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Keywords:

  • calcium;
  • dopamine receptor;
  • neurons;
  • signal transduction

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Recent evidence indicates the existence of a putative novel phosphatidylinositol-linked D1 dopamine receptor in brain that mediates phosphatidylinositol hydrolysis via activation of phospholipase Cβ. The present work was designed to characterize the Ca2+ signals regulated by this phosphatidylinositol-linked D1 dopamine receptor in primary cultures of hippocampal neurons. The results indicated that stimulation of phosphatidylinositol-linked D1 dopamine receptor by its newly identified selective agonist SKF83959 induced a long-lasting increase in basal [Ca2+]i in a time- and dose-dependent manner. Stimulation was observable at 0.1 μm and reached the maximal effect at 30 μm. The [Ca2+]i increase induced by 1 μm SKF83959 reached a plateau in 5 ± 2.13 min, an average 96 ± 5.6% increase over control. The sustained elevation of [Ca2+]i was due to both intracellular calcium release and calcium influx. The initial component of Ca2+ increase through release from intracellular stores was necessary for triggering the late component of Ca2+ rise through influx. We further demonstrated that activation of phospholipase Cβ/inositol triphosphate was responsible for SKF83959-induced Ca2+ release from intracellular stores. Moreover, inhibition of voltage-operated calcium channel or NMDA receptor-gated calcium channel strongly attenuated SKF83959-induced Ca2+ influx, indicating that both voltage-operated calcium channel and NMDA receptor contribute to phosphatidylinositol-linked D1 receptor regulation of [Ca2+]i.

Abbreviations used
ACSF

artificial cerebrospinal fluid

[Ca2+]i,

intracellular Ca2+ cAMP, cyclic AMP

CCE

capacitative calcium entry

CNQX

6-cyano-7-nitro quinoxaline-2,3-dione

IP3

inositol triphosphate

PLCβ

phospholipase Cβ

PKA

protein kinase A

CaMKII

calcium/çamodulin modulated kinase II

DARPP-32

(dopamine- and cyclic AMP-regulated phosphoprotein with molecular weight 32)

DMEM

Dulbecco's modified Eagle's medium

TTX

tetrodoxin

Dopamine, an important neurotransmitter in brain, exerts its action via dopamine receptors, which are known to belong to the G-protein-coupled receptor family. Calcium plays a critical role in mediating neuronal excitability and neuroplasticity. Regulation of calcium content in neuronal cells by dopamine has been reported in different experimental systems (Bergson et al. 2003; Lee et al. 2004; Neve et al. 2004). Both protein kinase A (PKA)-dependent and independent mechanisms have been proposed to mediate dopamine receptor-stimulated calcium signals (Tang et al. 2003; Neve et al. 2004). Recent evidence indicates that activation of dopamine receptors also activates phospholipase Cβ (PLCβ) through coupling to the Gq protein, resulting in hydrolysis of phosphatidylinositol into diacylglycerol and inositol triphosphate (IP3; Felder et al. 1989; Frail et al. 1993; Pacheco and Jope 1997; Lezano & Bergson., 2001; Lee et al. 2004; Wirtshafter and Osborn, 2005; Panchalingam and Undie 2005; Zhang et al. 2005). This stimulation of phosphatidylinositol hydrolysis appears to be mediated by a D1-like receptor as only selective D1 receptor agonist but not D2 receptor agonist can elicit this action, which is blocked by SCH23390, a selective D1 receptor antagonist (Undie et al. 1994). However, the stimulation of phosphatidylinositol hydrolysis by the D1-like receptor appears to be distinct from the D1A receptor because in D1A transfected PC12 cells there is no detectable D1 receptor-stimulated IP3 generation (Jin et al. 2002), and in D1A knock-out mice the stimulation of IP3 by D1 agonist in brain was still present even though the effect can be blunted by a D1 antagonist (Friedman et al. 1997). The characterization of this novel phosphatidylinositol-linked dopamine-stimulated pathway has been aided by the recent identification of a D1 receptor agonist, SKF83959, which selectively activates this pathway (Panchalingam & Undie, 2001, Jin et al. 2002). We have further demonstrated that activation of the phosphatidylinositol-linked dopamine receptor by SKF83959 stimulates CaMKII activity in the frontal cortex that is dependent on calcium and protein kinase C (Zhen et al. 2004). More recently, we demonstrated that SKF83959-mediated stimulation of PLC/IP3 pathway via this novel D1-like dopamine receptor is likely responsible for the anti-Parkinsonian action of the drug (Zhen et al. 2005).

Currently, the detail mechanisms by which SKF83959 regulates free Ca2+ in neurons remain unclear. The present study characterized SKF83959-regulated Ca2+ signaling by employing calcium imaging techniques in primary cultured hippocampal neurons. The results indicated that SKF83959 stimulated an increase in [Ca2+]i in hippocampal neurons via a D1-like dopamine receptor in a dose-dependent manner. The sustained increase in [Ca2+]i was found to depend on both intracellular calcium release via the action of the second messenger IP3 as well as on calcium influx through a voltage-operated calcium channel and a receptor-operated calcium channel.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Materials

6-Chloro-7, 8-dihydroxy-3-methyl-1-(3-methylphenyl)-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF83959) (±)-7-bromo-8-8-hydroxy-3 methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF83566), S(–)-raclopride, mesulergine HCl, scopolamine and prazosin were purchased from RBI (Natric, MA, USA). U-73122, U-73343, bucladesine (dibutyryl cyclic AMP [cAMP]), ATP, cyclopiazonic acid, nifedipine, CdCl2, dl-2-amino-5 phonovaleric acid, 6-cyano-7-nitroquinoxaline-2,3-dione, tetrodotoxin and thapsigargin were purchased from Sigma (St. Louis, MO, USA). Fura-2/AM was obtained from Biotium (Hayward, CA, USA) and B27 supplement from Gibco Invitrogen Corporation (Carlsbad, CA, USA) Other general agents were purchased from commercial suppliers. SKF83959 was prepared freshly with distilled water. Other agents were prepared as stock solutions with sterile water except nifedipine, U-73122, U-73343 and Fura-2/AM, which were dissolved in dimethylsulfoxide (DMSO) and stored at − 20°C. They were diluted to the final concentrations before application. The final concentration of DMSO was < 0.05%.

Cell culture

Neonatal SD (Sprague-Dawley) rats (day 0–3) of both sexes were obtained from the Experimental Animal Center of Tongji Medical College, Huazhong University of Science & Technology. The University Animal Welfare Committee approved the animal protocol used. Neurons were isolated as previously described (Chen et al. 1997; Bergson et al. 2003) with some modification. Briefly, hippocampi of newborn rats were dissected and rinsed in ice cold dissection buffer. Blood vessels and white matter were removed and tissues were incubated in 0.125% trypsin for 30 min at 37°C. Neurons were collected by centrifugation and resuspended in Dulbecco's modified Eagle's medium (DMEM) and F-12 supplement (1 : 1) (Gibco Invitrogen Corporation) with 10% fetal bovine serum (heat-inactivated, Hyclone), 2 mm l-glutamine (Sigma), and penicillin (100 U/mL)-streptomycin (100 U/mL). Cells were plated at a density of 104−105 per 35 mm2 on coverslides precoated with poly-l-lysine and kept at 37°C in a 5% CO2 incubator. After 24 h, the culture medium was changed to DMEM medium containing 2% B27 and 2 mmol/L glutamine. Astrocytes were minimized by treating the culture with cytarabine (10 μm) on day 3. The medium were replaced with fresh medium every 3 days. Experiments were performed on day 5–7.

Calcium imaging

Hippocampal neurons were washed three times with 1 μmol/L Fura-2/AM in artificial cerebrospinal fluid (ACSF, containing 140 mm NaCl, 5 mm KCl, 1 mm MgCl2, 2 mm CaCl2, 10 mm glucose and 10 mm HEPES, pH 7.3) then incubated in the same solution for 30 min at 37°C. In calcium-free experiments, EGTA (100 μm) was substituted for CaCl2. Before each experiment, the coverslides were mounted on a chamber positioned on the movable stage of an inverted Olympus IX-70 microscope equipped with a calcium imaging system (TILL Photonics GmbH, Gräfelfing, Germany), and superfused by ACSF for 10 min. Fura-2/AM loaded cells were illuminated at 340 nm for 150 ms and 380 nm for 50 ms at 1-s intervals using a TILL Polychrome monochromator. Fura-2 fluorescence emission was imaged at 510 nm by an intensified cooled charge coupled device (TILL Photonics GmbH) through a X-70 fluor oil immersion lens (Olympus) and a 460-nm long-pass barrier filter. F340/F380 fluorescence ratios were generated by TILLvisION 4.0 software. Paired F340/F380 fluorescence ratio images were acquired every second for [Ca2+]i. The intracellular free calcium concentration is presented as the ratio of the fluorescence signals obtained (340/380 nm). All experiments were repeated at least three times using different batches of cells.

Statistical analysis

Data are presented as mean ± SEM of the ratio of the fluorescence. Data were analyzed using SPSS 10.0 software. Student's t-test or one-way anova was used to test for significance. Differences were considered significant at p < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

SKF83959 elevated [Ca2+]i in rat hippocampal neurons in a dose-dependent manner

We employed the Ca2+ imaging technique to study the dynamic alteration of intracellular calcium mediated by SKF83959 in primary culture of rat hippocampal neurons. Application of 1 μm SKF83959 induced elevation of [Ca2+]i in these cells. This elevation was detectable in about 2–3min, and rapidly reached a plateau in 5 ± 2.13 min with an average maximal 96 ± 5.6% increase over unstimulated cells (n = 58). The increase in [Ca2+]i lasted at least 20 min (Fig. 1a). The effect of SKF83959 was significant at 0.1 μm (p < 0.05), and reached a maximal level at a concentration of 30 μm (Fig. 1b).

image

Figure 1.  Effect of SKF83959 on [Ca2+]i in primary cultured hippocampal neurons. Primary cultured hippocampal neurons were treated with 1 μm SKF83959 for designated times (a) or with various concentrations of SKF83959 for 20 min (b). [Ca2+]i increase was recorded as described in the Methods and expressed as F340/F380 nm ratio. (a) Time course of [Ca2+]i increase. SKF83959 stimulated [Ca2+]i increase by 96 ± 5.6%, n = 58. The arrow indicates the time of SKF83959 application. (b) Summary of the maximal response in [Ca2+]i for each dose of SKF83959 from three independent experiments. The basal F340/380 nm ratio without SKF83959 stimulation was 0.33 ± 0.03. *p < 0.05, **p < 0.01 compared to basal [Ca2+]i. (anova).

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D1-like dopamine receptor appears to mediate SKF83959-induced elevation of [Ca2+]i. Although SKF83959 is known to have a high affinity to the D1-like receptor, it also exhibits a moderate or weak affinity to D2 and other receptors. We therefore explored the potential role of the latter receptors in SKF83959-mediated calcium increase. As expected, preincubation of the D1 receptor-specific antagonist SKF83566 (0.1 μm) to the primary culture of hippocampal neurons prior to SKF83959 (1 μm) stimulation robustly reduced SKF83959-induced increase of [Ca2+]i(Fig. 2a). The maximal stimulation was reduced from 96 ± 5.6% (SKF83959 alone, n = 58) to 28.8 ± 2.8% (SKF83566 + SKF83959, n = 26). In contrast, application of the D2 receptor antagonist S (–)-raclopride (10 μm) did not significantly inhibit SKF83959-induced increase of [Ca2+]i (Fig. 2b), indicating that the SKF83959-induced stimulation of [Ca2+]i increase was mainly mediated by D1-like, but not D2-like, receptor. Indeed, application of other receptor antagonists such as the 5-HT1c and 5-HT2 antagonist mesulergine HCl (10 μm), the α-adrenergic blocker prazosin (1 μm) or the anticholinergic scopolamine (10 μm) did not produce any inhibition on SKF83959-stimulated elevation of free calcium in the neurons (Fig. 2c). These results indicate that selective stimulation of the D1-like dopamine receptor is responsible for SKF83959-induced increase of [Ca2+]i, presumably via phosphatidylinositol-linked D1 dopamine receptor.

image

Figure 2.  Phosphatidylinositol-linked D1 dopamine receptor mediates SKF83959-stimulated [Ca2+]i elevation. (a) Primary cultured hippocampal neurons were pretreated with 10 μm SKF83566 for 10 min prior to application of 1 μm SKF83959. [Ca2+]i signals were collected as F340/380 nm ratios, n = 36. The experiments were repeated three times with similar results and a representative experiment is shown. (b) Effect of D2 dopamine receptor antagonist S(–)-raclopride (10 μm) on SKF83959-induced [Ca2+]i. Arrow indicates time when SKF83959 was applied. (c) Summary of changes in [Ca2+]i with preincubation of 1 μm prazosine, 10 μm mesulergine HCl or 10 μm scopolamine for 10 min prior to SKF83959 stimulation. Data summarize three independent experiments. *p < 0.01 compared to unstimulated control.

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Both Ca2+ influx and intracellular calcium release pathways were involved in SKF83959-stimulated increase in the intracellular Ca2+. We examined the respective contribution of Ca2+ influx from extracellular compartment or the release from intracellular Ca2+ stores on SKF83959-mediated changes in calcium content in the neurons. In the absence of extracellular Ca2+, 1 μm SKF83959 was able to induce a significant increase of basal [Ca2+]i (52 ± 4.1% over unstimulated level, at 8 min, n = 60), although the magnitude of elevation (Fig. 3a) was less than that in the presence of extracellular Ca2+ (Fig. 1a). This suggests that calcium release from intracellular stores was partly responsible for the induced Ca2+ elevation. To further explore the role of extracellular Ca2+, we alternately superfused hippocampal neurons with Ca2+-containing and Ca2+-free media in the presence of SKF83959. As shown in Fig. 3b, SKF83959-induced [Ca2+]i increase was blunted while Ca2+-free medium was applied, but recovered again after reperfusion with Ca2+-containing medium. Again, it was noted that application of Ca2+-free medium did not completely block the increase in [Ca2+]i, consistent with calcium release from intracellular stores as noted in Fig. 3a. Thus, there two components of Ca2+ transit were involved. The data indicated that the phosphatidylinositol-linked D1 dopamine receptor-mediated Ca2+ release from intracellular stores has a major early onset and is a steadily rising component of Ca2+ elevation, whereas Ca2+ influx from extracellular compartment is additive to intracellular Ca2+ release. Furthermore, we examined the importance of Ca2+ release from intracellular stores in triggering the late component elevation of [Ca2+]i. SKF83959 failed to evoke a significant increase in [Ca2+]i after preincubation with 1 μm thapsigargin to deplete the intracellular calcium store in neurons bathed in Ca2+-containing medium (Fig. 3c). Similar results were obtained when cyclopiazonic acid, an inhibitor of sarcoplasmic/endoplasmic reticulum Ca2-ATPase was applied (data not shown). Taken together, these results indicated that the SKF83959-induced Ca2+ release from the intracellular calcium store is necessary and sufficient for triggering the late component of [Ca2+]i elevation via Ca2+ influx. Moreover, our data do not support the occurrence of capacitative calcium entry (CCE) as reported in hippocampal neurons, as depletion of intracellular calcium store by thapsigargin or cyclopiazonic acid did not by itself elicit any significant Ca2+ influx (Fig. 3c). CCE is therefore unlikely to be involved in SKF83959-induced Ca2+ influx.

image

Figure 3.  SKF83959-mediated increase in [Ca2+]i depends on release of Ca2+ from intracellular stores and Ca2+ influx. (a) Superfusion of cultured neurons was switched to a Ca2+-free solution when 1 μm SKF83959 was added. F340/380 nm ratio was measured. (b) [Ca2+]i increase was halted while Ca2+-free solution was applied and restored after switching back to Ca2+-containing solution during SKF83959-induced increase in [Ca2+]i. + Ca, Ca2+-containing ACSF applied. –Ca, Ca2+-free ACSF applied. (c) Application of 1 μm thapsigargin to deplete the intracellular calcium store completely blocked SKF83959-induced [Ca2+]i increase in hippocampal neurons (n = 17). Arrow indicates the time of SKF83959 application. The experiments were repeated at least three times and representative data are shown for each treatment.

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L-type calcium channel and receptor-operated calcium channel contributed to the maintenance of SKF83959-induced enhancement of [Ca2+]i

Although we demonstrated that Ca2+ influx from extracellular influx contributed to the late component of the SKF83959-induced [Ca2+]i elevation, the exact pathway involved in this regulation is unclear. To address this issue, we first employed Cd2+ and nifedipine, antagonists for calcium channels, to block these voltage-gated calcium channels. As shown in Fig. 4a, the late component of the SKF83959-induced increase of [Ca2+]i was greatly attenuated by nifedipine (Fig. 4a, left panel) and Cd2+ (Fig. 4a, right panel). The average [Ca2+]i stimulated by 1 μm SKF83959 was 48 ± 3%(n = 43) and 40 ± 5% (n = 15) for the nifedipine- and CdCl2-treated group, respectively, significantly less (p < 0.05) than the 92 ± 6.3% increase when SKF83959 was applied alone. We next checked the role of the receptor-operated calcium channels. As depicted in Fig 4b, 50 μm dl-2-amino-5 phonovaleric acid (Fig. 4b, left panel) attenuated the SKF83959-induced increase of [Ca2+]i, especially the late component of [Ca2+]i. Application of 6-cyano-7-nitroquinoxaline-2,3-dione (10 μm), a selective ®-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptor competitive antagonist induced partial suppression of the late increase of [Ca2+]i (Fig. 4b, right panel). This result indicated that activation of NMDA receptor, and to a lesser extent the AMPA receptor, also contributed to the calcium influx stimulated by SKF83959 in hippocampal neurons. Lastly, we tested whether voltage-operated Na+ channels played a role in the drug-stimulated calcium elevation. Preincubation of the hippocampal neurons with tetrodotoxin, a specific voltage-gated Na+ channel blocker, did not alter SKF83959-induced increase of [Ca2+]i (Fig. 4c). The maximal stimulation of [Ca2+]i by SKF83959 in the presence of tetrodotoxin was 73.8 ± 1.08% (n = 34), p > 0.05, compared with that of the SKF83959 only group (92 ± 6.3%).

image

Figure 4.  Both voltage-operated calcium channel and receptor-operated calcium channel contribute to Ca2+ influx. (a) Application of 10 μm nifedipine (left panel) or CdCl2 (right panel) 10 min prior to addition of 1 μm SKF83959 reduced the drug-stimulated [Ca2+]i. elevation. The maximal increase in [Ca2+]i for 1 μm SKF83959 alone was 92 ± 6.3% over control. The maximal stimulation of [Ca2+]i by 1 μm SKF83959 in the presence of nifedipine (48 ± 3%, n = 43) or CdCl2 (40 ± 5%, n = 15) was significantly lower (p < 0.05). (b) Pretreatment of 50 μm dl-2-amino-5 phonovaleric acid (APV) (left panel) 10 min prior to addition of 1 μm SKF83959 reduced the drug-stimulated [Ca2+]i elevation (p < 0.05). A slight inhibition was also observed when 10 μm 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (right panel) was applied. The maximal increase in [Ca2+]i over control was 57.2 ± 4.8% (n = 17) and 68 ± 7% (n = 32) for dl-2-amino-5 phonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione, respectively. (c) Application of 2 μm tetrodotoxin (TTX) did not significantly alter SKF83959-stimulated [Ca2+]i. All experiments were repeated at least three times with similar results.

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Phosphatidylinositol-linked D1 receptor-mediated activation of PLCβ is the major pathway for SKF83959-stimulated [Ca2 + ]i elevation

To identify the signaling pathway involved in SKF83959-induced Ca2+ release from intracellular stores, we applied U-73122, an inhibitor of PLCβ, to the primary cultured hippocampal neurons before and during SKF83959 stimulation. Addition of U-73122 resulted in blockade of the SKF83959-mediated increase of [Ca2+]i(Fig. 5a), whereas U-73343, an inactive analog of U-73122 (without inhibitory activity), did not alter SKF83959-stimulated [Ca2+]i elevation (data not shown). ATP, which is known to exhaust IP3-sensitive calcium stores, also completely blocked the drug-elicited [Ca2+]i elevation (Fig. 5b). In contrast, activation of PKA by cell-permanent dibutyryl cAMP (100 μm) did not significantly inhibit phosphatidylinositol-linked D1 receptor-stimulated Ca2+ (data not shown). Taken together, our data clearly indicate that SKF83959-stimulated Ca2+ elevation is mediated via a PLCβ- and IP3- dependent pathway.

image

Figure 5.  Activation of PLC/IP3 pathway is essential for SKF83959-stimulated [Ca2+]i increase. Primary cultured hippocampal neurons were pretreated with various drugs for 10 min prior to application of 1 μm SKF83959. Ca2+ signal was collected. (a) Pretreatment with 10 μm U73122. (b) Pretreatment with 100 μm ATP.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The present study demonstrated that SKF83959 stimulated increases in [Ca2+]i in a dose-dependent manner in primary cultured hippocampal neurons. The elevation of [Ca2+]i was detectable with 0.1 μm SKF83959, and a maximal [Ca2+]i increase was achieved with 30 μm SKF83959. The initial phase of the [Ca2+]i increase caused by SKF83959 was mediated by IP3-induced intracellular calcium release via activation of PLCβ-coupled D1-like dopamine receptor; whereas a late onset component of the [Ca2+]i increase required Ca2+ influx via a voltage-operated calcium channel and receptor-operated calcium channel. Furthermore, we demonstrated that SKF83959-stimulated increase in [Ca2+]i was dependent on PLCβ activation but not on PKA.

SKF83959 is a recently identified selective agonist for PLCβ-linked D1-like dopamine receptor. It exhibits the greatest potency among tested D1-dopamine receptor agonists including SKF81297 and SKF38393 with regard to stimulation of IP3 production in brain via a D1-like dopamine receptor (Panchalingam & Undie, 2001; Jin et al. 2002; Lee et al. 2004). Unlike other D1 dopamine receptor agonists, which stimulate both cAMP and IP3 production in the brain, SKF83959 elicits no stimulation of cAMP production, exerting its IP3 stimulation via a putative phosphatidylinositol-linked D1 dopamine receptor (Deveney and Waddington. 1995; Jin et al. 2002). We further demonstrated that SKF83959-induced activation of CaMKII and Cdk5 in brain slices depended on calcium (Zhen et al. 2004). Present results detailed the regulation and signaling pathway for SKF83959-stimulated Ca2+ elevation in primary cultured hippocampal neurons, thereby providing further support for the importance of phosphatidylinositol-linked D1 dopamine receptor in neuronal function. Considering the critical role of calcium and CaMKII in neuroplasticity, the potential importance of SKF83959-responsive phosphatidylinositol-linked D1 receptor in learning and memory is of great interest. Indeed, some IP3-coupled guanine nucleotide-binding regulatory protein-linked receptor activation induces only a rapid and transient Ca2+ increase (Lo et al. 2002); whereas SKF83959 stimulates [Ca2+]i elevation in a sustained and slow manner (Fig. 1a). It has been proposed that sustained or long-lasting increases in cellular [Ca2+]i functions differently than transient change in [Ca2+]i, and long-lasting increase of [Ca2+]I in neurons more likely induces gene modulation underlying neuroplasticity (Ross et al. 2005). Release of Ca2+ from intracellular stores has been linked to long-term potentiation (LTP), protein synthesis and dendritic growth (Yeckel et al. 1999; Raymond et al. 2000; Bergson et al. 2003). We recently also found that SKF83959 induces a long-term potentiation-like response in mouse hippocampal slices (unpublished data). Thus it is of particular interest to further study the functional role of SKF83959-mediated long-lasting elevation of intracellular Ca2+.

Dopamine D1- and D2-like receptors were both reported to alter Ca2+ dynamics in neurons, although apparent controversies exist. Ca2+ transit in primary cultures of neocortical and hippocampal neurons was activated by selective D1/D5 receptor agonists SKF81297 or SKF38393, but not by D2-like dopamine receptor agonists (Lezocano and Bergson, 2005; Bergson et al. 2003). On the other hand, stimulation of D2-like dopamine receptor on striatal neurons or D4 dopamine receptor on hippocampal neurons has been reported to cause IP3 production and Ca2+ release (Hernandez-Lopez et al. 2000; Gu and Yan, 2004). The discrepancy could relate to the differences in neuronal types or differences in experimental regiments. The signaling cascades mediating D1-like receptor stimulation of Ca2+ were proposed to involve both PKA-dependent and independent mechanisms (Bergson et al. 2003). Consistent with this, a hypothesis of crosstalk between the cAMP and IP3 pathways converging at the type-1 IP3 receptor in the dopamine-regulated Ca2+ signal in striatal medium spiny neurons has been recently proposed (Tang & Bezprozvanny 2004). However, it is known that SKF83959 does not elicit any cAMP stimulation (Cools et al. 2002; Jin et al. 2002). Indeed, application of cell-permeant cAMP, which activates PKA in primary cultured hippocampal neurons, did not elicit any alteration of SKF83959-induced [Ca2+]i increase, clearly indicating that PKA is not involved in the drug-stimulated Ca2+ elevation in hippocampal neurons. In agreement with the report (Lezocano & Bergson, 2005) that D1 receptor agonist SKF81297- or SKF83393-stimulated increases in [Ca2+]i in hippocampal neurons is dependent on PLCβ, a PLCβ- selective inhibitor also blocked SKF83959-induced [Ca2+]i increases (Fig. 5a) in the present study. These data indicate that activation of phosphatidylinositol-linked D1-dopamine receptor via PLCβ may be the common pathway for SKF81297, SKF83393 and SKF83959-regulated [Ca2+]i.

Capacitative calcium entry (CCE) via store-operated calcium channel is considered to be the major mechanism for influx of Ca2+ in a nonexcitable cell system. It is generally believed that CCE operation is nonexistent in neurons, although there were some disagreements (Putney 2003). Our data also indicate that CCE is not operative in the cultured hippocampal neurons. Thus, it is unlikely that SKF83959 employs this mechanism to stimulate Ca2+ influx in primary cultured hippocampal neurons. In contrast, application of dl-2-amino-5 phonovaleric acid, a selective NMDA receptor antagonist, greatly attenuated the SKF83959-induced increase in [Ca2+]i, particularly the late component of the increase (Ca2+ influx), indicating involvement of NMDA receptor activation. Our result is in agreement with a previous report that [Ca2+]i elevation stimulated by a high dose of dopamine (400 μm) in striatal neurons depends on coactivation of the NMDA receptor (Tang & Bezprozvanny 2004). Indeed, an interaction between the D1 dopamine receptor and the NMDA receptor has been established. The mechanisms underlying such an interaction are associated with alterations in cell excitability due to activation of Ca2+ conductances and/or phosphorylation of the NMDA receptor. (Cepeda and Levine 1998; Flores-Hernandez et al. 2002; Liu et al. 2004).

A previous study indicated that application of 400 μm of dopamine elicited repetitive Ca2+ transients (oscillations) in striatal medium spiny neurons (Tang and Bezprozvanny 2004). However, we have not observed noticeable Ca2+ oscillations in hippocampal neurons even at high dosages of SKF83959 (data not shown, also see Tang and Bezprozvanny 2004). Our data indicate that stimulation of phosphatidylinositol-linked D1 dopamine receptor by SKF83959 is not sufficient to elicit Ca2+ oscillations. It was found that phosphorylation of DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein with molecular weight 32) mediated by dopamine-stimulated PKA plays an important role in dopamine-induced Ca2+ oscillations (Tang and Bezprozvanny 2004). It is therefore conceivable that the failure of SKF83959 to induce Ca2+ oscillations in our study may result from a lack of stimulation of PKA by the drug. Interestingly, our previous study demonstrated that SKF83959 stimulates cdk5, a kinase known to phosphorylate DARPP-32 at Thr-75 (unlike DARPP-32 phosphorylation at Thr-34 by PKA). It is unclear whether the disparate regulatory effects on DARPP-32 by dopamine and SKF83959 account for the difference in the pattern of evoked Ca2+ transit.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We thank Dr Christopher Chan for critical reading of the manuscript. This work is supported by a bridge fund from Tonji Medical College to X. Zhen and a grant from National Science Foundation of China to J. Chen (No. 30425024).

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • Bergson C., Levenson R., Goldman-Rakic P. S. and Lidow M. S. (2003) Dopamine receptor-interacting proteins: the Ca (2+) connection in dopamine signaling. Trends Pharmacol. Sci. 24, 486492.
  • Cepeda C. and Levine M. S. (1998) Dopamine and N-methyl-D-aspartate receptor interactions in the neostriatum. Dev. Neurosci. 20, 118.
  • Chen J., Backus K. H. and Deitmer J. W. (1997) Intracellular calcium transients and potassium current oscillations evoked by glutamate in cultured rat astrocytes. J. Neurosci. 17, 72787287.
  • Cools A. R., Lubbers L., Oosten R. V. and Andringa G. (2002) SKF83959 is an antagonist of dopamine D1-like receptors in the prefrontal cortex and nucleus accumbens: a key to its antiparkinsonian effect in animals? Neuropharmacology 42, 237245.
  • Deveney A. M. and Waddington J. L. (1995) Pharmacological characterization of behavioral responses to SKF83959 in relation to ‘D1-like’ dopamine receptors not linked to adenylyl cyclase. Br. J. Pharmacol. 116, 21202126.
  • Felder C. C., Jose P. A. and Axelrod J. (1989) The dopamine D1 agonist, SKF82526, stimulates phospholipase-C activity independent of adenylate cyclase. J. Pharmacol. Exp. Ther. 248, 171175.
  • Flores-Hernandez J., Cepeda C., Hernandez-Echeagaray E., Calvert C. R., Jokel E. S., Fienberg A. A., Greengard P. and Levine M. S. (2002) Dopamine enhancement of NMDA currents in dissociated medium-sized striatal neurons: role of D1 receptors and DARPP-32. J. Neurophysiol. 88, 30103020.
  • Frail D. E., Manelli A. M., Witte D. G., Lin C. W., Steffey M. E. and Mackenzie R. G. (1993) Cloning and characterization of a truncated dopamine D1 receptor from goldfish retina: stimulation of cyclic AMP production and calcium mobilization. Mol. Pharmacol. 44, 11131118.
  • Friedman E., Jin L. Q., Cai G. P., Hollon T. R., Drago J., Sibley D. R. and Wang H. Y. (1997) D1-like dopaminergic activation of phosphoinositide hydrolysis is independent of D1A dopamine receptors: evidence from D1A knockout mice. Mol. Pharmacol. 51, 611.
  • Gu Z. and Yan Z. (2004) Bidirectional regulation of Ca2+/calmodulin-dependent protein kinase II activity by dopamine D4 receptors in prefrontal cortex. Mol. Pharmacol. 66, 948955.
  • Hernandez-Lopez S., Tkatch T., Perez-Garci E., Galarraga E., Bargas J., Hamm H. and Surmeier D. J. (2000) D2 dopamine receptors in striatal medium spiny neurons reduce L-type Ca2+ currents and excitability via a novel PLC[beta]1-IP3-calcineurin-signaling cascade. J. Neurosci. 20, 89878995.
  • Jin L., Goswami S., Gai G., Zhen X. and Friedman E. (2002) SKF83959 selectively regulates phosphatidylinositol-linked D1 dopamine receptors in rat brain. J. Neurochem. 85, 378386.
  • Lee S. P., So C. H., Rashid A. J., Varghese G., Cheng R., Lanca A. J., O'Dowd B. F. and George S. R. (2004) Dopamine D1 and D2 receptor Co-activation generates a novel phospholipase C-mediated calcium signal. J. Biol. Chem. 279, 35 67135 678.
  • Lezocano N. and Bergson C. (2005) D1/D5 dopamine receptors stimulate intracellular calcium release in primary cultures of neocortical and hippocampal neurons. J. Neurophysiol. 87, 21672175.
  • Liu J. C., DeFazio R. A., Espinosa-Jeffrey A., Cepeda C., De Vellis J. and Levine M. S. (2004) Calcium modulates dopamine potentiation of N-methyl-D-aspartate responses: electrophysiological and imaging evidence. J. Neurosci. Res. 76, 315322.
  • Lo K. J., Luk H. N., Chin T. Y. and Chueh S. H. (2002) Store depletion-induced calcium influx in rat cerebellar astrocytes. Br. J. Pharmacol. 135, 13831392.
  • Neve K. A., Seamans J. K. and Trantham-Davidson H. (2004) Dopamine receptor signaling. J. Recept. Signal Transduct. Res. 24, 165205.
  • Pacheco M. A. and Jope R. S. (1997) Comparison of [3H] phosphatidylinositol and [3H] phosphatidylinositol 4, 5-bisphosphate hydrolysis in postmortem human brain membranes and characterization of stimulation by dopamine D1 receptors. J. Neurochem. 69, 639644.
  • Panchalingam S. and Undie A. (2001) SKF83959 exhibits biochemical agonism by stimulating [35S]GTPgammaS binding and phosphoinositide hydrolysis in rat and monkey brain. Neuropharmacology 40, 826837.
  • Panchalingam S. and Undie A. (2005) Physicochemical modulation of agonist-induced [35s]GTPgammaS binding: implications for coexistence of multiple functional conformations of dopamine D1-like receptors. J. Recept. Signal Transduct. Res. 25, 125146.
  • Putney J. W. Jr (2003) Capacitative calcium entry in the nervous system. Cell Calcium 34, 339344.
  • Raymond C. R., Thompson V. L., Tate W. P. and Abraham W. C. (2000) Metabotropic glutamate receptors trigger homosynaptic protein synthesis to prolong long-term potentiation. J. Neurosci. 20, 969976.
  • Ross W. N., Nakamura T., Watanabe S., Larkum M. and Lasser-Ross N. (2005) Synaptically activated Ca2+ release from internal stores in CNS neurons. Cell. Mol. Neurosci. 25, 283295.
  • Tang T. S. and Bezprozvanny I. (2004) Dopamine receptor-mediated Ca2+ signaling in striatal neurons. J. Biol. Chem. 279, 42 08242 094.
  • Tang T. S., Tu H., Wang Z. and Bezprozvanny I. (2003) Modulation of type 1 inositol (1,4,5)-trisphosphate receptor function by protein kinase A and protein phosphatase 1. J. Neurosci. 23, 403415.
  • Undie A. S., Weinstock J., Sarau H. M. and Friedman E. (1994) Evidence for a distinct D1-like dopamine receptor that couples to activation of phosphoinositide metabolism in brain. J. Neurochem. 62, 20452048.
  • Wirtshafter D. and Osborn C. V. (2005) The atypical dopamine D1 receptor agonist SKF83959 inducesstriatal Fos expression in rats. Eur. J. Pharmacol. 528, 8894.
  • Yeckel M. F., Kapur A. and Johnston D. (1999) Multiple forms of LTP in hippocampal CA3 neurons use a common postsynaptic mechanism. Nat. Neurosci. 2, 625633.
  • Zhang Z. J., Jiang X. L., Zhang S. E., Hougha C., Li H., Chen J. G. and Zhen X. C. (2005) The paradoxical effects of SKF83959, a novel dopamine D1-like receptor agonist, in the rat acoustic startle reflex paradigm. Neurosci. Lett. 382, 134138.
  • Zhen X. C., Goswami S., Abdali S. A., Gil M., Bakshi K. and Friedman E. (2004) Regulation of cyclin-dependent kinase 5 and calcium/calmodulin-dependent protein kinase II by phosphatidylinositol-linked dopamine receptor in rat. Brain. Mol. Pharmacol. 66, 15001507.
  • Zhen X. C., Goswami S. and Friedman E. (2005) The role of the phosphatidylinositol-linked D1 dopamine receptor in the pharmacology of SKF83959. Pharmacol. Biochem. Behav. 80, 597601.