(Hypertension. 1997;29:83.)
© 1997 American Heart Association, Inc.
Research Articles (Issue 1, Part 1) |
Thrombin Inhibits Atrial Natriuretic Peptide Receptor Activity in Cultured Bovine Endothelial Cells
the Metabolic Research Unit and Department of Medicine, University of California at San Francisco.
Correspondence to David G. Gardner, Box 0540 1141 HSW, Metabolic Research Unit, University of California, San Francisco, CA 94143.
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Abstract |
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Thrombin and the atrial natriuretic peptide (ANP) possess a number of functionally antagonistic properties in vascular endothelial cells. Thus, regulatory interactions that modulate the activity of one or the other could have important sequelae with regard to cardiovascular homeostasis. Thrombin treatment effected a dose- and time-dependent reduction in ANP receptor activity (maximal 70% to 80% inhibition) in cultured bovine aortic endothelial cells. This resulted from a decrease in total receptor number as well as a modest reduction in the affinity of the receptor for its ligand. The inhibition was largely confined to the type C receptor population, in that thrombin had no effect on maximal type A receptor–linked cGMP accumulation. The protein kinase C–activating phorbol ester 12-O-tetradecanoylphorbol 13-acetate effected a similar reduction in binding activity; however, suppression of protein kinase C activity did not reverse the thrombin effect. Pretreatment of endothelial cells with cycloheximide did not completely prevent the thrombin-dependent inhibition, and thrombin did not effect a reduction in type C receptor mRNA levels, findings that argue for a postsynthetic inhibitory locus. The inhibition of receptor activity was effectively irreversible in that suspension of protein synthesis blocked the recovery of receptor density on the cell surface. Reduction in type C receptor density was accompanied by modest increases in the stability of ANP in the culture medium and enhancement of the cellular cGMP response to the peptide, particularly at low ligand concentrations. These findings demonstrate a potentially important interaction between these two agonist systems in regulating endothelial cell function within the vascular wall.
Key Words: atrial natriuretic factor • thrombin • receptors, atrial natriuretic factor • endothelium
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Introduction |
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The potent natriuretic hormone ANP lowers blood pressure and inhibits renin, aldosterone, and vasopressin secretion in a variety of animal and human models.1 Recent studies suggest that it may also play a role in controlling cell proliferation in the vascular wall,2 3 renal glomerulus,4 and central nervous system.5
ANP exerts its biological effects through high-affinity cell surface receptors. These receptors have been identified in a wide variety of tissues and cultured cells.1 To date, three NPRs (NPR-A, NPR-B, and NPR-C) have been identified. The three subtypes differ in terms of their ligand specificity and signal transduction. NPR-A binds to ANP with high affinity, whereas the C-type natriuretic peptide appears to be the natural ligand for NPR-B.6 NPR-C binds to each of the natriuretic peptides as well as some functionally inactive structural homologues.7
NPR-A is a 130-kD membrane protein that harbors intrinsic guanylate cyclase activity. It binds to ANP with high affinity (Kd=0.02 to 2.0 nmol/L) and specificity. NPR-C has a molecular weight of 60 to 65 kD and is the most abundant receptor subtype. In BAE cells, it constitutes 85% to 95% of the total receptor population.8 Its affinity for ANP is equal to that of NPR-A; however, it has a broader range of ligand specificity, binding to ring-deleted and truncated ANP analogues as well as brain natriuretic peptide and C-type natriuretic peptide. The broad ligand specificity and short cytoplasmic domain (38 amino acids) of NPR-C have led investigators to conclude that it functions as a clearance receptor, responsible for removing natriuretic peptides from circulating plasma. There is reasonably strong experimental support for this model.9 10 In addition, several lines of evidence suggest that NPR-C may have a signaling function of its own. The best evidence for this comes from studies which demonstrate that NPR-C reduces adenylyl cyclase activity in selected cellular and membrane preparations through a pertussis toxin–sensitive mechanism.11 12 In addition, cANF, a relatively selective ligand for NPR-C, has been shown to reduce [3H]thymidine incorporation (as an index of mitogenic activity) in cultured vascular smooth muscle,13 endothelial,3 glial,4 and cardiac mesenchymal14 cells. Taken together, these findings imply that NPR-C may operate in parallel with the guanylate cyclase–linked NPRs to regulate a number of important biological activities relevant to cardiovascular homeostasis.
A variety of stimuli have been shown to regulate cellular ANP receptor levels, including hormones (eg, ANP, angiotensin II, glucocorticoids, vasopressin, endothelin, and thyroid-stimulating hormone)15 16 17 18 19 and pharmacological agents with putative intracellular signaling capacity (eg, phorbol esters and cGMP).20 21 In BAE cells, the phorbol ester TPA effects a significant and sustained decrease in NPR-C levels as well as a more transient decrease in NPR-A activity.20 Thus, each of the major receptor populations in these cells (NPR-B is not expressed to an appreciable extent; see Reference 22) is negatively affected by PKC activation.
Thrombin is a serine protease best known for its role as a participant in the clotting cascade.23 It also has potent effects on platelet function and serves as a mitogenic stimulus for both vascular smooth muscle24 and endothelial25 cells. This latter property has aroused speculation as to the potential role of thrombin as a mediator of the intimal proliferation that accompanies vascular injury and atherosclerosis.26 In addition, thrombin increases vascular permeability to albumin27 and stimulates release of endothelin from vascular endothelial cells.28 It is believed to activate its receptor on target cells by effecting a proteolytic cleavage event at a specific site in the extracellular domain of the receptor.29 Once cleaved, the new amino-terminal segment (downstream of the cleavage site) functions as a tethered ligand to activate the receptor. Several investigators have confirmed this tethered ligand model using a 14–amino acid truncated peptide (SFLLRNPNDKYEPF), derived from the amino terminus of the thrombin-treated receptor, as a surrogate agonist to promote thrombin-dependent activity.30 31
ANP, as already pointed out, suppresses mitogenic activity in cultured vascular cells.2 3 It inhibits thrombin-induced changes in endothelial permeability32 as well as thrombin-induced endothelin-1 secretion from vascular endothelial cells.28 Therefore, at the level of the vasculature, ANP could function as a natural antagonist of thrombin-dependent activity. A shift in the delicate balance between ANP and thrombin-like agonists (eg, through alterations in ANP receptors) could lead to serious perturbation of the regulatory mechanisms that govern cardiovascular function. We designed the current study to address the latter aspect of the interactions between thrombin and ANP, specifically, the role of thrombin as a potential modulator of ANP receptor activity.
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Methods |
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Materials
ANP (rANF, 28 amino acids) and cANF (des-[rat, Gln18,Ser19,Gly20,Leu21,Gly22]-ANF-[4-23]-NH2) were purchased from Peninsula Laboratories. Thrombin, 3-isobutylmethylxanthine, and TPA were obtained from Sigma Chemical Co. Reagents for cGMP radioimmunoassay and [
-32P]dCTP were purchased from New England Nuclear Corp. Enriched calf serum was obtained from Gemini Bioproducts, Inc. Other reagents were obtained from standard commercial suppliers.
Cell Culture
BAE cells (second passage) were provided to us by Dr Jeffry Lansman. Passages 4 through 9 were used in the present study. Cells were grown to confluence in Dulbecco's modified Eagle's medium (DMEM) H-21 supplemented with 10% enriched calf serum, 2 mmol/L glutamine, and penicillin/streptomycin (100 IU/mL and 100 µg/mL, respectively) at 37°C in a 95% air/5% CO2 humidified atmosphere. Medium was changed every 48 to 72 hours before the experiment was begun.
For binding studies, cells were plated in 24-well plates at a density of 5x104 cells per well and grown to confluence in serum-containing medium. The cells were then transferred to serum-free medium containing 10% serum substitute,33 2 mmol/L glutamine, 100 IU/mL penicillin, 100 µg/mL streptomycin, 5 µg/mL insulin, 5 µg/mL transferrin, and 1 µg/mL vitamin B12 for 24 hours. Fresh medium, with or without the additives indicated, was then added to each of the individual cultures.
Measurement of ANP Binding
ANP was labeled with 125I by the chloramine T method and purified as described previously.34 The specific activity of the 125I-ANP was 300 to 700 µCi/µg (919 to 2142 Ci/mmol). Binding assays were performed in 24-well plates on confluent cell monolayers as described by Leitman et al,35 with slight modification. In brief, cells were washed twice with 1 mL PBS and incubated with 200 µL DMEM containing 10 mmol/L HEPES, 0.4% bovine serum albumin, and 0.8x10-9 to 1.0x10-9 mol/L 125I-ANP. After 30 minutes of incubation at 37°C, the cells were washed four times with cold PBS containing 0.2% bovine serum albumin and solubilized with 1 mL of 1N NaOH, and 900-µL aliquots were assayed for total bound radioactivity (total binding). Nonspecific binding was determined in parallel untreated or treated cells receiving identical amounts of 125I-ANP as well as 10-7 mol/L unlabeled ANP or cANF. Specific binding was computed as the difference between total and nonspecific binding and routinely represented 85% to 90% of total binding. For Scatchard analysis, 125I-ANP was added at increasing concentrations in the presence or absence of 10-7 mol/L unlabeled ANP. Kd and Bmax values were determined with a computer program described previously.36
In selected studies, cells were preincubated with the protein synthesis inhibitor cycloheximide (2 µmol/L) for 3 hours before addition of thrombin or control medium, either with or without a pretreatment period. This cycloheximide dose reduced the incorporation of 35S-methionine into trichloroacetic acid–insoluble cellular material by more than 92% (control: 44 230 cpm per well; cycloheximide: 3540 cpm per well; mean of six determinations).
To assess internalization of the receptor-ligand complex, we incubated cells with 1x10-9 to 3x10-9 mol/L 125I-ANP in the buffer system described above for 4 hours at 4°C. Cells were washed three times with cold PBS. Fresh binding buffer containing 10 mmol/L NH4Cl (to reduce receptor recycling), with or without thrombin, was added and the temperature was shifted to 37°C. At specific time points, the cells were washed three times with cold PBS, and the radioactivity of the total cellular, cell surface, or intracellular compartments was measured. Total cellular specific binding was determined by solubilizing the cells with 1N NaOH (1 mL) and counting a 900-µL aliquot. Binding on the cell surface was estimated from the amount of bound radioactivity stripped from the cell surface with two sequential 15-minute washes with cold acetic acid (0.2%)/NaCl (0.5 mol/L). Residual cellular binding after the acetic acid wash was solubilized with 1N NaOH and used for approximation of ligand sequestration in the intracellular compartment. Specific binding was determined by inclusion of 10-7 mol/L unlabeled ANP in parallel samples and subtraction of the nonspecific component from total receptor binding activity.
cGMP and ANP Measurements
Cells were grown to confluence in 24-well plates. After pretreatment with thrombin (for 24 hours), cells were washed four times with warm PBS and preincubated at 37°C for 10 minutes with 250 µL DMEM containing 10 mmol/L HEPES and 0.5 mmol/L isobutylmethylxanthine. Fresh medium containing the additives indicated in the presence or absence of ANP was added, and the incubation was continued for 10 minutes. Medium was recovered for ANP radioimmunoassay as described previously,34 and the incubation was terminated with 0.5 mL cold 10% trichloroacetic acid. After a 30-minute incubation at 4°C, cellular extracts were collected and centrifuged at 12 000 rpm for 15 minutes. The supernatant was extracted four times with water-saturated ether. cGMP was measured by radioimmunoassay using commercially available reagents. Both samples and standards were acetylated before assay.
Blot Hybridization Analysis
Cells were exposed to thrombin (or vehicle) for 8 and 24 hours. Cells were then collected, and RNA was extracted according to the method of Chirgwin et al.37 Total RNA (30 µg) was size-fractionated on a 1% agarose gel containing 2.2 mol/L formaldehyde, transferred by capillary action to a nitrocellulose filter in 10x SSC (1.5 mol/L sodium chloride and 0.15 mol/L sodium citrate) for 8 to 16 hours, and fixed to the filter by UV irradiation (DNA transfer lamp, Fotodyne Inc). The filter was then baked at 80°C for 1 hour and hybridized overnight at 42°C using conventional techniques.38 Blots were probed with a full-length (2.1-kb) bovine NPR-C receptor cDNA isolated as an HindIII-EcoRI fragment,39 after labeling with [-32P]dCTP using the random primer technique.38 Each blot was also probed with a labeled 1.3-kb Pst I fragment from the human GAPDH cDNA40 to control for loading and transfer of total RNA in individual samples. Autoradiograms were scanned by laser densitometry. NPR-C signal intensity was expressed relative to the GAPDH control.
Statistical Analysis
Data were subjected to one-way ANOVA and the Newman-Keuls test for significance. Data points represent mean±SE for a minimum of three determinations.
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Results |
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As shown in Fig 1, thrombin effected a dose- and time-dependent reduction in 125I-ANP binding to cultured BAE cell monolayers. The highest level of inhibition was seen at 3 U/mL. At this dose, reduction in binding activity was seen as early as 2 hours into the incubation, whereas lower doses required 5 hours of treatment to demonstrate the inhibition. In all instances, maximal inhibition was reached after 20 to 34 hours of exposure to the protease.
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To explore the nature of the reduction in binding activity, we performed a Scatchard analysis (Fig 2). Exposure of BAE cells to thrombin (3 U/mL) for 24 hours resulted in a significant decrease in total binding capacity (150±20 versus 63±6 fmol/mg protein, control versus thrombin) as well as a modest increase in Kd (0.04±0.005 versus 0.16±0.02 nmol/L, control versus thrombin), indicating that thrombin reduces 125I-ANP binding through both a decrease in receptor number and a modest decrease in the affinity of the receptor for its ligand. Using the NPR-C–selective ligand cANF as an unlabeled competitor, we found that more than 85% of receptors in these cells are of the NPR-C subtype (D.W.Z., unpublished observations, 1996), confirming the findings reported by others previously.8 By inference, the major portion of the reduction in receptor activity derives from suppression of the NPR-C receptor population.
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Because our thrombin preparation was not purified to homogeneity, it was possible that the effects observed were mediated by a nonthrombin contaminant (eg, serum-derived growth factor) rather than by thrombin itself. To explore this possibility, we investigated the effect of phenylalanyl-prolyl-arginyl-chloromethyl ketone (PPACK), a relatively selective thrombin inhibitor,41 on the thrombin-mediated suppression of 125I-ANP binding activity. As shown in Fig 3A, 5 µmol/L PPACK completely reversed the suppression of binding seen after 13 hours of thrombin treatment. After 22 hours of treatment, PPACK had a modest independent, and likely nonspecific, effect to reduce binding activity; however, the presence of thrombin failed to decrease binding further. These findings indicate that the reduction in binding is mediated by thrombin or a closely related activity that shares similar substrate specificity.
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P<.01 vs thrombin.A second concern related to the nature of the ligand itself. As noted above, thrombin is a serine protease that activates its receptor through a specific proteolytic cleavage event. It seemed quite possible that the observed reduction in 125I-ANP binding might reflect nonspecific proteolysis of the ANP receptor as opposed to an event signaled through the thrombin receptor. To address this issue, we used a synthetic peptide derived from the amino-terminal sequence of the activated (ie, thrombin-cleaved) thrombin receptor. This portion of the thrombin receptor is believed to function as the endogenous "ligand" driving receptor-signaled activity. This particular peptide (SFLLRNPNDKYEPF) has been shown to mimic the activity of thrombin in a number of different systems.29 30 31 High peptide concentrations are usually required to elicit these effects because the receptor has only limited affinity for this untethered ligand (a situation that is remedied in vivo by its covalent attachment to the receptor molecule). As shown in Fig 3B, the agonist peptide effected a dose-dependent reduction in 125I-ANP binding activity which approached that obtained with thrombin. This argues that the inhibitory activity of thrombin is mediated through activation of the thrombin receptor and not through nonspecific proteolysis of the ANP receptor.
In a number of systems, thrombin has been shown to activate phospholipase C, triggering phosphoinositide hydrolysis, with increases in intracellular calcium, diacylglycerol, and, putatively, PKC activity. The latter is of particular relevance because activation of PKC (eg, with phorbol esters) has been shown to suppress ANP binding activity in cultured BAE cells.20 We therefore asked whether the observed effect might operate through a PKC-dependent mechanism. To address this question, we pretreated cells for 24 hours with TPA, a maneuver that would be expected to downregulate PKC activity in these cells,42 and then challenged them with thrombin in the continued presence of TPA for an additional 24 hours. As shown in Fig 4, the thrombin effect was clearly detectable on the TPA-suppressed background. This observation was highly reproducible and suggests that thrombin and TPA operate through parallel but independent molecular circuitry to inhibit binding activity. We employed a second approach to probe the question of PKC dependence using the PKC-selective kinase inhibitor staurosporine. As shown in Fig 5, at concentrations that were reasonably effective in reversing TPA-dependent inhibition of binding activity (Fig 5A), the thrombin-dependent inhibition was not reversed (Fig 5B), again implying that thrombin exerts its effects through a non–PKC-dependent pathway. Similar findings were obtained with the more selective PKC inhibitor chelerythrine (Table 1).
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The rather prolonged time course of the thrombin-dependent inhibition of ANP binding activity raised the possibility that the inhibition might be mediated by an effect on NPR-C synthesis. To explore this possibility, we pretreated cells with the protein synthesis inhibitor cycloheximide at a dose sufficient to suspend more than 92% of new protein synthesis in these cells and asked whether thrombin would inhibit residual receptor activity (ie, demonstrate effects independent of receptor synthesis). As shown in Fig 6, cycloheximide treatment led to a time-dependent decrement in binding activity, presumably reflecting decay of preexisting receptor on the surface of these cells. On the basis of data accrued from three similarly generated curves, we estimate that the half-life of receptors in these cells is on the order of 6 to 12 hours. The addition of thrombin to the cycloheximide-treated cells reproducibly effected a small but significant decrement in binding activity (replicated in three independent experiments). This implies that at least a portion of the inhibitory activity of thrombin operates at a locus distal to synthesis. This is supported by the Northern analysis of NPR-C mRNA levels displayed in Fig 7. Thrombin did not reduce NPR-C mRNA levels, and hence template for new receptor synthesis, in these cells.
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After pretreatment with thrombin (3 U/mL) for 24 hours, BAE cells required approximately 10 hours in thrombin-free culture medium to restore ANP binding activity to control levels (Fig 8). More importantly, this restoration was completely blocked in the presence of cycloheximide. Thus, receptor depletion by thrombin appears to be an effectively irreversible process in that recovery of receptor activity requires de novo receptor synthesis.
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Receptor internalization (ie, downregulation) is a well-documented mechanism for depleting receptor populations on the cell surface. An increase in the internalization rate (ie, drawing receptors from the cell surface to the intracellular compartment) could contribute to the reduction in receptor availability seen after thrombin treatment. We examined the former possibility using a temperature shift protocol that follows the depletion (ie, internalization) of a preloaded surface receptor pool as the temperature is raised from 4°C to 37°C. As shown in Fig 9A, while thrombin effected a reduction in steady-state receptor levels (ie, lower levels of 125I-ANP binding at 4°C), internalization of cellular receptors proceeded at a rapid rate in both the control and thrombin-treated cells. Similar findings were obtained in a separate experiment when surface receptors were analyzed independently (Fig 9B), and there was no increase in intracellular 125I-ANP accumulation in the thrombin-treated cells to suggest enhanced sequestration of the internalized ligand (Fig 9C). A subsequent experiment using a shorter exposure to thrombin (3 hours) resulted in a near-identical decay profile for surface 125I-ANP binding in control versus thrombin-treated cells (data not shown). Taken together, these data argue that thrombin does not significantly affect the rate of NPR-C internalization in these cells.
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Finally, we attempted to assess the physiological sequelae of the thrombin-dependent reduction in NPR-C levels. Thrombin had no influence on maximal ANP-dependent cGMP generation in these endothelial cells, implying that this agent has little or no effect on the functional consequences of NPR-A occupancy by saturating concentrations of ligand (Table 2). However, since NPR-C has been implicated in playing a role in the clearance of natriuretic peptides from the circulation,9 10 we hypothesized that reduction of NPR-C levels would serve to increase available natriuretic peptide ligand in the extracellular compartment, particularly at low agonist concentrations, thereby increasing occupancy of available NPR-A receptors and amplifying the NPR-A–signaled events in the target cell. This is precisely what we observed. As shown in Table 2A, pretreatment for 24 hours with thrombin effected a threefold to fourfold increase in the cGMP response to ANP at ligand concentrations considerably below the Kd for the receptor. As ANP concentration was increased to 10-10 mol/L, the magnitude of the effect diminished, and at higher ligand concentrations, the effect disappeared. This follows the predicted model in that reduction in ANP clearance would be expected to have the greatest effect when ligand concentration is limiting and less effect when abundant ligand is present in the medium. Coincident measurement of medium ANP levels (Table 2B) provided further confirmation of the model. Modest increments (approximately twofold) in available ligand were seen at lower medium concentrations of peptide, but this disappeared as ligand concentrations were increased. Thus, it appears that the thrombin-mediated reduction in clearance receptors translates into an amplification of NPR-A activity, at least at low concentrations of ligand.
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Discussion |
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Thrombin and ANP display a number of antagonistic properties in the cardiovascular system. In addition to its role as a procoagulant, thrombin is a vasoconstrictor with mitogenic activity for vascular cells in culture.23 This latter property has led to speculation about its potential role in promoting smooth muscle hyperplasia in atherosclerosis,26 vascular injury,43 and some forms of hypertension. ANP is a potent vasodilator with antimitogenic activity in a variety of cell types, including vascular smooth muscle2 and endothelial3 cells. ANP has also been shown to antagonize thrombin-dependent transport of fluid and albumin across the vascular endothelium and to inhibit thrombin-dependent stimulation of endothelin release from vascular endothelial cells.28 32 In view of the seemingly antagonistic properties of ANP and thrombin, a second level of regulatory interaction (eg, regulation of the ANP signal transduction system by thrombin) carries with it the potential for significant perturbation of cardiovascular homeostasis.
We have shown that thrombin promotes a significant reduction in NPR levels in cultured BAE cells. Since the NPR-C receptors constitute more than 85% of the total receptor population in BAE cells,8 we can infer that the thrombin effect (50% to 80% inhibition) is largely targeted toward this receptor class. As a consequence, ligand degradation is decreased, most obviously at lower concentrations of the latter, which leads to an increase in NPR-A occupancy and consequent amplification of the ligand-dependent cGMP response. Such an increase in NPR-A–related activity might be considered as counterregulatory, that is, designed as a negative servomechanism to oppose or restrain the activities of thrombin. If one can extrapolate this activity to the whole animal, it could have important implications for cell growth in the vascular wall in pathological conditions such as atherosclerosis and hypertension.
The present study demonstrates that the thrombin-dependent suppression of NPR-C results from both a reduction in receptor number and a modest decrease in the affinity of the receptor for its ligand. The thrombin receptor, a seven-transmembrane domain protein, has been linked to activation of phospholipase C, with mobilization of intracellular calcium and increased production of diacylglycerol, an activator of PKC. As noted above, the PKC activator TPA is a well-documented inhibitor of NPR-C activity in these cells20 ; however, the thrombin-dependent inhibition does not appear to operate through PKC, raising the question of an alternate signaling pathway for thrombin in these cells. Such pathways (eg, signaling through a ras-dependent mechanism) have been invoked to explain thrombin-dependent activity in other systems.44 45
On the basis of mRNA analyses, there appears to be little evidence for a thrombin-mediated reduction in NPR-C synthesis. In fact, suspension of protein synthesis with cycloheximide does not completely eliminate thrombin-dependent inhibition of NPR-C binding activity. Similarly, there is little evidence for effects on receptor processing based on direct measurement of receptor-ligand internalization rates. Since studies of NPR-C internalization suggest that the half-time of receptor recycling is on the order of 30 minutes,46 we should have detected an appreciable effect on receptor internalization rate in our experiments. However, this does not preclude the possibility that a small percentage of receptors, not readily measurable with the techniques used here, could be extracted from the internalized pool during each cycle, with the cumulative effect on receptor depletion being manifest only after a significant number of cycles have been completed.
New receptor synthesis is required for restoration of NPR-C levels after thrombin removal. This conclusion is based on the cycloheximide recovery studies shown in Fig 8. This suggests that whatever mechanism is used to deplete NPR-C levels, it is effectively irreversible and that regeneration of the full receptor complement at the cell surface depends on de novo synthesis of new protein molecules rather than reprocessing of previously inactivated receptors to an active form.
Taken together, the data are most compatible with a model in which thrombin acts through a posttranscriptional mechanism to target the clearance receptor for extraction from the functional receptor pool. This reduction in clearance receptor occupancy and consequent increase in available ligand results in an increase in guanylyl cyclase–linked receptor activity at low ligand concentrations. These findings suggest that thrombin can modulate the activity of a relevant physiological antagonist in a cultured cell system. The role of thrombin as a mediator of vascular hyperplasia or hypertrophy and the physiological importance of its modulatory role with regard to NPR activity remain to be established in an in vivo model.
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Acknowledgments |
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This work was supported by HL-35753 and HL-45637 from the National Institutes of Health and by a National Research Service Award fellowship award to D.Z. We are grateful to Shaun Coughlin for his gift of the thrombin agonist peptide and Gordon Porter, who provided the bovine NPR-C cDNA used in these studies.
Received July 8, 1996;
first decision July 30, 1996; first decision August 16, 1996;
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