Protein Kinase C Modulation of Cardiomyocyte Angiotensin II and Vasopressin Receptor Desensitization
Abstract Angiotensin II (Ang II) and arginine vasopressin (AVP) increased intracellular free Ca2+ concentration [Ca2+]i and/or the [Ca2+]i transient rate (CaTR) in cultured neonatal rat cardiomyocytes. These agents increased membrane-bound protein kinase C (PKC) with peak activity at 5 and 10 minutes, respectively. Two-minute exposure to Ang II produced homologous desensitization to a repeated stimulation with Ang II and heterologous desensitization to AVP. Two-minute exposure to AVP also produced homologous desensitization to AVP but not heterologous desensitization to Ang II. When the AVP exposure time was increased from 2 to 10 minutes coincident with maximal AVP-mediated PKC activation, heterologous desensitization to Ang II was also observed. Acute activation (15 minutes) of PKC by phorbol 12-myristate 13-acetate (PMA) blocked responsiveness to both Ang II and AVP. When PKC activation was inhibited by 20 hours of prior exposure to PMA, as confirmed by PKC assay, homologous desensitization of Ang II still occurred, confirming an alternative mechanism(s) for homologous desensitization in the cardiomyocytes. In contrast, 20-hour PMA suppression of PKC markedly diminished the ability of the cardiomyocytes to exhibit AVP-mediated heterologous desensitization for Ang II. These data indicate that PKC activation plays a primary role in mediating vasopressin V1 receptor–induced heterologous desensitization of the Ang II receptor and participates in a hierarchy of two or more kinase systems mediating homologous desensitization of the Ang II receptor in cardiomyocytes.
Angiotensin II and AVP exert positive inotropic and/or chronotropic effects on the myocardium.1 2 3 These agonist effects have been demonstrated in several species and appear to function predominantly by G protein receptor–mediated activation of PLC.4 5 6 7 Inositol trisphosphate generated by PLC activation increases [Ca2+]i and together with cogenerated diacylglycerol activates PKC.8 9
Receptors associated with heterotrimeric G protein transduction are susceptible to both homologous and heterologous desensitization. Ang II and AVP receptors share these properties, yet little is known concerning the biochemical events mediating these properties.10 Abdellatif et al11 examined the effect of Ang II on cardiomyocyte desensitization and concluded that this phenomenon was mediated solely at the level of the Ang II receptor and was independent of PKC. They examined heterologous desensitization by comparing Ang II receptor activation to that of the α1-adrenergic receptor, two receptors whose agonists are different in structure and function.
These provocative studies did not directly address the question as to the role of PKC activation in the cardiomyocyte response to Ang II, to the effect of these stimuli or of PKC on [Ca2+]i, or to the probability that more than one modality for homologous desensitization may exist in these cells after activation by Ang II. The basis for the present study is to examine the effects of activation and suppression of PKC activity on homologous and heterologous desensitization of cardiomyocyte responsiveness to repeated exposure to Ang II and AVP.
All culture media were from Gibco-BRL, Inc, except FCS, which was from Sigma Chemical Co. Fura 2-AM was purchased from Molecular Probes, Inc. PMA, P12-MA, [IIe5] Ang II, AVP, insulin, transferrin, and antibiotics were purchased from Sigma. Assay reagents for measurement of PKC were obtained from Gibco-BRL.
Preparation of Neonatal Rat Cardiomyocytes
Pregnant Sprague-Dawley rats were obtained from Sasco, Inc, Omaha, Neb. Cardiomyocytes were isolated from 2-day-old pups by a previously published technique.1 The cells were resuspended in McCoy’s modified 5A medium containing 6% FCS and incubated for 20 minutes at 37°C in 100-mm Petri dishes to enrich the cardiomyocyte fraction before counting and plating. The medium was supplemented with 6% FCS, 8.3 μmol/L insulin, 637 nmol/L transferrin, 145 nmol/L sodium selenite, 100 000 IU/L penicillin, and 6.86 μmol/L streptomycin. For the fluorescent microcytometry studies, 300 000 cells were plated into 30-mm wells containing sterile ethanol-washed 25-mm round glass coverslips. The cells were cultured in a humidified incubator in 5% CO2 and air at 37°C. The medium was discarded after 24 hours and replaced with one containing 1.0% FCS and the supplements. This was changed every 48 hours. However, in each case the medium was changed on the night before the actual study.
Measurement of [Ca2+]i
Loading with Fura 2-AM
Dual-excitation single-emission fluorescence microcytometry was performed with modifications of our previously described technique.1 Fura 2-AM solution (with 1 mmol/L stock solution in dimethyl sulfoxide) was added to medium for a final concentration of 0.5 μmol/L fura 2-AM. The cells were incubated at 37°C in a 5% CO2 incubator for 20 minutes. The culture medium was then replaced with a balanced ionic medium containing (in mmol/L) Na+ 152, K+ 5.6, Ca2+ 1.5, Mg2+ 1, H2PO4 1, HCO3− 26, Cl− 133.6, and D+ glucose 11, pH 7.35. The coverslips were placed in a temperature-controlled (37°C) cell chamber mounted on a Nikon Diaphot microscope. The chamber volume of 330 μL was superfused with medium at 1.25 mL/min, which provided a 4× exchange of medium per minute. An SLM DMX-1000 spectrofluorometer connected to the microscope was used to produce a rapidly alternating (300-Hz) 340/380-nm excitation and to detect the emission at 510 nm. The analog electronic signal, proportional to the ratio (R) of light intensity measured at 340 and 380 nm excitation wavelengths, was stored and collected for analysis on an IBM-compatible personal computer after digitization. Calculation of [Ca2+]i was performed as previously described.1 After loading, the cells were treated with either ionomycin (2 μmol/L), digitonin, or BrA23187 and either 1.5 mmol/L Ca2+ or 20 mmol/L EGTA to determine the ratio at either maximal (Rmax) or minimal (Rmin) intracellular free Ca2+. These values were then used to normalize a standard curve. A calibration was performed each day when the cells were being studied to minimize day-to-day variation of the data.
The field of view was centered on a group of two to four contiguous cells that beat synchronously. Basal fluorescence was recorded after a 5-minute stabilization period. The cells were superfused for an additional 20 seconds and the buffer was switched to one containing the specific stimulus. If the cells were pretreated immediately before the study (15 minutes with PMA or P12-MA), this concentration was introduced before or during the last 15 minutes of the loading period. If a second stimulation was to be performed, the first agonist-containing buffer was replaced by one containing buffer alone for a washout period and the second exposure was made at the proper time interval by using a manual valve system.
PKC activity was measured in neonatal rat cardiomyocytes at day 6 of culture in Primaria-coated 100-mm Petri (Falcon) dishes. The cells were washed with warm, fresh buffer and incubated with the test substance for the chosen time intervals. Each 100-mm Petri dish was washed with iced PBS ×3 and aspirated dry. The cells were scraped in the presence of 0.5 mL iced (4°C) extraction buffer containing 20 mmol/L Tris, pH 7.5, 0.5 mmol/L EDTA, 0.5 mmol/L EGTA, 105 μmol/L leupeptin, 7.7 μmol/L aprotinin, and 10 mmol/L mercaptoethanol. This was homogenized with a polytron at 12 000 rpm×30 seconds at 4°C, centrifuged at 85 000g for 1 hour at 4°C, and then the supernatant (cytosol) was placed on ice. The pellet was dissolved in 0.5 mL extraction buffer containing 0.5% Triton X-100, set on ice for 30 minutes, and then centrifuged at 85 000g for 1 hour at 4°C. The supernatant containing the solubilized membrane fraction was removed and also stored on ice at 4°C until PKC assay on the same day. Protein concentrations of the cytosol and membrane fractions were measured by spectrophotometry using bicinchoninic acid (BCA) protein assay reagents (Pierce Chemicals).
PKC was assayed12 in a final volume of 50 μL containing 10 μg total protein from the cytosol or pellet fractions, 0.5 μmol/L PMA, 345 μmol/L phosphatidyl serine, 1.0% Triton X-100–mixed micelles, 20 μmol/L γ32P ATP (3000 Ci/mol) stock solution, 20 μmol/L MgCl2, 1 μmol/L CaCl2, 50 μmol/L acetylated-myelin basic protein substrate, and 20 μmol/L Tris, pH 7.5, in the absence or presence of 20 μmol/L PKC pseudosubstrate inhibitor (PKC 19-36). After incubation at 30°C for 10 minutes, 25 μL was removed and spotted onto a phosphocellulose disk. This was immersed in 33% glacial acetic acid and washed for 30 minutes. A second wash in 15% glacial acetic acid was made for 1 hour. The phosphocellulose disks were then placed in scintillation vials containing 10 mL Universal ES (ICN Biomedicals) and counted for 32P incorporation (Beckman Instruments, LSC 1701). Results were expressed as picomoles per minute per microgram protein and then as percent increase over baseline to normalize multiple samples.
Binding to Whole-Cell Preparations
To determine whether phorbol esters altered Ang II binding to cultured cardiomyocytes, studies were performed on 5- to 7-day-old cultured cells in 35-mm six-well plates. After two washes with prewarmed 37°C medium containing no FCS, 150 000 cpm (45.1 fmol/well) of 125I–Ang II (2000 Ci/mmol, NEN-DuPont, Inc) was added under various conditions for 0 to 60 minutes at various temperatures of 37°C, 20°C, and 4°C, as well as in the presence or absence of phorbol ester analogues. The cells were washed with iced PBS ×3, scraped, and counted in a gamma scintillation instrument (Minimed, Inc). Aliquots were measured for total protein as previously described.
Specific and nonspecific bindings were measured in the absence or presence of 10−5 mol/L Ang II. The specific binding data are expressed as percent of the control tubes. Membrane-bound 125I–Ang II was estimated by washing the cells for 10 minutes in a glycine buffer, pH 3, before scraping of the cells. Both the cell fraction and an aliquot of the glycine wash were counted and corrected for volume and expressed as percent of the control tubes.
Statistical analysis was performed, after testing for normality and variance, by one-way ANOVA for comparison between treatment groups (SigmaStat, version 1.0, Jandel Scientific). Student’s t test for paired observations was used as appropriate. Significance was ascribed to values of P<.05 using the two-tailed test.
Measurement of [Ca2+]i by Ratiometric Microfluorometry
We have previously demonstrated that Ang II at concentrations from 10−10 to 10−8 mol/L produced an acute rise in [Ca2+]i in spontaneously contracting cultured neonatal rat cardiomyocytes.1 In the present studies, we used higher concentrations of Ang II (10−7 mol/L) and AVP (10−6 mol/L) to maximally activate PKC and produce maximal and rapid receptor desensitization in the cells. Fig 1A⇓ shows a typical [Ca2+]i and CaTR response after stimulation of the cultured rat cardiomyocytes for 2 minutes with Ang II 10−7 mol/L. Fig 1B⇓ shows the lack of a [Ca2+]i or CaTR response to a second Ang II exposure of these same cells after a 5-minute washout period. Fig 1C⇓ and 1D⇓ shows the [Ca2+]i response to two consecutive exposures of the cells to AVP 10−6 mol/L. The rise in [Ca2+]i after AVP is variable, but there is a consistent, positive chronotropic (CaTR) response. Exposure to AVP 10−6 mol/L desensitized the cells to any additional chronotropic response during the second AVP exposure (Fig 1D⇓). Ang II 10−7 mol/L completely desensitized the cells to a subsequent exposure to AVP 10−6 mol/L (Fig 1E⇓ and 1F⇓). In contrast, exposure to AVP 10−6 mol/L for 2 minutes failed to desensitize the cardiomyocyte response to Ang II 10−7 mol/L (Fig 1G⇓ and 1H⇓).
The mean diastolic and systolic [Ca2+]i and CaTRs for these experiments are shown in Fig 2⇓. There was a significant rise in the diastolic and systolic [Ca2+]i with Ang II (Fig 2A⇓, P<.0005) but not for AVP during the first agonist exposure period (Fig 2C⇓ and 2D⇓). Both agonists produced a positive chronotropic response during the first exposure (Fig 2A⇓ through 2E). There was no significant rise in [Ca2+]i observed during the second of two Ang II exposures and no increase in the CaTR (Fig 2A⇓), indicating that desensitization was virtually complete. When the cells were exposed to AVP 10−6 mol/L for 2 or 10 minutes, followed by a 5-minute washout period, and then reexposed to AVP 10−6 mol/L, there was no significant change in the CaTR during the reexposure period and desensitization occurred (data not shown). Thus, Ang II and AVP both demonstrated significant homologous desensitization.
Two-minute exposures to Ang II 10−7 mol/L consistently blocked the [Ca2+]i and CaTR responses to AVP 10−6 mol/L (Fig 2B⇑), but 2-minute exposure to AVP 10−6 (or 10−7) mol/L did not produce significant heterologous desensitization to subsequent exposure to Ang II 10−6 mol/L (Fig 2C⇑). When the exposure time to AVP 10−6 (or 10−7) mol/L was increased to 10 minutes, followed by a 5-minute washout period, there was significant desensitization to Ang II 10−7 mol/L (Fig 2D⇑).
When the cardiomyocyte PKC activity was depressed by preincubation of the cells for 20 hours with PMA 10−6 mol/L, their immediate response to AVP 10−6 mol/L was not altered (Fig 2E⇑). In contrast, AVP-mediated (10-minute) desensitization of these cells to a subsequent exposure to Ang II 10−7 mol/L was blocked by PKC suppression. Both the CaTR (P<.05) and [Ca2+]i (P<.0005) rose above baseline after the Ang II stimulation (Fig 2E⇑), in contrast to that observed without PKC suppression (Fig 2D⇑). Pretreatment with the inactive phorbol esters 4-α-phorbol and P12-MA failed to desensitize the [Ca2+]i or chronotropic responses (data not shown).
Measurement of PKC Activity
To examine the time course and relative effects of Ang II, AVP, and PMA on PKC activation in the cardiomyocytes, we measured the effect of these agonists on PKC activity in 6-day cultured cells grown in 60-mm Petri dishes. PKC activity was measured in both cytosol- and membrane-bound fractions to determine the relative fraction of active PKC during each treatment phase. PKC activity was measured after exposure to Ang II and AVP (Fig 3⇓). There was a 120% increase over baseline values in membranous PKC activity after exposure to Ang II 10−6 mol/L (P=.02). Peak activity was observed at 5 minutes, with a sharp decline observed thereafter despite continued exposure to the agonist. The rise in PKC activity after AVP was delayed (Fig 3⇓), peaked at 10 minutes (to 150% over baseline, P=.01), and rapidly declined, with a secondary rise observed after 20 minutes (data not shown). PMA 10−8 to 10−6 mol/L produced a dramatic dose-dependent increase in PKC activation, with peak values observed at 15 to 20 minutes (Fig 4A⇓) and declined thereafter. Fig 4B⇓ shows the percent suppression of membranous PKC activity after 20 hours of PMA 10−6 mol/L. These cells were washed and reexposed for 15 minutes to fresh PMA 10−6 mol/L. There was no significant rise in PKC activity, confirming the marked suppression of PKC in these cells.
To determine the effect of PKC activation on homologous and heterologous desensitization by these agonists, we performed two sets of experiments designed to either activate or suppress PKC activity before agonist exposure. The cardiomyocytes were loaded with fura 2-AM for 20 minutes and PMA 10−6 mol/L was added 5 minutes later so that PKC activation would peak when the cells were ready for examination. These pretreated cells were mounted in the microscope perfusion chamber, stabilized for 5 minutes in buffer alone, and studied as before with a repeated stimulation with Ang II (Fig 5A⇓). The effect of PMA 10−6 mol/L on the [Ca2+]i response to Ang II 10−7 mol/L was examined. Acute exposure to PMA was associated with a rise in the mean diastolic and systolic [Ca2+]i and effectively blocked any significant rise in [Ca2+]i to Ang II during either the first or second exposure (Fig 5B⇓). A similar desensitizing effect of acute PMA activation of PKC on AVP-mediated [Ca2+]i responsiveness was also observed (data not shown).
The cardiomyocytes were preincubated with PMA 10−6 mol/L for 20 hours to suppress PKC activity. These cells had normal Ca transients and demonstrated significant and occasionally prolonged [Ca2+]i responses on their first exposure to Ang II 10−7 mol/L. Despite marked suppression of PKC activity, homologous desensitization was observed since the cells failed to respond to their second exposure to Ang II (Fig 5C⇑). Homologous desensitization after AVP also was not blocked by suppression of PKC activity (data not shown).
Cardiomyocyte Ang II Binding
To determine whether prior treatment of the cardiomyocytes by PMA desensitized the cardiomyocyte to Ang II by diminishing Ang II–receptor binding, we performed a series of whole-cell studies on 5- to 7-day-old cultured rat cardiomyocytes in 35-mm six-well plates. There was a significant increase in total and “specific” binding over baseline when the cells were acutely pretreated for 15 minutes with PMA 10−6 mol/L (Fig 6⇓). Pretreatment with OAG, the membrane-permeable analogue of diacylglycerol, which is also a potent activator of PKC, did not reproduce this effect, and neither did pretreatment with the inactive phorbol esters 4-α-phorbol or P12-MA (data not shown). This enhanced binding, therefore, appears to be specifically related to phorbol ester–mediated activation of PKC, a feature that is not shared when PKC is activated by the diacylglycerol analogue OAG or by phorbol esters that fail to activate PKC.
We could not demonstrate a phorbol ester–mediated increase in Ang II binding at 22°C or at 4°C (data not shown). A series of studies was performed in which the cells were again pretreated with either buffer (control) or PMA. After the binding period, the cells were washed for 10 minutes in iced glycine buffer at pH 3, followed by washing and counting. Seventy percent of the apparent binding of 125I–Ang II in the whole-cell control studies was acid resistant. After acute exposure to PMA, a similar percentage, 71.9%, was acid resistant despite the 40% increase in total binding.
There have been significant differences in opinion as to the relation of fluorescence-derived [Ca2+]i estimates compared with absolute concentrations measured by other techniques. The value of the present data is directed toward measurement of relative changes in ionized free calcium from baseline and does not necessarily represent absolute concentrations. It should be noted, however, that these basal values are comparable to several other studies that used neonatal cardiomyocytes.6 13 14
We have previously demonstrated that cultured neonatal rat cardiomyocytes possessed both AT1 and AT2 receptor subtypes.1 Activation of AT1 but not AT2 receptors was required to produce Ang II–mediated changes of [Ca2+]i and contraction frequency. Prior exposure of the cells to Ang II for 5 minutes or more blunted or minimized the response of these cells to a repeated Ang II stimulus, demonstrating the presence of homologous desensitization. Likewise, acute (15-minute) pretreatment of these cells with PMA, known to activate PKC, effectively blocked the Ang II–induced rise in [Ca2+]i as did activation of PKC by the diacylglycerol analogue OAG. These data supported the hypothesis that acute desensitization of Ang II–mediated effects occurred after activation of PKC.
Abdellatif et al11 reported that the rate of cellular contractility and total inositol phosphate accumulation after Ang II were diminished after exposure of cardiomyocytes to this same agonist despite long-term phorbol ester–induced suppression of PKC activation. They proposed a model analogous to the β2-adrenergic system wherein homologous desensitization occurred through a specific phosphorylation of the Ang II–occupied receptor. Heterologous desensitization by Ang II of the α1-receptor agonist phenylephrine appeared to be mediated by activation of PKC.
In the present study, we have compared and contrasted the effects of Ang II and AVP, two peptides that share vasoactive properties through G protein receptor–mediated activation of PLC. Both agonists demonstrate homologous desensitization as demonstrated by a diminished [Ca2+]i and/or CaTR after exposure to the agonist. In each case, initial stimulation with either Ang II, AVP, or PMA led to activation of Ca/lipid–sensitive PKC activity within 1 to 2 minutes and peak levels were observed at 5 to 10 minutes, respectively. This time course for peak activity is consistent with that observed for maximal desensitization. Peak PKC activity after PMA 10−6 mol/L was significantly greater than the more modest activation associated with Ang II 10−7 mol/L or AVP 10−6 mol/L. Since the inactive phorbol esters 4-α-phorbol and P12-MA failed to alter the [Ca2+]i or chronotropic responses, it would appear that phorbol-mediated effects not related to PKC activation (ie, direct membrane effects) are unlikely.
In addition to demonstrating the desensitizing effect of PKC on Ang II–modulated changes in [Ca2+]i, our data with long-term suppression (20 hours) of PKC by PMA confirm those of Abdellatif et al11 that another mechanism is operative. After 20-hour continuous exposure to PMA 10−6 mol/L, PKC activity was suppressed to 19% of baseline and was unresponsive to subsequent activation by acute reexposure to PMA 10−6 mol/L. Those cells with long-term PMA-suppressed PKC had either a normal or occasionally exaggerated [Ca2+]i and/or chronotropic responses to Ang II or AVP. Our data directly measuring PKC activation and the effect of the agonists on [Ca2+]i support a role for both PKC and an as-yet-unidentified mechanism for inducing homologous desensitization in these cells.
With the use of PKC-suppressed cardiomyocytes, AVP 10−6 mol/L failed to desensitize the cellular response to a subsequent exposure to Ang II 10−7 mol/L, since there was a rise of [Ca2+]i and an increase in the CaTR with the second stimulus. This supports the hypothesis that PKC serves an important and probably defining role in mediating heterologous desensitization of the Ang II receptor after exposure to AVP. These data are complementary to those of Savage et al,15 who reported that activation of PKC was important in desensitization of the vasopressin receptor in cultured WRK1 cells.
To determine whether alterations in receptor binding might account for changes observed after pretreatment of the cells with phorbol ester, we measured whole-cell binding of 125I–Ang II. Acute PMA pretreatment of the cells increased specific 125I–Ang II binding approximately 40% over non–phorbol-pretreated cells. This increase could not be attributed to nonspecific membrane effects of the phorbol esters since the inactive phorbol esters (4-α-phorbol and P12-MA) failed to increase this binding. The increase in total binding was equally distributed to the acid-resistant (internalized) and surface-binding fractions, supporting the possibility that PMA activation of PKC increases the total number of available binding sites. In contrast, activation of PKC by preincubation with OAG, a more physiological, non–phorbol ester activator of PKC, failed to increase the total binding or internalization of the Ang II receptors. However, OAG acutely blocked the Ang II–mediated increase in [Ca2+]i, as did PMA.1 It may be possible to reconcile these observations if exposure to PMA and OAG results in slightly different PKC activation states that mediate phosphorylation of differing target proteins. Ang II receptor sequestration and diminished surface binding have been observed16 17 and thought to be necessary for desensitization in cultured vascular smooth muscle. The present study indicates that PMA-mediated desensitization to Ang II occurred despite an increase in the number of Ang II surface receptors in these cultured neonatal cardiomyocytes.
In summary, the present studies confirm and extend previous findings1 18 19 20 of an important role for PKC activation as a component of the Ang II receptor–PLC transduction system. PKC activation serves as a powerful inhibitor of persistent Ang II–mediated changes in [Ca2+]i. It acts as one of two or more biochemical processes whose function is to limit the rise in [Ca2+]i after prolonged exposure to Ang II. PKC presumably acts in this fashion by phosphorylating Ca2+ regulatory proteins at the cell membrane, including the receptor itself. A second and independent regulatory process must also be active since homologous desensitization is retained when PKC activity is suppressed. We have provided direct evidence that PKC activation acts as a powerful mediator of AVP-induced heterologous desensitization of the Ang II receptor in neonatal cardiomyocytes. This heterologous desensitization between two related vasoactive peptides may be of clinical significance in conditions such as congestive heart failure by limiting the impact of marked elevations of Ang II and AVP on intracellular Ca homeostasis.
Note added in proof. Since submission of this manuscript, Tang et al21 have reported that Chinese Hamster ovary cells transfected with the type1B Ang II (AT1B) receptor are partially desensitized by PKC. They provided evidence that an additional PKC-independent pathway also contributes to desensitization. This report supports the data and conclusions reached in the present article, which uses rat cardiomyocytes.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|[Ca2+]i||=||intracellular free calcium concentration|
|CaTR||=||[Ca2+]i transient rate|
|FCS||=||fetal calf serum|
|PKC||=||protein kinase C|
|PMA||=||phorbol 12-myristate 13-acetate|
Support for this work was provided by the Department of Veterans Affairs Medical Center, Oklahoma City, Okla; Oklahoma Center for Molecular Medicine; Oklahoma Center for the Advancement of Science & Technology, project #HR2-050; Barbara Green; and the W.K. Warren Foundation. Drs Cofie, Dogan, and Koshida were postdoctoral fellows during work on this study. Jana Porter provided secretarial support.
- Received August 18, 1995.
- Revision received September 26, 1995.
- Accepted September 26, 1995.
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