Mannose 6-Phosphate Receptor–Mediated Internalization and Activation of Prorenin by Cardiac Cells
Abstract The binding and internalization of recombinant human renin and prorenin (2500 μU/mL) and the activation of prorenin were studied in neonatal rat cardiac myocytes and fibroblasts cultured in a chemically defined medium. Surface-bound and internalized enzymes were distinguished by the addition of mannose 6-phosphate to the medium, by incubating the cells both at 37°C and 4°C, and by the acid-wash method. Mannose 6-phosphate inhibited the binding of renin and prorenin to the myocyte cell surface in a dose-dependent manner. At 37°C, after incubation at 4°C for 2 hours, 60% to 70% of cell surface-bound renin or prorenin was internalized within 5 minutes. Intracellular prorenin was activated, but extracellular prorenin was not. The half-time of activation at 37°C was 25 minutes. Ammonium chloride and monensin, which interfere with the normal trafficking and recycling of internalized receptors and ligands, inhibited the activation of prorenin. Results obtained with cardiac fibroblasts were comparable to those in the myocytes. This study is the first to show experimental evidence for the internalization and activation of prorenin in extrarenal cells by a mannose 6-phosphate receptor–dependent process. Our findings may have physiological significance in light of recent experimental data indicating that angiotensin I and II are produced at cardiac and other extrarenal tissue sites by the action of renal renin and that intracellular angiotensin II can elicit important physiological responses.
A local renin-angiotensin system in the heart is often invoked to explain the long-term beneficial effects of angiotensin-converting enzyme inhibitor treatment on postinfarction cardiac remodeling and failure and in left ventricular hypertrophy.
Experimental studies of the isolated perfused rat heart have indeed demonstrated that the heart is capable of producing Ang I and Ang II.1 2 However, attempts to provide evidence that the cardiac production of these peptides depends on in situ synthesized renin have failed so far. Rather, perfusion experiments in the isolated rat heart, as well as studies of the effects of nephrectomy on the cardiac tissue levels of Ang I and II in the pig, indicate that the local production of these peptides depends on kidney-derived renin.1 2 3
Uptake of renin from the circulation by vascular tissues and by the heart has been reported and appears to contribute to blood pressure control.3 4 5 Binding of rat renin to rat cardiac and other tissue membranes has also been reported, as has binding of human renin to human umbilical vein endothelial cells.6 7 8 A recent study has demonstrated specific receptor binding of human renin to cultured human mesangial cells.9
Recombinant human renin synthesized by Xenopus oocytes and mouse L cells contains phosphomannosyl residues that are recognized by specific receptors (mannose 6-phosphate receptors), as does human prorenin synthesized by CHO cells.10 11 Two different mannose 6-phosphate receptors have been identified; one is dependent on divalent cations, the other not. Both mannose 6-phosphate receptors function in the process of intracellular lysosomal enzyme sorting, and the cation-independent receptor is also capable of binding and internalizing extracellular lysosomal enzymes.12 13
The aim of the present study was to explore the possibility that cardiac myocytes and fibroblasts are capable of binding and internalizing renin and prorenin and that these processes are mannose 6-phosphate receptor–mediated. We also addressed the possibility that receptor-mediated endocytosis of prorenin results in its activation.
Trypsin (type III), bovine serum albumin, monensin, mannose 6-phosphate, mannose 1-phosphate, and glucose 6-phosphate were from Sigma Chemical Co. Fetal calf serum, horse serum, penicillin, and streptomycin were purchased from Boehringer-Mannheim. DMEM and Medium 199 were from Gibco, Life Technologies. Six-well tissue plates were obtained from Costar.
All experiments were performed according to the regulations of the Animal Care Committee of Erasmus University, Rotterdam, Netherlands, in accordance with the Guiding Principles in the Care and Use of Animals approved by the American Physiological Society.
Primary cultures of neonatal rat ventricular cardiomyocytes and fibroblasts were prepared as described before.14 Briefly, ventricles from newborn 1- to 3-day-old Wistar rats were minced, and cells were isolated in eight subsequent trypsinization steps at 30°C. Noncardiomyocytes were separated from the cardiomyocytes by differential preplating. Cardiomyocytes were seeded in 6-well plates at 1.5×106 cells per well, giving a confluent monolayer of spontaneously beating cells after 24 hours. The preplated cells (fibroblast fraction) were passaged after 4 days to 6-well dishes at 0.75×106 cells per well. The cells were maintained in a humidified incubator at 37°C and 5% CO2 in air, in growth medium consisting of DMEM and Medium 199 (4:1, vol/vol), supplemented with 5% fetal calf serum, 5% horse serum, 100 U penicillin/mL, and 100 μg streptomycin/mL for 72 hours. The incubations with renin and prorenin (see below) were carried out under serum-free conditions. Before the start of each experiment, cells were washed with 3 mL warm (37°C) PBS (140 mmol/L NaCl, 2.6 mmol/L KCl, 1.4 mmol/L KH2PO4, 8.1 mmol/L Na2HPO4, pH 7.4). The cells were then preincubated either at 37°C or 4°C for 30 minutes with 0.9 mL incubation medium consisting of DMEM and Medium 199 (4:1, vol/vol), supplemented with 1% (wt/vol) bovine serum albumin.
Prorenin and Renin
Recombinant human prorenin was a kind gift of Dr W. Fischli (Hoffmann-LaRoche, Basel, Switzerland). It was produced in CHO cells transfected with a vector containing human prorenin cDNA. It was partially purified to remove traces of renin by Cibacron Blue Sepharose affinity chromatography.15 The intrinsic renin activity of the prorenin preparation, prior to proteolytic activation, was <2% of the activity after complete proteolytic activation. After proteolytic activation, the prorenin preparation contained approximately 8.5×106 μU/mL renin. Recombinant human renin was prepared by the proteolytic activation of the recombinant human prorenin with immobilized trypsin.3 15 Sepharose-bound trypsin (final concentration, 0.25 mg trypsin/mL) was added to the prorenin preparation, and the mixture was kept at 4°C for 72 hours. Trypsin was then removed by centrifugation. The prorenin and renin preparations were stored at −70°C.
Incubation of Cells With Renin or Prorenin at 37°C or 4°C
After preincubation at 37°C or 4°C for 30 minutes in 0.9 mL serum-free medium (see above), experiments were started by the addition of 0.1 mL of a dilution of recombinant human renin or prorenin to the medium (final concentration in the wells was approximately 2500 μU/mL). Cells were then incubated at 37°C or 4°C. Incubations were also performed in the presence of mannose 6-phosphate (0.01 μmol/mL to 50 μmol/mL), mannose 1-phosphate (1μmol/mL), glucose 6-phosphate (1 μmol/mL), ammonium chloride (50 μmol/mL), or monensin (0.01 μmol/mL).
At the end of the incubation period, the culture medium was removed and quickly frozen on dry ice. Each well was washed three times with 3 mL ice-cold PBS. Renin and prorenin were not detectable in the last PBS wash. Cells were then lysed in 0.5 mL ice-cold PBS containing 0.2% Triton X-100, and the cell lysates were quickly frozen on dry ice. Medium and cell lysate were stored at −70°C until assayed for renin and prorenin.
To determine the internalized renin or prorenin, the acid-wash method was used.16 Briefly, after the cells had been washed three times with 3 mL ice-cold PBS, the cells were incubated at 4°C with 1 mL of an acid solution containing 50 mmol/L glycine and 150 mmol/L NaCl, pH 3.0. After 10 minutes the acid solution was removed, and the cells were washed three times with 3 mL ice-cold PBS. Cells were then lysed in 0.5 mL ice-cold PBS containing 0.2% Triton X-100, as described above.
To determine the prorenin that was bound to mannose 6-phosphate receptors on the cell surface, prorenin was measured in the culture medium before and after its displacement from the receptor by mannose 6-phosphate. For this purpose, the cells were preincubated at 37°C or 4°C with prorenin (2500 μU/mL) for 2 hours. The cells were then washed three times with 3 mL ice-cold PBS. After the last wash, 1 mL of fresh incubation medium, free of serum and renin and prorenin, containing mannose 6-phosphate (final concentration, 10 μmol/mL) was added, and the incubations were continued at the same temperature as during the preincubation. After this second incubation period, the cells were washed and lysed as described above.
Incubation of Cells at 4°C Followed by Incubation at 37°C
To study the kinetics of renin and prorenin internalization and prorenin activation in more detail, cells were incubated with renin or prorenin (final concentration, approximately 2500 μU/mL) for 2 hours at 4°C. After this period, free renin and prorenin were removed by washing the cells three times with 3 mL ice-cold PBS. After the last wash, 1 mL of fresh incubation medium at 37°C without renin and prorenin was added, and the cells were incubated at 37°C. The incubations were terminated by washing the cells with ice-cold PBS. The cells were then lysed as described above. Medium and cell lysate were assayed for renin and prorenin. Internalized renin and prorenin were determined with the acid-wash method as described above.
Renin and Prorenin Measurements
Measurements of renin were made with the enzyme-kinetic assay.3 15 Briefly, 100 μL sample was incubated for 3 hours with saturating amounts of sheep renin substrate at 37°C and pH 7.4 in the presence of serine protease and angiotensinase inhibitors. The generated Ang I was quantified by radioimmunoassay. Results were expressed as μU/mL using the international World Health Organization human kidney renin standard, lot 68/356 (WHO International Laboratory for Biological Standards and Control, Potter Bar, Hertfordshire, UK), as a reference. The Ang I–generating activity we measured in cells that had been exposed to renin in the culture medium represents “true” renin; the specific inhibitor remikiren,17 at a concentration of 10−8 mol/mL, caused complete inhibition of the Ang I–generating activity of the lysates from these cells.
Prorenin is the inactive biosynthetic precursor of renin. The prorenin-to-renin conversion in vivo is a proteolytic process by which the propeptide is cleaved from the renin part of the molecule. In vitro, prorenin can be activated both nonproteolytically (eg, by exposure to low pH) and proteolytically by various enzymes. The Ang I–generating activity we measured in the lysates of cells that had been exposed to inactive prorenin in the culture medium represents prorenin that was activated by the cells. This cell-activated prorenin may or may not be identical with renin. We also measured “total” prorenin, ie, prorenin that was activated by the cells plus prorenin that was not activated by the cells. For these measurements, the samples were incubated for 48 hours at 4°C with plasmin (0.5 casein units/mL) before the incubation with sheep renin substrate.3 The preincubation with plasmin caused complete proteolytic activation of prorenin. Plasmin from the activation step was inactivated by the serine protease inhibitor aprotinin (final concentration, 100 kallikrein-inhibiting units/mL) that had been added to the incubation medium of the Ang I–generating step.
Prorenin that could be measured without preincubation of the samples with plasmin is referred to as “cell-activated” prorenin. Prorenin that was measured in samples that had been preincubated with plasmin is referred to as “total” prorenin. Mannose 6-phosphate, ammonium chloride, and monensin in the concentrations we used had no influence on the renin and prorenin assays.
Data were compared using Student’s t test for unpaired observations. A value of P<.05 was considered to be significant.
Figs 1⇓ (top) and 2 (top) show the amounts of cell-associated renin and prorenin after 4 hours of incubation of cardiac myocytes at either 4°C or 37°C with renin or prorenin, respectively. At 4°C the cells bound both renin and prorenin, but internalization occurred only at 37°C. The cell-associated renin or prorenin after 4 hours of incubation amounted to about 5% of the total quantity in the culture medium both at 4°C and 37°C. After incubation at 37°C, most of the cell-associated prorenin was resistant to the acid-wash and was in an activated form, whereas at 4°C prorenin was washed away by acid and remained inactive.
Fig 3⇓ shows the increase in cell-associated prorenin over time in the myocytes at 37°C. Cell-associated prorenin reached a maximum in 3 to 4 hours. It was 120±37 μU/1.5×106 cells (mean±SD, n=9) at 4 hours of incubation, and more than 90% was in an activated form at that time. When at 2 hours of incubation at 37°C, the prorenin-containing medium was renewed and the cells were incubated for another 2 hours at 37°C, the cell-associated prorenin rose to levels well above the maximum obtained without renewal of the medium, and again more than 90% was in an activated form. The cell-associated prorenin at 4 hours of incubation, 2 hours after renewal of the medium, was 160±41% of the level at 4 hours of incubation without renewal of the medium (n=5, P<.05 for difference from 100%).
When the incubations at 37°C were carried out in the presence of mannose 6-phosphate (10 μmol/mL), cell-associated prorenin at 4 hours of incubation was 24±8 μU/1.5×106 cells (n=5, P<.01 for difference from incubations in the absence of mannose 6-phosphate). Fig 4⇓ shows the effect of different concentrations of mannose 6-phosphate on the levels of cell-associated prorenin at 37°C and 4°C; 50% inhibition was reached at a mannose 6-phosphate concentration in the order of 0.1 μmol/mL. Mannose 1-phosphate (n=3) and glucose 6-phosphate (n=3) at a concentration of 1 μmol/mL were without effect (data not shown).
Fig 5⇓ shows the effect of mannose 6-phosphate on myocytes that had been incubated for 2 hours with prorenin at either 4°C or 37°C. After incubation with prorenin, the cells were washed with ice-cold PBS and incubated at 4°C or 37°C, respectively, for 2 hours in serum-free medium to which mannose 6-phosphate (final concentration, 10 μmol/mL) but not prorenin had been added. After preincubation with prorenin at 4°C, mannose 6-phosphate displaced the cell-associated prorenin into the medium. No displacement by mannose 6-phosphate was observed after preincubation with prorenin at 37°C. Mannose 6-phosphate had similar effects after preincubation with renin (data not shown). These observations are in agreement with the results obtained with the acid-wash method. The two sets of results indicate that at 4°C the cell-associated renin and prorenin remain surface-bound, whereas at 37°C most of it appears to be internalized.
The marked reduction in cell-associated prorenin in response to mannose 6-phosphate was not seen when instead of mannose 6-phosphate, ammonium chloride (50 μmol/mL) or monensin (0.01 μmol/mL) had been added to the medium (Fig 6⇓). Both ammonium chloride and monensin, however, inhibited the cellular activation of prorenin. In the control incubations, the fraction of cell-associated prorenin that was in an activated form rose from 30% after 30 minutes to 90% after 4 hours. This was reduced to 10% and 35%, respectively, by ammonium chloride and to 5% and 10% by monensin (Fig 7⇓, left panel).
Fig 8⇓ and Fig 9⇓ show the results of experiments in which the myocytes were preincubated with either renin or prorenin at 4°C for 2 hours and then incubated at 37°C without renin or prorenin. At 37°C, after the incubation at 4°C, approximately 70% of cell surface-bound renin and 60% of cell surface-bound prorenin were internalized within 5 minutes, and most of the remainder was released into the medium. Results for renin and prorenin were not significantly different. Intracellular prorenin was activated, but most, if not all, extracellular prorenin remained inactive. The half-time of intracellular prorenin activation was approximately 25 minutes. There was no evidence that prorenin, once internalized, was released by the cells back into the medium.
Experiments identical to those performed in the cardiac myocytes, as shown in Fig 1⇑ (top), Fig 2⇑ (top), and Fig 7⇑ (left), were carried out with the use of cardiac fibroblasts. Results of the two sets of experiments were very similar (Fig 1⇑, bottom; Fig 2⇑, bottom; and Fig 7⇑, right). The kinetics of internalization and activation of prorenin by cardiac fibroblasts at 37°C, after preincubation at 4°C for 2 hours, were also similar to those observed in the myocytes (results not shown). At 37°C, after the incubation at 4°C, 42±19% of the cell surface-bound prorenin was internalized within 5 minutes (n=5), and the half-time of intracellular prorenin activation was approximately 25 minutes, as it was in the myocytes. Release of internalized prorenin from the fibroblasts back into the culture medium could not be detected, which also corresponds with our observations in the myocytes. As in the myocytes, the binding of renin and prorenin to the fibroblasts was inhibited by mannose 6-phosphate.
This study indicates that renin and prorenin are internalized by cardiac myocytes and fibroblasts and that prorenin, after its internalization, is rapidly activated. At 4°C both renin and prorenin bound to the cell surface, and at 37°C both were rapidly internalized. It appears therefore that neither the catalytic domain of the enzyme nor the propeptide domain are essential for the processes of binding and internalization. There was no indication that renin and prorenin, once internalized, were released into the culture medium. The acid-wash method that we used to distinguish between surface-bound and internalized renin or prorenin has been validated for a number of peptide hormone receptors and their ligands, including lysosomal enzymes carrying the mannose 6-phosphate signal. Exposure to low pH causes rapid dissociation of these enzymes from cell-surface mannose 6-phosphate receptors.12 To check whether the acid-wash method is also applicable to the cell binding of prorenin, the cells were incubated with prorenin at 4°C. At this low temperature, the internalization process is known to be effectively inhibited. We found that after incubation with prorenin for 2 hours, the cell-associated prorenin was completely removed from the cells by the acid-wash. In contrast, when, after incubation with prorenin at 4°C, the cells were incubated at 37°C with fresh medium without prorenin, nearly all cell-associated prorenin became resistant to the acid-wash within 5 minutes (the first measurement point). Thus, it appears that the acid treatment of the cells did not affect the nonsurface-bound, ie, internalized, prorenin. The cell-surface binding of renin and prorenin at 4°C and its internalization at 37°C were confirmed by our observations on the effects of mannose 6-phosphate. After incubation with prorenin for 2 hours at 4°C, the cell-associated prorenin could be displaced into the medium by the addition of mannose 6-phosphate to the medium, whereas no such displacement was observed after incubation with prorenin for 2 hours at 37°C. The results were similar when the cells were incubated with renin instead of prorenin.
Our results strongly suggest that the internalization is a mannose 6-phosphate receptor–dependent process. The inhibition of prorenin internalization by mannose 6-phosphate was specific and saturable, with an IC50 in the order of 0.1μmol/mL. This corresponds with other mannose 6-phosphate receptor–mediated responses.18 19 Our finding that most of the cell surface–bound renin and prorenin after preincubation with these enzymes at 4°C was internalized within 5 minutes of incubation at 37°C is also in agreement with published studies of other ligands that bind to cell-surface mannose 6-phosphate receptors via the mannose 6-phosphate signal.20
Approximately 5% of the renin or prorenin from the culture medium was bound and internalized by the cardiac cells. This low percentage may be related to the fact that only a small percentage of the renin and prorenin molecules carries the mannose 6-phosphate signal.11 21 That this, rather than the number of cell receptors, is the rate-limiting factor is also suggested by our experiments in which the prorenin-containing culture medium was renewed after 2 hours of incubation at 37°C, at a time when the amount of internalized prorenin was at 85% of its maximum. Renewal of the medium in these experiments caused a rise of intracellular prorenin to levels well above the maximum that was reached without renewal of the medium. These results have to be confirmed by binding studies, using radiolabeled prorenin.
The effects of mannose 6-phosphate that we observed were different from the inhibitory effects of ammonium chloride and monensin. Mannose 6-phosphate in the incubation experiments with prorenin at 37°C reduced the cellular concentrations of both total and cell-activated prorenin to equally low levels, whereas ammonium chloride and monensin had a much greater effect on the cellular level of cell-activated prorenin than on the cellular level of nonactivated prorenin. This supports the view that mannose 6-phosphate inhibits the binding of prorenin to cell-surface mannose 6-phosphate receptors, whereas ammonium chloride and monensin primarily act on events that follow the binding to these receptors. Ammonium chloride and monensin are known to interfere with the normal intracellular trafficking and the recycling and lysosomal degradation of internalized receptors and ligands.22
This study provides the first evidence of intracellular activation of prorenin, derived from the extracellular fluid, by extrarenal cells. The evidence in our study that a precursor protein is converted into a biologically active agent by a mannose 6-phosphate receptor–mediated and endocytosis-dependent process is not an isolated finding. It has been demonstrated for a number of precursor proteins, including the aspartyl protease cathepsin D.23 Cathepsin D is a lysosomal enzyme showing a high degree of structural homology with renin.24 Transforming growth factor-β, which may modulate the growth-promoting actions of Ang II,25 is another example. This factor is produced in a latent form by vascular endothelial and smooth muscle cells and is converted into the active form in cocultures of these cells by a mannose 6-phosphate receptor–dependent process.19 The mannose 6-phosphate receptor that is involved in the endocytosis-dependent activation of both procathepsin and the latent form of transforming growth factor-β is of the cation-independent type, which is identical with the insulin-like growth factor II receptor.19 23 It seems logical to assume that this is also the receptor that mediates the internalization and activation of prorenin which we observed in the present study.
Our findings may have physiological significance in light of the experiments by Swales’ group (Thurston et al4 )in rats more than 15 years ago. Their experiments showed that the slow decrease in blood pressure after nephrectomy followed the same time course as the decrease in vascular renin and contrasted with the rapid decrease in circulating renin. In recent years, a series of perfusion studies using the isolated rat Langendorff heart and isolated rat hindquarters have provided direct evidence for the local production of Ang I and II.1 2 26 Angiotensin production in these studies was dependent on the presence of renin in the perfusion fluid.
Binding of renin to cell-surface receptors may not only be the first step in endocytosis of the renin-receptor complex but may also transduce other membrane-associated or intracellular events. Receptor-mediated binding of human renin to human renal mesangial cells in culture has been reported to cause an increase in [3H]thymidine incorporation and an increase in the concentration of plasminogen activator inhibitor-1 antigen in the conditioned medium.9 The concentration of renin at which these responses were observed was in the order of 100 000 fmol/mL. This is much higher than the concentration used in our experiments, which was 2500 μU/mL or approximately 50 fmol/mL. Even a concentration of 50 fmol/mL is very high; the normal concentrations of renin and prorenin in human plasma are approximately 0.5 fmol/mL and 5 fmol/mL, respectively.27 However, the lower in vivo concentrations may be sufficient because, in the in vivo situation, the cells are continuously exposed to prorenin carrying the mannose 6-phosphate signal, whereas in vitro this prorenin disappears from the culture medium because of uptake by the cells.
Cellular binding and internalization of renin and prorenin and intracellular activation of prorenin may lead to local levels of enzyme activity that are higher than those in the extracellular fluid. If angiotensinogen is internalized via bulk fluid endocytosis concurrently with the receptor-mediated endocytosis of renin and prorenin, a scenario is provided for intracellular Ang I generation. Ang II is known to stimulate plasminogen activator inhibitor-1 production by renal mesangial cells.28 The reported increase in plasminogen activator inhibitor-1 antigen in response to the binding of renin to these cells9 would therefore fit in the view that this is an Ang II–mediated effect. However, the question of whether Ang I and/or II are formed within the cells is highly controversial. It is possible that the reported responses of mesangial cells to the cellular binding of renin are independent of Ang I and II formation.
Our observations on the internalization of renin and prorenin and the intracellular activation of prorenin warrant further study addressing this issue. Internalized Ang II has a long half-life compared with circulating Ang II.29 It rapidly accumulates in cardiac and vascular muscle cell nuclei,30 where it may bind to chromatin and may influence transcriptional processes that are related to the growth-promoting effects of Ang II.31 Finally, recent experiments, in which Ang II was injected into cardiac myocytes and vascular smooth muscle cells, also favor the concept that intracellular Ang II may serve important functions.32 33 Studies addressing the possibility of intracellular angiotensin generation may therefore hold promise.
Selected Abbreviations and Acronyms
|CHO||=||chinese hamster ovary|
|DMEM||=||Dulbecco’s modified Eagle’s medium|
This study was supported by the Netherlands Heart Foundation, research grant 96.019.
- Received May 5, 1997.
- Revision received May 19, 1997.
- Accepted July 9, 1997.
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