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(Circulation Research. 1997;80:551-556.)
© 1997 American Heart Association, Inc.

Articles

Differential Desensitization of Thromboxane A₂ Receptor Subtypes

Masao Yukawa¹, Ryoji Yokota¹, Robert T. Eberhardt, Laila von Andrian, , J. Anthony Ware

From the Vascular Biology Unit, Cardiovascular Division, Beth Israel Hospital and Harvard Medical School, Boston, Mass.

Correspondence to J. Anthony Ware, MD, Cardiovascular Division, Albert Einstein College of Medicine, 1300 Morris Park Blvd, Bronx, NY 10461. E-mail jaware{at}aecom.yu.edu

	Abstract

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Abstract Two subtypes of the thromboxane A₂ (TxA₂) receptor (TxA₂R-E and TxA₂R-P), which differ in their alternatively spliced cytoplasmic tails, have been identified. The initial concentration of the TxA₂ mimetic IBOP required to reduce peak intracellular Ca²⁺ concentration ([Ca²⁺]_i) induced by a second addition of IBOP (100 nmol/L) was similar (IC₅₀ for TxA₂R-E and TxA₂R-P, 0.46±0.16 and 0.40±0.07 nmol/L) in fibroblasts overexpressing either the TxA₂R-E or -P subtype. Although the number of TxA₂ binding sites decreased in TxA₂R-P cells after prolonged stimulation with a TxA₂ mimetic, those in the TxA₂R-E cells increased markedly. To determine whether the mechanism for desensitization differs between subtypes, the effect of activation of protein kinase C (PKC) or cAMP-dependent kinase on TxA₂-induced [Ca²⁺]_i mobilization was measured. Forskolin reduced the IBOP-induced peak [Ca²⁺]_i in neither TxA₂R-E nor TxA₂R-P cells; however, treatment with phorbol esters (IC₅₀, 0.57±0.70 nmol/L) strongly prevented IBOP-mediated [Ca²⁺]_i rise in TxA₂R-E but not in TxA₂R-P cells. Desensitization of TxA₂R-E by phorbol esters was prevented by the PKC inhibitor calphostin C or by downregulation of PKC-. Thus, the response of TxA₂R-E to prolonged stimulation differs from that of TxA₂R-P in both the regulation of the number of binding sites and the mechanism for desensitization; agonists that activate PKC- might interfere with TxA₂R-E–mediated signaling.

Key Words: protein kinase C • prostaglandin • eicosanoid • desensitization • downregulation

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The eicosanoid TxA₂ is released from activated platelets and the vessel wall and causes platelet aggregation and release of granule contents,^{1 2} constriction and hypertrophy of vascular smooth muscle,³ and release of prostacyclin by endothelial cells.^{4 5} These functions are mediated by surface receptors for TxA₂² that are members of the G protein–linked receptor family; two such receptors have been cloned, one from megakaryocytic and placenta sources⁶ (TxA₂R-P, also called TxA₂R-⁷ ) and a second from a vascular endothelial library⁸ (TxA₂R-E, also called TxA₂R-ß⁷ ). These receptors differ in their alternately spliced cytoplasmic tails^{6 7 8} and are expressed in different tissues.⁸ Recently, these differences in cytoplasmic domains have been shown to confer association with different G proteins, suggesting that each receptor utilizes different signal transduction pathways.⁷

An important cellular function mediated by the cytosolic domains of G protein–linked receptors is desensitization,^{9 10} in which the receptor, after agonist stimulation, becomes refractory to further stimulation.^{11 12} Desensitization of the TxA₂ receptor can be either homologous (ie, caused by an initial preincubation with TxA₂ or an analogue^{11 12 13 14 15} ) or heterologous (ie, caused by another stimulatory agonist^{12 14} ). One mechanism for desensitization is loss of binding sites, called downregulation, that occurs after prolonged stimulation with TxA₂ mimetics and other agonists in platelets and vascular smooth muscle cells.^{14 15 16 17} Desensitization can also occur by uncoupling of the receptor from its downstream effectors without loss of binding sites^{11 14} ; one mechanism for this in other G protein–linked receptors is phosphorylation of the cytoplasmic domains by serine-threonine kinases such as the ß-adrenergic receptor kinase or other G-protein receptor kinases.¹⁸ In addition, activation of PKC can uncouple the TxA₂ receptor as well^{12 17} and thus prevent G-protein–mediated activation of downstream effectors. In the cytoplasmic tails of TxA₂R-P and TxA₂R-E, there are several differences in serines and threonines that might be phosphorylated by one or more kinases,^{6 7 8} thus suggesting that their mechanisms of desensitization may differ as well. In this study, we created cell lines of CHO fibroblasts in which each of the TxA₂ receptors was overexpressed, and we used measurements of TxA₂-mediated intracellular Ca²⁺ concentration ([Ca²⁺]_i) to determine whether the mechanisms for desensitization differed between receptor subtypes.

	Materials and Methods

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Preparation of Stable Lines of CHO Cells
Complementary DNAs encoding either TxA₂R-E (isolated as described previously⁸ ) or TxA₂R-P (a generous gift of Drs M. Hirata and S. Narumiya, Kyoto University) were cloned into pcDNA1/Neo vector (Invitrogen) and transfected by lipofectin (Gibco/BRL) into CHO cells. The 5'-untranslated region of the TxA₂R-E and TxA₂R-P cDNAs were identical for 207 bp immediately 5' to the start codon; TxA₂R-P had an additional 26-bp sequence, which contained no start or stop codon, to 5' to the 207-bp sequence. The 3'-untranslated region sequences in both cDNAs were the same (73 bp). Stable clones of each type as well as vector-only controls were selected by neomycin resistance. Multiple clones were studied for each experiment; qualitative results did not differ among clones expressing each type of receptor.

Intracellular Calcium Measurement in CHO Cells
CHO cells stably transfected with either TxA₂R-E or TxA₂R-P were harvested and loaded with fura 2-AM as described previously.¹⁹ Cells were washed once and resuspended in modified HEPES-Tyrode's buffer (in mmol/L: HEPES 10 [pH 7.4], NaCl 129, KCl 2.8, NaHCO₃ 8.9, KH₂PO₄ 1.8, MgCl₂ 0.8, dextrose 5.6, and CaCl₂ 1). Fluorescence intensity was induced by excitation wavelengths of 340 nm (I₃₄₀) and 380 nm (I₃₈₀) and measured at an emission wavelength of 505 nm, which was monitored with a LS50B luminescence spectrometer (Perkin-Elmer), and the data were analyzed by the FL Data Manager program (Perkin-Elmer). Changes in [Ca²⁺]_i were induced by the addition of the TxA₂ mimetic IBOP ([1S-[1,2(Z),3ß(1E,3S*),4]]-7-[3-[3-hydroxy-4-[4-iodophenoxy]-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid). Calibration was performed as previously described^{19 20} ; R_max and R_min were obtained by addition of 40 µmol/L digitonin and subsequently 20 mmol/L EGTA for each preparation of cells. Data are presented as the mean±SD of at least three determinations; values of P<.01 by unpaired t test, or by ANOVA in the case of multiple comparisons, were regarded as significant.

Determination of the TxA₂R Binding Sites
Subconfluent CHO cells (TxA₂R-E or TxA₂R-P) were incubated with either the TxA₂ mimetic U46619 (5-heptenoic acid, 7-[6-(3-hydroxy-1-octenyl)-2-oxabicyclo[2.2.1]hept-5-yl]-,[1R-[1,4,5ß(Z),6(1E,3S*)]]-) (1 µmol/L) or vehicle (ethanol) for 24 hours. Cells were washed twice with PBS and harvested with collagenase. After another two washes, cells were resuspended in the binding buffer (9 vol modified HEPES-Tyrode's buffer, 1 vol 3.8% sodium citrate), and 2.5x10⁵ cells in 100 µL were incubated with 50 nmol/L of the TxA₂ receptor antagonist ³H-SQ29548 ([5,6-³H(N)]-5-heptenoic acid, 7-[3-[[2-[(phenylamino) carbonyl] hydrazino] methyl]-7-oxabicyclo [2.2.1] hept-2-yl]-,[1S(1,2(Z),3,4)]-) at room temperature for 30 minutes.²¹ For the Scatchard analysis, six different concentrations of ³H-SQ29548 (ranging from 2 to 100 nmol/L) were used. The reaction was stopped by addition of 4 mL ice-cold washing buffer (20 mmol/L HEPES, pH 7.4, 0.38% sodium citrate) and subsequent filtration with GF/B filter paper with a cell harvester (Brandel). After another two washes with washing buffer, the filter paper was placed in scintillation liquid (EcoLite ICN), and bound radioactivity was measured. Specific binding was obtained by subtracting nonspecific binding in the presence of excess amounts of cold SQ29548 (20 µmol/L).

Northern Analysis
CHO cells overexpressing either TxA₂R-E or TxA₂R-P were incubated with either the TxA₂ mimetic U46619 (1 µmol/L) or vehicle (ethanol) for 24 hours; total RNA was extracted from each cell type and processed as previously reported.^{8 22} Briefly, 20 µg of total RNA sample was loaded for electrophoresis on a 1.5% agarose gel with formaldehyde and transferred to a nylon membrane. ³²P-labeled human TxA₂R cDNA probe, based on a sequence from the consensus region present in both subtypes, was used for hybridization. The signals were quantified by densitometric analysis with a Hewlett-Packard Scanjet Plus Scanner.

Immunoblot Analysis of CHO Cell Lysate
In studies in which PKC was downregulated, subconfluent CHO cells were incubated with either DMSO or PMA 100 nmol/L for 24 hours. The cells were washed twice with PBS and scraped into lysis buffer (in mmol/L: HEPES 50 [pH 7.4], NaCl 150, EDTA 5, EGTA 5, PMSF 1, and leupeptin 0.1, and 1% Triton X-100). After brief sonication, the cell lysate was centrifuged at 10 000g for 10 minutes. The supernatant of the total lysate was applied to SDS-PAGE, followed by transfer to a PVDF membrane (Immobilon-P, from Millipore). Protein on the membrane was analyzed by immunodetection with anti-PKC isoenzyme–specific antibodies from either Santa Cruz (, ßI, ßII, , , ) or Transduction Laboratories (, , µ, , ) and visualized by enhanced chemiluminescence (ECL Amersham).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
In initial studies, clonal populations of CHO cells in which either TxA₂R-P or -E was expressed were loaded with the Ca²⁺-sensitive fluorophore fura 2 and then stimulated with the endoperoxide analogue IBOP. Comparison of the concentration-response relationship revealed no differences between TxA₂R-P and TxA₂R-E in their ability to respond to the TxA₂ mimetic (Fig 1A). The EC₅₀ was 6.8±2.1 or 8.5±3.0 nmol/L for IBOP for TxA₂R-P or TxA₂R-E, respectively. To determine whether differences in homologous desensitization could be detected, each of the clonal populations was preincubated for 10 minutes with increasing quantities of IBOP and then stimulated with a second concentration of IBOP (100 nmol/L) to determine whether the point at which the peak [Ca²⁺]_i is diminished by preincubation differs according to the receptor subtype (Fig 1B). These studies revealed no significant differences between these receptors, with an IC₅₀ of 0.46±0.16 or 0.40±0.07 nmol/L in TxA₂R-P or TxA₂R-E, respectively, indicating that the alternatively spliced cytoplasmic tail in the TxA₂ receptor does not alter its ability to undergo homologous desensitization.

View larger version (9K): [in this window] [in a new window]   Figure 1. A, Concentration-dependent [Ca2+]i mobilization induced by a thromboxane A2 mimetic. Peak [Ca2+]i in CHO cells transfected with either TxA2R-E () or TxA2R-P () caused by addition of various concentrations of agonists is shown as percentage of maximum peak [Ca2+]i obtained with each agonist. Results shown are mean±SD of five experiments. B, Homologous desensitization of TxA2R-E and TxA2R-P. Peak [Ca2+]i in CHO cells transfected with either TxA2R-E () or TxA2R-P () in response to IBOP after a 10-minute incubation with various concentrations of IBOP is shown as percentage of control (initial IBOP of 0 nmol/L in ethanol). Results shown are mean±SD of four separate experiments in two different clonal populations of CHO cells expressing each receptor subtype.

To determine whether prolonged TxA₂ stimulation also altered the number of TxA₂ binding sites, CHO cells overexpressing each receptor were incubated with another endoperoxide analogue (U46619), 1 µmol/L for 24 hours. The number of binding sites in TxA₂R-P cells, as assessed by ³H-SQ29548, decreased significantly with TxA₂ incubation (from 46.8±2.4 to 30.1±2.8 fmol/10⁶ cells) (Fig 2); further experiments demonstrated that the reduction in TxA₂R-P binding sites did not occur until at least 3 hours of incubation (data not shown). In contrast, the number of binding sites in TxA₂R-E cells not only did not decrease but actually significantly increased (from 50±3.1 to 118.3±6.3 fmol/10⁶ cells). The CHO cells transfected with the vector alone did not demonstrate specific ³H-SQ29548 binding either with or without the addition of U46619. Scatchard analysis revealed no significant change in the affinity of SQ29548 to either receptor subtype after 24 hours of incubation with U46619; the K_ds for control and U46619-treated TxA₂R-E cells were 12.6 and 10.7 nmol/L, respectively, and the K_ds for control and U46619-treated TxA₂R-P cells were 14.0 and 14.1 nmol/L, respectively. Thus, neither the affinity of SQ29548 to TxA₂R-E nor that to TxA₂R-P was changed by incubation with TxA₂ mimetic. Along with the increase in the number of binding sites, the expression of mRNA encoding TxA₂R-E was modestly but significantly upregulated (1.8±0.1-fold) after 24 hours of U46619 treatment, whereas the level of TxA₂R-P mRNA did not change significantly (1.1±0.1-fold). These results suggest that loss of binding sites is one possible mechanism for TxA₂-induced desensitization in TxA₂R-P cells but not in TxA₂R-E cells.

View larger version (11K): [in this window] [in a new window]   Figure 2. Changes in binding sites induced by 24-hour incubation with a thromboxane A2 mimetic. Data are shown as mean±SD of quadruplicate determinations from a single clone and are representative of more than three separate experiments in different clones of each receptor subtype. *P<.01, **P<.001 vs vehicle.

Next, we tested whether agents that enhanced activation of serine-threonine kinases would uncouple each subtype of the TxA₂ receptor from IBOP-induced rises in [Ca²⁺]_i. To test the effects of PKC activation, increasing quantities of the PKC-activating phorbol ester PMA were added for 1 minute before addition of IBOP to fura 2–loaded CHO cells that overexpressed either TxA₂R-P or TxA₂R-E. These studies revealed that TxA₂R-P was only mildly sensitive to even high concentrations of PMA, with an 20% drop in [Ca²⁺]_i at 1 µmol/L; in contrast, TxA₂R-E was very sensitive to PMA-induced uncoupling, with an IC₅₀ of 0.57±0.70 nmol/L (Fig 3A). This difference was not due to a loss of binding sites, the numbers of which were unchanged at 24 hours of incubation with PMA (data not shown). In contrast, addition of the adenylate cyclase mimic forskolin (1 µmol/L), which elevates levels of cAMP and thus activates cAMP-dependent kinase, did not reduce IBOP-induced peak [Ca²⁺]_i and in fact caused a slight increase in both receptor subtypes (Fig 3B). These results suggest that phosphorylation of a substrate for PKC but not cAMP-dependent kinase on serine or threonine residues is capable of uncoupling the [Ca²⁺]_i rise mediated by only TxA₂R-E.

View larger version (12K): [in this window] [in a new window]   Figure 3. Effect of PMA and forskolin on IBOP-induced peak [Ca2+]i. A, [Ca2+]i mobilization with IBOP (100 nmol/L) after preincubation with various concentrations of PMA for 1 minute in CHO cells transfected with either TxA2R-E () or TxA2R-P (), expressed as a percentage of that obtained with preincubation with 0 nmol/L PMA in DMSO. Data shown are from five experiments obtained in a single clonal population. B, Cells were preincubated with forskolin (1 µmol/L for 10 minutes). Peak [Ca2+]i induced by IBOP (100 nmol/L) after forskolin is expressed as a percentage of that obtained in IBOP-treated samples preincubated with vehicle. Data shown are mean±SD of six determinations from two independent experiments.

To determine whether these effects demonstrated by PMA resulted from its interaction with PKC, the effect of inhibiting PKC with the inhibitor calphostin C on an IBOP-mediated [Ca²⁺]_i rise was determined (Fig 4A and 4B). Calphostin C (150 µmol/L) partially reversed the PMA-induced inhibition of the [Ca²⁺]_i rise in TxA₂R-E but had no effect above that produced by the solvent in the CHO cells expressing TxA₂R-P. To examine further the importance of PKC in promoting selective uncoupling of TxA₂R-E, downregulation experiments were performed in which CHO cells expressing either subtype of TxA₂ receptor received prolonged incubation with PMA (100 nmol/L, 24 hours) and then treated with a protocol similar to that in Fig 3A, in which cells were first treated with PMA 10 nmol/L and then stimulated with IBOP (Fig 4C). These experiments showed that the PMA-induced reduction in [Ca²⁺]_i rise that follows IBOP interaction with TxA₂R-E but not that seen in TxA₂R-P cells was restored by PKC downregulation, thus indicating further that PKC can uncouple signaling processes in a receptor subtype–specific manner.

View larger version (17K): [in this window] [in a new window]   Figure 4. Effect of PKC inhibitor calphostin C on PMA-induced reduction of IBOP-induced peak [Ca2+]i. TxA2R-E (A)– or TxA2R-P (B)–transfected CHO cells were incubated with either calphostin C 150 µmol/L (tracing b) or DMSO (tracings a and c) for 15 minutes before 1-minute treatment with either PMA (10 nmol/L) (b and c) or DMSO (a). Changes in [Ca2+]i induced by IBOP (100 nmol/L) added at time 0 was monitored as described in "Methods." Tracings shown are from a single experiment representative of three similar independent experiments of each. C, TxA2R-E– or TxA2R-P–transfected CHO cells were incubated with either 100 nmol/L PMA (open bars) or DMSO (solid bars) for 24 hours. [Ca2+]i mobilization with IBOP (100 nmol/L) after 1-minute preincubation with PMA (10 nmol/L) is shown as percentage of that with 1-minute preincubation with vehicle (DMSO). Results are shown as mean±SD of six determinations from two separate experiments. *P<.01, **P<.001 vs vehicle.

The above studies suggested that one (or more) PKC isoenzyme that is sensitive to both phorbol ester and calphostin C interacts with TxA₂R-E. To identify possible candidates for this isoenzyme, immunoblotting with PKC-specific antibodies was performed on CHO cells both before and after prolonged incubation with PMA to downregulate PKC. CHO cells were found to express PKCs , ß, and ; PKCs , , , , , µ, and were not expressed in sufficiently high levels to be detected by this method. Prolonged incubation with PMA resulted in decreased expression of PKC- in whole CHO cell lysates but did not reduce overall expression of either PKC-ß or PKC- (Fig 5). (In addition to the disappearance of PKC-, a change in PKC-ß was also observed; an immunoreactive doublet was recognized in unstimulated cells, and a single, more prominent band was noted after treatment with phorbol ester. Such a phenomenon has been observed with other isoenzymes and cell types (see Reference 23²³ for example) and is thought to reflect an altered electrophoretic mobility due to autophosphorylation or reaction with a putative PKC kinase.²⁴ ) Thus, these data suggest that the effects of PKC on uncoupling TxA₂R-E from the subsequent [Ca²⁺]_i rise may reflect the actions of a single isoenzyme, PKC-.

View larger version (57K): [in this window] [in a new window]   Figure 5. Immunoblot of CHO cells for expression of PKC isoenzymes after downregulation. CHO cells overexpressing TxA2R-E (lanes 1 and 2) and TxA2R-P (lanes 3 and 4) were treated with either PMA 100 nmol/L (lanes 2 and 4) or DMSO (lanes 1 and 3) for 24 hours. Expression of PKC- (a), PKC-ßI (b), and PKC- (c) was detected; in lanes 2 and 4, each blot shows results with CHO cells treated with prolonged PMA incubation for downregulation. Results shown are from a single experiment representative of three independent experiments. Arrows indicate size of an 83-kD band of prestained protein standard (Bio Rad) run on same gel (not shown).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The results of this study reveal that the mechanisms of desensitization differ between the two subtypes of the TxA₂ receptor in at least two respects: TxA₂R-E, rather than becoming downregulated, actually becomes upregulated with prolonged stimulation; and the activation of PKC effectively uncouples Ca²⁺ mobilization from the interaction of TxA₂ with TxA₂R-E but not TxA₂R-P. Interruption of TxA₂-mediated signal transduction is not a general property of serine-threonine kinases, since cAMP activation does not produce an effect specific for a receptor subtype. These findings demonstrate that these receptor subtypes interact differently with mediators distal to each receptor and utilize different signal transduction pathways, as also suggested by the recent report that TxA₂ receptor subtypes interact with different G protein -subunits.⁷

The ability of PKC activation to uncouple TxA₂R-E–induced Ca²⁺ mobilization in these experiments appears to result from the action of a single isoenzyme, PKC-. (It should be noted, however, that the possibility that another, as yet undiscovered, PKC isoenzyme in CHO cells that is also downregulated by PMA mediates these effects cannot be ruled out.) PKC- is expressed in a wide variety of tissues, including endothelium,²⁵ platelets,^{19 22} and vascular smooth muscle,²⁶ all of which express TxA₂R-E as well^{7 8} (M.Y. and J.A.W., unpublished observations, 1995). Overexpression of PKC- has previously been shown to downregulate a receptor tyrosine kinase, the EGF receptor, by an unknown mechanism,²⁷ but isoenzyme-specific regulation of G protein–linked receptor function has not been reported. Members of the PKC family have differing substrates and activators, and increasing evidence demonstrates that they can selectively mediate distinct intracellular functions.²⁸ PKC- is regulated both by lipid products such as diacylglycerol and by elevations in [Ca²⁺]_i, suggesting that Ca²⁺ mobilization may be required before PKC activation can uncouple TxA₂-induced activation when it occurs in response to other physiological agonists. TxA₂-induced elevations of [Ca²⁺]_i are closely correlated with homologous desensitization,^{11 14} but whether they are required for it to occur is not clear. Since it is well established that PKC can become activated in platelets treated with TxA₂ mimetics,¹⁴ it would seem likely that PKC- activation may be a feature of homologous desensitization; it is not known, however, whether PKC- specifically becomes activated after these agonists. A PKC- uncoupling mechanism might also explain heterologous desensitization to agonists such as thrombin or PAF, which also activate PKC.¹²

The mechanism by which PKC- uncouples TxA₂-mediated signaling is unknown. One possibility is that PKC- directly phosphorylates the cytoplasmic tail of TxA₂R-E but not that of TxA₂R-P, since phosphorylation of the cytoplasmic tail by serine-threonine kinases is a major mechanism for desensitization.²⁹ Such a model seems unlikely, however; both PKC and cAMP-dependent kinase can phosphorylate recombinant forms of the carboxyl tail of TxA₂R-P.¹⁷ Furthermore, recent preliminary evidence has indicated that TxA₂R-P is phosphorylated after agonist addition, and such phosphorylation was not blocked by PKC inhibitors.³⁰ A more likely possibility is that PKC- might phosphorylate a G protein and thus interfere with signal transmission; a particularly good candidate for such a role is G_i, which has previously been shown to be phosphorylated by PKC.³¹ G_i and G_q are subunits that are activated by TxA₂R-E; TxA₂R-P, on the other hand, is associated with G_q and possibly with G_s,⁷ neither of which is known to become phosphorylated by PKC.

There are at least two possible physiological or clinical implications of our findings. The number of TxA₂ binding sites has been shown to decrease in platelets and vascular smooth muscle that have been exposed to endoperoxide analogues.^{14 15 16 17} In contrast, in disease states such as myocardial infarction or pregnancy-induced hypertension, in which circulating levels of TxA₂ are elevated,^{32 33} the number of binding sites is elevated rather than decreased.^{34 35} Our finding that the number of binding sites after stimulation is elevated in TxA₂R-E but reduced in TxA₂R-P suggests a model in which the augmentation in total binding sites results from a selective increase in number of TxA₂R-E during these disease states. Additionally, PKC-mediated uncoupling of the TxA₂ receptor participates in heterologous desensitization by agonists such as thrombin,¹⁰ a powerful PKC activator in many cell types that also formed in excess amounts during thrombotic states.³⁶ Since TxA₂ induces release of the vasodilating antiaggregant prostacyclin from the endothelium, in which TxA₂R-E but not TxA₂R-P is expressed,^{4 5 8} it is possible that agonist-induced uncoupling of the function of this receptor subtype contributes to platelet aggregation and vasoconstriction induced by thrombin or other agonists. With the development of reagents specific for TxA₂ receptor subtypes, testing these possibilities should become feasible.

	Selected Abbreviations and Acronyms

 

CHO = Chinese hamster ovary PKC = protein kinase C PMA = phorbol 12-myristate 13-acetate TxA2 = thromboxane A2 TxA2R = thromboxane A2 receptor

	Acknowledgments

 
This work was supported by HL-47032 and HL-51043 to Dr Ware.

	Footnotes

 
¹ The first two authors contributed equally to this study.

Received November 27, 1996; accepted February 3, 1997.

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