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Neurochemistry International 49 (2006) 698–707

Involvement of G-protein bg subunits on the influence of inhibitory

a2-autoreceptors on the angiotensin AT1-receptor modulation of

noradrenaline release in the rat vas deferens

Carlos Talaia a, Gloria Queiroz a,*, Helder Pinheiro a,Daniel Moura b, Jorge Goncalves a

a Laboratory of Pharmacology, CEQOFFUP, Faculty of Pharmacy, University of Porto,

Rua Anıbal Cunha, 164, 4050-047 Porto, Portugalb Institute of Pharmacology and Therapeutics, Faculty of Medicine, University of Porto,

Alameda Hernani Monteiro, 4200-319 Porto, Portugal

Received 9 March 2006; received in revised form 12 June 2006; accepted 7 July 2006

Available online 7 September 2006

Abstract

The influence of a2-autoreceptors on the facilitation of [3H]-noradrenaline release mediated by angiotensin II was studied in prostatic portions

of rat vas deferens preincubated with [3H]-noradrenaline. Angiotensin II enhanced tritium overflow evoked by trains of 100 pulses at 8 Hz, an effect

that was attenuated by the AT1-receptor antagonist losartan (0.3–1 mM), at concentrations suggesting the involvement of the AT1B subtype. The

effect of angiotensin II was also attenuated by inhibition of phospholipase C (PLC) and protein kinase C (PKC) indicating that prejunctional

AT1-receptors are coupled to the PLC–PKC pathway.

Angiotensin II (0.3–100 nM) enhanced tritium overflow more markedly, up to 64%, under conditions that favor a2-autoinhibition, observed

when stimulation consisted of 100 pulses at 8 Hz, than under poor a2-autoinhibition conditions, only up to 14%, observed when a2-adrenoceptors

were blocked with yohimbine (1 mM) or when stimulation consisted of 20 pulses at 50 Hz. Activation of PKC with 12-myristate 13-acetate (PMA,

0.1–3 mM) also enhanced tritium overflow more markedly under strong a2-autoinhibition conditions. Inhibition of Gi/o-proteins with pertussis

toxin (8 mg/ml) or blockade of Gbg subunits with the anti-bg peptide MPS-Phos (30 mM) attenuated the effects of angiotensin II and PMA.

The results indicate that activation of AT1-receptors coupled to the PLC–PKC pathway enhances noradrenaline release, an effect that is

markedly favoured by an ongoing activation of a2-autoreceptors. Interaction between a2-adrenoceptors and AT1-receptors seems to involve the bg

subunits released from the Gi/o-proteins coupled to a2-adrenoceptors and protein kinase C activated by AT1-receptors.

# 2006 Elsevier Ltd. All rights reserved.

Keywords: Noradrenaline release; AT1-receptors; Protein kinase C; Gi/o-coupled receptors; Gbg subunits; Receptor crosstalk

Release of noradrenaline from sympathetic nerve terminals

may be modulated (facilitated or inhibited) by activation of

Abbreviations: AC, adenylyl cyclase; Rp-cAMPS, Rp-adenosine-30,50-cyc-

lic-monophosphorothioate triethylammonium; 9-CP-Ade, 9-(cyclopentyl)-ade-

nine; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; PTX, pertussis toxin; PMA,

phorbol 12-myristate 13-acetate; 4-M-PMA, phorbol-12-myristate-13-acetate

4-O-methyl ether; PKC, protein kinase C; PKA, protein kinase A; PLC,

phospholipase C; Ro 32-0432, bisindolylmaleimide XI; TEA, tetraethylamo-

nium; U-73122, 1-[6-[((17b)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]-

hexyl]-1H-pyrrole-2,5-dione; U-73343, 1-[6-[((17b)-3-methoxyestra-

1,3,5[10]-trien-17-yl)amino]hexyl]-2,5-pyrrolidinedione; Brimonidine, 5-

bromo-N-(2-imidazolin-2-yl)-6-quinoxalinamine

* Corresponding author. Tel.: +351 22 2078 970; fax: +351 22 2078 969.

E-mail address: gloria@ff.up.pt (G. Queiroz).

0197-0186/$ – see front matter # 2006 Elsevier Ltd. All rights reserved.

doi:10.1016/j.neuint.2006.07.002

presynaptic receptors; the activation of one type of receptor

may modify the effect mediated by another (Boehm and

Kubista, 2002), a mutual influence that may involve receptors

coupled to different signalling pathways.

Facilitation of noradrenaline release mediated by b2-

adrenoceptors, which are coupled to the Gs–adenylyl cyclase

(AC)–protein kinase A (PKA) pathway, may be attenuated by

simultaneous activation of Gi/o-protein coupled receptors

(Majewski and Rand, 1981; Johnston and Majewski, 1986;

Apparsundaram and Eikenburg, 1995; Queiroz et al., 2003)

whereas facilitation mediated by bradykinin B2 receptors,

which are coupled to the Gq/11-phospholipase C (PLC)–protein

kinase C (PKC) pathway, seems to require always the

simultaneous activation of presynaptic Gi/o-protein coupled

C. Talaia et al. / Neurochemistry International 49 (2006) 698–707 699

Fig. 1. Experimental protocol. Tissues were incubated with [3H]-noradrenaline

([3H]NA; 0.1 mM), transferred to superfusion chambers and superfused with

medium containing the drugs indicated in the Section 1.2. In some experiments,

before incubation with [3H]NA, tissues were incubated with PTX (8 mg/ml) or

the anti-bg peptide (30 mM, MPS-Phos) or the respective solvents, which were

tested in the same experiment, in parallel control tissues. Up to five trains of 100

pulses at 8 Hz or 20 pulses at 50 Hz (S0–S4) were applied, every 30 min. S0 was

applied at t = 30 min of the superfusion but was not used for determination of

tritium overflow (not shown). Superfusate samples were collected at 5 min

intervals, from t = 55 min onwards. Ordinates represent tritium outflow as

fraction of the tissue tritium content at the onset of the respective collection

period. The area of the peaks represents tritium overflow evoked by electrical

stimulation (S1–S4). Agonists were added to the superfusion medium as

indicated. Values represent the fractional rate of tritium outflow from sol-

vent-treated tissues stimulated with trains 100 pulses at 8 Hz.

receptors (Cox et al., 2000; Trendelenburg et al., 2003; Mota

and Guimaraes, 2003). However, inconsistent effects have been

published on the influence of Gi/o-protein coupled receptors on

the effects of other Gq/11-PLC–PKC coupled receptors, such as

the angiotensin AT1-receptors. Angiotensin II mediated

facilitation of noradrenaline release was reported to be

enhanced (rabbit heart: Starke and Schumann, 1972; guinea

pig atria: Brasch et al., 1995; mouse tissues: Cox et al., 2000;

Trendelenburg et al., 2003; hearts of newborn rats: Mota and

Guimaraes, 2003), attenuated (rabbit pulmonary arteries: Costa

and Majewski, 1988) or not influenced (rat-tail artery; Mota

et al., 2000) by activation Gi/o-coupled receptors, such as the

a2-adrenoceptors.

Interaction between presynaptic receptors, although may not

be a general rule, may represent an important mechanism of fine

regulation of transmitter release under physiological conditions

and may also have important pathophysiological implications.

For example, in spontaneous hypertensive rats, hypertension

may be caused by an augmented crosstalk between receptors

activated by vasoactive peptides, such as angiotensin or

vasopressin, and a2-adrenoceptors in the renal microcirculation

(Jackson et al., 2001; Gao et al., 2003). The mechanisms

involved in such interactions are not fully elucidated, but some

evidences indicate it may occur at some step of the signalling

pathways to which the receptors are coupled (Starke, 2001).

The vas deferens has been widely used as a model to study

the postganglionic sympathetic transmission and its regulation.

In this tissue, noradrenaline release is modulated by several

presynaptic autoreceptors such as a2- and P2-receptors (von

Kugelgen et al., 1993) and by heteroreceptors such as the b2-

adrenoceptors, activated by blood born adrenaline and

adenosine A1-, A2A- and A2B-receptors, activated by locally

formed adenosine (Goncalves et al., 1996; Queiroz et al., 2003,

2004). Some of these receptors have been shown to interact. For

example, facilitation of noradrenaline release mediated by

adenosine A2-receptors, but not that mediated by b2-

adrenoceptors, is enhanced by tonic activation of Gi/o-protein

coupled receptors (Queiroz et al., 2003; Talaia et al., 2005). In

the rat vas deferens, angiotensin II also modulates sympathetic

transmission, through activation of AT1-receptors (Cox et al.,

1995; Sum et al., 1996). However, the contribuition of a

prejunctional modulation of noradrenaline release and the

influence of other prejunctional receptors on a putative release

facilitatory effect of angiotensin II in this tissue have not been

investigated. The present study aimed to investigate the effect

of angiotensin II on noradrenaline release, in the prostatic

portion of rat vas deferens, and whether the effect is influenced

and how it is influenced by activation of presynaptic

Gi/o-protein coupled receptors (i.e. a2-adrenoceptors).

1. Experimental procedures

1.1. Materials and solutions

The following drugs were used: levo-[ring-2,5,6-3H]-noradrenaline

([3H]NA), specific activity 53.0 Ci mmol�1 was from DuPont NEN (Garal,

Lisboa, Portugal); desipramine hydrochloride (DMI), 8-cyclopentyl-1,3-dipro-

pylxanthine (DPCPX), yohimbine hydrochloride, angiotensin II human,

5-bromo-N-(2-imidazolin-2-yl)-6-quinoxalinamine (brimonidine), phorbol

12-myristate 13-acetate (PMA), phorbol-12-myristate-13-acetate 4-O-methyl

ether (4-M-PMA), bisindolylmaleimide XI hydrochloride (Ro 32-0432), 9-

cyclopentyladenine (9-CP-Ade), Rp-adenosine-30,50-cyclic-monophosphor-

othioate triethylammonium (Rp-cAMPS), 1-(6-(((17b)-3-methoxyestra-1,3,5

(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U-73122), 1-(6-(((17b)-

3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-2,5-pyrrolidinedione (U-

73343), pertussis toxin (PTX) and tetraethylamonium (TEA) were from Sigma

(Sintra, Portugal); losartan was from (Merck Portuguesa, Lisbon, Portugal) and

the anti-bg peptide AAVALLPAVLLALLAVTDQLGEDFFAVDLEAFL-QEF-

GLLPEKE (MPS-Phos) was from AnaSpec, Inc. (San Jose, USA). Stock

solutions of drugs were prepared with DMSO or distilled water and kept at

�20 8C. Solutions of drugs were prepared from aliquots of stock solutions that

were diluted in buffer immediately before use. The final concentration of

DMSO was 0.02%, being tested in parallel control tissue preparations.

1.2. Experimental protocol

Adult male Wistar rats (250–300 g; IBMC, Porto, Portugal) were used.

Animal handling and experiments were conducted according to the guidelines

of the European Communities Council Directive (86/609/EEC). Animals were

killed by cervical dislocation and exsanguination. Prostatic portions of vas

deferens were dissected out, cleaned of connective tissue and divided in portions

of 13 � 5 mg weight. The procedures used to label tissue preparations with

[3H]NA and to evaluate changes on electrically evoked tritium overflow as an

indicator of changes on neuronal noradrenaline release have been previously

described (e.g. Queiroz et al., 2004). Tissue preparations were incubated in 2 ml

of Krebs solution containing [3H]NA (0.1 mM; specific activity of

53.0 Ci mmol�1) for 40 min at 37 8C. In experiments with the anti-bg peptide

MPS-Phos or PTX, incubation conditions were adapted according to the

literature (Lai et al., 1983; von Kugelgen et al., 1993; Chang et al., 2000)

and tissues were incubated with anti-bg peptide MPS-Phos (30 mM) or PTX

(8 mg/ml) for 120 min and in the last 40 min period [3H]NA was also present

(Fig. 1). PTX was pre-activated by incubation with dithiothreitol (DTT) for

C. Talaia et al. / Neurochemistry International 49 (2006) 698–707700

30 min at 37 8C (Kaslow et al., 1987). Control tissues, tested in parallel (in the

same experiment), were incubated with the respective solvents (PBS for anti-bg

peptide and 5 mM DDT for PTX).

Individual preparations were then transferred to superfusion chambers

where they were held between platinum electrodes 7 mm apart, and superfused

with [3H]NA free medium at a constant flow rate of 1 ml min�1. A stimulator

(Hugo Sachs Elektronic-Type 215, March-Hungstetten, Germany), operating in

the constant current mode, was used for electrical field stimulation with square

wave pulses of 1 ms width and 50 mA current strength, yielding a voltage drop

of 20 V per chamber. The stimulation periods consisted of 100 pulses at 8 Hz or

20 pulses at 50 Hz. Up to five stimulation periods were applied. A primer

stimulation period, applied at t = 30 min (t = 0 min was the onset of super-

fusion) was not used for determination of tritium overflow; unless otherwise

indicated, subsequent stimulation periods were applied at t = 60 min (S1), t = 90

(S2), t = 120 (S3) and t = 150 (S4) min. Superfusate samples were collected at

5 min intervals, from t = 55 min onwards (Fig. 1). At the end of each experi-

ment, tritium content was determined in superfusate samples and in tissue

preparations by scintillation spectrometry (Beckman LS 6500, Beckman

Instruments, Fullerton, USA).

The solution used for incubation and superfusion contained (mM): NaCl

118.6, KCl 4.70, CaCl2 2.52, MgSO4 1.23, NaHCO3 25.0, glucose 10.0,

ascorbic acid 0.3 and dissodium EDTA 0.031; it was continuously gassed with

a mixture of 95% O2, 5% CO2 and maintained at 37 8C. Desipramine (0.4 mM;

to inhibit neuronal uptake of noradrenaline) and DPCPX (0.1 mM; to block

adenosine A1-receptors) were added at the beginning of superfusion and kept

throughout. When indicated, yohimbine (a2-adrenoceptor antagonist), U-73122

or U-73343 (phospholipase C inhibitor and its inactive analogue, respectively),

were also added at the beginning of superfusion and kept throughout. Unless

otherwise indicated, enzyme inhibitors and losartan were added to the super-

fusion medium 25 and 60 min before S2, respectively, and kept until the end of

the experiment; other drugs were added to the superfusion medium as indicated:

angiotensin II was added 10 min before Sn, PMA or its inactive analogue 4-M-

PMA were added 15 min before Sn and all were kept until the end of the

respective stimulation period. In tissues pre-incubated with the anti-bg peptide

MPS-Phos and PTX, or the respective solvents, angiotensin II, PMA or

isoprenaline, yohimbine or brimonidine were all tested in the same tissue

preparations: angiotensin II on S2, PMA on S3 and yohimbine on S4 (added

20 min before S4 and kept until the end of the experiment). In a set of these

experiments, isoprenaline instead of PMA, and brimonidine instead of yohim-

bine were assayed; they were added 6 min before S3 or before S4, respectively

and kept until the end of the stimulation period. TEA, when tested was added

25 min before S2 and kept until the end of the experiment.

Table 1

Basal tritium outflow (b1) and electrically evoked tritium overflow (S1) from contr

Drugs present Basal tritium

(b1; % of tissu

Throughout superfusion

100 pulses/8 Hz

Solvent 0.119 � 0.005

Yohimbine (1 mM) 0.152 � 0.038

20 pulses/50 Hz

Solvent 0.125 � 0.030

During incubation

Solvent (DTT; 5 mM) 0.270 � 0.056

DTT + PTX (8 mg/ml) 0.317 � 0.040

Solvent (PBS) 0.122 � 0.004

PBS + MPS-Phos (30 mM) 0.127 � 0.010

Preparations were incubated with [3H]-noradrenaline and superfused with medium c

throughout superfusion or present during the incubation). Pertussis toxin (PTX) and th

phosphate buffer saline (PBS), were present during the incubation period (see Section

(S0–S4). Presented values were obtained from control tissue preparations not expo

immediately before S1. Evoked tritium overflow was calculated by subtracting the

subsequent to the first stimulation period (S1), and was expressed as percentage of

means � S.E.M. for (n) tissue preparations. Significant differences from basal outfl* P < 0.05.

1.3. Evaluation of drug effects on evoked tritium overflow

Tritium outflow was calculated as fraction of the tissue tritium content at the

onset of the respective collection period (fractional rate of outflow, min�1). Drug

effects on basal tritium outflow were evaluated by the bn/b1 ratios and expressed

as percentage of change from the respective mean ratio obtained in the appro-

priate control; bn was the fractional rate of outflow in the 5 min period before S2–

S4 (b2–b4, respectively); b1 was the fractional rate of outflow in the 5 min period

before S1. Tritium overflow evoked by electrical stimulation was calculated by

subtracting the estimated basal outflow from the total outflow observed during

and in the 10 min period subsequent to each stimulation period and was expressed

as percentage of the total tritium present in the tissue at the onset of the

stimulation (Fig. 1). Effects of drugs added after S1 on tritium overflow were

estimated by ratios of the overflow elicited by S2–S4 (Sn) and the overflow elicited

by S1 (Sn/S1). Sn/S1 values obtained in individual experiments in which a test

compound ‘‘A’’ was added after S1 were calculated as a percentage of change

(increase or decrease) from the respective mean ratio obtained in the appropriate

control group (solvent instead of ‘‘A’’). When interaction of ‘‘A’’, added after S1,

and a drug ‘‘B’’ either added after S1 or at the beginning of superfusion was

studied, the ‘‘appropriate control’’ was a group in which B alone was used.

1.4. Presentation of data and statistical analysis

Results are expressed as means � standard errors of the mean (S.E.M.); n is

the number of tissue preparations. Statistical analysis of the effect of drugs on

basal tritium outflow and on electrically evoked tritium overflow was performed

by using the unpaired Student’s t-test or one-way analysis of variance (ANOVA)

followed by Dunnett’s multiple comparison test. P values lower than 0.05

(P < 0.05) indicate significant differences.

2. Results

2.1. General observations

The fractional rate of basal tritium outflow immediately

before S1 (b1) and the electrically evoked tritium overflow

elicited by S1 from prostatic portions of rat vas deferens in

different experimental conditions are shown in Table 1. Under

the experimental conditions used, calcium-omission from

ol preparations of the prostatic portion of rat vas deferens

outflow

e tritium min�1)

Evoked tritium overflow

(S1; % of tissue tritium)

(34) 0.210 � 0.011 (34)

(16) 0.837 � 0.064 (16)*

(8) 0.161 � 0.039 (8)*

(8)* 0.211 � 0.019 (8)

(8)* 0.285 � 0.025 (8)

(6) 0.161 � 0.012 (6)

(10) 0.174 � 0.014 (10)

ontaining DMI (0.4 mM) plus DPCPX (0.1 mM) and the drugs indicated (added

e anti-bg peptide (MPS-Phos) or the respective solvents: dithiothreitol (DTT) or

1.2). Up to five trains of 100 pulses at 8 Hz or 20 pulses at 50 Hz were applied at

sed to any drug after S1; b1 refers to tritium outflow during the 5 min period

estimated basal outflow from total outflow observed during and in the 10 min

the total tritium present in the tissue at the onset of the stimulation. Values are

ow or from tritium overflow in the presence of solvent at 100 pulses at 8 Hz.

C. Talaia et al. / Neurochemistry International 49 (2006) 698–707 701

superfusion medium abolished tritium overflow but not basal

outflow (not shown). The adenosine A1 receptor antagonist

DPCPX (0.1 mM) was present throughout superfusion in all

experiments to prevent the influence of tonic activation of

inhibitory adenosine A1 receptors.

Tissues were stimulated with trains of 100 pulses at 8 Hz or

20 pulses at 50 Hz. The stimulation conditions used were

selected in order to provide different levels of tritium overflow

and ongoing a2-autoinhibition (Singer, 1988). Electrical

stimulation with trains of 20 pulses at 50 Hz, elicited an

overflow of tritium which was about 67% of that elicited

by trains of 100 pulses at 8 Hz (see Table 1). The level of

ongoing a2-autoinhibition was assessed by the effect of the

a2-adrenoceptor antagonist yohimbine (1 mM) on tritium

overflow. Stimulation with trains of 100 pulses at 8 Hz

elicited an overflow of tritium that was much higher when the

a2-adrenoceptor antagonist yohimbine (1 mM) was present,

indica-ting a marked ongoing a2-autoinhibition under these

Fig. 2. Effect of angiotensin II on electrically evoked tritium overflow from

prostatic portion of rat vas deferens in the presence of solvent, the AT1 receptor

antagonist losartan, the PLC inhibitor U-73122 or its inactive analogue U-

73343, the protein kinase C inhibitor Ro 32-0432, the adenylyl cyclase inhibitor

9-CP-Ade, or the protein kinase A inhibitor cAMPS-Rp. Tissues were stimu-

lated with three trains (S0–S2) of 100 pulses at 8 Hz. Angiotensin II was added

10 min before S2 and was kept until the end of respective stimulation period. U-

73122 and U-73343 were added at the beginning of superfusion and were kept

throughout; other enzyme inhibitors were added 20 min before S2 and kept until

the end of the experiment. In experiments with losartan, S2 was applied at min

90 of superfusion and the antagonist was added 60 min before S2 and kept

throughout. Ordinates, tritium overflow expressed as percentage of change from

the respective control (see Section 1.3). Values are means � S.E.M. from 5 to 25

tissue preparations. Significant differences from respective control: *P < 0.05;

from the effect of angiotensin II in the presence of solvent: +P < 0.05.

stimulation conditions (Table 1). When added 20 min before S2,

yohimbine (1 mM) caused an enhancement of tritium overflow

of 524 � 29% (n = 12; P < 0.05). Tritium overflow elicited by

trains of 20 pulses at 50 Hz was under less a2-autoinhibition

since 1 mM yohimbine, added 20 min before S2, caused an

enhancement of tritium of 170 � 17% (n = 6; P < 0.05).

In some experiments, tissues were incubated with PTX

(8 mg/ml) and MPS-Phos (30 mM; Table 1). None of these

drugs changed tritium overflow but PTX caused an increase on

basal outflow. Drugs present throughout or added after S1 did

not change basal outflow and bn/b1 ratios were close to unity

(not shown).

2.2. Effects of angiotensin II on the modulation of tritium

overflow

In the vas deferens, angiotensin II has been shown to

enhance sympathetic transmission (Cox et al., 1995; Sum et al.,

1996). The question remains if enhancement of noradrenaline

release mediated by prejunctional receptors also contributes to

the enhancement of sympathetic transmission. This question

was addressed in the present study by investigating the effects

of angiotensin II on tritium overflow elicited by trains of 100

pulses at 8 Hz. As shown in Fig. 2, angiotensin II (30 nM)

markedly enhanced electrically evoked tritium overflow. The

AT1-receptor antagonist losartan (0.3–1 mM) did not change

basal outflow or evoked tritium overflow (not shown), but

markedly attenuated the enhancement of tritium overflow

caused by angiotensin II (Fig. 2), indicating that AT1-receptors

are involved in the facilitation of noradrenaline release.

Angiotensin AT1-receptors are usually coupled to the Gq/11–

PLC–PKC pathway (De Gasparo et al., 2000). The possibility

that the PLC–PKC pathway was involved in the observed AT1-

receptor-mediated increase of tritium overflow was investigated

by testing the effects of the PLC inhibitor U-73122, of its inactive

analogue U-73343 and of the PKC inhibitor Ro 32-0432 on the

enhancement of tritium overflow caused by angiotensin II. The

involvement of the AC-PKA pathway, whose activation may

also cause presynaptic facilitation of transmitter release, was

also investigated by testing the influence of the AC inhibitor

9-CP-Ade and the PKA inhibitor Rp-cAMPS.

Basal outflow, but not tritium overflow, was increased when

the PLC inhibitor U-73122 (1 mM) was added throughout

superfusion: b1 = 0.159 � 0.009 (n = 6; P < 0.05) and

S1 = 0.172 � 0.015 (n = 6), when compared with b1 and S1

values in the presence of solvent (Table 1). However, this effect

cannot be ascribed to PLC inhibition since it also occurred in

the presence of the inactive analogue U-73343 (1 mM):

b1 = 0.245 � 0.011 (n = 6; P < 0.05), compared with b1 values

obtained in the presence of solvent (Table 1). As observed with

the PLC inhibitor, tritium overflow was not changed by the

inactive analogue U-73343: S1 = 0.175 � 0.023 (n = 6). The

basal outflow and evoked tritium overflow remained constant

throughout the experiments; b2/b1 was 0.93 � 0.04 (n = 6) and

0.94 � 0.03 (n = 6) for U-73122 and U-73342, respectively,

and the S2/S1 ratios were 1.03 � 0.03 (n = 6) and 0.98 � 0.04

(n = 6) for of U-73122 and U-73342, respectively. Inhibitors of

C. Talaia et al. / Neurochemistry International 49 (2006) 698–707702

PKC, AC and PKA, when tested alone, did not change basal

outflow or tritium overflow (not shown).

Enhancement of tritium overflow elicited by angiotensin II

(30 nM) was markedly attenuated by the PLC inhibitor U-

73122 (1 mM) and by the PKC inhibitor Ro 32-0432 (1 mM)

and was not changed by U-73343 (1 mM; Fig. 2). Inhibition of

the AC-PKA pathway with the AC inhibitor 9-CP-Ade

(100 mM) or with the PKA inhibitor Rp-cAMPS (30 mM)

did not change the enhancement of tritium overflow caused by

angiotensin II (30 nM; Fig. 2).

2.3. Effects of angiotensin AT1-receptors under different

levels of ongoing a2-autoinhibition

In several sympathetically innervated tissues, the level of

ongoing activation of a2-autoreceptors has been shown to

Fig. 3. Effect of angiotensin II on electrically evoked tritium overflow from the

prostatic portion of rat vas deferens under different levels of a2-autoinhibition:

100 pulses at 8 Hz (marked a2-autoinhibition) or 100 pulses at 8 Hz plus

yohimbine (1 mM) and 20 pulses at 50 Hz (poor a2-autoinhibition). Tissues

were stimulated with five trains (S0–S4) of 100 pulses at 8 Hz or 20 pulses at

50 Hz. Yohimbine (1 mM), when present, was added at the beginning of

superfusion and kept throughout. Angiotensin II was added 10 min before

S2–S4, at increasing concentrations and kept until the end of respective

stimulation period. Ordinates, tritium overflow expressed as percentage change

from the respective control (see Section 1.3). Values are means � S.E.M. from

eight tissue preparations. Significant differences from respective control:*P < 0.05; from the effect of angiotensin II at 100 pulses at 8 Hz: +P < 0.05.

influence the release-enhancing effects of angiotensin II.

Therefore, the influence of a2-autoreceptor activation on the

AT1-receptor-mediated enhancement of tritium overflow was

investigated in order to verify whether this influence occurs in

the rat vas deferens.

When tissues were stimulated with trains of 100 pulses at

8 Hz, angiotensin II (0.3–100 nM) enhanced tritium overflow in

a concentration-dependent manner (Fig. 3). The maximal

enhancement caused by angiotensin II was about 67%

(Fig. 3). The estimated EC50 (i.e., the concentration that caused

half of that enhancement, calculated by interpolation from the

nearest points of the concentration–response curve; Fig. 3) was

about 2 nM. The enhancement of tritium overflow caused by

Fig. 4. Effect of the protein kinase C activator PMA and of its inactive analogue

4-M-PMA, on electrically-evoked tritium overflow from prostatic portion of rat

vas deferens in the presence of different levels of a2-autoinhibition: 100 pulses

at 8 Hz (marked a2-autoinhibition) or 100 pulses at 8 Hz plus yohimbine

(1 mM) and 20 pulses at 50 Hz (poor a2-autoinhibition). Tissues were stimu-

lated with five trains (S0–S4) of 100 pulses at 8 Hz or 20 pulses at 50 Hz, in

experiments with 4-M-PMA only Three trains (S0–S2) were applied. Yohimbine

(1 mM), when present, was added at the beginning of superfusion and kept

throughout. PMA was added 15 min before S2–S4 at increasing concentrations,

and kept until the end of respective stimulation period. 4-M-PMA (3 mM) was

added 15 min before S2 and kept until the end of respective stimulation period.

Ordinates, tritium overflow expressed as percentage change from the respective

control (see Section 1.3). Values are means � S.E.M. from 5 to 10 tissue

preparations. Significant differences from respective control: *P < 0.05; from

the effect of PMA at 100 pulses at 8 Hz: +P < 0.05.

C. Talaia et al. / Neurochemistry International 49 (2006) 698–707 703

angiotensin II was attenuated in the presence of the a2-

adrenoceptor-antagonist yohimbine (1 mM) and when tissues

were stimulated under conditions that also led to poor ongoing

a2-autoinhibition (20 pulses at 50 Hz, Fig. 3), indicating that

effects mediated by prejunctional angiotensin AT1-receptors on

tritium overflow are also influenced by the ongoing activation of

a2-autoreceptors, and the existence of an interaction between

AT1-receptors and a2-adrenoceptors in this tissue.

2.4. Mechanism of interaction between angiotensin AT1

and a2-adrenoceptors

In order to elucidate the molecular mechanisms of the

interaction between AT1-receptors and a2-adrenoceptors, the

effect of several drugs that interfere with the signalling pathways

to which these receptors are coupled was tested. The effects of

two compounds that disrupt the Gi/o-proteins, PTX and the anti

bg-peptide MPS-Phos, were investigated on the modulation of

tritium overflow mediated by a2-adrenoceptors and on the

effects of angiotensin II and PMA, which activate the AT1-

transduction pathway at the receptor or PKC level, respectively.

The results obtained indicate that prejunctional AT1-

receptores are coupled to the PLC–PKC pathway. Therefore,

the influence of a direct activator of PKC on tritium overflow,

evoked under different levels of ongoing a2-autoinhibition (see

Section 2.1) was tested. The PKCactivator PMA (0.1–3 mM), but

not its inactive analogue 4-M-PMA, enhanced, in a concentra-

tion-dependent manner, tritium overflow evoked by 100 pulses at

8 Hz (Fig. 4). The effect of PMA, like that caused by angiotensin

II, was attenuated by blockade of a2-adrenoceptors with 1 mM

yohimbine or when tissues were stimulated under conditions

Fig. 5. Effect of the a2-adrenoceptor agonist brimonidine (A) and the a2-adrenocep

prostatic portions of rat vas deferens incubated with PTX or the anti-bg peptide MPS

up to five trains (S0–S4) of 100 pulses at 8 Hz. Angiotensin II was added 10 min befo

results on Fig. 6); brimonidine or yohimbine were added 6 and 20 min before S4, re

Ordinates, tritium overflow expressed as percentage change from the respective contr

Significant differences from respective control: *P < 0.05; from the effect of yohimbi+P < 0.05.

that also led to poor ongoing a2-autoinhibition (20 pulses at

50 Hz; Fig. 4).

Incubation of the tissues with PTX (8 mg/ml), at a concen-

tration that hasbeen shown to uncouple Gi/o-proteins in this tissue

(Lai et al., 1983; von Kugelgen et al., 1993) prevented the

decrease on tritium overflow caused by the a2-adrenoceptor

agonist brimonidine (1 mM; Fig. 5A). A similar effect was

observed when tissues were incubated with the anti-bg peptide

MPS-Phos, an inhibitor of Gbg subunits (Chang et al., 2000; Orr

et al., 2002), at a concentration shown to prevent effects mediated

by Gbg subunits (30 mM; Chang et al., 2000). Furthermore,

yohimbine (1 mM) enhanced tritium overflow by preventing

ongoing activation of a2-autoreceptors (Fig. 5B) but this effect

was much smaller in tissues that were previously incubated with

PTX and MPS-Phos (Fig. 5B). Taken together, these results

indicate that, under these experimental conditions, PTX and

MPS-Phos are inhibiting the pathway to which the a2-adre-

noceptors are coupled.

The enhancement of tritium overflow caused by angiotensin II

(30 nM) and PMA (1 mM), but not that caused by the b-

adrenoceptor agonist isoprenaline, was markedly attenuated in

tissues treated with PTX (8 mg/ml; Fig. 6). Isoprenalinewas used

asnegative control, in this set of experiments because it facilitates

noradrenaline release by activation of b2-adrenoceptors that are

not coupled to Gq-proteins and the effect they mediate is not

influenced by ongoing activation of a2-adrenoceptors (Talaia

et al., 2005).

The anti-bg peptide MPS-Phos (30 mM) also attenuated

the enhancement of tritium overflow caused by angiotensin II

(30 nM) or PMA (1 mM; Fig. 6). However, MPS-Phos failed

to change the enhancement of tritium overflow caused by the

tor antagonist yohimbine (B), on electrically evoked tritium overflow from the

-Phos or the respective solvents (see Section 1.2). Tissues were stimulated with

re S2, PMA or isoprenaline were added 15 and 6 min before S3, respectively (see

spectively, and all were kept up to the end of the respective stimulation period.

ol (see Section 1.3). Values are means � S.E.M. from 6 to 12 tissue preparations.

ne in tissues incubated the respective solvents of PTX and of the anti-bg peptide,

C. Talaia et al. / Neurochemistry International 49 (2006) 698–707704

Fig. 6. Effects of angiotensin II, PMA, isoprenaline and TEA, on electrically

evoked tritium overflow from prostatic portion of rat vas deferens incubated

with PTX or anti-bg peptide (anti-bg) or the respective solvents (see Section

1.2). Tissues were stimulated with up to five trains (S0–S4) of 100 pulses at 8 Hz.

Angiotensin II was added 10 min before S2, PMA or isoprenaline were added 15

and 6 min before S3, respectively, and all were kept up to the end of the

respective stimulation period. TEA was added 20 min before S2 and kept

throughout. Only in experiments with TEA, yohimbine (1 mM) was present

throughout superfusion. Ordinates, tritium overflow expressed as percentage of

change from the respective control (see Section 1.3). Values are mean-

s � S.E.M. from 5 to 8 tissue preparations. Significant differences from

respective control (solvent): *P < 0.05; from the effect of angiotensin II,

PMA, isoprenaline and TEA in tissues incubated with the respective solvents

of PTX and of the anti-bg peptide, +P < 0.05.

non-selective K+-channel blocker TEA (1 mM; Fig. 6),

excluding the possibility that MPS-Phos attenuates the effects

of any drug that enhances tritium overflow.

3. Discussion

The electrically evoked tritium overflow from tissue

preparations pre-incubated with [3H]NA has been shown to

reflect action potential-evoked neuronal release of noradrena-

line (Starke, 1977). Therefore, changes in tritium overflow

elicited by drugs, from the prostatic portions of rat vas deferens

pre-incubated with [3H]NA, were assumed to reflect changes in

neuronal release of noradrenaline, as observed in previous

studies (Queiroz et al., 2004).

In the rat vas deferens, angiotensin II facilitates the

purinergic component of sympathetic transmission through

activation of AT1-receptors (Cox et al., 1995; Sum et al., 1996).

The present study shows that angiotensin II also facilitates

noradrenaline release, an effect that may contribute to the

overall facilitation of sympathetic transmission. This facilita-

tion was blocked by the selective AT1-receptor antagonist

losartan (Duncia et al., 1992), although concentrations much

higher than those needed to antagonise the constrictor response

to angiotensin II were needed. In our experiments the blockade

of the prejunctional angiotensin II effect was obtained only

after incubation for 60 min with 0.3 mM losartan. This agrees

with the suggestion that prejunctional angiotensin AT1-

receptors belong to the AT1B subtype, thus requiring higher

concentrations of losartan to be blocked (Pinheiro et al., 2002;

Nap et al., 2004; Guimaraes and Pinheiro, 2005).

Angiotensin AT1-receptors are generally coupled to the

Gq/11–PLC–PKC pathway, the same transduction mechanism

activated by AT1-receptors that mediate a facilitation of

noradrenaline release in the rat vas deferens. This conclusion is

supported by the observation that the effect of angiotensin II

was attenuated by inhibition of PLC with U-73122 and by

inhibition of PKC with Ro 32-0432. Both compounds have

been used at concentrations shown to selectively inhibit PLC

and PKC, respectively (Wirkner et al., 2000; Wilkinson et al.,

1993). The possibility that prejunctional angiotensin AT1-

receptors might also be coupled to the AC-PKA pathway was

excluded since the effect of angiotensin II was not changed by

inhibition of AC or PKA, with 9-CP-Ade and Rp-cAMPS,

respectively.

In the rat vas deferens, facilitation of noradrenaline by

adenosine A2A-receptors, which are coupled to the PLC–PKC

pathway and by adenosine A2B-receptors, which are coupled to

the AC-PKA pathway requires, or at least is much amplified by

activation of the release inhibitory a2-adrenoceptors (Queiroz

et al., 2003; Talaia et al., 2005). The possibility that activation

a2-autoreceptors may also influence the facilitation of nora-

drenaline mediated by angiotensin AT1-receptors was investi-

gated by studying the effects of angiotensin II on noradrenaline

release elicited under different conditions of ongoing a2-

autoinhibition. The level of a2-autoinhibition was assessed by

the enhancement of noradrenaline release caused by blockade of

a2-adrenoceptors with yohimbine, tested at 1 mM, a concentra-

tion that is more than 10-fold its pA2 value (Bylund and Ray-

Prenger, 1989). When tissues were stimulated with trains of 100

pulses at 8 Hz, noradrenaline release was under more marked

influence of a2-autoreceptors than when tissues were stimulated

with trains of 20 pulses at 50 Hz, since yohimbine enhanced

more markedly noradrenaline release evoked by trains of 100

pulses at 8 Hz than by trains of 20 pulses at 50 Hz. Stimulation

with brief trains of pulses applied at high frequency (20 pulses at

50 Hz), does not favour activation of a2-autoreceptors (Singer,

1988; Starke, 2001). Poor ongoing a2-autoinhibition conditions

were also created by stimulating the tissues with trains of 100

pulses at 8 Hz in the presence of the a2-adrenoceptor antagonist

yohimbine (1 mM), at a concentration that, in the experimental

conditions used, caused maximal facilitation of noradrenaline

release.

Angiotensin II enhanced noradrenaline release more

markedly when tissues were stimulated with trains of 100

pulses at 8 Hz (strong ongoing a2-autoinhibition conditions),

than when a2-adrenoceptors were blocked with yohimbine, or

when release was evoked by trains of 20 pulses at 50 Hz (both

conditions lead to poor ongoing a2-autoinhibition). The

possibility that noradrenaline release was already maximal

when a2-adrenoceptors were blocked, preventing further

C. Talaia et al. / Neurochemistry International 49 (2006) 698–707 705

increases from being observed, has been excluded because the

b-adrenoceptor-agonist isoprenaline caused a facilitation of

noradrenaline of the same magnitude with or without a2-

adrenoceptor blockade (Talaia et al., 2005). Furthermore, two

different conditions that lead to poor a2-autoinhibition also lead

to an attenuation of the angiotensin II effect, indicating that in

this tissue, AT1-receptors and a2-adrenoceptors interact in a

way that angiotensin AT1-receptor-mediated facilitation of

noradrenaline release requires, or is amplified, by an ongoing

activation of a2-adrenoceptors. A similar interaction has been

previously described in other sympathetically innervated

tissues (rabbit heart; Starke and Schumann, 1972; guinea pig

atria; Brasch et al., 1995; mouse atria; Cox et al., 2000; mouse

tissues; Trendelenburg et al., 2003; rat heart; Mota and

Guimaraes, 2003). However it does not seem to be a general

phenomena since in some tissues facilitation of noradrenaline

release by angiotensin II is not influenced (rat tail artery; Mota

et al., 2000) or is attenuated (rabbit pulmonary artery; Costa and

Majewski, 1988) by activation of a2-autoreceptors; therefore

the understanding of the molecular mechanism behind this

interaction may contribute to explain this variability.

Angiotensin AT1-receptors are coupled to the Gq/11–PLC–

PKC pathway. Facilitation of noradrenaline release caused by

direct activation of PKC, like facilitation caused by angiotensin

II, was smaller under poor a2-autoinhibition conditions (100

pulses at 8 Hz plus yohimbine or 20 pulses at 50 Hz). Evidence

that enhancement of tritium overflow caused by PMA is

mediated by PKC was provided by a previous observation that

the PKC inhibitor Ro 32-0432 prevents the release enhancing

effect of PMA (Queiroz et al., 2004). Since interaction is also

observed when the Gq/11–PLC–PKC pathway is activated at

PKC level, the interaction between the a2-adrenoceptors and

angiotensin AT1-receptor-signalling pathways must occur

mainly at steps downstream PKC activation. However,

involvement of other steps located upstream PKC activation

cannot be excluded since blockade of a2-adrenoceptors or

inactivation of Gi/o-proteins with PTX attenuated more

markedly the facilitation of noradrenaline release caused by

angiotensin II than that caused by direct activation of PKC.

Previous studies suggest that interaction between Gi/o-

protein coupled receptors and Gq11-protein coupled receptors

may occur at multiple steps of the signalling pathways, namely:

(i) enhancement of PLC activity by bg subunits released from

Gi/o-proteins (Blank et al., 1992; Banno et al., 1998; Selbie and

Hill, 1998); (ii) PKC removal of Ca2+-channel-inhibition

mediated by the bg subunits released from Gi/o-proteins

(Swartz, 1993; Hamid et al., 1999; Barrett and Rittenhouse,

2000; Lee et al., 2004); (iii) PKC-mediated desensitisation of

the Gi/o-proteins (Murthy et al., 2000) or the Gi/o-coupled

receptors (Fredholm and Lindgren, 1988; Liang et al., 1998).

The present study indicates an involvement of G-protein bg

subunits on this interaction. The anti-bg peptide MPS-Phos, a

cell permeable peptide that prevents the effects of bg subunits

(Chang et al., 2000; Orr et al., 2002) attenuated the facilitation

of noradrenaline release caused by angiotensin II. The main

source of bg subunits seem to be the Gi/o-proteins activated by

a2-adrenoceptors because MPS-Phos also attenuated the

release enhancing effects caused by direct activation of

PKC, which bypasses activation of Gq11-proteins and PLC

coupled to the angiotensin AT1-receptors. The effect of the

MPS-Phos seems to be selective for bg subunits because it did

not changed the effect of TEA that enhances noradrenaline

release by a mechanism independent of G-protein activation

(Kirpekar et al., 1976). Furthermore, bg subunits are key

players in the interaction observed because MPS-Phos did not

change the facilitation of noradrenaline release mediated by b-

adrenoceptors, which are coupled to G-proteins but do not

interact with the a2-adrenoceptors (Talaia et al., 2005).

Influence of bg subunits on the PLC–PKC signalling

pathway has been described upstream or downstream PKC

activation. Upstream PKC, a possible step at which interaction

may occur is the activation of PLC. It has been shown that bg

subunits released from Gi/o-proteins, upon activation of Gi/o-

protein-coupled receptors, can enhance activation of PLCb by

the Gaq subunits released from Gq11-coupled receptors (Blank

et al., 1992; Banno et al., 1998; Selbie and Hill, 1998)

contributing to trigger or amplify the effect mediated by

angiotensin AT1-receptors. A possible step downstream PKC

activation is the regulation of the opening-closure rate of

voltage-gated Ca2+-channels, a key-step in prejunctional

modulation of transmitter release and one of the targets of

the a2-adrenoceptor-mediated inhibition of noradrenaline

release (Hamid et al., 1999). The bg subunits, released from

Gi/o-proteins, inhibit Ca2+ influx and transmitter release, an

effect that can be attenuated by PKC (Swartz, 1993; Hamid

et al., 1999; Barrett and Rittenhouse, 2000; Lee et al., 2004) and

this may provide a mechanism by which PKC can cause a

facilitation of noradrenaline release that depends on the level of

ongoing a2-autoinhibition.

Irrespectively of the step at which interaction occurs, the

common link is the involvement of the bg subunits released by

activation of Gi/o-coupled receptors. The observation that MPS-

Phos also attenuated facilitation of noradrenaline release

caused by direct activation of PKC allows to conclude that: (i)

the main source of the bg subunits involved are released by

activation of Gi/o proteins; (ii) the interaction must involve steps

downstream PKC activation.

Modulation of neurotransmitter release by presynaptic

receptors was an interesting finding and changed the way

neuronal communication was understood. The release inhibi-

tory receptors are viewed as a logical mechanism for a neuron

to control the amount of neurotransmitter required, but the

physiological role of facilitatory receptors is still not yet very

clear. One possibility is that they can work to select the more

convenient cotransmitter combination (Goncalves et al., 1996;

Driessen et al., 1996). The interactions like those reported in the

present study may indicate another function for these receptors:

that they may represent a mechanism to control the effects of

the release inhibitory receptors, preventing excessive inhibi-

tion.

Interaction between prejunctional facilitatory and inhibitory

receptors involved in the modulation of noradrenaline release

have been described in several tissues, and also in the tissue

used in the present study. However, interactions do not follow a

C. Talaia et al. / Neurochemistry International 49 (2006) 698–707706

Fig. 7. Model proposed to explain the influence of a2-adrenoceptors on the

modulation of noradrenaline release by angiotensin AT1-receptors. Activation

of angiotensin AT1-receptors leads to activation of PLC and PKC. Activation of

a2-adrenoceptors, which are coupled to Gi/o-proteins, leads to the release of bg

subunits that may enhance activation of PLC (Blank et al., 1992; Banno et al.,

1998) or inhibit the secretory machinery (Blackmer et al., 2001) and the opening

of N-type Ca2+-channels (Delmas et al., 1999). PKC enhances noradrenaline

release by acting on secretory machinery (Shoji-Kasai et al., 2002) and/or the N-

type Ca2+-channels, removing the inhibition mediated by the bg subunits, and

thus causing desinhibition (facilitation) of transmitter release (Hamid et al.,

1999).

constant pattern. It is possible that the occurrence of

interactions between receptors depends on the cellular

organization of receptors or cluster of receptors, that have

common molecular targets, whose function they regulate, or

that share common intracellular signalling proteins (Oh and

Schnitzer, 2001; Hur and Kim, 2002; Razani et al., 2002).

In conclusion, in the prostatic portion of the rat vas deferens,

angiotensin AT1-receptor-activation facilitates noradrenaline

release and this may contribute to the enhancement of

sympathetic transmission reported in this tissue. The angio-

tensin AT1-receptor-mediated facilitation of noradrenaline

release is greatly enhanced by an ongoing activation of a2-

adrenoceptors and the mechanisms proposed to explain such

interaction involves steps mainly downstream PKC activation

and the bg subunits released upon activation of Gi/o-protein

coupled a2-adrenoceptors (Fig. 7).

Acknowledgements

This work was supported by FCT (I&D n. 226/94) and

POCI/SAU-FCT/60714/2004.

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