Introduction

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The serotonin (5-HT)_1A and 5-HT_1B 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-HT_1A and 5-HT_1B 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-HT_1A and 5-HT_1B 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-HT_1A/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-HT_1A and 5-HT_1B 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-HT_1A and 5-HT_1B receptors (Berg et al., 1994b, 1996; Evans et al., 2001). Receptor-mediated activation of PLA₂ reduces the responsiveness of the 5-HT_1B 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-HT_1B system (Berg et al., 1994b, 1996). On the other hand, the 5-HT_1A receptor system is more dynamically regulated. As for the 5-HT_1B receptor, activation of the PLA₂ reduces 5-HT_1A receptor responsiveness; however, unlike the 5-HT_1B receptor, PLC-mediated increases in [Ca²⁺]_i enhance 5-HT_1A 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-HT_1A receptors, there was no role for PKC activation in regulation of 5-HT_1A agonist efficacy. This lack of a role for PKC was surprising in that the 5-HT_1A receptor is a target for PKC-mediated phosphorylation, and PKC activation with phorbol esters reduces 5-HT_1A 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-HT_1A and 5-HT_1B receptor system responsiveness in response to activation of P_2Y purinergic receptors. In contrast to coactivation of P_2Y receptors, pretreatment of cells with the P_2Y agonist ATP reduced responsiveness of the 5-HT_1A, but not 5-HT_1B, receptor system in a PKC-dependent manner. Furthermore, the data suggest that the PKC effect on 5-HT_1A agonist responsiveness is dependent upon upstream activation of phospholipase D (PLD). These data underscore important differences in the regulation of 5-HT_1A and 5-HT_1B receptor systems by phospholipid signaling cascades.

	Materials and Methods

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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-HT_1A receptors 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-HT_2C receptors (Berg et al., 1994b) and which have been transfected stably to express h5-HT_1A receptors (~1.3 pmol/mg protein) (Evans et al., 2001). Cells were maintained in minimal essential medium- 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-HT_1A and 5-HT_1B agonist-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-HT_1A receptor antagonist p-MPPF (10 µM; K_B = 1.2 nM) was used to distinguish 5-HT_1B receptor responses from those mediated by 5-HT_1A 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 (***).

	Results

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CHO-K1 cells express naturally purinergic receptors of the P_2Y subtype (Iredale and Hill, 1993). Consistent with our previous reports (Berg et al., 1994b; Evans et al., 2001), coactivation of P_2Y receptors with ATP reduced the capacity of the 5-HT_1B 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-HT_1A receptor system (Fig. 1). However, the action of P_2Y receptor activation on 5-HT_1A and 5-HT_1B 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-HT_1A receptor system was reduced by about 55%, whereas the responsiveness of the 5-HT_1B receptor system to agonist stimulation was not changed (Fig. 2). The ATP-pretreatment effect on the 5-HT_1A agonist response gradually reversed as the time interval between P_2Y receptor activation and the test of 5-HT_1A receptor system responsiveness was increased (Fig. 2). The responsiveness of the 5-HT_1B receptor system was not changed by any of the pretreatment time periods.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Effect of coincubation with either 5-HT1A or 5-HT1B receptor agonists and the P2Y receptor 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-HT1B receptor function, cells were pretreated with 10 µM p-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.

View larger version (18K): [in this window] [in a new window]   Fig. 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-HT_2C receptor expressed in CHO-2C/1A cells reduces both 5-HT_1A and 5-HT_1B agonist efficacy because of a greater capacity of 5-HT_2C 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-HT_1A, but not 5-HT_1B, agonist responsiveness produced by coactivation of the 5-HT_2C receptor, further illustrating differences in the capacity of the 5-HT_1A and 5-HT_1B receptor systems to be regulated by phospholipid signaling cascades.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Effect of ATP pretreatment on 5-HT2C receptor-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-HT_1A receptor is a target for PKC-mediated phosphorylation, and PKC activation with phorbol esters reduces 5-HT_1A 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-HT_1A agonist efficacy in response to coactivation of P_2Y receptors (Evans et al., 2001). However, as shown in Fig. 4, the reduction in responsiveness of the 5-HT_1A receptor system produced by pretreatment with ATP was blocked by the protein kinase inhibitor staurosporine and by the selective PKC inhibitor bisindolylmaleimide.

View larger version (33K): [in this window] [in a new window]   Fig. 4.   Effect of ATP pretreatment on 5-HT1A receptor 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 P_2Y 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-HT_1A agonist responsiveness after ATP pretreatment was blocked in the presence of ethanol, but not in the presence of the secondary alcohol isopropanol.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Effect of ATP pretreatment on 5-HT1A receptor 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-HT_1A 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-HT_1A agonist responsiveness produced by activation of PKC with the phorbol ester PDBu.

View larger version (22K): [in this window] [in a new window]   Fig. 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

	Discussion

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Previously, we reported that the responsiveness of the 5-HT_1A receptor system can be altered by coactivation of receptors that couple to phospholipid signaling cascades (e.g., 5-HT_2C and purinergic P_2Y) (Evans et al., 2001). 5-HT_1A 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-HT_1A agonist efficacy is enhanced by increased [Ca²⁺]_i derived from receptor-mediated activation of PLC. The net effect on 5-HT_1A 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-HT_1A 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-HT_1A agonist efficacy in response to coactivation of 5-HT_2C or P_2Y 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-HT_1A receptor system but that the effect is highly dependent upon the timing of receptor activation. Consistent with our previous study, coactivation of P_2Y receptors did not alter 5-HT_1A agonist responsiveness. However, when the P_2Y receptor was activated for 5 min, 5 min before testing the responsiveness of the 5-HT_1A receptor system, the 5-HT_1A agonist response was reduced. If the time interval between P_2Y and 5-HT_1A receptor activation was increased to 15 or 30 min, the effect was abolished, suggesting that the effect on 5-HT_1A responsiveness is rapidly reversible. The reduction in the 5-HT_1A agonist response by P_2Y 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., , I, II, and ) are stimulated by both calcium and 1,2-diacylglycerol or phorbol esters; the novel PKC isoforms (i.e., , , , and ) are activated by diacylglycerol or phorbol esters yet are calcium-independent; and the atypical PKC isoforms (i.e., , , and ) 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) P_2Y 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-HT_1A 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-HT_1A receptor system responsiveness, suggesting the effect of P_2Y 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-HT_1A 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-HT_1A 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-HT_1A 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-HT_1A receptor system. These results are consistent with studies that have shown that activation of PKC with phorbol esters directly phosphorylates the 5-HT_1A receptor and reduces 5-HT_1A 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., PKC), whereas PLD-derived diacylglycerol is associated with activation of calcium-independent isoforms (e.g., PKC) (Ha and Exton, 1993). CHO cells have been reported to express PKC , , , , and  (Megson et al., 2001), which would suggest that the isoform(s) of PKC that mediate the reduction in responsiveness of the 5-HT_1A receptor system in response to P_2Y receptor activation could be  or .

Although the 5-HT_1A and 5-HT_1B 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-HT_1A, but not 5-HT_1B, receptor system is regulated by increases in [Ca²⁺]_i. Herein, we found that P_2Y-mediated PKC activation, as a result of ATP pretreatment, reduced 5-HT_1A, but did not alter 5-HT_1B, receptor system responsiveness. This agrees with our previous report that 5-HT_1B agonist 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-HT_1A and 5-HT_1B receptor systems is that ATP pretreatment blocked the effect of 5-HT_2C receptor activation on 5-HT_1A, but not 5-HT_1B, receptor signaling. This specificity for the 5-HT_1A receptor is consistent with the notion that the 5-HT_1A receptor itself is the target for the ATP pretreatment effect, likely receptor phosphorylation by PKC.

As mentioned above, both 5-HT_1A and 5-HT_1B 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-HT_1A receptor system in response to ATP pretreatment. Unlike the 5-HT_1A receptor system, in response to P_2Y receptor activation the 5-HT_1B 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-HT_1A agonist responses, the responsiveness of the 5-HT_1B receptor system was not affected. In fact, none of the pretreatment conditions altered 5-HT_1B agonist efficacy. These data suggest that the effect of the cyclooxygenase-dependent metabolite of arachidonic acid on 5-HT_1B responsiveness either requires a longer period of P_2Y receptor activation (>5 min) or requires coactivation of both receptor systems.

In summary, activation of the P_2Y receptor reduces the 5-HT_1A receptor system responsiveness in a manner that is dependent upon the temporal pattern of receptor activation. Furthermore, the mechanism for this reduced responsiveness is P_2Y-mediated stimulation of PLD, which leads to activation of PKC. It is likely that phosphorylation of the 5-HT_1A receptor by PKC, as shown by others (Raymond, 1991), reduces its signaling in response to agonist activation. In contrast, the 5-HT_1B receptor system is not regulated in the same time-dependent manner by P_2Y receptor activation. Reduced responsiveness of the 5-HT_1B receptor system requires coactivation of the P_2Y and 5-HT_1B receptors. These studies underscore the differences in the 5-HT_1A and 5-HT_1B receptor systems and emphasize the importance of time as a variable in signal transduction.