©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Synergistic Interaction of Y1-Neuropeptide Y and -Adrenergic Receptors in the Regulation of Phospholipase C, Protein Kinase C, and Arachidonic Acid Production (*)

Lisa A. Selbie , Karen Darby , Carsten Schmitz-Peiffer , Carol L. Browne , Herbert Herzog , John Shine , Trevor J. Biden (§)

From the (1) Garvan Institute of Medical Research, St. Vincent's Hospital, Sydney, New South Wales, 2010 Australia

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Neuropeptide Y (NPY) and norepinephrine, found co-localized in sympathetic neurons innervating blood vessels, exert synergistic responses on vasoconstriction. To examine the signaling mechanisms involved, free of complications associated with mixed receptor populations, we have established a stable Chinese hamster ovary cell line expressing both Y1-NPY and -adrenergic receptors. Occupation of either receptor species, with 100 nM peptide YY (PYY) or 10 µM phenylephrine (PE), respectively, resulted in a rapid increase in the cytoplasmic free calcium concentration ([Ca]) as assessed with Fura-2/AM. The rise due to PYY, but not that due to PE, was abolished by pretreatment with pertussis toxin. Both responses were largely maintained in the absence of extracellular Ca, but abolished by prior depletion of intracellular Ca pools with either thapsigargin or 2,5-di-(t-butyl)-1,4-benzohydroquinone. Using cells prelabeled with myo-[H]inositol, PE promoted a rapid (5 s) rise in inositol 1,4,5-trisphosphate (Ins(1,4,5)P) as analyzed by anion-exchange high pressure liquid chromatography, whereas the response to PYY (first significant at >15 s post-stimulation) was too slow to play a causative role in Ca mobilization. Combination of PE and PYY resulted in increases in [Ca]which were at best additive, whereas they promoted a clearly synergistic rise in Ins(1,4,5)P at both 15 and 60 s. Co-stimulation also resulted in a synergistic activation of both protein kinase C (PKC) and [H]arachidonic acid release. In either instance PYY alone was without effect. The potentiation of arachidonic acid release was abolished by depletion of cellular PKC following chronic treatment with phorbol esters. It is suggested that the ability of PYY to mobilize Ca in an Ins(1,4,5)P-independent fashion minimizes the functional importance of the capacity to potentiate PE-stimulated Ins(1,4,5)P generation. Instead the major conseqences of the synergistic activation of phospholipase C are mediated via PKC, the other route of the signaling pathway.


INTRODUCTION

Neuropeptide Y (NPY)() is an abundant peptide hormone and neurotransmitter that is important in mediating anxiety responses and the regulation of food intake. It also plays a major role in the control of cardiovascular function and vascular tone, acting through both the peripheral and central nervous systems (1, 2, 3, 4) . The presence of NPY at high concentrations in the nerve terminals surrounding blood vessels, particularly arteries, suggests that it influences vascular resistance and tissue perfusion. NPY acts through both pre- and postsynaptic mechanisms on the heart and cardiovascular system. Presynaptically the peptide exerts long lasting inhibitory effects on the release of norepinephrine and acetylcholine by sympathetic and parasympathetic nerves. At the postsynaptic site, NPY has been shown to act as a potent vasoconstrictor in its own right, as well as to potentiate the constrictor effects of numerous pressor agents, including norepinephrine, with which it is found co-localized in sympathetic neurons (1, 2, 3, 4) .

Receptors for NPY are present in a wide variety of tissues including atrial cells, coronary and cerebral blood vessels, and in aortic and vascular smooth muscle cell lines. These correspond to at least three receptor subtypes, designated Y1, Y2, and Y3, as identified using receptor-specific peptide agonists (4, 5, 6) . Whereas the presynaptic responses are mediated chiefly through Y2 receptors, the pressor effects of NPY are due predominantly to interaction with the Y1 species (4-6). The latter receptor has recently been cloned from several species and revealed to be a member of the family of G protein-coupled receptors (7, 8) . This is consistent with earlier functional studies, using a variety of tissues and cell lines, which suggested that NPY receptors coupled to second messenger systems via an interaction with one or more PT-sensitive G proteins (9, 10, 11, 12) . Both Y1 and Y2 receptors couple to an inhibition of adenylate cyclase (4, 5, 6) . In addition, occupation of Y1 receptors has been well documented to promote a rise in [Ca]in vascular smooth muscle cells (11, 13) , human erythroleukemia (HEL) cells (10, 14) , and neuroblastoma SK-N-MC cells (15) . The coupling of a single NPY receptor species to the two intracellular signaling systems has been confirmed by heterologous expression of the Y1 receptor (7, 8) .

Because of the essential role that Ca plays in the regulation of vasoconstriction (16, 17) , the effects of NPY on [Ca]are likely to be more important in that regard than those on cAMP. These NPY-induced Ca responses involve mobilization of Ca from intracellular stores and a consequent Ca influx through non-voltage-gated channels (7, 8, 10, 11, 13, 14). By far the best understood means of linking Ca mobilization with G protein-coupled receptors is via the activation of PtdInsP hydrolysis and release of the intracellular messengerIns(1,4,5)P (18). This reaction, catalyzed by PLC, also liberates diacylglycerol, the endogenous activator of PKC (19) . However, the mechanism underlying the Y1-NPY receptor-stimulated rise in [Ca]remains unclear with Ins(1,4,5)P being clearly implicated in only one (14) of a number of studies (10, 11, 12, 13, 20) . In addition, there is very little understanding of the signaling pathways through which Y1-NPY and -adrenergic receptors interact synergistically to promote vasoconstriction. A potentiation of PLC activation by NPY has been reported in some (20, 21) but not all (13) studies. Furthermore, in experiments with arterial preparations, a requirement for Ca influx through voltage-gated Ca channels has sometimes (22, 23) but not always (24, 25) been reported. These discrepancies might reflect drawbacks inherent in the use of arterial preparations which contain mixed populations of both cell type and receptor species. For example, in rabbit aorta, -adrenergic receptors couple predominantly to Ca influx, whereas occupation of -receptors causes a phasic contraction associated with PtdInsP hydrolysis and Ca mobilization (26). To obviate these difficulties we sought to explore some of the mechanisms underlying the synergistic interactions of NPY and PE using a defined model system. This consisted of a single cell type (CHO cell) in which the Y1-NPY and -adrenergic receptor subtypes were heterologously expressed.


EXPERIMENTAL PROCEDURES

Materials

All media and materials for tissue culture were purchased from Cytosystems, Castle Hill, NSW, Australia, and radiochemicals from DuPont NEN. NPY and its related peptides, as well as peptides for PKC assays, were obtained from Auspep Pty. Ltd., Parkville, Victoria, Australia. HPLC columns and P81 phosphocellulose paper were from Whatman. TLC plates were from Merck. Fura-2/AM was supplied by Molecular Probes, Eugene, OR. All other biochemicals were from Sigma or Boehringer Mannheim.

Cell Culture and Transfection

Cells (CHO K1:ATCC:CCL 61) were maintained in 5% CO in Dulbecco's modified Eagle's medium/Ham's F-12 medium with 2 mM glutamine, 100 IU of penicillin, streptomycin at 100 µg/ml, and 10% fetal calf serum. CHO cells were co-transfected using a modified calcium phosphate method with the hamster -adrenergic receptor cDNA in a pMT2 vector (27) and the human Y1-NPY receptor cDNA in the pcDNA Neo vector (7) . Stably transfected cell populations were selected with neomycin (G418). Cells were harvested by treatment with 0.2% EDTA in PBS and immediately used in the binding assay or functional assays described.

Binding Assay

Stable transfected cells were selected and assayed for their ability to bind radiolabeled PYY and prazosin, an antagonist at the -adrenergic receptor. PYY and the Y1 receptor-specific ligand NPY(Leu,Pro) were used to compete for the binding of I-labeled PYY, and PE for the binding of [H]prazosin. Transfected cells were harvested with EDTA/PBS and washed twice in the assay buffer (50 mM Tris-HCl, pH 7.4, 2 mM CaCl, 5 mM KCl, 120 mM NaCl, 1 mM MgCl, and 0.1% bovine serum albumin). Washed cells (1 10) were incubated in 0.5 ml of assay buffer in the presence of labeled agonist and increasing concentrations of competitors for 1 h at room temperature. After centrifugation for 4 min in a microcentrifuge, the tips of the tubes containing the pellets were cut off and placed in plastic tubes. Radioactivity associated with the pellets was counted for 1 min in a gamma counter. Receptor numbers were calculated as 17,900 and 19,000 per cell for Y1-NPY and -adrenergic receptors, respectively. Measurement of [Ca]-Cells expressing the human Y1-NPY and hamster -adrenergic receptors were suspended in loading medium (modified RPMI, 10 mM Hepes, 1% newborn fetal calf serum) and incubated in a spinner flask at 37 °C for 2.5 h at 10 cells/ml. Cells were then treated with 1 µM Fura-2/AM for 30 min at 37 °C, washed twice with loading medium, and resuspended at 5 10 cells/ml. Immediately prior to use, an aliquot of 2 10 cells was resuspended in 2 ml of modified KRB buffer (135 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO, 1.2 mM KHPO, 5 mM NaHCO, 1 mM CaCl, 2.8 mM glucose, and 10 mM Hepes, pH 7.4) containing 1 µM sulfinpyrazone (28). Fluorescence recordings were made on a Hitachi fluorescence spectrometer (F4010) at 340 nm (excitation) and 505 nm (emission) with slit widths of 5 nm and a response time of 2 s. All fluorescence traces are representative of at least three separate experiments and were calibrated as described previously (28) using the published formulas (29).

Measurement of Inositol Phosphate Production

Cells were labeled with myo-[H]inositol (10 µCi/ml) for 48 h under culture conditions, then harvested and transferred to spinner culture as described above. After 2 h they were washed and resuspended at 2 10 cells/ml in 0.5-ml aliquots of modified KRB buffer containing 0.1% bovine serum albumin. Experimental additions were made as doubly concentrated stock solutions in 0.5 ml of the same buffer. Reactions were stopped with the addition of 100% trichloroacetic acid (100 µl), and the solutions were vortexed and left on ice for 20 min. After centrifugation for 4 min in a microcentrifuge, the supernatant was removed and extracted three times with 5 ml of diethyl ether. After removal of cations with Dowex 50W-X8, inositol phosphates in the aqueous extracts were separated and analyzed by anion-exchange HPLC using a Partisphere PAC column eluted with ammonium phosphate (pH 3.8). The exact gradient and relative elution times of Ins(1,3,4)P and Ins(1,4,5)P were exactly as described previously (28) . Eluted fractions (0.5 ml) were mixed with 4 ml of scintillant, and radioactivity was determined by liquid scintillation spectrometry. Measurement of [H]Arachidonic Acid Release-Cells (10/ml) were labeled with [H]arachidonic acid (1.3 µCi/ml) for 3 h at 37 °C in spinner culture. They were then washed, resuspended, and incubated with experimental additions exactly as described for the inositol phosphate studies. After incubation the experimental tubes were chilled on ice, and the cells then pelletized using a microcentrifuge. Supernatant (0.5 ml) was transferred to a scintillation tube with 5 ml of scintillant, and radioactivity was determined. For confirmation of the release of arachidonic acid, incubations were stopped with the addition of 1.9 ml of ice-cold chloroform/methanol/HCl (100:200:2, v/v), and lipids were extracted with further addition of chloroform and HO. Additional arachidonic acid (5 µg) was added to aid in recovery during the extractions. The lower lipid-containing phase was dried under nitrogen, resuspended in chloroform, and applied to thin layer chromatography on Silica Gel 60 F 20 20-cm, 0.2-mm thick TLC plates in ethyl acetate:isooctane:acetic acid:HO (9:5:2:1) or benzene:chloroform:methanol (80:15:5). Free arachidonic acid was identified by comigration with a known standard and staining with iodine crystals or Coomassie Blue stain, and the appropriate spot was scraped from the plate and quantified by scintillation counting.

Measurement of PKC Activity

CHO cells (approximately 2 10) were plated out 24 h prior to the experiment in 9.6 cm wells. Medium was aspirated, and the cells were washed with 2 ml of PBS. Incubations were carried out in 1 ml of modified KRB buffer with either 100 nM PYY, 10 µM PE, both agonists or vehicle. After 15 s, medium was aspirated, and the cells were washed once with ice-cold PBS and scraped into 1.5-ml centrifuge tubes with 0.4 ml of homogenization buffer (20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 0.5 mM EGTA, 1 mM dithiothreitol, 200 µg/ml (w/v) leupeptin, 2 mM benzamidine, and 2 mM phenylmethylsulfonyl fluoride). The cells were extracted by three rapid freeze-thaw cycles, and the extracts were subjected to centrifugation at 100,000 g for 30 min at 4 °C in a Beckman 70.1 Ti rotor, to give cytosolic and particulate fractions. The particulate fraction was further extracted, after briefly rinsing with ice-cold PBS, by resuspension in 0.2 ml of homogenization buffer containing 0.1% Triton X-100. Following centrifugation, the 100,000 g supernatant from this suspension, corresponding to the original membrane associated PKC, was assayed for activity. Samples (10 µl) were assayed in a final volume of 50 µl, containing 24 mM MOPS, pH 7.5, 0.04% Triton X-100, 1 mM CaCl, 120 nM cyclic AMP-dependent protein kinase inhibitor peptide (rabbit sequence), 0.5 mg/ml histone HIIIS, 100 µM [-P]ATP (150 cpm/pmol), and 5 mM magnesium acetate, in the absence or presence of 125 µg/ml phosphatidylserine and 2.5 µg/ml dioctanoylglycerol. Lipids, at 5 mg/ml in chloroform/methanol (19:1), had been dried under nitrogen and sonicated into 100 mM MOPS (pH 7.5), 1% Triton X-100 until clear before addition to the assay buffer. After 10 min at 30 °C, assays were terminated with the addition of 10 µl of 150 mM unlabeled ATP, and samples were spotted onto P81 paper, washed with orthophosphoric acid, and counted for Cerenkov radiation (30) . PKC was determined as the additional activity observed in the presence of the lipid activators.

Statistics

All results are presented as mean ± S.E. of the mean. Statistical significance was assessed using Student's t test for unpaired data.

RESULTS

NPY and Adrenergic Receptor-induced Increases in [Ca]-Y1R-1BAR/CHO K1 cells, stably expressing the Y1-NPY and -adrenergic receptors, were loaded with the calcium-sensitive dye Fura-2/AM. In all of the following experiments PYY, which is less hydrophobic than NPY, was used as a selective agonist for the Y1-NPY receptor (7) . Consistent with earlier studies (7) , PYY (100 nM) caused a transient increase in [Ca]in CHO K1 cells expressing the Y1-NPY receptor (Fig. 1a). This was largely unaffected by the removal of extracellular Ca and is therefore primarily due to the mobilization of Ca from intracellular stores (Fig. 1b). Occupation of the -adrenergic receptor with 1 µM PE also resulted in an immediate increase in [Ca], but this was followed by only a slight decline from the peak level. Extracellular Ca was essential for the sustained phase (Fig. 1b), suggesting that it was due to Ca influx. Y1R-1BAR/CHO cells pretreated with PT (200 ng/ml) were barely responsive to PYY, whereas both Ca mobilization and Ca influx due to PE were virtually unaffected (Fig. 1, c and d). This suggests that the Y1 and -adrenergic receptors couple to rises in [Ca]via distinct G proteins, which show differential sensitivities to PT.


Figure 1: Calcium responses in CHO cells expressing Y1-NPY and -adrenergic receptors. After loading for 30 min with 1 µM Fura-2/AM, cells were washed and resuspended at 2.0 10 cells/ml in KRB buffer. Fluorescence was measured using cells maintained under continuous stirring in a cuvette at 37 °C. For further details, see ``Experimental Procedures.'' Changes in [Ca] were measured using cells incubated in the presence (Panels a and c) or absence (b and d) of 1 mM extracellular CaCl and that had been pretreated overnight with 200 ng/ml pertussis toxin (c and d) or not (a and b). Gaps in the recordings are due to the opening of the compartment to make additions. Arrows mark the point of addition of PYY (100 nM) or PE (1 µM).



The next series of experiments were undertaken to examine the interactive effects of PYY and PE on [Ca]. This was tested in Fura-2/AM loaded Y1R-1BAR/CHO cells using three different experimental protocols: co-addition of the two receptor agonists; prior addition of PE followed by PYY; or addition of PYY first followed by PE. The results of these experiments are summarized in Fig. 2. Co-stimulation with PYY (100 nM) and PE (100 nM or 1 µM) resulted in [Ca]increases which were not significantly greater than the sums of the responses to both agonists alone (top panel). Using the alternative experimental protocols, no effect of pretreating the cells with 1 µM PE was seen on the subsequent rises in [Ca]induced by a range of PYY concentrations (middle panel); nor did prior addition of PYY (100 nM) affect the responses to various doses of PE (bottom panel). Taken together these results suggest that PYY and PE exert effects on [Ca]which are at best additive, but clearly not synergistic.


Figure 2: Effect of co-stimulation of Y1-NPY and -adrenergic receptors on [Ca] in transfected CHO cells. Cells were loaded with Fura-2/AM and [Ca] measured as described in the legend to Fig. 1. Top panel, average increases in [Ca] in cells stimulated with PYY (100 nM) either alone, or in the combined presence of PE (100 nM or 1 µM). Middle panel, dose-dependence of average increases in [Ca] versus PYY concentration in cells that had, or had not, been prestimulated with 1 µM PE. Where appropriate, PYY was added as soon as the PE response had plateaued out. Bottom panel, dose dependence of average increases in [Ca] versus PE concentration in cells that had, or had not, been prestimulated with 100 nM PYY. Each column or data point represents the average of duplicate determinations from at least three experiments.



Y1-NPY Receptor-stimulated PLC Activity and Potentiation of Adrenergic Receptor PLC Stimulation

Mobilization of Ca from intracellular stores occurs most frequently as a result of Ins(1,4,5)P generation (18) . When added to Y1R-1BAR/CHO cells, prelabeled with myo-[H]inositol, PE (10 µM) induced an increase in Ins(1,4,5)P which was rapid in onset (5 s) and sustained for at least 5 min (Fig. 3). In marked contrast, the rise in Ins(1,4,5)P stimulated by PYY was not significantly different from control until 60 s after addition. This demonstrates clear differences in the mechanisms by which the -adrenergic and Y1 receptors couple to PLC. Most importantly, the rise in Ins(1,4,5)P seen under these conditions appears to be too slow to explain the PYY-stimulated Ca mobilization which peaks within 10-15 s (see Fig. 1 )


Figure 3: Time courses of Ins(1,4,5)P generation in transfected CHO cells. Cells prelabeled for 48 h with [H]inositol were stimulated for various times with either PYY (100 nM) or PE (10 µM). Inositol phosphates in cell extracts were analyzed by anion-exchange HPLC. For further details, see ``Experimental Procedures.'' Each point represents the average of at least duplicate determinations from two (PE) or three (PYY) independent experiments. The earliest significant rises occurred at 5 s (p < 0.02) and 60 s (p < 0.005) for PE and PYY, respectively. Results are expressed as a percentage of the zero time control which averaged 69 cpm (n = 8).



We next examined the effects of combined addition of PYY and PE on PLC activity (Fig. 4). One minute of stimulation with both agonists raised Ins(1,4,5)P to a level which was approximately 1.4-fold higher than simple summation of the individual responses. This potentiation was even more apparent at the 15 s time point where PYY was unable to exert any significant effect by itself but almost doubled the response to PE (p < 0.05 versus PE alone). This was a true potentiation of PLC activity, rather than an inhibition of Ins(1,4,5)P degradation, since the latter's metabolites, Ins(1,3,4)P and Ins(1,4)P, were also augmented under these conditions (). The potentiation of PE-stimulated Ins(1,4,5)P generation by PYY is noteworthy in view of the fact that the Ca responses, under corresponding circumstances, were no more than additive (cf. Fig. 2). This is most probably explained by the ability of PYY to generate a robust Ca mobilization independently of Ins(1,4,5)P, which would tend to minimize the functional relevance of the potentiation of Ins(1,4,5)P generation.


Figure 4: Effect of combined addition of PE and PYY on Ins(1,4,5)P generation. Transfected CHO cells were stimulated for either 15 s or 1 min in the presence of PYY (100 nM) and PE (10 µM) either alone or in combination. Ins(1,4,5)P was measured as described in the legend to Fig. 3 and under ``Experimental Procedures.'' Each column represents the average of at least duplicate determinations from two independent experiments. Results are expressed as a percentage of the control response which averaged 56 and 50 cpm at 15 s and 1 min, respectively.



The simplest explanation for the observed potentiation of PLC activity would be that it is secondary to PYY-induced Ca mobilization. To investigate this possibility intracellular Ca pools were depleted by sequential addition of the chelating agent EGTA and the Ca-ionophore ionomycin. Under these conditions the capacity of either PYY or PE to raise [Ca]was completely blocked (Fig. 5, upper panel). Although slightly attenuated as compared to the response in non-depleted cells, addition of PE alone to Ca-depleted cells still resulted in a 2.5-fold stimulation of PtdInsP hydrolysis (Fig. 5, lower panel). Similarly, PYY increased PE-stimulated Ins(1,4,5)P production from approximately 260% of control to 400% in the depleted cells, as compared to a rise from 290 to 480% in the non-pretreated cells. Therefore, the synergistic activation of PLC is only partially inhibited under circumstances in which the rise in [Ca]is abolished. This indicates that, although the PYY-induced Ca mobilization might, under normal circumstances, contribute to the potentiation of PLC activity, it is not the primary mechanism underlying that potentiation.


Figure 5: Effect of Ca depletion on the potentiation of PE-stimulated Ins(1,4,5)P generation by PYY. Transfected CHO cells were preincubated in KRB medium for 10 min and then stimulated with PE (10 µM) in the presence or absence of PYY (100 nM) for 15 s. Cells depleted of Ca were treated during the preincubation for 3 min with 2 mM EGTA, and then for a further 4 min with 100 nM ionomycin, prior to addition of the receptor agonists. Upper panel, representative [Ca] trace showing depletion of intracellular stores. Lower panel, Ins(1,4,5)P responses in non-depleted and Ca-depleted cells. Ins(1,4,5)P was measured as described in the legend to Fig. 3 and under ``Experimental Procedures.'' Each column represents the average of duplicate determinations from one experiment representive of two.



Further Characterization of Y1-NPY Receptor-coupled Ca Mobilization

Because an increase in Ins(1,4,5)P does not appear to account for the rise in [Ca]due to PYY, we next sought evidence for the involvement of either cyclic ADP-ribose (31) or sphingosine 1-phosphate (32) , both recently proposed Ca mobilizing agents (Fig. 6). Cyclic ADP-ribose has been shown to act on those Ca stores, present in certain cell types, which are depleted by ryanodine and caffeine (33) . However, these agents were without effect in CHO cells when added either individually (not shown) or in combination (Fig. 6b). This suggests that the appropriate stores are not present and, hence, that cyclic ADP-ribose is unlikely to be a mediator of the Ca mobilization due to PYY in CHO cells. Sphingosine 1-phosphate is a metabolite of sphingosine formed in fibroblasts in response to growth factor stimulation (34) . When added to permeabilized fibroblasts it brings about a rapid release of Ca from a non-mitochondrial store (35) . Addition of DL-threo-erythro-sphingosine, a competitive inhibitor of sphingosine 1-phosphate formation (34) , to the Y1R-1BAR/CHO cells resulted in complex effects on [Ca]: an initial decrease, followed by a transient rise (Fig. 6c). However, the inhibitor exerted no effect on the ability of PYY to raise [Ca], suggesting that formation of sphingosine 1-phosphate was not involved in the mediation of that response (Fig. 6c).


Figure 6: Evidence against the involvement of cyclic ADP-ribose and sphingosine 1-phosphate in the mobilization of intracellular Ca by PYY. Measurements of [Ca] in transfected CHO cells were performed as described in the legend to Fig. 1 and under ``Experimental Procedures.'' Arrows mark the point of addition of 100 nM PYY, 50 µM ryanodine, 5 mM caffeine, and 10 µMDL-threo-erythro-sphingosine.



Because PE and PYY appear to act via different mechanisms, we reasoned that it might be possible to distinguish between the intracellular stores from which they release Ca. The fungal alkaloid thapsigargin acts as an inhibitor of certain intracellular calcium ATPases and has been widely used to deplete Ins(1,4,5)P-sensitive Ca pools (36) . Another compound, tBuHQ, acts through a similiar mechanism but is possibly more selective than thapsigargin in terms of the stores upon which it acts (37). When added to CHO cells, both thapsigargin and tBuHQ caused a transient rise in [Ca], after which neither PYY nor PE were effective, suggesting that the PYY and PE responses are functionally indistinguishable at this level (Fig. 7). This finding, together with the demonstrated absence of a caffeine-sensitive store in CHO cells (Fig. 6b), suggests that PYY and PE might mobilize Ca from the same intracellular pool, albeit using different mechanisms.


Figure 7: Depletion of the PYY and PE-sensitive intracellular Ca pools by thapsigargin and tBuHQ. Measurements of [Ca] in transfected CHO cells were performed as described in the legend to Fig. 1. and under ``Experimental Procedures.'' Arrows mark the point of addition of 100 nM PYY, 1 µM PE, 100 nM thapsigargin, and 20 µM tBuHQ.



Y1-NPY Receptor-mediated Augmentation of Adrenergic Receptor-stimulated Protein Kinase C Activity

Because of the results described above, which down-played the functional relevance of the synergistic increase in Ins(1,4,5)P generation during co-stimulation with PYY and PE, it was important to determine whether the potentiation of PtdInsP hydrolysis might be manifest as a potentiation of PKC activity, the other arm of the signaling pathway. As shown in Fig. 8, a 15-s stimulation with PE-increased membrane-associated PKC activity in Y1R-1BAR/CHO cells by approximately 20% (p < 0.05), whereas PYY alone exerted no statistically significant effect (p > 0.1). However, in the presence of PYY, the PE response was augmented by almost threefold (p < 0.005 versus PE alone). This clearly demonstrates that the potentiation of PLC activity due to co-stimulation with PE and PYY does have functional significance in terms of the activation of PKC, if not with respect to the mobilization of Ca.


Figure 8: Effects of PYY and PE on PKC activity. Transfected CHO cells were incubated for 15 s with PPY (100 nM) and PE (10 µM) either alone or in combination. They were then extracted, and particulate fractions were prepared and assayed for PKC activity as described under ``Experimental Procedures.'' PKC activity is expressed as a percentage of unstimulated control which corresponded to 482 pmol/min phosphotransferase activity per mg of protein. Results were normalized using background activity observed in the absence of lipid activators and are means of six independent experiments, each assayed in triplicate.



Y1-NPY Receptor-mediated Augmentation of Adrenergic Receptor-stimulated PLA Activity

In addition to rises in [Ca]and PKC activity, an increase in arachidonic acid, generated by action of the enzyme PLA, has also been proposed as an important event in the regulation of vasoconstriction (16) . Furthermore, receptors coupling through PT-sensitive G proteins have previously been shown to stimulate PLA activity in CHO cells (38) . We therefore sought to determine whether this was also the case for PYY. Using Y1R-1BAR/CHO cells prelabeled with [H]arachidonic acid, PYY alone was unable to stimulate PLA activity (Fig. 9, top). In contrast PE stimulated arachidonic acid release by approximately 80% above basal, and this response was significantly augmented (p < 0.001) in the presence of PYY (Fig. 9, top). Using cells pretreated with PT, PE-stimulated PLA activity was slightly enhanced, but PYY was no longer able to potentiate the PE response (Fig. 9, top). This potentiation of arachidonic acid release was due to an increase in the maximal response rather than in sensitivity to PE (Fig. 9, bottom).


Figure 9: Effects of PE and PYY on arachidonic acid production. Transfected CHO cells were prelabeled for 3 h with [H]arachidonic acid, washed, and resuspended at 10 cells/ml in KRB buffer. Arachidonic acid released into the medium over 15 min was measured in response to PE (10 µM) or PYY (100 nM) either alone or in combination. For further details, see ``Experimental Procedures.'' Each column or data point represents the average of four independent experiments each assayed in triplicate. Upper panel, cells were cultured overnight in the presence or absence of 200 ng/ml pertussis toxin. Results are expressed as a percentage of the basal release which averaged 4688 and 4323 cpm (n = 12) for untreated and PT-pretreated cells, respectively. Lower panel, dose dependence of arachidonic acid release versus PE concentration in the presence or absence of PYY (100 nM). Results are expressed as a percentage on the incremental response due to 10 µM PE which averaged 7211 cpm (n = 12).



NPY Receptor-mediated Augmentation of Adrenergic Receptor-mediated Arachidonic Acid Production Is Secondary to PKC Activation

To determine the mechanism underlying the potentiation of PE-induced arachidonic acid production by PYY, Y1R-1BAR/CHO cells were pretreated overnight with the phorbol ester TPA, to deplete them of PKC activity. As shown in Fig. 10, the ability of PE alone to generate arachidonic acid was partially impaired in the depleted cells indicating that, under normal circumstances, the PE response is mediated as a result of both PKC activation and the direct coupling of the receptor to PLA. However, the capacity of PYY to potentiate the arachidonic acid production initiated by PE was entirely abolished in PKC-depleted cells.


Figure 10: Effect of the down-regulation of PKC activity on the synergistic activation of arachidonic acid release by PYY and PE. Transfected CHO cells were cultured overnight in the presence or absence of 1 µM TPA and then labeled with [H]arachidonic acid as described in the legend to Fig. 9. Arachidonic acid released into the medium over 2 min was measured in response to PE (10 µM) or PYY (100 nM) either alone or in combination. For further details, see ``Experimental Procedures.'' Results are expressed as a percentage of basal which averaged 3308 and 2818 cpm (n = 9) in the untreated and TPA pretreated cells respectively. Each column or data point represents the average of three independent experiments each assayed in triplicate.



DISCUSSION

In many blood vessels the pressor actions of PE, acting through -adrenergic receptors, are potentiated by NPY (1, 2, 3, 4) . The aim of the current study was to examine the mechanisms underlying this potentiation using a model system in which the Y1-NPY and -adrenergic receptors were heterologously co-expressed in CHO cells. The unique advantage of the current system is that it avoids potentially confusing results due to the presence of multiple subtypes of NPY or adrenergic receptors. Moreover, the fact that our findings are consistent with, but greatly clarify and extend, those obtained using other models of NPY receptor signaling, suggests that their discussion in the context of vasoconstriction is warranted. However, it should be stressed that additional mechanisms are almost certainly operative in vivo where the integration of signals from multiple receptor species and mixed cell populations would be involved.

The current study demonstrates that in transfected CHO cells the Y1-NPY receptor subtype couples, via a PT-sensitive G protein, to mobilization of Ca from a thapsigargin-depletable intracellular store. This is consistent with results obtained using a variety of cells expressing endogenous Y1-NPY receptors (10, 11, 15, 39, 40) . However, the role of Ins(1,4,5)P in this process has been controversial, and a clear resolution of this controversy is an important consequence of the present study. Although NPY has been shown to stimulate PtdInsP hydrolysis presynaptically (5, 6) , the only evidence implicating Y1 receptors in this process was obtained in a single study using HEL cells (14) . This was inconsistent with an earlier study using the same cells (10) as well as experiments performed on pig splenic tissue (41) , vascular smooth muscle cells (13), porcine cultured aortic smooth muscle cells (12) , rabbit pulmonary artery (42) , rat tail artery (21) , and SK-N-MC cells (12, 15) . While it is possible that species, tissue, or receptor subtype differences might be involved, the simplest explanation for these discrepancies is methodological: in the earlier study supporting a role for Ins(1,4,5)P its levels were measured by anion-exchange HPLC (14) ; in most of the negative reports inositol phosphates were measured by cruder chromatographic techniques unable to resolve individual isomers. In one study a mass assay was used, but time points longer than 60 s were not investigated (11) . In our hands the stimulation of inositol phosphates, due to PYY alone, and the potentiation of the PE responses were not seen if the cell extracts were simply analyzed by batch elution from Dowex anion-exchange columns (not shown). We can thus conclude that PYY clearly does stimulate PtdInsP hydrolysis, albeit weakly. However, analysis of the time courses of Ins(1,4,5)P generation further revealed that the PYY response was much slower in onset than that due to PE and is therefore probably not coupled closely to occupation of the Y1 receptor. The process involved in this delayed activation of PtdInsP hydrolysis is unknown, but is not simply a consequence of the rise in [Ca], since the Ca mobilizing agent thapsigargin was unable to stimulate PLC in these cells (not shown). More importantly, the delay in the rise in Ins(1,4,5)P clearly precludes involvement of this second messenger in the Y1 receptor-mediated mobilization of intracellular Ca stores which, like the response to PE, peaked within several seconds of agonist addition.

In addition to Ins(1,4,5)P, a number of other molecules potentially involved in the release of Ca from intracellular pools have been put forward in recent years. The most prominent of these is cyclic ADP-ribose which appears to act through those intracellular Ca channels that bind the pharmacological agent, ryanodine. These channels, intimately involved in the process of Ca-induced Ca release, were originally described as components of the sarcoplasmic reticulum, but are now known to be present on the delimiting membranes of intracellular Ca pools in a number of non-muscle cell types (18) . The cells used in the current study did not appear to contain ryanodine-sensitive Ca stores, which is consistent with an earlier study reporting the lack of immunologically detectable ryanodine receptor protein in CHO cells (43) . These results would therefore argue against the involvement of cyclic ADP-ribose in mediating the Ins(1,4,5)P-independent Ca mobilization initiated by occupation of the Y1 receptor. It should be mentioned that this conclusion is in apparent conflict with that of a previous study, using HEL cells, in which it was shown that NPY mobilizes Ca from a ryanodine-sensitive store (40) . This conclusion was based on the observation that NPY promoted a 20% higher rise in [Ca]after prior addition of ryanodine than in its absence. Strictly speaking these results could be explained by a ryanodine-sensitive reuptake of a small portion of the released Ca. The store responsible for this reuptake need not be the same as that from which the Ca was released. The results presented here clearly indicate that a ryanodine-sensitive Ca store is not necessary for the demonstration of Ca mobilization following occupation of Y1-NPY receptors.

A less well characterized Ca-mobilizing agent is sphingosine 1-phosphate, which has been implicated in the proliferative responses of growth factors in fibroblasts (32, 35) . These responses were blocked by DL-threo-erythro-sphingosine, a competitive inhibitor of sphingosine 1-phosphate formation (34) . However, this inhibitor was without effect on the Ca response to PYY in CHO cells. Another candidate to be ruled out was arachidonic acid, which releases Ca from certain cell types in vitro(44) . However, the concentration of arachidonic acid in CHO cells was not increased by addition of PYY. Therefore the coupling of Y1-NPY receptors to intracellular Ca mobilization does not appear to involve Ins(1,4,5)P, cyclic ADP-ribose, sphingosine 1-phosphate, or arachidonic acid, and hence the underlying mechanism remains obscure.

NPY has been previously shown to synergize with other agonists promoting PtdInsP hydrolysis in aortic smooth muscle cells (20) and segments of rat tail arteries (21) , but not rabbit pulmonary artery vascular smooth muscle (13) . As discussed above these differences may be methodological or related to differential expression of NPY receptor subtypes. Our results clearly demonstrate that occupation of the Y1-NPY receptor does potentiate the activation of PLC by PE. This occurs within 15 s of agonist addition and is therefore more rapid in onset than the rise in Ins(1,4,5)P due to PYY alone. The synergistic response was still apparent in Ca-depleted cells, arguing against the possibility that it might be explained by a potentiation of PLC activity secondary to PYY-induced Ca mobilization. The mechanism underlying this phenomenon is therefore unclear, but probably involves some sort of cross-talk between the Y1 and -adrenergic receptors. This is not uncommon between two classes of receptor where, as is the case here, one couples to PLC most probably via the subunit of Gq/11, and the other interacts with a PT-sensitive G protein (28, 45) . Potentiation of PtdInsP hydrolysis under those circumstances has been shown to require continued occupation of both receptor species, suggestive of a dynamic interaction occurring early in the process of receptor/effector coupling rather than, for example, simple covalent modification of one of the signaling molecules (28). The most obvious site for cross-talk would be at the level of the G proteins, and it is therefore noteworthy that the widely expressed PLC 3 isoform has been shown to be activated in vitro by both q/11, and subunits potentially associated with PT-sensitive G protein subunits (46, 47) .

A major thrust of the current study was directed toward determining which of the two pathways, acting downstream of PtdInsP hydrolysis, is potentially the more important in mediating the physiological consequences of the synergistic interaction between NPY and PE. This has not been directly addressed previously. Several findings suggest that PKC activation, rather than Ca mobilization, is the more important in this instance. First, no significant potentiation of receptor-induced increases in [Ca]was observed in the transfected CHO cells, despite potentiation at the level of Ins(1,4,5)P accumulation. Indeed, the Ca responses were no more than additive. This apparent discrepancy is most probably explained by the capacity of PYY to mobilize Ca in a manner independent of Ins(1,4,5)P production. This separate effect of PYY would tend to minimize the functional importance of the potentiation of PE-stimulated Ins(1,4,5)P generation. Second, and in contrast to the lack of synergism in the observed Ca responses, occupation of Y1 receptors clearly did potentiate the PE-stimulated activation of PKC. This extends a previous study using cultured aortic smooth muscle cells, in which NPY enhanced the phosphorylation of an endogenous PKC substrate, but only in the presence of a primary agonist (angiotensin II) coupled to PLC activation (20) . Since no Ca responses were measured in that study, no conclusions concerning the relative importance of the two pathways in mediating the synergistic response could be made. This is probably due to the fact that, until recently, the obvious importance of a rise in [Ca]for the regulation of vasoconstriction has tended to overshadow the contributions of other pathways (16, 17) . However, there is now growing evidence for an involvement of PKC in this process. For example, the activities of calponin and and caldesmon, both involved in the cross-bridging of actin filaments, are regulated by PKC phosphorylation (48).

Indeed our results demonstrate that PKC activation is necessary for at least one process acting downstream of the Y1 receptor: potentiation of the PE-stimulated activation of PLA. Although the primary response was only slightly attenuated by PKC depletion, consistent with previous studies (49) showing a close coupling between adrenergic receptors and PLA in CHO cells, the potentiation of arachidonic acid release due to concomitant stimulation with PYY was clearly secondary to PKC activation. Delineation of the site of action of PKC in this process was not an aim of the current study, but it could be at the PLA enzyme itself, or upstream of the activation of the mitogen-activated protein kinase which is now recognized as an modulator of PLA activity (50) . Whatever the precise mechanism, the potentiation of PLA activity by PYY is likely to be relevant since arachidonic acid has been postulated to play a role in the regulation of vasoconstriction by inhibiting the enzyme responsible for dephosphorylation of myosin light chains (16) . In functional terms this is the same as stimulating phosphorylation through the action of the Ca calmodulin-dependent myosin light chain kinase, and it results in activation of the myosin Mg ATPase and contraction of actin filaments (16, 17) . The net effect is to render the contractile apparatus more sensitive to a given rise in [Ca]. Potentiation of arachidonic acid production would thus be an efficient (but not the sole) means of linking synergistic increases in PKC activity to vasoconstriction. An involvement of Y1-NPY receptors in the activation of PLA has only been inferred previously from a study measuring prostaglandin synthesis in vascular endothelial cells (51) . The underlying processes were not investigated. However, it has been demonstrated that other receptor species coupling to PT-sensitive G proteins are capable of potentiating the arachidonic acid release initiated by a primary stimulus in CHO cells, without exerting any stimulation by themselves (52). As was the case with PYY, the synergistic responses were secondary to activation of PKC (52) .

In conclusion, we have used CHO cells heterologously expressing the Y1-NPY and -adrenergic receptors in order to delineate possible mechanisms underlying the potentiation of vasoconstriction seen when these receptors are both occupied in vivo. We have demonstrated a potentiation of PLC activity using this model, but suggest that the physiological consequences of this are not evenly distributed between the downstream pathways of Ca mobilization and PKC activation. Thus a synergistic response was seen with PKC but not at the level of Ca mobilization. Furthermore, the synergistic activation of PLA, an event closely linked to regulation of the contractile apparatus, was absolutely dependent on the presence of PKC. We conclude that potentiation of PKC activity is important for the synergistic responses occurring downstream of the occupation of Y1-NPY and -adrenergic receptors.

  
Table: Y1-NPY and -adrenergic receptor-induced inositol phosphate accumulation

Transfected CHO cells were stimulated for 1 min with PYY (100 nM) or PE (10 µM) either alone or in combination. Results (n=3) are expressed as percent of the unstimulated inositol phosphate levels which averaged 58, 97, and 50 cpm for Ins(1,4)P, Ins(1,3,4)P, and Ins(1,4,5)P, respectively.



FOOTNOTES

*
This work was supported by the National Health and Medical Research Council, the National Heart Foundation of Australia, the Cooperative Research Center for Biopharmaceutical Research, and the R. T. Hall Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Garvan Institute of Medical Research, 384 Victoria St., Sydney 2010, Australia. Tel.: 612-361-2050; Fax: 612-332-4876.

The abbreviations used are: NPY, neuropeptide Y; PYY, peptide YY; PE, phenylephrine; G protein, heterotrimeric GTP-binding protein; Fura-2/AM, Fura-2 acetoxymethyl ester; PLC, phospholipase C; PLA, phospholipase A; TPA, 12-O-tetradecanoylphorbol-13-acetate; CHO, Chinese hamster ovary; tBuHQ, 2,5-di-(t-butyl)-1,4-benzohydroquinone; PtdInsP, phosphatidylinositol 4,5-bisphosphate; Ins(1,4,5)P, inositol 1,4,5-trisphosphate; Ins(1,3,4)P, inositol 1,3,4-trisphosphate; Ins(1,4)P, inositol 1,4-bisphosphate; [Ca], cytoplasmic free Ca concentration; PBS, phosphate-buffered saline; KRB, Krebs-Ringer bicarbonate; MOPS, 3-[N-morpholino)propanesulfonic acid; PKC, protein kinase C; HPLC, high performance liquid chromatography; PT, pertussis toxin.


ACKNOWLEDGEMENTS

We thank Vikki Falls and Yvonne Hort for technical assistance, Robert Graham for helpful advice and generous provision of the -adrenergic receptor cDNA, and Joe Lynch for useful comments on the manuscript.


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