Temporal Regulation of Agonist Efficacy at 5-Hydroxytryptamine (5-HT)1A and 5-HT1BReceptors

  1. Kelly A. Berg,
  2. Kenda L. J. Evans1,
  3. Jodie D. Cropper and
  4. William P. Clarke
  1. Department of Pharmacology, University of Texas Health Science Center, San Antonio, Texas
  1. Kelly A. Berg, Department of Pharmacology-7764, University of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX 78229-3900. E-mail: berg{at}uthscsa.edu
 
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Abstract

Coactivation of purinergic (P2Y) receptors reduces agonist efficacy at serotonin1B (5-HT1B), but not 5-HT1A receptors. Herein, we report that pretreatment for 5 min with the P2Y receptor agonist ATP reduced agonist responsiveness at the 5-HT1A, but not at the 5-HT1B, receptor. The effect of ATP pretreatment on the 5-HT1A receptor response rapidly reversed within a 10 min time frame between P2Y receptor and 5-HT1Areceptor activation. ATP pretreatment effects on 5-HT1Aagonist responsiveness were blocked by the protein kinase inhibitors staurosporine and bisindolylmaleimide, suggesting that the ATP-mediated temporal regulation involves activation of protein kinase C (PKC). Moreover, the temporal effect of ATP was blocked by incubation with 1% ethanol, suggesting that consequences of phospholipase D (PLD) activation play a role. ATP pretreatment blocked the inhibitory effect produced by 5-HT2C receptor activation on the 5-HT1A, but not the 5-HT1B, receptor response, suggesting that the 5-HT1A receptor itself was the target for PLD/PKC action. Finally, ethanol did not block the reduction in responsiveness of the 5-HT1A receptor system produced by activation of PKC with phorbol ester treatment, suggesting that PKC activation lies downstream of PLD. Taken together, these data suggest that activation of P2Y receptors can reduce responsiveness of the 5-HT1A receptor system via a PLD/PKC-dependent mechanism that is highly dependent upon the temporal pattern of receptor activation. Moreover, this work underscores the importance of time as a variable in receptor signaling cross talk and serves to further illustrate differences between the 5-HT1A and 5-HT1B receptor systems.

The serotonin (5-HT)1A and 5-HT1B receptors are seven-transmembrane spanning receptors that inhibit adenylyl cyclase activity via pertussis toxin-sensitive G proteins (i.e., Gi/Go) in brain tissue (De Vivo and Maayani, 1985; Bouhelal et al., 1988; Schoeffter and Hoyer, 1989) and heterologous expression systems (Raymond, 1991; Adham et al., 1992; Hamblin et al., 1992; Albert et al., 1996). Both 5-HT1A and 5-HT1B receptors are autoreceptors on serotonergic neurons and thus play pivotal roles in the regulation of serotonergic neurotransmission (Pineyro and Blier, 1999; Knobelman et al., 2000). Alterations in the responsiveness of 5-HT1A and 5-HT1B receptor systems, and consequently of serotonergic neurotransmission, have been implicated in the etiology of a variety of psychiatric disorders, including anxiety, depression, obsessive-compulsive disorders, schizophrenia, and eating disturbances (Lucki, 1998). Furthermore, drugs that influence serotonergic neurotransmission have proven to be valuable therapeutic agents (Blier and de Montigny, 1999). Accordingly, knowledge of how the responsiveness of the 5-HT1A/1B receptor systems can be regulated may lead to better understanding of the role these receptor subtypes play in various physiological and pathological conditions and aid in the development of new clinically useful drugs.

Several reports have indicated that the responsiveness of the 5-HT1A and 5-HT1B receptor systems to agonist activation can be regulated by intracellular cross talk mechanisms coupled to heterologous receptor systems (Raymond, 1991; Harrington et al., 1994; Lembo and Albert, 1995; Hensler et al., 1996). For example, we recently found that coactivation of receptors coupled to phospholipid signaling cascades [phospholipase C (PLC) and phospholipase A2 (PLA2)] can alter agonist efficacy at 5-HT1A and 5-HT1B receptors (Berg et al., 1994b, 1996; Evans et al., 2001). Receptor-mediated activation of PLA2 reduces the responsiveness of the 5-HT1B receptor system via a cyclooxygenase-sensitive metabolite of arachidonic acid that targets adenylyl cyclase. Consequences of PLC signaling (PKC and [Ca2+]i) do not alter the 5-HT1B system (Berg et al., 1994b, 1996). On the other hand, the 5-HT1A receptor system is more dynamically regulated. As for the 5-HT1Breceptor, activation of the PLA2 reduces 5-HT1A receptor responsiveness; however, unlike the 5-HT1B receptor, PLC-mediated increases in [Ca2+]i enhance 5-HT1A agonist efficacy. The net effect of coactivation of a receptor that couples to both PLA2 and PLC depends upon the relative capacity of the receptor to produce arachidonic acid versus increase [Ca2+]i (Evans et al., 2001). Interestingly, in these experiments in which heterologous receptors were coactivated with 5-HT1A receptors, there was no role for PKC activation in regulation of 5-HT1A agonist efficacy. This lack of a role for PKC was surprising in that the 5-HT1A receptor is a target for PKC-mediated phosphorylation, and PKC activation with phorbol esters reduces 5-HT1A receptor signaling (Raymond, 1991; Harrington et al., 1994; Lembo and Albert, 1995;Hensler et al., 1996).

Time is an important variable in biological systems, however, relatively little is known about the time dependence of changes in responsiveness elicited by cross talk mechanisms between receptor systems. In this study, we explored the time course of regulation of 5-HT1A and 5-HT1B receptor system responsiveness in response to activation of P2Y purinergic receptors. In contrast to coactivation of P2Y receptors, pretreatment of cells with the P2Y agonist ATP reduced responsiveness of the 5-HT1A, but not 5-HT1B, receptor system in a PKC-dependent manner. Furthermore, the data suggest that the PKC effect on 5-HT1A agonist responsiveness is dependent upon upstream activation of phospholipase D (PLD). These data underscore important differences in the regulation of 5-HT1Aand 5-HT1B receptor systems by phospholipid signaling cascades.

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Materials and Methods

Materials.

N,N-Dipropyl-5-carboxamidotryptamine (dp-5-CT), 5-carboxamidotryptamine (5-CT), and (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane (DOI) were purchased from Sigma/RBI (Natick, MA); 125I-cAMP tracer was from PerkinElmer Life Sciences (Boston, MA), and anti-cAMP antibody was from ICN Pharmaceuticals (Costa Mesa, CA). Forskolin, staurosporine, and bisindolylmaleimide were purchased from Calbiochem (La Jolla, CA). Rolipram was a generous gift from Berlex Laboratories (Cedar Knolls, NJ) and 4-(2′-methoxy-)-phenyl-1-[2′-(N-2"-pyridyl)-p-fluorobenz-amido]ethyl-piperazine (p-MPPF) was a generous gift from Dr. Hank Kung (University of Pennsylvania, Philadelphia, PA). All tissue culture reagents and Hanks' balanced salt solution were purchased from Invitrogen (Carlsbad, CA). All other drugs and chemicals (reagent grade) were purchased from Sigma-Aldrich (St. Louis, MO).

Transfection and Cell Culture.

CHO-1A cells are a CHO-K1 clonal cell line that expresses stably h5-HT1Areceptors at a density of approx 130 fmol/mg protein as determined by BMY-7378 sensitive, [3H](±)-8-hydroxy-dipropylaminotetralin saturation binding (Evans et al., 2001). CHO-2C/1A cells are a clonal cell line expressing approximately 200 to 250 fmol/mg protein h5-HT2C receptors (Berg et al., 1994b) and which have been transfected stably to express h5-HT1Areceptors (∼1.3 pmol/mg protein) (Evans et al., 2001). Cells were maintained in minimal essential medium-α formulation supplemented with 5% fetal bovine serum and 50 μg/ml G418 (CHO-1A) or 125 μg/ml zeocin + 300 μg/ml hygromycin (CHO-2C/1A). For all experiments, cells were seeded into 24-well, 15-cm, or T175 tissue culture vessels at a density of 4 × 104 cells/cm2. After 24 h, cells were washed with Hanks' balanced salt solution and placed into Dulbecco's modified Eagle's medium/F-12 (1:1) with 5 μg/ml insulin, 5 μg/ml transferrin, 30 nM selenium, 20 nM progesterone, and 100 μM putrescine (serum-free media) and grown for an additional 24 h before experimentation.

Inhibition of cAMP Accumulation.

5-HT1A and 5-HT1Bagonist-mediated inhibition of forskolin-stimulated cAMP accumulation was determined by measuring the inhibition of cAMP accumulated in response to 1 μM forskolin (15 min, 37°C) in the presence of the phosphodiesterase inhibitor rolipram (0.1 mM) as described previously (Berg et al., 1994a). The selective 5-HT1Areceptor antagonist p-MPPF (10 μM;KB = 1.2 nM) was used to distinguish 5-HT1B receptor responses from those mediated by 5-HT1A receptors (Evans et al., 2001). Cellular cAMP content was measured by radioimmunoassay and normalized to protein content, which was measured according to the method of Lowry et al. (1951).

Data Analysis.

For cAMP accumulation experiments, data from each experiment were normalized by defining the cAMP response to 1 μM forskolin as 100%. Statistical comparisons of treatment effects were done where appropriate with the Student's t test (paired). For experiments where multiple comparisons were made, one-way analysis of variance followed by Newman-Keuls post hoc test was used. Asterisks (*) denote statistically significant p values <0.05 (*), <0.01 (**), and <0.001 (***).

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Results

CHO-K1 cells express naturally purinergic receptors of the P2Y subtype (Iredale and Hill, 1993). Consistent with our previous reports (Berg et al., 1994b; Evans et al., 2001), coactivation of P2Y receptors with ATP reduced the capacity of the 5-HT1B receptor agonist 5-CT to inhibit forskolin-stimulated cAMP accumulation by about 40%, but did not alter agonist (dp-5-CT) responsiveness at the 5-HT1A receptor system (Fig.1). However, the action of P2Y receptor activation on 5-HT1A and 5-HT1B receptor system responsiveness was reversed when the receptors were activated in a different temporal sequence. When cells were treated with ATP for 5 min, followed by a quick wash and further incubation without ATP for 5 min (subsequently referred to herein as “the ATP pretreatment paradigm”), agonist responsiveness at the 5-HT1A receptor system was reduced by about 55%, whereas the responsiveness of the 5-HT1B receptor system to agonist stimulation was not changed (Fig.2). The ATP-pretreatment effect on the 5-HT1A agonist response gradually reversed as the time interval between P2Y receptor activation and the test of 5-HT1A receptor system responsiveness was increased (Fig. 2). The responsiveness of the 5-HT1B receptor system was not changed by any of the pretreatment time periods.

Figure 1
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Figure 1

Effect of coincubation with either 5-HT1Aor 5-HT1B receptor agonists and the P2Yreceptor agonist ATP on forskolin-stimulated cAMP accumulation (FScA). cAMP accumulation was measured in CHO cells expressing stably h5-HT1A receptors (CHO-1A) incubated with forskolin (1 μM, 15 min, 37°C) with or without either dp-5-CT (10 nM, 5-HT1A) or 5-CT (5 nM, 5-HT1B) in the presence or absence of 1 mM ATP as indicated. To assess 5-HT1Breceptor function, cells were pretreated with 10 μMp-MPPF for 15 min to block 5-HT1A receptor activation. Data shown are expressed as the percentage of inhibition of FScA by either dp-5-CT or 5-CT and represent the mean ± S.E.M. of four experiments. The presence of p-MPPF did not alter FscA, which was 67 ± 9 and 46 ± 2 pmol/mg of protein in the presence of vehicle or ATP, respectively. ∗∗, p< 0.01.

Figure 2
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Figure 2

Effect of ATP pretreatment on agonist efficacy at either 5-HT1A or 5-HT1B receptors. CHO-1A cells were treated with vehicle (distilled H2O) or 1 mM ATP for 5 min at 37°C, washed twice quickly, and further incubated (37°C) for 5, 15, and 30 min as indicated. After this treatment paradigm, inhibition of forskolin-stimulated cAMP accumulation (FscA) (15 min, 37°C) by dp-5-CT (10 nM, 5-HT1A) or 5-CT (5 nM, 5-HT1B), in the presence of 10 μM p-MPPF, was measured. Data shown are expressed as the percentage of control (no pretreatment) inhibition of FscA by either dp-5-CT or 5-CT and are mean ± S.E.M. of four experiments. FScA after vehicle or ATP pretreatment was 63 ± 6 and 51 ± 4 pmol/mg of protein, respectively, and did change with increased time intervals between pretreatment and 5-HT1A/1B agonist challenge. ∗,p < 0.05

Coactivation of the 5-HT2C receptor expressed in CHO-2C/1A cells reduces both 5-HT1A and 5-HT1B agonist efficacy because of a greater capacity of 5-HT2C receptor activation to stimulate PLA2 versus PLC (Evans et al., 2001). As shown in Fig. 3, the ATP pretreatment paradigm blocked the reduction in 5-HT1A, but not 5-HT1B, agonist responsiveness produced by coactivation of the 5-HT2C receptor, further illustrating differences in the capacity of the 5-HT1A and 5-HT1B receptor systems to be regulated by phospholipid signaling cascades.

Figure 3
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Figure 3

Effect of ATP pretreatment on 5-HT2Creceptor-mediated cross talk regulation of agonist efficacy at 5-HT1A and 5-HT1B receptors. CHO cells transfected stably with both h-5-HT1A and h5-HT2C receptors (CHO-2C/1A) were treated with vehicle (distilled H2O) or 1 mM ATP for 5 min (37°C), washed, and further incubated at 37°C for 5 min. After this pretreatment paradigm, the inhibition of forskolin-stimulated cAMP accumulation (FscA) (15 min, 37°C) by the 5-HT1A receptor agonist dp-5-CT (10 nM) (A) or 5-HT1B receptor agonist 5-CT (5 nM) (B) in the presence of 10 μM p-MPPF, was measured in the presence and absence of maximal concentrations of the 5-HT2C receptor agonist DOI (1 μM). Data shown are expressed as the percentage of inhibition of FScA by dp-5-CT and 5-CT and are the mean ± S.E.M. of three to five experiments. A, for vehicle-pretreated cells, FScA was 68 ± 8 and 56 ± 7 pmol/mg protein in the absence and presence of DOI, respectively. For ATP-pretreated cells, FScA was 55 ± 10 and 42 ± 10 pmol/mg of protein absence of presence of DOI, respectively. B, for vehicle-pretreated cells, FScA was 78 ± 21 and 87 ± 13 pmol/mg of protein in the presence and absence of DOI, respectively. For ATP-pretreated cells, FScA was 84 ± 22 and 72 ± 13 pmol/mg of protein in the absence and presence of DOI, respectively. ∗, p < 0.05, ∗∗, p < 0.01 compared with vehicle control and †, p < 0.01 compared with DOI control.

The 5-HT1A receptor is a target for PKC-mediated phosphorylation, and PKC activation with phorbol esters reduces 5-HT1A receptor signaling (Raymond, 1991;Harrington et al., 1994; Lembo and Albert, 1995; Hensler et al., 1996), although PKC is not involved in mediating the reduction in 5-HT1A agonist efficacy in response to coactivation of P2Y receptors (Evans et al., 2001). However, as shown in Fig. 4, the reduction in responsiveness of the 5-HT1Areceptor system produced by pretreatment with ATP was blocked by the protein kinase inhibitor staurosporine and by the selective PKC inhibitor bisindolylmaleimide.

Figure 4
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Figure 4

Effect of ATP pretreatment on 5-HT1Areceptor agonist efficacy measured in the presence of the PKC inhibitors staurosporine (SSP) or bisindolmaleimide (BIS). cAMP accumulation was measured in CHO-1A cells incubated with forskolin (1 μM, 15 min, 37°C) with or without 10 nM dp-5-CT in the presence or absence of 1 mM ATP. SSP (1 μM) or 1 μM BIS were present 5 min before ATP pretreatment, during the wash, and 5-min challenge interval. Data shown represent the mean ± S.E.M. of four experiments. Forskolin-stimulated cAMP accumulation (FscA) was 92 ± 7 and 90 ± 6 pmol/mg of protein for vehicle-pretreated and ATP-pretreated cells, respectively. ∗∗, p < 0.01

It is well known that PKC activation is a major consequence of activation of PLC. However, more recently it has been shown that stimulation of PKC may also be a consequence of activation of other phospholipases, such as PLA2 and PLD (Jaken, 1996; Exton, 1997), and each of these phospholipases can be activated by P2Y receptors (Briley et al., 1994; Berg et al., 1999; Evans et al., 2001). In the presence of primary alcohols such as ethanol, but not secondary alcohols, the production of the primary product of PLD activation, phosphatidic acid, is blocked (Exton, 1998; Liscovitch et al., 2000). As shown in Fig.5, the reduction in 5-HT1A agonist responsiveness after ATP pretreatment was blocked in the presence of ethanol, but not in the presence of the secondary alcohol isopropanol.

Figure 5
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Figure 5

Effect of ATP pretreatment on 5-HT1Areceptor agonist efficacy measured in the presence of inhibition of phospholipase D signaling by ethanol (ETOH). cAMP accumulation was measured in CHO-1A cells incubated with forskolin (1 μM, 15 min, 37°C) with or without 10 nM dp-5-CT in the presence or absence of 1 mM ATP. ETOH (1%) or the secondary alcohol isopropanol (ISO, 1%) was present 5 min before ATP treatment, during the wash, and 5-min challenge interval. Data shown represent the mean ± S.E.M. from three to nine experiments. The presence of alcohol did not alter forskolin-stimulated cAMP accumulation (FscA), which was 185 ± 19 and 156 ± 7 pmol/mg of protein for vehicle-pretreated and for ATP-pretreated cells, respectively. ∗∗, p < 0.01

Receptor-mediated activation of PLD has been shown to involve both PKC-dependent and -independent mechanisms (for review, see Exton, 1997). To determine whether PKC activation occurred upstream or downstream to that of PLD, we tested whether the reduction of 5-HT1A receptor system responsiveness by direct activation of PKC with phorbol ester was sensitive to the presence of ethanol. As shown in Fig. 6, ethanol did not block the reduction in 5-HT1A agonist responsiveness produced by activation of PKC with the phorbol ester PDBu.

Figure 6
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Figure 6

Regulation of 5-HT1A agonist efficacy by phorbol ester treatment measured in the presence and absence of ETOH. After incubation for 5 min (37°C) with or without 1% ETOH, cells were treated for 15 min (37°C) with or without the phorbol ester PDBu (1 μM). Inhibition of forskolin-stimulated cAMP accumulation (FscA) (15 min, 37°C) by the 5-HT1A receptor agonist dp-5-CT (100 nM) was measured. Data are expressed as the percentage of control dp-5-CT-mediated inhibition of forskolin-stimulated cAMP accumulation and represent the mean ± S.E.M. (n = 7). The presence of ETOH did alter FscA, which was 96 ± 9 and 114 ± 16 pmol/mg of protein in the absence and presence of PDBu, respectively. The presence of ETOH did alter the inhibition of FScA by dp-5-CT which was 50 ± 6 and 17 ± 6% in the absence and presence of PDBu, respectively. ∗∗, p < 0.01

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Discussion

Previously, we reported that the responsiveness of the 5-HT1A receptor system can be altered by coactivation of receptors that couple to phospholipid signaling cascades (e.g., 5-HT2C and purinergic P2Y) (Evans et al., 2001). 5-HT1A agonist efficacy is reduced by a cyclooxygenase-dependent metabolite of arachidonic acid that is derived from receptor-mediated activation of PLA2. On the other hand, 5-HT1A agonist efficacy is enhanced by increased [Ca2+]iderived from receptor-mediated activation of PLC. The net effect on 5-HT1A receptor system responsiveness is based upon the relative changes in arachidonic acid production versus [Ca2+]i release elicited by phospholipid-coupled receptor activation. Interestingly, although PKC is generally thought to be a consequence of PLC activation and PKC activation with phorbol ester phosphorylates the 5-HT1A receptor and reduces its responsiveness (Raymond, 1991; Harrington et al., 1994; Lembo and Albert, 1995;Hensler et al., 1996), PKC does not seem to play a role in reducing 5-HT1A agonist efficacy in response to coactivation of 5-HT2C or P2Y receptors (Evans et al., 2001).

Herein, we have found that receptor-mediated PKC activation can play a role in regulating the responsiveness of the 5-HT1A receptor system but that the effect is highly dependent upon the timing of receptor activation. Consistent with our previous study, coactivation of P2Yreceptors did not alter 5-HT1A agonist responsiveness. However, when the P2Y receptor was activated for 5 min, 5 min before testing the responsiveness of the 5-HT1A receptor system, the 5-HT1A agonist response was reduced. If the time interval between P2Y and 5-HT1A receptor activation was increased to 15 or 30 min, the effect was abolished, suggesting that the effect on 5-HT1A responsiveness is rapidly reversible. The reduction in the 5-HT1A agonist response by P2Y receptor activation is likely mediated by PKC activation because inhibitors of PKC (staurosporine and bisindolylmaleimide) blocked the ATP pretreatment effect.

Currently, 11 isoforms of PKC have been identified that are divided into three classes based on structural properties and cofactor requirements. Classical PKC isoforms (i.e., α, βI, βII, and γ) are stimulated by both calcium and 1,2-diacylglycerol or phorbol esters; the novel PKC isoforms (i.e., δ, ε, η, and ς) are activated by diacylglycerol or phorbol esters yet are calcium-independent; and the atypical PKC isoforms (i.e., ζ, λ, and ι) are also calcium-independent but are not activated by either diacylglycerol or phorbol esters (Mellor and Parker, 1998). Receptor-mediated diacylglycerol production characteristically occurs in a biphasic manner, such that an initial transient increase in diacylglycerol levels is due to PLC-mediated phosphatidylinositol lipid hydrolysis, whereas a delayed and more sustained increase in diacylglycerol occurs as a result of PLD-mediated phosphatidylcholine lipid hydrolysis and subsequent conversion of phosphatidic acid to diacylglycerol by a phosphatidate phosphohydrolase (Jaken, 1996; Exton, 1997) P2Y receptors have been shown to couple to both PLC (Berg et al., 1996, 1999; Selbie et al., 1997) and PLD (Briley et al., 1994) in CHO cells. To examine the role of PLD in the ATP pretreatment-mediated reduction in 5-HT1A agonist responsiveness, CHO cells were treated with ATP in the presence of ethanol, which blocks the production of phosphatidic acid and the subsequent production of diacylglycerol (Exton, 1998; Liscovitch et al., 2000). Ethanol blocked the ATP pretreatment-mediated reduction in 5-HT1A receptor system responsiveness, suggesting the effect of P2Y receptor activation is mediated by PLD.

Receptor-mediated PLD activation has been shown to involve both PKC-dependent as well as PKC-independent mechanisms involving small molecular weight G proteins such as Rho and ARF (Exton, 1998;Liscovitch et al., 2000). Thus, it is possible that PLD may lie upstream or downstream of PKC. Receptor-mediated activation of PKC could lead to stimulation of PLD with the direct effect on the 5-HT1A receptor system being mediated by consequences of PLD activation (e.g., phosphatidic acid). Phosphatidic acid targets a number of enzymes, including various protein kinases (including PKC), G protein receptor kinases, and a novel serine/threonine protein kinase, phosphatidic acid-activated protein kinase (McPhail et al., 1999); thus, perhaps a component of the 5-HT1A receptor system could be a direct or indirect target of phosphatidic acid. However, our data suggest that PLD is upstream of PKC because the reduction in 5-HT1A agonist responsiveness elicited by direct activation of PKC with phorbol ester was not blocked by ethanol. Taken together, these data suggest that pretreatment with ATP leads to activation of PLD, which results in a rapidly reversible, PKC-mediated reduction in the responsiveness of the 5-HT1Areceptor system. These results are consistent with studies that have shown that activation of PKC with phorbol esters directly phosphorylates the 5-HT1A receptor and reduces 5-HT1A agonist responsiveness (Raymond, 1991;Lembo and Albert, 1995).

Some studies have suggested that activation of diacylglycerol-sensitive PKC isoforms is dependent on the source of diacylglycerol. For example, diacylglycerol derived from PLC activation results in stimulation of calcium-dependent isoforms (e.g., PKCα), whereas PLD-derived diacylglycerol is associated with activation of calcium-independent isoforms (e.g., PKCε) (Ha and Exton, 1993). CHO cells have been reported to express PKC α, δ, ε, ι, and ζ (Megson et al., 2001), which would suggest that the isoform(s) of PKC that mediate the reduction in responsiveness of the 5-HT1Areceptor system in response to P2Y receptor activation could be δ or ε.

Although the 5-HT1A and 5-HT1B receptors share a high level of sequence homology and have similar signal transduction systems, it is becoming clear that in addition to similarities, there are significant differences between these receptor systems, especially in their capacity to be regulated. Previously, we found that the responsiveness of both receptor systems is reduced by a cyclooxygenase-dependent metabolite of arachidonic acid derived from activation of PLA2. However the 5-HT1A, but not 5-HT1B, receptor system is regulated by increases in [Ca2+]i. Herein, we found that P2Y-mediated PKC activation, as a result of ATP pretreatment, reduced 5-HT1A, but did not alter 5-HT1B, receptor system responsiveness. This agrees with our previous report that 5-HT1Bagonist efficacy is not altered by activation of PKC with phorbol ester and highlights the significant differences between these two receptor systems. Further evidence for differences between the 5-HT1A and 5-HT1B receptor systems is that ATP pretreatment blocked the effect of 5-HT2C receptor activation on 5-HT1A, but not 5-HT1B, receptor signaling. This specificity for the 5-HT1A receptor is consistent with the notion that the 5-HT1A receptor itself is the target for the ATP pretreatment effect, likely receptor phosphorylation by PKC.

As mentioned above, both 5-HT1A and 5-HT1B receptor system responsiveness is reduced by coactivation of the PLA2 signaling cascade (Berg et al., 1996; Evans et al., 2001). However, the PLA2-arachidonic pathway does not seem to play a role in reducing the responsiveness of the 5-HT1Areceptor system in response to ATP pretreatment. Unlike the 5-HT1A receptor system, in response to P2Y receptor activation the 5-HT1B receptor system seems to be exclusively regulated by PLA2, not by consequences of PLC activation (PKC or Ca2+) (Berg et al., 1996,1998). With ATP pretreatment conditions that reduced 5-HT1A agonist responses, the responsiveness of the 5-HT1B receptor system was not affected. In fact, none of the pretreatment conditions altered 5-HT1B agonist efficacy. These data suggest that the effect of the cyclooxygenase-dependent metabolite of arachidonic acid on 5-HT1B responsiveness either requires a longer period of P2Y receptor activation (>5 min) or requires coactivation of both receptor systems.

In summary, activation of the P2Y receptor reduces the 5-HT1A receptor system responsiveness in a manner that is dependent upon the temporal pattern of receptor activation. Furthermore, the mechanism for this reduced responsiveness is P2Y-mediated stimulation of PLD, which leads to activation of PKC. It is likely that phosphorylation of the 5-HT1A receptor by PKC, as shown by others (Raymond, 1991), reduces its signaling in response to agonist activation. In contrast, the 5-HT1B receptor system is not regulated in the same time-dependent manner by P2Y receptor activation. Reduced responsiveness of the 5-HT1B receptor system requires coactivation of the P2Y and 5-HT1B receptors. These studies underscore the differences in the 5-HT1A and 5-HT1B receptor systems and emphasize the importance of time as a variable in signal transduction.

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Footnotes

  • ↵1 Current address: Texas Biotechnology Corporation, Departments of Pharmacology and HTS, 7000 Fannin St., 20th Floor, Houston, TX 77030-5400.

  • This work was supported by U.S. Public Health Service Grants DA 09094 (to K.A.B.), MH 57441 (to W.P.C.), and MH 48125.

  • DOI: 10.1124/jpet.102.042564

  • Abbreviations:
    5-HT
    5-hydroxytryptamine, serotonin
    PLC
    phospholipase C
    PLA2
    phospholipase A2
    PKC
    protein kinase C
    [Ca2+]i
    intracellular calcium concentration
    BMY 7378
    8-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-8-azaspirol(4,5)decane-7,9-dione dihydrochloride
    PLD
    phospholipase D
    dp-5-CT
    dipropyl 5-carboxamidotryptamine
    5-CT
    5-carboxamidotryptamine
    DOI
    (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane
    p-MPPF
    4-(2′-methoxy-)-phenyl-1-[2′-(N-2"-pyridyl)-p-fluorobenz-amido]ethyl-piperazine
    CHO
    Chinese hamster ovary
    PDBu
    phorbol dibutyrate
    • Received August 6, 2002.
    • Accepted August 30, 2002.
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References

  1. ↵
    1. Adham N,
    2. Romanienko P,
    3. Hartig P,
    4. Weinshank RL,
    5. Branchek T
    (1992) The rat 5-hydroxytryptamine1B receptor is the species homologue of the human 5-hydroxytryptamine1D beta receptor. Mol Pharmacol 41:1–7.
    Abstract
  2. ↵
    1. Albert PR,
    2. Lembo P,
    3. Storring JM,
    4. Charest A,
    5. Saucier C
    (1996) The 5-HT1A receptor: signaling, desensitization and gene transcription. Neuropsychopharmacology 14:19–25.
    CrossRefMedlineWeb of Science
  3. ↵
    1. Berg KA,
    2. Clarke WP,
    3. Chen Y,
    4. Ebersole BJ,
    5. McKay RD,
    6. Maayani S
    (1994a) 5-Hydroxytryptamine type 2A receptors regulate cyclic AMP accumulation in a neuronal cell line by protein kinase C-dependent and calcium/calmodulin-dependent mechanisms. Mol Pharmacol 45:826–836.
    Abstract
  4. ↵
    1. Berg KA,
    2. Clarke WP,
    3. Sailstad C,
    4. Saltzman A,
    5. Maayani S
    (1994b) Signal transduction differences between 5-hydroxytryptamine type 2A and type 2C receptor systems. Mol Pharmacol 46:477–484.
    Abstract
  5. ↵
    1. Berg KA,
    2. Maayani S,
    3. Clarke WP
    (1996) 5-Hydroxytryptamine2C receptor activation inhibits 5-hydroxytryptamine1B-like receptor function via arachidonic acid metabolism. Mol Pharmacol 50:1017–1023.
    Abstract
  6. ↵
    1. Berg KA,
    2. Maayani S,
    3. Clarke WP
    (1998) Interactions between effectors linked to serotonin receptors. Ann NY Acad Sci 861:111–120.
    CrossRefMedline
  7. ↵
    1. Berg KA,
    2. Stout BD,
    3. Cropper JD,
    4. Maayani S,
    5. Clarke WP
    (1999) Novel actions of inverse agonists on 5-HT2C receptor systems. Mol Pharmacol 55:863–872.
    Abstract/FREE Full Text
  8. ↵
    1. Blier P,
    2. de Montigny C
    (1999) Serotonin and drug-induced therapeutic responses in major depression, obsessive-compulsive and panic disorders. Neuropsychopharmacology 21:91S–98S.
    Medline
  9. ↵
    1. Bouhelal R,
    2. Smounya L,
    3. Bockaert J
    (1988) 5-HT1B receptors are negatively coupled with adenylate cyclase in rat substantia nigra. Eur J Pharmacol 151:189–196.
    CrossRefMedlineWeb of Science
  10. ↵
    1. Briley EM,
    2. Lolait SJ,
    3. Axelrod J,
    4. Felder CC
    (1994) The cloned vasopressin V1a receptor stimulates phospholipase A2, phospholipase C and phospholipase D through activation of receptor-operated calcium channels. Neuropeptides 27:63–74.
    CrossRefMedlineWeb of Science
  11. ↵
    1. De Vivo M,
    2. Maayani S
    (1985) Inhibition of forskolin-stimulated adenylate cyclase activity by 5-HT receptor agonists. Eur J Pharmacol 119:231–234.
    CrossRefMedline
  12. ↵
    1. Evans KJE,
    2. Cropper JD,
    3. Berg KA,
    4. Clarke WP
    (2001) Mechanisms of regulation of agonist efficacy at the 5-HT1A receptor by phospholipid-derived signaling components. J Pharmacol Exp Ther 297:1025–1035.
    Abstract/FREE Full Text
  13. ↵
    1. Exton JH
    (1997) Phospholipase D: enzymology, mechanisms of regulation and function. Physiol Rev 77:303–320.
    Abstract/FREE Full Text
  14. ↵
    1. Exton JH
    (1998) Phospholipase D. Biochim Biophys Acta 1436:105–115.
    Medline
  15. ↵
    1. Ha KS,
    2. Exton JH
    (1993) Differential translocation of protein kinase C isozymes by thrombin and platelet-derived growth factor. A possible function for phosphatidylcholine-derived diacylglycerol. J Biol Chem 268:10534–10539.
    Abstract/FREE Full Text
  16. ↵
    1. Hamblin MW,
    2. Metcalf MA,
    3. McGuffin RW,
    4. Karpells S
    (1992) Molecular cloning and functional characterization of a human 5-HT1B serotonin receptor: a homologue of the rat 5-HT1B receptor with 5-HT1D-like pharmacological specificity. Biochem Biophys Res Commun 184:752–759.
    CrossRefMedlineWeb of Science
  17. ↵
    1. Harrington MA,
    2. Shaw K,
    3. Zhong P,
    4. Ciaranello RD
    (1994) Agonist-induced desensitization and loss of high-affinity binding sites of stably expressed human 5-HT1A receptors. J Pharmacol Exp Ther 268:1098–1106.
    Abstract/FREE Full Text
  18. ↵
    1. Hensler JG,
    2. Cervera LS,
    3. Miller HA,
    4. Corbitt J
    (1996) Expression and modulation of 5-hydroxytryptamine1A receptors in P11 cells. J Pharmacol Exp Ther 278:1138–4115.
    Abstract/FREE Full Text
  19. ↵
    1. Iredale PA,
    2. Hill SJ
    (1993) Increases in intracellular calcium via activation of an endogenous P2-purinoceptor in cultured CHO-K1 cells. Br J Pharmacol 110:1305–1310.
    MedlineWeb of Science
  20. ↵
    1. Jaken S
    (1996) Protein kinase C isozymes and substrates. Curr Opin Cell Biol 8:168–173.
    CrossRefMedlineWeb of Science
  21. ↵
    1. Knobelman DA,
    2. Kung HF,
    3. Lucki I
    (2000) Regulation of extracellular concentrations of 5-hydroxytryptamine (5-HT) in mouse striatum by 5-HT(1A) and 5-HT(1B) receptors. J Pharmacol Exp Ther 292:1111–1117.
    Abstract/FREE Full Text
  22. ↵
    1. Lembo PM,
    2. Albert PR
    (1995) Multiple phosphorylation sites are required for pathway-selective uncoupling of the 5-hydroxytryptamine1A receptor by protein kinase C. Mol Pharmacol 48:1024–1029.
    Abstract
  23. ↵
    1. Liscovitch M,
    2. Czarny M,
    3. Fiucci G,
    4. Tang X
    (2000) Phospholipase D: molecular and cell biology of a novel gene family. Biochem J 345:401–415.
  24. ↵
    1. Lowry OH,
    2. Rosebrough NJ,
    3. Farr AL,
    4. Randall RJ
    (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275.
    FREE Full Text
  25. ↵
    1. Lucki I
    (1998) The spectrum of behaviors influenced by serotonin. Biol Psychiatry 44:151–162.
    CrossRefMedlineWeb of Science
  26. ↵
    1. McPhail LC,
    2. Waite KA,
    3. Regier DS,
    4. Nixon JB,
    5. Qualliotine-Mann D,
    6. Zhang WX,
    7. Wallin R,
    8. Sergeant S
    (1999) A novel protein kinase target for the lipid second messenger phosphatidic acid. Biochim Biophys Acta 1439:277–290.
    Medline
  27. ↵
    1. Megson AC,
    2. Walker EM,
    3. Hill SJ
    (2001) Role of protein kinase Cα in signaling from the histamine H(1) receptor to the nucleus. Mol Pharmacol 59:1012–1021.
    Abstract/FREE Full Text
  28. ↵
    1. Mellor H,
    2. Parker PJ
    (1998) The extended protein kinase C superfamily. Biochem J 332:281–292.
  29. ↵
    1. Pineyro G,
    2. Blier P
    (1999) Autoregulation of serotonin neurons: role in antidepressant drug action. Pharmacol Rev 51:533–591.
    Abstract/FREE Full Text
  30. ↵
    1. Raymond JR
    (1991) Protein kinase C induces phosphorylation and desensitization of the human 5-HT1A receptor. J Biol Chem 266:14747–14753.
    Abstract/FREE Full Text
  31. ↵
    1. Schoeffter P,
    2. Hoyer D
    (1989) 5-Hydroxytryptamine 5-HT1B and 5-HT1D receptors mediating inhibition of adenylate cyclase activity. Pharmacological comparison with special reference to the effects of yohimbine, rauwolscine and some beta-adrenoceptor antagonists. Naunyn-Schmiedeberg's Arch Pharmacol 340:285–292.
    MedlineWeb of Science
  32. ↵
    1. Selbie LA,
    2. King NV,
    3. Dickenson JM,
    4. Hill SJ
    (1997) Role of G-protein beta gamma subunits in the augmentation of P2Y2 (P2U)receptor-stimulated responses by neuropeptide Y Y1 Gi/o-coupled receptors. Biochem J 328:153–158.
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  1. doi: 10.1124/jpet.102.042564 JPET January 1, 2003 vol. 304 no. 1 200-205
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