5-HT1A and histamine H1 receptors in HeLa cells stimulate phosphoinositide hydrolysis and phosphate uptake via distinct G protein pools.

Regulation of phosphate uptake was studied in a HeLa cell line after transfection with DNA encoding the human 5-HT1A receptor. In these cells, 5-HT stimulates sodium-dependent phosphate uptake via protein kinase C activation. Endogenous histamine H1 receptors (739 +/- 20 fmol/mg protein) were identified with [3H]pyrilamine. Histamine (i) stimulated phosphoinositide hydrolysis (EC50 = 8.6 +/- 4.1 microM), (ii) activated protein kinase C (2.4-fold increase in activity), and (iii) increased phosphate uptake (EC50 = 3.2 +/- 1.8 microM) by increasing maximal transport (Vmax(basal) = 6.2 +/- 0.3 versus Vmax(histamine) = 9.1 +/- 0.4) without changing the affinity of the transport process for phosphate. Prolonged treatment with 16 microM phorbol 12-myristate 13-acetate completely blocked protein kinase C activation and markedly attenuated the stimulation of phosphate uptake induced by histamine, establishing that 5-HT and histamine stimulate phosphate uptake through the common pathway of protein kinase C activation. The linkages of the histamine H1 and 5-HT1A receptors to G protein pools were assessed in two ways. (i) The stimulation of phosphoinositide hydrolysis, protein kinase C activity, and phosphate uptake associated with histamine were insensitive to pertussis toxin, whereas those associated with 5-HT were very sensitive to pertussis toxin. (ii) The stimulation of phosphoinositide hydrolysis, protein kinase C activity, and phosphate uptake induced by histamine and 5-HT were additive. These findings suggest that distinct receptor types can stimulate phosphoinositide hydrolysis, protein kinase C, and phosphate uptake in an additive fashion through distinct pools of G proteins in a single cell type.

= 3.2 2 1.8 p~) by increasing maximal transport ( Vmsx(basslf = 6.2 f 0.3 uersus Vmax(~istemiae) = 9.1 f 0.4) without changing the affinity of the transport process for phosphate. Prolonged treatment with 16 p~ phorbol 12-myristate 13acetate completely blocked protein kinase C activation and markedly attenuated the stimulation of phosphate uptake induced by histamine, establishing that 5-HT and histamine stimulate phosphate uptake through the common pathway of protein kinase C activation. The linkages of the histamine HI and 5-HTIA receptors to G protein pools were assessed in two ways. (i) The stimulation of phosphoinositide hydrolysis, protein kinase C activity, and phosphate uptake associated with histamine were insensitive to pertussis toxin, whereas those associated with 5-HT were very sensitive to pertussis toxin. (ii) The stimulation of phosphoinositide hydrolysis, protein kinase C activity, and phosphate uptake induced by histamine and 5-HT were additive. These findings suggest that distinct receptor types can stimulate phosphoinositide hydrolysis, protein kinase C, and phosphate uptake in an additive fashion through distinct pools of G proteins in a single cell type.
Sodium-dependent phosphate uptake, which is a component of many mammalian cell systems (1)(2)(3)(4)(5)(6)(7)(8)(9) is subject to dynamic regulation by various hormones, receptors, second messengers and kinases and thus serves as a useful example of modifiable cellular transport. The regulation of phosphate PKJ cell line, both kinases appear to mediate the stimulation of phosphate uptake (16,25). We have utilized the HeLa cell line, because it represents a unique model for the regulation of sodium-dependent phosphate uptake in that the effects of the two kinases can be more easily distinguished.
HeLa cells contain an endogenous histamine HI receptor (26). We have previously demonstrated that recombinant human 5 -H T 1~ receptors stably expressed in these cells stimulate phosphate uptake primarily through activation of PKC (22). Because histamine HI receptors are classically linked to the stimulation of phosphoinositide hydrolysis through a pertussis toxin-insensitive G protein pool, it was of interest to examine whether histamine would stimulate sodium-dependent phosphate uptake in these cells and to compare the mechanism to that of 5-HT-mediated stimulation of phosphate uptake. We therefore predicted (i) that the histamine HI receptor would also stimulate sodium-dependent phosphate uptake in these cells and if so (ii) that these effects would be mediated through a distinct pool of G proteins than that used by the 5-HT1, receptor. (31.5 Ci/mmol) were from Du Pont-New England Nuclear. Protein kinase C DNA probes were from American Type Culture Collection. Other reagents were from Sigma or Bio-Rad.
Phosphate Uptake-Cells were grown in six-well dishes. Before the uptake studies were performed, the medium was replaced with Earle's solution (143 mM Na+, 5.4 mM K' , 0.8 mM M2+, 1.8 mM ca2+, 125 mM CI-, 15 mM Hepes, 5 mM glucose, pH 7.4) and allowed to equilibrate at 37 "C for 30 min. Unless otherwise indicated, 10 min prior to measurement of phosphate uptake, the Earle's solution was replaced with Earle's solution with or without the various drugs or hormones and incubated at 37 "C. Following that, the Earle's solution was replaced with transport media (either Earle's solution or sodiumfree choline-substituted Earle's solution) containing 32P04 and various concentrations of total phosphate (0.02-2 mM for kinetic studies; 0.2 mM for all other studies), and cells were incubated at 37 "C for an additional 3 min. The solutions were then aspirated, and the cell monolayer washed rapidly three times with ice-cold sodium free choline-substituted Earle's solution. Cells were dissolved in 2 ml of 0.1 N NaOH, and aliquots were collected for measurements of protein (28) and "PO4. Uptakes were linear between 1 and 20 min.
Time Course of Histamine Stimulation of Phosphate Uptake-Experiments to delineate the time course of histamine effects on phosphate uptake were performed as above at a fixed phosphate concentration of 0.2 mM. All incubations were performed in Earle's solution at 37 "C with 100 p~ histamine or vehicle (Earle's) for 1-60 min. Uptakes at 1 and 3 min were performed for one minute to more accurately reflect dynamic changes in the uptake rate. Values for these time points were multiplied by three so that comparisons with other uptake rates (expressed as nanomoles/mg protein/3 min) could be made. All other uptakes were for 3 min.
Formation of Inositol Phosphates-Cells were grown in six-well dishes and equilibrated for 24 h in regular medium supplemented with 5 pCi/ml of my~-[~H]inositol (14.6 Ci/mmol). After washing with phosphate-buffered saline (PBS), cells were incubated for 30 min in PBS containing 20 mM LiCl (37 "C). The medium was then replaced by fresh medium supplemented with various concentrations of the drugs. After 15 min the reaction was terminated by aspiration and addition of 1 ml of 0.4 M perchloric acid. The lysate was neutralized and used for the measurement of [3H]inositolphosphates using ion-exchange chromatography with Dowex-AGlX8 resin (0.8 ml, 100-200 mesh) in the formate phase (29,30).
Intact Cell Phosphorylations-PKC activation was assessed by measuring the phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) protein (formerly referred to as the 80-87-kDa protein) utilizing the method of Blackshear et al. (31-33) as described previously (22). Cells were grown in 100-mm dishes. The medium was removed, and cells were washed three times with 4 ml of incubation buffer (168 mM NaC1, 4.7 mM KCI, 1.4 mM CaCI2, 800 p M MgS04, 25 mM NaHC03, 10 mM D-glucose, 10 mM Hepes, pH 7.4) at 37 "C. Two ml of incubation buffer supplemented with 1% (w! v) bovine serum albumin (fraction V, cell culture grade) and 100 pC1 :'"PO4/ml were then added to each dish. After 2 h, cells were incubated in the presence of the hormones for 15 min. The buffer was then aspirated and cells washed twice with 4 ml of ice-cold incubation buffer and then scraped with a rubber policeman into 1 ml of 50 mM Tris/HCI, 50 mM benzamidine, 10 mM EDTA, 100 mM NaF, 5 mM dithiothreitol, and 0.25 M sucrose, pH 7.4. The cells were transferred to a plastic tube, homogenized and centrifuged at 12,000 X g for 20 min. The cytosolic fraction was centrifuged at 100,000 X g for 40 min. The pellet was discarded and the cytosolic proteins in the supernatant precipitated with 20% trichloroacetic acid followed by centrifugation at 12,000 X g for 30 min. The resulting pellet was washed once by resuspension in acetone (-20 "C) followed by centrifugation at 12,000 X g for 30 min. Samples were taken up in 9 M urea, matched for equal amounts of total counts (counts incorporated into the MARCKS protein represented a small fraction of total counts incorporated into * J. P. Middleton, unpublished observation. the cells), and subjected to sequential isoelectric focusing and SDSpolyacrylamide gel electrophoresis and autoradiography as described (32).
Down-regulation of Protein Kinase C Actiuity-Cells near confluence (-70-90%) were washed three times with serum-free DMEM and then incubated for 18 h with DMEM supplemented with penicillin (100 units/ml), streptomycin (100 pg/ml), and bovine serum albumin (1 mg/ml; fraction V, cell culture grade) in the presence or absence of 16 ~L M phorbol 12-myristate 13-acetate (PMA) (22, 31). Cells were washed three times with DMEM and then incubated in bovine serum albumin-supplemented DMEM without PMA for 1 h. Uptake studies or intact cell phosphorylations were then performed as above.
Pertussis Toxin Treatment-Cells were treated with various doses of pertussis toxin added to the regular medium for various times. Before further studies were undertaken, cells were treated as for phosphate uptake or phosphoinositide hydrolysis studies in pertussis toxin-free solutions.
ADP-ribosylation-After treatment of cell monolayers with pertussis toxin, cells were washed three times with ice-cold PBS and then scraped into ice-cold lysis buffer (5 mM Tris, pH 7.4, 10 mM EDTA, 2 mM EGTA, supplemented with 5 pg/ml each of leupeptin, benzamidine, soybean trypsin inhibitor, and pepstatin A), homogenized, and centrifuged at 37,000 X g for 15 min. The pellet (-200 kg of membrane protein) was washed three times by resuspension and centrifugation in lysis buffer. ADP-ribosylation was performed by a modification of the procedure of Sternweis and Robishaw (34) by resuspending the pellets to a final volume of 100 p1 in 25 mM Tris, pH 8.0, 100 mM NaC1, 1 mM dithiothreitol, 2.5 mM MgCI2, 0.5 mM EDTA, 10 mM thymidine, 10 p~ NAD, 100 p M ATP, 10 pM GTP, 5 pg of pre-activated pertussis toxin, and 5 pCi of [32P]NAD, and incubating at 37 "C with shaking for 1 h. The membranes were centrifuged at 37,000 X g for 15 min and washed four times by resuspension and centrifugation in lysis buffer. Pellets were suspended in 30 pl of 40 mM Tris, pH 6.8, 1% cholate, 0.2 mM dithiothreitol, and 1% SDS, warmed to 90 "C for 5 min, cooled and mixed with 10 pl of 10 mM N-ethylmaleimide, and incubated at room temperature for 30 min. Sample buffer (160 pl of 40 mM Tris, pH 6.8, 1% SDS, 5% P-mercaptoethanol, and 30% glycerol) was added, and the samples heated at 100°C for 5 min. Electrophoresis was performed through discontinuous 32-cm 12% polyacrylamide gels (1.3% crosslinked with N,N'-methylene bisacrylamide) as described by Laemmli (35). Gels were washed extensively in 10% glacial acetic acid, 20% methanol, 70% water, dried, and subjected to autoradiography.
PHIPyrilarnine Binding-Cell membranes were obtained by hypotonic lysis as described above. Aliquots of membranes were resuspended in Hanks' balanced salt solution supplemented with 5 mM histidine and various concentrations of [3H]pyrilamine (5 nM to 1 pM) in the absence or presence of 100 p M chlorpheniramine for 30 min at 4 "C. Assays were terminated by vacuum filtration through Whatman GF/C filters (pre-soaked with 3% aqueous polyethylenimine for 1 h at 4 "C to reduce nonspecific binding) followed by three washes with ice-cold PBS supplemented with 5 mM histidine as described by Mitsuhashi and Payan (36). Data from saturation and competition binding assays were analyzed by computer using a nonlinear least squares weighted fit as described by DeLean (37).
Northern Blot Analysis-Northern blot analysis was performed as described previously (38). Cells were washed three times with ice-cold PBS and total cellular RNA extracted by the method of Chirgwin et al. (39). RNA was denatured and fractionated by electrophoresis on 1% agorose gels containing formaldehyde (40) and then transferred to a nitrocellulose membrane and baked at 80 "C in a vacuum oven for 2 h. The membrane was prehybridized at 42 "C for 16 h in 50% formamide, 2 X SSC (1 X SCC: 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 50 mM NaP04, 0.1% SDS, 10 X Denhardt's solution, and 400 pg/ml sheered salmon sperm DNA. Hybridization to human a, P, and y protein kinase C isoenzyme probes was done under the same conditions using 2 X lo6 counts/ml of probe labeled by nick translation with [a-"PIdCTP to a specific activity of 6 X 10' counts/pg of DNA as recommended by the manufacturer (Bethesda Research Laboratories). After hybridization for 16 h, the filters were washed twice for 20 min with 0.2 X SSC and 0.1% SDS at room temperature and then twice for 1 h at 65 "C. The filters were then exposed to Kodak X-AR film at -70 "C for 3-6 days using intensifying screens. RNA band sizes were estimated by comparison with an RNA ladder (Bethesda Research Laboratories).
Statistics-Analysis of kinetic data was by nonlinear regression using simple weighting of individual va!ues (ENZFITTER, Elsevier-BIOSOFT, Cambridge, United Kingdom). The half-maximal values for stimulation of phosphate uptake or phosphoinositide hydrolysis by the various agents were determined by nonlinear least squares regression analysis (LIGAND, Elsevier-BIOSOFT). Statistical comparisons were by paired or unpaired t test. The Bonferroni correction for multiple comparisons was used if indicated in the figure legend.  (Fig. 1B). The magnitude of this stimulation of sodium-dependent phosphate uptake is similar to that observed with 5-HT in these cells (22). The stimulation of phosphate uptake by histamine was rapid, reaching maximal values after 10-15 min of incubation and returning toward base line within 60 min (Fig. 2). The time course for the stimulation of phosphate uptake correlates very well with those previously described in these cells for 5-HT, phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-dibutyrate (PDBu), and sn-1,2-dioctylglycerol (DiC8) (22) and is consistent with the involvement of a kinase in this process.

Histamine
Histamine Stimulates Phosphate Uptake uia Histamine HI Receptors-HeLa cells have been reported to have histamine HI receptors which regulate potassium channels (26) and intracellular Ca2+ levels (41). Three series of studies were performed to establish that the histamine-induced stimulation of phosphate uptake is mediated through the histamine  FIG. 3. Pharmacology of the histamine-induced stimulation of phosphate uptake matches that of the histamine HI receptor. Cells were incubated with histamine for 5-10 min in the absence or presence of the histamine HI antagonists chlorpheniramine (Chlor), pyrilamine (Pyril), and doxepin (Dor) or the histamine Hz antagonist cimetidine (Cimet). The 5-HTIA receptor antagonists pin-dolo1 (100 WM) and spiperone (100 PM) did not reduce the histamineinduced stimulation of phosphate uptake (n = three separate experiments performed in duplicate for each drug, not shown). Experiments were performed three to five separate times in duplicate a t a fixed phosphate concentration of 0.2 mM in HeLa cells.
HI receptor and not through the transfected 5-HTlA receptor or other endogenous receptor subtype. These experiments were necessary to rule out an interaction with 5 -H T I~ receptors because of their high density (~2 . 8 f 0.6 pmol/mg protein) in these cells and the high doses of histamine utilized in these experiments. First, histamine (100 PM) increased phosphate uptake 39.8 f 4.3% in nontransfected HeLa cells which do not express 5-HT receptors linked to adenylyl cyclase or phosphoinositide hydrolysis (22, 27) (n = 3 experiments performed in triplicate, p < 0.01). Second, a series of antagonists were utilized to characterize the pharmacology of the effect of histamine in HeLa cells. As shown in Fig. 3, the histamine HI receptor antagonists chlorpheniramine, pyrilamine, and doxepin significantly inhibited the histamine-induced stimulation of phosphate uptake, whereas the histamine HP receptor antagonist cimetidine did not. Similarly, the 5 -H T I~ receptor antagonists pindolol and spiperone did not (not shown). Similar results were obtained in assays of phosphoinositide hydrolysis (not shown).
Third, the existence of endogenous histamine HI receptors was confirmed by binding studies performed with [3H]pyrilamine (Fig. 4). Saturation analysis (Fig. 4A) revealed that ['HI pyrilamine binds to a single class of binding sites (740 f 20 fmol/mg protein) with a Kd of 164 f 6 nM. This affinity is somewhat lower than that reported in brain tissue (42, 43) but very similar to that reported in DDTI-MF-2 cells (36)  expected for the histamine HI receptor (Fig. 4B).
Histamine Stimulates Phosphate Uptake in the Presence of 8-Br-CAMP-We have shown previously that agents which stimulate or mimic cAMP accumulation in HeLa cells inhibit phosphate uptake (23). Histamine (up to 1 mM) does not lower basal or forskolin-stimulated whole cell cAMP or membrane adenylyl cyclase in these cells over a 30 min period ( n = 3 for both assays, not shown). To further establish that the histamine effect on phosphate uptake occurs independent of any potential effect on adenylyl cylcase, we examined the effects of histamine on phosphate uptake in the presence of 8-Br-CAMP. In this regard, 100 p~ 8-Br-CAMP decreased phosphate uptake from 2.1 f 0.3 nmol/mg protein/3 min to 1.7 f 0.2 nmol/mg protein/3 min a t a phosphate concentration of 0.2 mM  3 f 4.9). These findings indicate that histamine stimulates phosphate uptake by a mechanism other than inhibition of adenylylcyclase.
PKC Mediates the Histamine HI Receptor-induced Stimulation of Phosphate Uptake-We have demonstrated previously that agents which activate PKC stimulate phosphate uptake in HeLa cells and that the 5-HT-induced stimulation of phosphate uptake is primarily mediated through PKC (22, 23). Because the histamine H, receptor is classically linked to hydrolysis of phosphoinositides, diacylglycerol-induced activation of PKC seemed the most likely pathway for the stimulation of phosphate uptake. Data supportive of this hypoth-esis are as follows. There is close concordance of the ECso values for the stimulation of phosphate uptake (3.2 f 1.8 p M ) and total inositol phosphates (8.6 f 4.1 p~) .
Second, 100 pM histamine activates PKC in these cells as measured by the intact cell phosphorylation of the MARCKS protein (formerly known as the 80-87-kDa protein) (Fig. 5). This assay showed that histamine stimulated phosphorylation of the MARCKS protein by 2.4 & 0.2-fold ( n = 3, p < 0.05). Third, depletion of PKC by overnight treatment with 16 p~ PMA attenuates the histamine-induced stimulation of phosphate uptake (Fig.  6). For these studies, effective down-regulation of PKC was confirmed by lack of effect of 1.6 p~ PMA on phosphate uptake or phosphorylation of the MARCKS protein ( Fig. 6 and not shown). Although the effects of prolonged high-dose PMA treatment are likely very complex, collectively, these results support the hypothesis that histamine stimulates of five separate experiments (*, p < 0.05; **, p < 0.01, Bonferroni correction used). There was no significant difference in basal phosphate uptake between cells pre-treated or not with PMA. phosphate uptake via activation of PKC. We cannot exclude an effect of PMA on other elements of the signal transduction system which could lead to a blunted responsiveness. In this regard, the actions of histamine are similar to those of 5-HT, which activates PKC and stimulates phosphate uptake in these cells (22).
Histamine and 5-HT Responses Have Differential Sensitivity to Pertussis Toxin-The 5-HTlA receptor activates phosphoinositide hydrolysis and stimulates phosphate uptake in HeLa cells through a pertussis toxin-sensitive G protein (IC50 = 10 ng/ml for 4 h) (22,27). In the current study, treatment with various doses of pertussis toxin (up to 1 pg/ml for 24 h) had no effect on histamine (100 pM)-induced phosphate uptake (Fig. 7A). Such treatment also had no effect on the histamine-induced stimulation of phosphoinositide hydrolysis as measured by accumulation of total inositol phosphates a t 15 min (Fig. 7B), strongly suggesting that the effects of histamine and 5-HT are mediated through functionally distinct pools of G proteins in HeLa cells. Confirmation that pre-treatment with pertussis toxin (100 ng/ml for 4 h) was Effects of pertussis toxin treatment on 5-HT and histamine-induced increase in phosphate uptake and phosphoinositide hydrolysis. Cells were preincubated with various concentrations of pertussis toxin (depicted on the horizontal axis as nanograms/ml) for 4 h in DMEM supplemented with serum and antibiotics as under "Experimental Procedures" and then washed three times with DMEM. A, for phosphate uptakes, cells were then pre-equilibrated in Earle's solution and uptakes performed a t a phosphate concentration of 0.2 mM in the absence or presence of 10 pM 5-HT or 100 p~ histamine. Stimulation over basal by histamine was 43.3 f 5.2% and by 5-HT was 36.3 f 4.3% for these experiments. The effect of pertussis toxin treatment is depicted in the vertical axis as the normalized percent of the maximal stimulation for each hormone. The data for 5-HT have been published previously (22). The data represent the mean f S.E. from three separate experiments. Basal phosphate uptakes did not differ between pertussis toxintreated cells and untreated cells (not shown). E, for phosphoinositide hydrolysis experiments, cells were treated exactly as described under "Experimental Procedures." Data from three experiments are presented as means f S.E. subsequently normalized to represent percent of the maximal stimulation for each hormone (88.5 f 14.8% for 5-HT, 56.2 f 5.9% for histamine for these experiments). Since the cells were pretreated with 20 mM LiCl, the total inositolphosphates are assumed to represent phospholipase C activity. C, ADP-ribosylation of washed cell membranes derived from cells pretreated or not with various doses of pertussis toxin was done as described under "Experimental Procedures." Depicted is a representative autoradiogram (exposed for 12 h a t -80 "C) which shows a dose-responsive decrease in pertussis toxin catalyzable (42 kDa) ADP-ribosylation substrate in membranes derived from intact cells preincubated with pertussis toxin. Pertussis toxin dose is depicted on the horizontal axis and apparent molecular weight on the uertical axis. When exposed for longer periods of (>48 h) time, a small amount of substrate was detectable at 10 ng/ml but not a t higher doses. adequate to eliminate all pertussis toxin substrate available for ADP-ribosylation is presented in Fig. 8C.
Histamine and 5-HT Have Additive Effects on Phosphoinositide Hydrolysis and Phosphate Uptake-To further confirm that the histamine HI receptor and the 5-HTIA receptor couple to distinct G proteins in HeLa cells, we examined the additivity of the histamine and 5-HT-induced stimulations of phosphoinositide hydrolysis and phosphate uptake. As shown in Fig. 8A, the stimulatory effects of the two agonists on phosphoinositide hydrolysis were completely additive. Maximal stimulation was 88 f 7% for histamine alone and 202 f 12% for histamine in the presence of 10 p~ 5-HT. Maximal stimulation for 5-HT alone was 106 f 10% and for 5-HT in the presence of 1 mM histamine was 206 f 4%. Half-maximal stimulatory doses for phosphoinositide hydrolysis were as follows: histamine alone (8.6 f 4.1 p~) or +lo0 p M 5-HT (12.0 & 3.2 p~) , 5-HT alone (290 f 70 nM) or +1 mM histamine (260 f 50 nM). As shown in Fig. 8B, a similar additivity was shown for phosphate uptake. Phosphate uptake was stimulated 29 f 6.2% by histamine alone and 50.1 f 8.9% in the presence of 100 FM 5-HT. Maximal stimulation was 32.6 f 6.6% by 5-HT alone and 51.0 & 4.5% in the presence of 1 mM histamine. Half-maximal stimulatory doses for phosphate uptake were as follows: histamine alone (3.2 f 1.8 p M ) or +lo0 p M 5-HT (2.0 f 2.2 pM), 5-HT alone (990 f 200 nM) or +1 mM histamine (760 & 140 nM). The observations that the stimulatory effects of histamine and 5-HT on phosphoinositide hydrolysis and phosphate uptake are additive in HeLa cells support the notion that the histamine HI and 5-HTIA receptors couple to distinct pools of G proteins in these cells.

Histamine and 5-HT Have Additive Effects on MARCKS
Protein Phosphorylation-If the stimulation of phosphate transport by both agonists is due to activation of protein kinase C, one would expect that phosphorylation of the MARCKS protein would be additive. Data presented in  8. Additivity of the effects of histamine and 5-HT on phosphate uptake and phosphoinositide hydrolysis. Experiments for phosphoinositide hydrolysis ( A ) and phosphate uptake ( E ) were performed as described in Fig. 2 and under "Experimental Procedures." Dose-response curves for histamine (0) and 5-HT (0) were generated. Then dose-response curves were generated for histamine in the presence 100 p~  and for 5-HT in the presence of 1 mM histamine (0). Values depicted represent the means f S.E. of three to five separate experiments performed in duplicate or triplicate. 9 support this notion. In A , a plateau of stimulation is demonstrated for both agonists, suggesting that activation of protein kinase C by either agonist reaches a maximal effect, as would be expected from the dose-response curves for phosphoinositide hydrolysis presented in Fig. 8. B of Fig. 9 demonstrates that the phosphorylation of the MARCKS protein induced by 5-HT and histamine are also additive, even when maximal doses of both agents are utilized. The stimulation (over basal) induced by 1 mM histamine was 69 f 17%, by 10 PM 5-HT was 100 f 8%, and for both agents together was 177 f 14%. These values compare with the previously published 170 f 40% increase induced by 1.6 p M PMA (22).
Northern Blot Anulysis of Protein Kinase C Subtypes Present in HeLa Cells- Fig. 10 demonstrates that RNA derived from HeLa cells cross-hybridizes with human DNA probes for three subtypes of PKC (a, p, 7 ) . Each transcript is ~3 . 0 -3.4 kilobases in size, consistent with previously published data (38, 44). It is unlikely that these bands represent crosshybridization of one probe with mRNA for another PKC subtype, because under the same stringent washing conditions (0.2 X SSC, 0.1% SDS, 65 "C), we have observed no detectable cross-hybridization of these probes with mRNA for other PKC subtypes from other human cell lines? Although PKCy is generally regarded as brain-specific (45) (44). The current data suggest that there are multiple subtypes of PKC expressed in HeLa cells and that PKC-7 may be expressed in multiple forms of human malignancies, as well as in brain.

DISCUSSION
The regulation of sodium-dependent phosphate uptake has been the subject of considerable recent interest. Previous studies have demonstrated that these regulatory processes are remarkably complex. Of particular importance are the respective roles of PKA and PKC and of the receptors and second messengers which activate them. For example, distinct hormones may exert similar effects on phosphate transport in the same cell. Specificity at this level may be conferred by the complement of receptor subtypes present in a given cell type. Specificity or diversity of these effects may be conferred at the level of the G proteins, second messengers, kinases, or even at the level of the sodium-dependent transporter itself, depending on what potential sites of kinase interaction and/ or dynamic phosphorylation are present. Several examples have been described in which a given hormone may activate two distinct second messenger cascades which regulate phosphate transport synergistically. For example, in OK cells, parathyroid hormone activates phosphoinositide hydrolysis and increases cAMP accumulation; both events may inhibit phosphate transport in these cells, although the relative importance of each individual pathway is still unresolved (15,24). In LLC-PK, cells, calcitonin both activates phosphoinositide hydrolysis and increases cAMP accumulation, leading to a stimulation of phosphate uptake (16). In this case, diacylglycerol-induced activation of PKC appears to be the more important pathway (16). In both cases, it is unclear whether multiple or single subtypes of receptors for parathyroid hormone or calcitonin are present or if single receptor subtypes are linked to the distinct second messenger cascades through the same or different G proteins. In this respect, distinct G proteins may exert opposing effects on phosphate transport in the same cell. In OK cells, a2-adrenergic receptors, which activate a "Gi-like" G protein, inhibit the parathyroid hormone-induced decrease in phosphate uptake, which is presumably mediated through G, (18). In the current study, we explored the possibility that two different hormones could alter phosphate transport additively via the same or different G proteins in HeLa cells.
The finding that endogenous histamine HI receptors present in HeLa cells stimulate phosphate uptake via PKC activation supports the hypothesis that receptors classically linked to phosphoinositide hydrolysis will stimulate phosphate uptake in these cells. The close concordance of the maximal degree of stimulation of both phosphoinositide hydrolysis and phosphate uptake induced by both histamine and 5-HT further verifies the close linkage between these two processes in HeLa cells (22). It is now known that there are several distinct G proteins which can activate phosphoinositide hydrolysis (so-called G, proteins). That the histamine effects are mediated through a different G, protein pool than those linked to 5-HT in HeLa cells is supported by the additivity of phosphoinositide hydrolysis PKC activation, and phosphate uptake and the differential sensitivity to pertussis toxin. The additive effect on phosphate uptake seemed to plateau at high doses (Fig. 8), implying saturability of a process distal to phosphoinositide hydrolysis. At least in the case of the 5 -H T I~ receptor, which is expressed at very high density in these cells (2.8 f 0.6 pmol/mg protein), one might predict that under conditions of maximal stimulation by agonist (100 pM 5-HT), the G protein pool utilized by the 5 -H T 1~ receptor should be fully activated (46). Therefore, receptors which utilize the same G proteins would not further stimulate phosphoinositide hydrolysis, whereas receptors which use distinct G proteins would augment the 5-HTlA receptor-induced stimulation of phosphoinositide hydrolysis. This prediction is reasonable if the pertussis toxinsensitive G, protein is not present in great excess to its linked receptors. It also assumes that phospholipase(s) C are present in excess of the various G protein pools capable of coupling to phosphoinositide hydrolysis. Recent studies using cells that express various levels of recombinant muscarinic acetylcholine receptors (46) or a,-adrenergic receptors (47) support one aspect of this model. In both cases, increasing the numbers of receptors expressed per cell increased activation of phosphoinositide hydrolysis to a maximal point, but eventually the degree of stimulation of phosphoinositide hydrolysis reached a plateau. The observations that the stimulatory effects of histamine and 5-HT on phosphoinositide hydrolysis, PKC activity, and phosphate uptake are additive in HeLa cells, coupled with the differential sensitivity of both processes to pertussis toxin, support the notion that the histamine HI and 5-HTIA receptors couple to distinct pools of G proteins in these cells.
These studies further illustrate that distinct pools of G, can exist in a single cell type (45, 48) and have important ramifications regarding the multiplicity of control mechanisms for the physiological transport of phosphate and, perhaps, other solutes. These G, proteins can be distinguished by their sensitivity to pertussis toxin (46,49), which uncouples certain G proteins from receptors by catalyzing the ADP-ribosylation of the G protein a-subunits (50-52). The presence of distinct G, proteins in a single cell which can couple selectively to different receptors may be a mechanism by which common cellular responses such as phospholipase C activation can be compartmentalized. Such an arrangement also provides a mechanism by which cellular effectors can be further activated under conditions of maximal stimulation of a single receptor (or G protein) subtype.
The differential sensitivity of the 5 -H T I~ and histamine HI receptors to pertussis toxin is consistent with that observed for these receptors in other cell lines or tissues. The cellular effects of 5-HTIA receptor activation are typically sensitive to pertussis toxin (53), whereas most of those associated with the histamine HI receptor are not (41, 54). The differential sensitivity of the G, proteins linked to phosphoinositide hydrolysis and stimulation of phosphate uptake in HeLa cells most likely relates to structural differences between the G, proteins. In that regard, the a-subunit of a G protein which exhibits a high degree of primary amino acid sequence identity with the Gi-like G proteins has been described (55, 56). This a-subunit lacks a carboxyl terminus cysteine residue which is the pertussis toxin-catalyzed ADP-ribosylation acceptor site (57). This region may also be involved in the coupling of receptors or effector enzymes to G protein (57).
It is not clear from the current studies whether the differential pertussis toxin sensitivity of G, proteins expressed in HeLa cells is due to differences in the primary amino acid sequences of the a-subunits or to different post-translational modifications of the G proteins which cause subtle structural differences which influence parameters essential for coupling of G, proteins to receptors or the phospholipase C effector enzymes and sensitivity to pertussis toxin. Moreover, it is also not known whether these distinct G, proteins couple to the same or different phospholipase(s) C. The existence of at least five subtypes of phospholipase C has been documented (58). A similar heterogeneity of PKC subtypes (at least seven) also has been described (59). Our data support the presence of mRNA for at least three subtypes of PKC (a, p, y ) in HeLa cells (Fig. 10). Therefore, there remains the possibility that the compartmentalization of the signal transduction pathways linked to histamine and 5-HT in HeLa cells extends beyond the G, proteins and may include isoforms of PKC. However, we have not directly addressed this hypothesis in the current studies.
A central concern in cellular signaling is the mechanism by which specificity of responses can be conferred by a limited repertoire of machinery necessary for signal transduction. In the case of sodium-dependent phosphate transport, both additive (15, 16, 24) and opposing (22, 27) regulation of PKA and PKC have been described. At the level of the G protein, opposite regulation by receptors putatively linked to G, and Gi has been described (18). The current studies document that additive regulation of phosphate transport can occur via distinct pools of G proteins, thus adding to the complexity of interactions between components of the cellular machinery which modulate the transport of phosphate. Thus, the regulation of phosphate transport can be augmented or antagonized at multiple points in the signal transduction cascade, including interactions with distinct pools of G proteins.