 7, 8: succinate dehydrogenase activity staining; lanes 9, 10: ATPase activity staining. Odd numbers, left SN (control); even numbers, right SN (6-OHDA injection). B) As in panel A, with odd numbers indicating left SN and even numbers right SN (6-OHDA injection + osmotic minipump with melatonin).


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  		Table 3. OXPHOS activity in rat nigral tissue: effect of 6-OHDA lesiona


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A key finding of the present study is that systemic administration of melatonin corrects a hemi-Parkinson condition in rats caused by intranigral application of the catecholaminergic neurotoxin 6-OHDA. There is considerable conflict regarding the protective potential of melatonin in PD. Early reports suggested improved motor activity in PD patients given high doses of melatonin (37 , 38) , although later studies either failed to replicate such therapeutic effects (39 , 40) or actually demonstrated worsening by melatonin (41) . More recent observations suggest that melatonin may directly effect motor system function. In fact, administration of the pineal hormone affected locomotor activity (42 , 43) , blocked L-dopa-induced movement disorder (38) , and inhibited apomorphine-induced rotation (44 , 45) . As melatonin is a lipophilic antioxidant with potent free radical-scavenging properties (19 , 23 , 46) , it could conceivably decrease the oxidative stress suggested to occur in PD and which is intensified by L-DOPA therapy (12 , 47 , 48) .

To explore further a role for melatonin in PD, we used the stereotaxic intranigral injection of 6-OHDA, a widely accepted experimental model for PD (4 , 5) . Lesion function was verified by administering the dopaminergic agonist apomorphine, which induced a contralateral rotation behavior reflecting an action at supersensitive, denervated dopamine receptors within the striatum of the lesioned site (6 7 8) . Apomorphine reduction in ipsilateral turning was likely caused by residual dopamine in the nonlesioned side. BN-PAGE histochemical analysis was used to provide quantitative evaluation of OXPHOS enzyme activities in nigral tissue (34) . In 6-OHDA-treated nigra, a 20% decrease in mitochondrial complex I activity was detected. Similar reductions have been reported after 6-OHDA application in vitro (49) and in vivo (50) , and in rats treated chronically with L-DOPA (51) . Complex I activity was reduced in postmortem nigral tissue from patients treated with L-DOPA (15 , 52) . It is noteworthy that mitochondrial complex I is of critical importance in the control of oxidative phosphorylation. In isolated brain mitochondria, a 25% reduction in enzyme activity is sufficient to impair ATP synthesis (53) . Further, a significant reduction in complex IV activity was reported in experimental PD (14) . In the present model no significant decrease in complex IV activity was observed. However, in isolated brain mitochondria, a 70% loss of complex IV activity is needed to produce major reductions in oxygen consumption and ATP synthesis (53) .

Inappropriate activation of apoptosis by dopamine and/or its oxidation products has been hypothesized to initiate nigral cell loss in PD (54 55 56) . Elevated ROS may participate in 6-OHDA neurotoxicity, as evidenced by reductions in brain GSH (e.g., -22% in striatum) and loss in SOD activity (-22% in striatum) (57 58 59) . Furthermore, 6-OHDA appears to be more toxic to complex I than 1-methyl-4-phenylpyridinium ion (MPP+) (49) . Inhibition of complex I stimulates mitochondrial production of superoxide free radicals (60) , hydrogen peroxide, and hydroxyl radicals (63) and may also trigger apoptotic mechanisms (1) .

Melatonin prevents neuronal apoptosis triggered by ROS (26) and brain injury caused by singlet oxygen (62) , and is effective in reducing kainic acid (27) and hydrogen peroxide (63) -induced lipid peroxidation in brain homogenates. Moreover, melatonin maintains glutathione homeostasis and protects against loss of glutathione reductase activity in kainate-treated rats (22) . The neuroprotective effects of melatonin appear to be mediated by the antioxidant capacity of this pineal hormone (64) . In the present study, a bolus of melatonin (5 mg/kg) given immediately after 6-OHDA stereotaxic injection failed to modify apomorphine-induced contralateral rotations. This is consistent with the short (20 min) biological half-life of melatonin (64) . Increasing melatonin bioavailability (1.2 mg/day for 7 days), however, clearly decreased the severity of hemi-Parkinson conditions caused by 6-OHDA. Apomorphine administered 14 days postlesion significantly decreased contralateral turning in these rats. BN-PAGE histochemical analysis of melatonin-treated animals revealed a sparing of mitochondrial complex I activity when compared to contralateral (untreated) nigra. Plasma concentrations of 1660 ± 240 pg/ml melatonin, i.e., ~80- and 30-fold above normal daytime (20±6.4 pg/ml) (65) and night (50±4.7 pg/ml) (65) values, respectively, are thus able to protect rats from injury by unilateral stereotaxic injection of 6-OHDA into substantia nigra.

In summary, prolonged melatonin bioavailability in 6-OHDA-treated rats produced a significant recovery from lesion-induced motor deficits. Melatonin also prevented a loss in mitochondrial complex I activity. Together with previous reports (66 , 67) , the data indicate that melatonin exerts a potent antioxidant action on the nigrostriatal dopaminergic system. While melatonin may be of potential use in treating neurological disorders associated with oxidative stress (e.g., PD), this lipophilic hormone has access to all cells and intracellular body compartments (68) and may be capable of producing pharmacological actions also at the genomic level (69) .


   ACKNOWLEDGMENTS
 
This work was partially supported by MURST 1998_prot. 9805089988_007 and by ‘Programma Biotecnologie: azioni previste dalla Legge No. 95 del 29–3-1995’.

Received for publication March 6, 2000. Revision received June 19, 2000.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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