Dopamine induces intracellular Ca2+ signals mediated by alpha(1B)-adrenoceptors in rat pineal cells

Ž. European Journal of Pharmacology 430 2001 9–17 www.elsevier.comrlocaterejphar Dopamine induces intracellular Ca2qsignals mediated by a-adrenoceptors in rat pineal cells 1B Elkin Rey a, Francisco J. Hernandez-Dıaz b, Pedro Abreub, Rafael Alonso b, Lucıa Tabaresa,) ´´ ´ aDepartment of Physiology and Biophysics, School of Medicine, UniÕersity of SeÕille, AÕda. Sanchez Pizjuan, 4, 41009 SeÕille, Spain ´´ bDepartment of Physiology, School of Medicine, UniÕersity of La Laguna, Tenerife, Spain Received 3 May 2001; received in revised form 16 July 2001; accepted 27 July 2001 Abstract We have studied the functional interaction of dopamine with a-adrenoceptor subtypes by measuring intracellular Ca 2 qlevels in 1 pineal cells, a cell type where adrenoceptors are well characterized. We show that dopamine induces transient intracellular Ca 2 qsignals in only 70% of cells responding to phenylephrine. Dopamine-induced Ca 2 qsignals desensitise faster than Ca 2 qtransients elicited with phenylephrine and are selectively blocked by desipramine, imipramine, and a-adrenoceptor antagonists. These results suggest that 1B dopamine induced Ca 2 qsignals are mainly due to the activation of one subtype of a-adrenoceptor, the a.q2001 Elsevier Science 1B B.V. All rights reserved. Keywords: Dopamine; a-Adrenoceptor; Pineal cell; Catecholamine 1. Introduction Noradrenaline binds at least to 10 different adrenoceptors, which either activate or inhibit adenylate cyclase, or are coupled to phospholipase C. All adrenoceptors share three conserved residues within their transmembrane segments, which are thought to be the minimal structure for ligand binding. This residues are aspartate in the third transmembrane segment which may act as a counterion for the amine moiety in biogenic amines, and two serines in the fifth transmembrane segment thought to be involved in the recognition of the two hydroxyl groups of adrenaline Ž. and noradrenaline Strader et al., 1987, 1989 .On the other hand, dopamine binds to a different family of receptors formed by at least five subtypes. Dopamine receptors, which are mainly coupled to the activation or the inhibition of adenylate cyclase, share with adrenoceptors the same three conserved residues within their transmembrane segments for the ligand binding. This is not surprising given the structural similarity between adrenaline, noradrenaline and dopamine. The possibility that all three catecholamines may indistinctly activate dopamine receptors and adrenoceptors has )Corresponding author. Tel.: q34-954-55-1768; fax: q34-954-55- 1769. Ž. E-mail address: [email protected] L. Tabares . been long timed suspected. So far, b-adrenergic-like effects of dopamine have been demonstrated both on the Ž vascular system and the heart Rajfer et al., 1988; Oue- . draogo et al., 1998 . Similarly, in human platelets, it has been shown that dopamine increases intracellular cAMP Ž levels through the activation of b-adrenoceptors Anfossi . et al., 1993 and, in rat adipocytes, dopamine lowers glucose uptake through the activation of b-adrenoceptors 3 Ž. Lee et al., 1998 . We have studied the effect of dopamine on intracellular Ca2qlevels in rat pinealocytes, a cell type having a- and b-adrenoceptors and where the intracellular pathways activated by noradrenaline have been deeply studied. Our results show that dopamine, at low micromolar concentrations, is able to elicit intracellular Ca2qincrements due to the activation of a-adrenoceptors. The pharmacological characterisation of receptors mediating the Ca2qincrement triggered by dopamine points to the preferential activation of the a-adrenoceptor over other asubtypes. 1B 1 2. Materials and methods 2.1. Cell dissociation Pineal glands were obtained from male Wistar rats Ž. 100–150 g housed in a temperature-controlled room 0014-2999r01r$ - see front matter q2001 Elsevier Science B.V. All rights reserved. Ž. PII: S0014-2999 01 01250-X () E. Rey et al.rEuropean Journal of Pharmacology 430 2001 9–1710 Ž. 23"38C and maintained on a 12:12-h light–dark cycle Žy2. 60–80 mW cm . For each experiment, six glands were Ž washed and cleaned in cold Ringer solution containing in mM: NaCl, 145; KCl, 2.7; and HEPES, 10; pH 7.25 . NaOH , and transferred to fresh solution with 0.5 mgrml protease where they were shaken for 30 minutes. The tissue was then washed twice in Dulbecco’s Modified Ž. Eagle’s Medium BioWhittaker, Verviers, Belgium supplemented with 5% vrv foetal bovine serum, 50 u.i.rml penicillin and 50 mgrml streptomycin, mechanically dispersed in clean medium and plated onto poly-L-lysine coated cover-slips. Cells were kept in a CO incubator 2 Ž. Ž . 5% at 36.5 8C until use 1–3 days later . 2.2. Solutions and chemicals Ringers and test solutions were continuously applied by superfusion. The bars marked on the figures indicate the Ž time at which the solutions were switched dead time 2–5 .Ž. s . The composition of the solution used was in mM : NaCl, 145; KCl, 2.7; CaCl , 10; MgCl , 1 and HEPES, 22 Ž. 10; glucose 10–20, pH 7.25–7.35 NaOH . In some experiments, CaCl was omitted and 1 mM EGTA added to 2Ž. the solution. Ascorbic acid 5 mM was included in all solutions to prevent catecholamine oxidation. Salts were Ž. purchased from Sigma Madrid, Spain , agonists and antagonists were obtained from Research Biochemicals Inter- Ž. national Natik, MA, USA . Drugs were prepared immediately before use. All experiments were performed at room Ž. temperature 20–22 8C. 2.3. Ca2qmeasurements Measurements of intracellular Ca2qin single cells were Ž. done using the acetoxymethyl AM ester, membrane per- 2qŽ meable form, of the Ca indicator dye Fura-2 Fura2r . AM . Cells were incubated during 5–10 min with 2 mM Ž. Fura 2rAM Molecular Probes, OR, USA at 36.5 8C and subsequently washed in Fura-2-free solution for a minimum of 10 min. Ca2qmeasurements, carried out on the Ž. stage of an inverted microscope Zeiss, Axiovert 35 , were Ž. done as described by Almers and Neher 1985 and Marın ´ Ž. et al. 1996 . Briefly, the light from a 75-W Xenon arc lamp was alternately filtered by means of a rotating wheel at 357 and 380 nm. The filtered light was guided through a filter transmission 430-nm dichroic mirror and its emission passed through a long pass 470-nm filter before projection w2qx onto the photomultiplier tube. Ca was calculated on- i Ž. 2qŽ. Fig. 1. Dopamine DA induced intracellular Ca increments in pineal cells. A Prolonged dopamine exposure produced a response consisting of a Ž. Ž. transient peak that decline to a sustained phase above the basal level. B Short dopamine exposure elicited only the first peak. C In the presence of Ž. 2qŽ. EGTA 1 mM , and without Ca -added in the extracellular solution, the transient response was maintained, whereas the plateau was not elicited. D Relationship between the amplitude of the Ca2qspike and dopamine concentration. The curve was fitted by Michaelis–Menten type equation of the form: 2q2qwxwx Ca sCa DA rDA qK,ns116, Ks5mM. dopamine concentration in A, B and C was 5 mM; recordings from three different cells. max d d () E. Rey et al.rEuropean Journal of Pharmacology 430 2001 9–17 11 line from the ratio of the emission at 357-nm excitation to Ž. that at 390-nm according to Grynkiewicz et al. 1985 . 3. Results 3.1. Dopamine induces Ca2qmobilisation The effect of dopamine exposure in pineal cells was analysed by single-cell Ca2qrecording. Dopamine induced transient intracellular Ca2qsignals in 132 out of 189 cells Ž. 70% responding to phenylephrine, an a-adrenoceptor 1 agonist that mediates Ca2qincrements in pineal cells Ž. Sugden et al., 1987; Marın et al., 1996 . Dopamine in- ´ duced intracellular Ca2qincrements consisting of a tran- Ž. sient peak followed by a plateau Fig. 1A . The plateau phase was absent when dopamine was applied for F20 s Ž. Fig. 1B or when the recording was made in an extracellu- 2q-Ž. lar solution without Ca added plus 1 mM EGTA ns9 Ž. Fig. 1C , suggesting that the dopamine-induced transient response was mostly due to mobilisation of intracellular Ca2q, whereas the sustained phase was dependent on extracellular Ca2q. The peak amplitude of the transient Ca2qsignal increased with dopamine concentration, in the range of 1–50 mM. The relationship between the amplitude of the Ca2q spike and dopamine concentration was obtained from ex- Ž. periments performed in 116 cells Fig. 1D . The mean amplitude of the Ca2qsignal at saturating dopamine concentrations ranged between 200 and 240 nM, and half maximal amplitude was reached at a dopamine concentration of 5 mM. 3.2. Dopamine receptor agonists do not induce Ca2q mobilisation In order to study the receptor types implicated in the dopamine-mediated Ca2qresponses, the effects of differ- Ž. ent dopamine receptor agonists were tested. Neither R q- Ž. SKF-38393 25–200 mM, ns12; Fig. 2A , a dopamine Ž D -like receptor agonist, nor Quinpirole 100 mM, ns3, 1. e.g. Fig. 2B , a D -like dopamine receptor agonist, was 2 able to elicit any response on intracellular Ca2q. The same Ž result was observed with either bromocriptine 50 mM, .Ž. ns5 or apomorfine 50 mM, ns4 , dopamine D -like 2 and D rD -like receptor agonists, respectively. The lack 12 of effect of dopamine receptor agonists on intracellular Ca2qlevels indicates that the Ca2qresponses induced with dopamine in pineal cells are not mediated through dopamine receptors. 3.3. Block of the dopamine-mediated Ca2qincrements with prazosin and other catecholamine receptor antagonists In order to test whether dopamine-induced Ca2qre- sponses were affected or not by prazosin, an a-adrenoc- 1 w2qx Fig. 2. Lack of effect on Ca of dopamine receptor agonists in two iŽ. Ž. cells. Application of 50 mM SKF-38393 A or 100 mM quinpirole B did not induce Ca2qresponses, while exposure to 50 mM dopamine elicited robust signals; recordings from two different cells. eptor antagonist, cells were exposed repetitively to dopa- Ž. mine 5 mM in the presence or the absence of the antagonist. Dopamine-mediated Ca2qsignal was not af- Ž. fected by 1 nM prazosin Fig. 3A and C , while it was Ž completely abolished by a concentration of 10 nM Fig. 3B .2q and C . Within this range, Ca response peak amplitudes were inversely proportional to the prazosin concentration Ž. used Fig. 3C, ns16 . The percent of inhibition of the Ca2qresponses as a function of the prazosin concentration was calculated after correcting for the desensitisation component resulting from repetitive dopamine stimulation Ž. white bars in Fig. 3C . The corrected values are repre- Ž. sented in Fig. 3D triangles . From the curve fitting of the data points, half inhibition of the response was obtained at 3 nM prazosin, a value which is close to Kreported for i Ž. a-adrenoceptors 0.14–1.2 nM in rat and human tissues 1 Ž. O’Malley et al., 1998 . The effect of dopamine receptor antagonists on dopamine-mediated Ca2qincrease was also studied. Haloperidol, classically a dopamine D rD receptor antag- 21 onist, and spiperone, an antagonist for D -type dopamine 2 receptors, inhibited Ca2qresponses at relatively low concentrations. Fig. 3D shows the percent of inhibition of the Ca2qresponse as a function of the antagonist concentrations. Half inhibition values were 15 nM with haloperidol () E. Rey et al.rEuropean Journal of Pharmacology 430 2001 9–1712 2qŽ. Ž . Fig. 3. Inhibition of dopamine-mediated Ca signals by different catecholamine antagonists. A, B Responses to the application of dopamine 5 mM Ž. Ž. Ž. 2qŽ. before, during or after the application of prazosin at 1 nM A or 10 nM B in two cells. C Normalized peak Ca amplitudes mean"S.E.M. in Ž. 2q response to three consecutive applications of dopamine 5 mM in the presence or absence of prazosin. Sizes of white bars correspond to Ca signal amplitudes in response to dopamine in control experiments without prazosin. Height of black bars represents the amplitudes of the Ca2qresponses to the Ž. second application of dopamine in the presence of different concentrations of prazosin 1, 2, 5 or 10 nM, ns16 . Strip bar amplitudes correspond to the recovery of the Ca2qresponses during the third application of dopamine after washing out prazosin. In all cases, dopamine exposures lasted 15–20 s and Ž. 2qŽ. were repeated every 3 min. D Percent of inhibition of the Ca signals elicited by dopamine 5 mM as a function of the concentration of prazosin Ž. Ž. Ž. Ž. ns16 , haloperidol ns26 , spiperone ns20 and SCH-23390 ns22 . Ž. Ž Fig. 3D, squares, ns20 and 20 nM with spiperone Fig. .Ž. 3D, diamonds . We also tested the effect of R q-SCH- 23390, a D -type dopamine receptor antagonist, on 1 dopamine-induced Ca2qresponses. This compound was much less efficient in producing inhibition of the response Ž. Fig. 3D, circles , with an IC of 1.7 mM SCH-23390. 50 Therefore, the higher inhibiting potency of prazosin, as compared to the others antagonists, suggests that the Ca2q response to dopamine was mediated by the activation of a-adrenoceptors, and that both haloperidol and spiperone 1 inhibited, with relatively high affinity, this receptor subtype. To test this last possibility, cells were stimulated with phenylephrine in the presence of different concentrations Ž. of haloperidol Fig. 4 . As with dopamine, haloperidol inhibited the Ca2qresponses to phenylephrine with a similar potency. To study whether or not dopamine and phenylephrine activate the same subtypes of a-adrenoceptors, the effects 1 of successive pulses of these agonists applied to the same Ž. cell were compared. In most cells, dopamine 5 mM and Ž. 2q phenylephrine 1 mM elicited Ca transients of similar form and duration, although the amplitudes of the Ca2q signal obtained with dopamine were usually smaller that Ž. those obtained with phenylephrine Fig. 5A . In 30% of the cells recorded, only phenylephrine elicited a Ca2q Ž. response Fig. 5B . No morphological differences were observed under the microscope between cells responding or not to dopamine. In cells responding both to dopamine and phenylephrine, experiments with repetitive applica- Ž. tions of a high concentration of dopamine 50 mM and a Ž. relatively low phenylephrine concentration 1 mMrevealed a different rate of desensitisation. Panels C and D in Fig. 5 show two examples where such a protocol was used. In all cases, the peak amplitude of the dopamine-mediated Ca2qresponses decreased with successive applications, while those induced by phenylephrine were maintained Ž. ns5 . A negative effect of dispersion enzymes on membrane receptors was rejected since the same results were obtained in one experiment in cells that had not been treated with protease. These results suggest that dopamine and phenylephrine activate different a-adrenoceptor sub- 1 types. To test further this possibility, we explored the effect of different selective a-adrenoceptor antagonists on dopa- 1 () E. Rey et al.rEuropean Journal of Pharmacology 430 2001 9–17 13 2qŽ.Ž. Fig. 4. Intracellular Ca responses to phenylephrine PE . A Re- Ž. sponses to the application of phenylephrine 1 mM before, during of Ž. Ž. after the application of haloperidol 1 mM in one cell. B Normalized 2qŽ. peak Ca amplitudes mean"S.E.M. in response to phenylephrine Ž.Ž. 15–20 s exposure, every 3 min. in control white bars , in the presence Ž. of haloperidol 1, 10, 100 or 1000 nM during the second application of Ž. phenylephrine black bars, ns24 , and in respond to the third application Ž. of phenylephrine after washing out haloperidol stripped bars, ns24 . Ž. 2q C Percent of inhibition of the Ca signals as a function of the Ž. concentration of haloperidol ns24 . mine-mediated Ca2qresponses. Risperidone is about 120- Ž. fold more selective for a-adrenoceptors Ks2.3 nM 1B i Ž.Ž than for a-adrenoceptors Ks280 nM Sleight et al., 1A i . 1993 . In our experiments with risperidone, the IC was 50 -10 nM when the response was elicited by dopamine, and about 25 times higher when the stimulus was phenylephrine. On the contrary, WB4101 and benoxathian have been reported to have much higher affinity for a-than 1A Ž for a-adrenoceptors Sugden et al., 1996; Yan et al., 1B . 1998 . In our experiments, when the stimulus was dopamine, the IC with WB4101 was 10 nM, and 1 nM 50 with benoxathian, in the range of that expected for a- 1B Ž. adrenoceptors Sugden et al., 1996 . To explore further if the dopamine-induced Ca2qincre- ments were mediated by a-adrenoceptors, cells were 1B incubated with either 10 or 50 mM chloroethylclonidine, an alkylating agent, preferentially of a-adrenoceptors, 1B during 10–60 min. Thereafter, the cells were washed and Ž. stimulated either with dopamine 5 mM or phenylephrine Ž. 1mM . In those cells stimulated with dopamine, the inhibition of the response was complete with 50 mM Ž. chloroethylclonidine Fig. 6A . A minor concentration of chloroethylclonidine, 10 mM, decreased the response to 44%. In cells stimulated with phenylephrine, the inhibition with 10 and 50 mM chloroethylclonidine was less, 49.7% Ž. and 69.3%, respectively Fig. 6B . These results also suggest that dopamine is more selective activating a-adren- 1B oceptors than phenylephrine, which has been reported to be similarly potent at all three a-adrenoceptors subtypes 1 Ž. Minneman et al., 1994 . Besides a-adrenoceptor antagonists, desipramine also 1B 2qŽ inhibited the dopamine-mediated Ca response ns26, . Fig. 7 . This drug is used in clinic as antidepressant, possibly due to its inhibitory effect at low concentrations Ž on serotonine and noradrenaline transporters Buck and . Amara, 1994 . In our experiments, the inhibition of the dopamine effect on intracellular Ca2qby desipramine was Ž concentration-dependent in the range 100–1000 nM IC50 . s250 nM , while no inhibition was obtained within this range when the stimulus was phenylephrine or noradrena- Ž. line Fig. 7C–E . Imipramine, other tricyclic antidepressant, at 1000 nM inhibited 90% of the response to dopamine, while it did not affect the response to phenyl- Ž. ephrine ns15, data not shown . 4. Discussion In this work, we describe the capability of dopamine to trigger Ca2qsignals in pineal cells. Ca2qtransients resulted from the release of Ca2qfrom intracellular stores and the subsequent Ca2qentry to the cytosol from the Ž. extracellular medium Fig. 1 . Half maximum responses were obtained with 5 mM dopamine. This value is within the activation range of dopamine receptor subtypes D and 1 D , but higher than the EC for receptor subtypes D and 2504 Ž. D submicromolar affinity , and much higher than the 5Ž.Ž EC for subtype D nanomolar affinity Gingrich and 50 3 . Caron, 1993 . Although all cloned dopamine receptors are known to be only coupled to adenylate cyclase, there are several reports describing the activation of the phosphatidylinositol turnover and the generation of Ca2qsig- Ž nals by dopamine in some tissues Kebabian and Calne, 1979; Stoof and Kebabian, 1981; Onali et al., 1984; Felder et al., 1989a,b; Mahan et al., 1990; Vallar et al., 1990; . Undie and Friedman, 1990 . In bovine pineal cells, D and 1 D dopamine receptors have been identified with radioli- 2Ž gand binding assays Govitrapong et al., 1984; Simon- . neaux et al., 1990 . However, our results point against the presence of dopamine receptors coupled to phospholipase C in rat pinealocytes since, in our hands, the dopamine () E. Rey et al.rEuropean Journal of Pharmacology 430 2001 9–1714 2qŽ. Ž . Ž . Fig. 5. Intracellular Ca responses to dopamine and phenylephrine. A Example of a cell that responded to dopamine 5 mM and phenylephrine 1 mM. Ž. Ž . Ž . B Example of a cell that only responded to phenylephrine. C, D Recordings from two cells with fast desensitisation to dopamine 50 mM and not to Ž. phenylephrine 1 mM. receptor agonists SKF-38393, bromocriptine, quinpirole and apomorphine, at saturating concentrations, were inefficient to increase intracellular Ca2q. Furthermore, the selective a-adrenoceptor antagonist prazosin inhibited with 1Ž. 2q high affinity low nanomolar the Ca increment mediated by dopamine. The fact that some dopamine receptor Ž. antagonists haloperidol, spiperone, or SCH-23390 also inhibited the effect of dopamine, although at a lower affinity than that of prazosin, is not surprising given the capability of these drugs to also bind to a-adrenoceptors. 1 It must be pointed out that the opposite has also been Ž described i.e. the activation of dopamine receptors by . noradrenaline since in cat retinal ganglion cells, noradrenaline has been shown to activate D dopamine recep- 2 Ž. tors Robbins et al., 1988 . The finding that in all cells tested the a-adrenoceptor 1 agonist phenylephrine induced Ca2qrelease from intracellular stores, but not all cells increased intracellular Ca2q in response to dopamine, suggests that dopamine action is mainly mediated through the activation of an a-adrenoc- 1 eptor subtype. This hypothesis was further confirmed by desensitising experiments. With successive applications of saturating dopamine concentrations, the Ca2qresponses progressively decreased, while those elicited with phenylephrine were maintained or decreased with a much slower time course. To ascertain what a-adrenoceptor subtype 1 was mainly activated by dopamine, we used both the alkylating agent chloroethylclonidine, which preferentially inactivates a-adrenoceptors, and three specific subtype 1B antagonists. The results obtained suggest that dopamine mainly activates a-adrenoceptors over other subtypes. In 1B rat pineal cells, a- and a-adrenoceptors have been 1A 1B identified by radioligand binding and reverse transcription- Ž. polymerase chain reaction analysis Sugden et al., 1996 , which is compatible with our results. However, whether normally both receptor subtypes do exist in all pinealocytes or whether in some cells only one subtype is expressed, thereby resulting in a heterogeneous population of pinealocytes, remains to be further clarified. A physiological role of dopamine on rat pineal cells has previously been proposed. In bovine pineal glands, dopamine is thought to have a regulatory role on melatonin Ž synthesis and secretion Govitrapong et al., 1989; Simon- . neaux et al., 1991 . The existence of pineal dopaminergic innervation from central origin has also been postulated based on the presence of tyroxine hydroxylase activity in rat pineal glands after bilateral denervation from the supe- Ž. rior cervical ganglia Hernandez et al., 1994 . In addition, it has been suggested that not all the dopamine accumulated in sympathetic nerve endings innervating the pineal gland is further metabolised to noradrenaline. This suggestion is based on the finding that only part of the fibres () E. Rey et al.rEuropean Journal of Pharmacology 430 2001 9–17 15 Ž. 2qŽ. Ž. Fig. 7. A, B Intracellular Ca responses to dopamine 5 mM in presence or absence of desipramine DES , 100 or 500 nM, respectively, in two cells. Ž. Ž . Ž .Ž. Ž .Ž . C Response to phenylephrine 1 mM in the absence or presence of desipramine 500 nM . D Response to noradrenaline NA 1 mM in the presence Ž.Ž. Ž or absence of desipramine 1 mM . E Relationship between the inhibition of the response to catecholamines dopamine 5 mM, IC s250 nM, ns26; 50 . phenylephrine 1 mM, IC )1000 nM, ns13 and desipramine concentration. In all cases, drug application lasted 15 s. Numbers in brackets represent 50 number of experiments. () E. Rey et al.rEuropean Journal of Pharmacology 430 2001 9–1716 Fig. 6. Inhibition of catecholamines-mediated Ca2qresponses by 10 or Ž. 50 mM chloroethylclonidine. A Responses to the application of Ž. Ž dopamine 5 mM of cells preincubated with chloroethylclonidine ns .Ž. Ž . 21 . B Responses to the application of phenylephrine 1 mM of cells Ž. preincubated with chloroethylclonidine ns30 . displaying catecholamine fluorescence also shows dopa- Ž mine-b-hydroxylase-like immunoreactivity Schroder and ¨ . Vollrath, 1985 . The fact that dopamine can activate adrenoceptors and that noradrenaline may also activate dopamine receptors in Õitro opens the interrogation whether this phenomenon also occurs in ÕiÕo. In such case, the question emerges whether the term cathecholaminergic receptors has a genuine physiological meaning. On the other hand, specific functional interactions between different neurotransmitter Ž receptor subfamilies have been recently reported Liu et . al., 2000 . Taken together, these findings indicate that neurotransmitter-membrane receptor cross-talk may represent a more widespread phenomenon than previously expected. 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