Natriuretic Peptides Suppress Protein Kinase C Activity to Reduce Evoked Dopamine Efflux from Pheochromocytoma(PC12) Cells
Abstractj&vl, 百拇医药
The observation that natriuretic peptides and protein kinase C activators influence evoked neurotransmitter efflux by diametrically opposed mechanisms prompted an investigation of the influence of natriuretic peptides on protein kinase C activity and the potential involvement of this pathway in neuromodulatory responses to natriuretic peptides. C-Type natriuretic peptide attenuated both evoked dopamine efflux and protein kinase C activity in a concentration-dependent manner consistent with a 10% diminution in protein kinase C activity producing a 4.6–6.2% reduction in evoked dopamine efflux. The ability of C-type natriuretic peptide to suppress evoked dopamine efflux was abolished by treatment with the protein kinase C inhibitors chelerythrine (10 µM) and staurosporine (10 nM). Both chelerythrine and staurosporine attenuated protein kinase C activity at the concentrations used. The natriuretic peptide C receptor (NPR-C) appeared to mediate the attenuation of protein kinase C activity, because the effect was mimicked by a pentadecapeptide fragment of the NPR-C, and the effect of C-type natriuretic peptide was attenuated by an antibody generated against the same region of the receptor. These data suggest that C-type natriuretic peptide attenuates neurotransmitter efflux by a mechanism involving suppression of neuronal protein kinase C activity via an interaction with the NPR-C.
Introductionse}t, 百拇医药
NATRIURETIC PEPTIDES were discovered as water-soluble components of atria that induce diuretic and natriuretic responses (1). Natriuretic peptides subsequently were identified as cyclic peptides consisting of approximately 20–40 amino acids containing a 17-member ring (2). Four major forms have been identified, including atrial natriuretic peptide (2), B-type natriuretic peptide (3), C-type natriuretic peptide (4), and urodilatin (5). These peptides interact with at least three receptors, two of them particulate guanylyl cyclases termed natriuretic peptide A or B receptors (NPR-A or NPR-B) (6, 7). Both atrial and B-type natriuretic peptide interact preferentially with NPR-A, and C-type natriuretic peptide interacts preferentially with NPR-B (8, 9). All four naturally occurring natriuretic peptides are ligands for the third natriuretic peptide receptor, NPR-C, which is a truncated receptor lacking guanylyl cyclase activity and containing only a 37-amino-acid extension into the cytosol (10, 11). The guanylyl cyclase receptors are thought to mediate renal and vascular effects of natriuretic peptides. NPR-C has been implicated in divergent signaling pathways, such as a suppression of adenylyl cyclase activity (12), activation of phospholipase C (13) and attenuation of mitogen-activated protein kinase (14). This investigation centers on the signal transduction pathway accounting for neuromodulatory effects of natriuretic peptides, a pathway that has been attributed to both NPR-C activation (15) and guanylyl cyclase activation (16). This information is critical to defining the mechanisms accounting for the marked effect of the natriuretic peptide endocrine system on blood pressure.
Natriuretic peptides were observed to reduce evoked neurotransmitter efflux in adrenergic tissue (17) shortly after their discovery. The mechanism accounting for the inhibition involves the NPR-C, based on the fact that agents solely interacting with the NPR-C mimic the effect of naturally occurring natriuretic peptides (15). Furthermore, some endogenous natriuretic peptides neuromodulate in the absence of a detectable elevation of guanylyl cyclase activity (18). A knockdown of NPR-C levels in PC12 cells also eliminates neuromodulatory effects of natriuretic peptides (19). Additional evidence involves a pentadecapeptide fragment of the NPR-C reproducing the effects of applied natriuretic peptides in permeabilized PC12 cells (20). Finally, an antibody generated against the active pentadecapeptide region of the NPR-C prevents the effects of natriuretic peptides in permeabilized PC12 cells (20). These data provide a strong rationale for considering the NPR-C to be the neuromodulatory natriuretic peptide receptor, but the NPR-A also has been invoked as this receptor (16).
The postreceptor mechanism accounting for the neuromodulatory effect of natriuretic peptides in PC12 cells involves a desensitization of the exocytotic apparatus to calcium (21). The natriuretic peptides increase calcium influx induced by a depolarizing concentration of potassium in PC12 cells (21). This curious mechanism of action is remarkably opposite that of protein kinase C activators, which augment evoked neurotransmitter efflux by sensitizing neurotransmitter efflux to calcium while reducing calcium influx into PC12 cells (22, 23, 24). These diametrically opposed actions of natriuretic peptides and protein kinase C activators formed the rationale for an investigation of the influence of natriuretic peptides on protein kinase C activity and the involvement of protein kinase C in neuromodulatory effects of natriuretic peptides. The specific receptor involvement was explored with an active peptide fragment of the NPR-C and with an antibody generated against this peptide. The specific hypothesis tested in this manuscript is that natriuretic peptides activate the NPR-C to attenuate protein kinase C activity, leading to a reduction in evoked dopamine efflux.
Materials and Methods], http://www.100md.com
Cell culture], http://www.100md.com
Pheochromocytoma cells were grown in DMEM supplemented with 5% horse serum and 10% fetal calf serum. Cells were cultured on 25-cm2 culture flasks in a 5% carbon dioxide atmosphere. Cells were used for experiments at passages 18–38 at a cell number varying from 1–3 million. The cells were used 7–10 d after differentiation with 200 ng/ml 7S nerve growth factor (Sigma, St. Louis, MO) combined with reduction of serum content to 1% fetal bovine serum and elimination of horse serum. The differentiated cells were grown on collagen-coated flasks.], http://www.100md.com
Dopamine efflux experiments in intact cells], http://www.100md.com
Dopamine efflux was induced by exposing the cells to a depolarizing concentration of potassium chloride for 5 min. The constituents of the depolarizing buffer included the following: 76 mM sodium chloride, 40 mM potassium chloride, 25 mM sodium bicarbonate, 1.2 mM sodium phosphate monobasic, 0.5 mM magnesium chloride, 2.5 mM calcium chloride, and 10 mM glucose. After exposure to the high potassium buffer, the cell supernate was poured into tubes containing 60 mg alumina to extract catecholamines. Cells were suspended in 1 ml perchloric acid (0.05 N) and sonicated to release intracellular catecholamines. Cellular debris was pelleted by centrifuging at 5000 x g, and the supernate was incubated with 60 mg alumina after addition of 1 ml Tris buffer (3 M) to raise the pH. Both released and cellular catecholamines were exposed to the alumina for 10 min with agitation, followed by a 10-min incubation with water and 10 min with perchloric acid (0.2 N). The perchloric acid fraction was collected for injection into a Spectraphysics (Thermo Separation Products, San Jose, CA) SP20 high performance liquid chromatograph with electrochemical detection to determine the dopamine content of each fraction. Dihydroxybenzylamine (10 ng) was included as an internal standard to assess recovery. All samples were corrected for recovery. The total cellular dopamine was calculated by summing the amount released with the amount remaining in the cells after the experiment. The percent release was calculated by dividing the amount released by the total cellular dopamine. Norepinephrine was detected inconsistently; therefore, only results with dopamine are reported.
Experiments were conducted to examine the effect of C-type natriuretic peptide in the presence of chelerythrine (10 µM; Sigma), staurosporine (10 nM; Sigma), or their diluents. Chelerythrine was dissolved in water, and staurosporine was dissolved in dimethylsulfoxide.[]am, http://www.100md.com
Cellular permeabilization[]am, http://www.100md.com
Cells were permeabilized with 10 µM digitonin for 5 min. The digitonin was dissolved in an intracellular buffer consisting of the following: 157 mM potassium glutamate, 10 mM HEPES, 5 mM magnesium sulfate, and 5 mM ATP. This procedure was used in protein kinase C assays and in dopamine efflux experiments examining the effects of either intracellular regions of the NPR-C or antibodies generated against intracellular regions of the NPR-C. In the latter experiments both calcium chloride (4 mM) and EGTA (4 mM) were included in the intracellular buffer to yield a predicted free calcium concentration of 10 µM (25).[]am, http://www.100md.com
Protein kinase C assay
Protein kinase C activity was measured in permeabilized cells using the colorimetric PKC Spinzyme assay kit (Pierce Chemical Co., Rockford, IL) with glycogen synthase as the enzyme substrate. After permeabilization, the cells were scraped from 10 culture flasks, pelleted at 1000 x g, and suspended in intracellular buffer containing calcium and EGTA. Tetradecanoylphorbol ester (10 µM) was used as an additional stimulus for protein kinase C activity. Peptides, including C-type natriuretic peptide, and a pentadecapeptide representing an intracellular region of the NPR-C with neuromodulatory activity, were tested for their influence on protein kinase C activity by including them in the assay. The effect of an antibody reacting with the NPR-C also was ascertained. The antibody was included at a concentration of 10 µg/ml. The reaction was terminated after 20 min by placing the reaction mixture on Spinzyme separation units with the addition of phosphopeptide binding buffer, as recommended by the manufacturer. After two cycles of phosphopeptide binding buffer addition and two cycles of phosphopeptide elution buffer addition, the absorbance at 570 nm was determined to indicate the amount of protein kinase C activity. The difference between absorbance in reactions performed at 4 and 37 C was interpreted as the amount of protein kinase C activity, and results are reported as raw absorbance data.
Dopamine efflux from permeabilized cells{x, 百拇医药
Dopamine efflux from permeabilized cells was stimulated by inclusion of calcium (4 mM). The extraction procedure and measurement were identical to those described for intact cells. Control efflux of dopamine was designated as the difference between dopamine efflux in the presence of EGTA alone (4 mM) and efflux in the presence of both EGTA (4 mM) and calcium chloride (4 mM). The combination yields a predicted free calcium concentration of 10 µM (24).{x, 百拇医药
Guanylyl cyclase assay{x, 百拇医药
Guanylyl cyclase activity was measured indirectly by determining the cGMP concentrations of cells after exposure to C-type natriuretic peptide in the presence of protein kinase C inhibitors. The assay was conducted in depolarizing buffer supplemented with isobutylmethylxanthine (2.5 mM) to suppress phosphodiesterase activity. cGMP was extracted with 1 ml ethanol. Cellular debris was pelleted at 1000 x g, the ethanol supernatant was evaporated, and cyclic nucleotides were resuspended in assay buffer. cGMP levels were quantified by RIA using a TRK.500 kit (Amersham Pharmacia Biotech, Arlington Heights, IL).
Peptides and antibodies|vnv5, http://www.100md.com
C-Type natriuretic peptide was purchased from Peninsula Laboratories, Inc. (Belmont, CA). A peptide representing the active region of the NPR-C was purchased from Chiron Corp. (Raleigh, NC). This peptide consisted of the following amino acids, arranged in an amino to carboxyl terminus orientation: RKKYRITIERRNHQE. A scrambled peptide was used as a negative control. The scrambled peptide had the following sequence: TYNHERRIRKIQEKR. An antibody reacting with the active NPR-C pentadecapeptide was generated in rabbits by Research Genetics, Inc. (Huntsville, AL). The antibody titer increased to a maximum of 12,300, and the antibody was partially purified on a protein A-Superose column as previously reported (20). The peptides and antibody were used to test whether the effects of C-type natriuretic peptide on evoked dopamine efflux and protein kinase C activity were mediated by NPR-C.|vnv5, http://www.100md.com
Statistical comparisons|vnv5, http://www.100md.com
Curves were compared by ANOVA for repeated measures. Individual values were compared by paired t test, with appropriate adjustments for multiple comparisons. Linear regression analyses were performed using Cricket Graph. P 0.05 was considered statistically significant.*-!5n, http://www.100md.com
Results*-!5n, http://www.100md.com
C-Type natriuretic peptide both suppressed protein kinase C activity and evoked dopamine efflux in a concentration-dependent manner, as shown in Fig. 1. The suppression of protein kinase C activity exceeded 60%, with a clear maximum at a C-type natriuretic peptide concentration of 10 nM. Evoked dopamine efflux also was reduced maximally at 10 nM C-type natriuretic peptide, and the maximum effect was 26 ± 4% attenuation. The reductions in protein kinase C activity were statistically significant at peptide concentrations of 1, 10, and 100 nM. Evoked dopamine efflux was suppressed to a statistically significant level at C-type natriuretic peptide concentrations of 0.1, 1, and 10 nM. The concentration of peptide producing a half-maximal effect averaged 0.22 ± 0.08 nM for protein kinase C activity and 0.63 ± 0.28 nM for evoked dopamine efflux. The 50% effective concentrations (EC50) were not significantly different for the two responses (P = 0.21).
fig.ommitteed#1y$', http://www.100md.com
Figure 1. Effect of C-type natriuretic peptide (CNP) on protein kinase C (PKC) activity and evoked dopamine efflux. All symbols represent the mean ± SE. The number of preparations per group was 18 for evoked dopamine efflux and 9 for PKC activity, except for the value at 100 nM, where the numbers were 9 and 2, respectively. Asterisks indicate a statistically significant difference from control (**, P < 0.01; *, P < 0.05) determinted by paired t test with appropriate correction for multiple comparisons. The inset demonstrates the correlation between PKC activity and evoked dopamine efflux at the various concentrations of C-type natriuretic peptide. The correlation is statistically significant (P < 0.001).#1y$', http://www.100md.com
The relationship between C-type natriuretic peptide effects on protein kinase C activity and evoked dopamine efflux is depicted in the inset to Fig. 1. The regression analysis describing this relationship is statistically significant (P < 0.001) and predicts that a 100% reduction in protein kinase C activity would reduce evoked dopamine efflux to 10.0% of the total cell content. This represents a 46% reduction from the dopamine efflux induced by the depolarizing stimulus in the absence of natriuretic peptides (i.e. 18.6% of total cell dopamine content). Thus, a 10% reduction in protein kinase C activity correlates with a 4.6% reduction from control levels of evoked dopamine efflux.
Both chelerythrine and staurosporine were used to test the relevance of protein kinase C inhibition to neuromodulatory effects of C-type natriuretic peptide. As shown in Fig. 2, chelerythrine suppressed protein kinase C activity approximately 71% and attenuated evoked dopamine efflux 32%. Staurosporine produced a similar inhibitory effect on protein kinase C activity, reducing it 67%, but only marginally affected evoked dopamine efflux, reducing it approximately 12%. These data for protein kinase C activity show that the protein kinase C inhibitors used in this study were effective at the concentrations used. The chelerythrine effect on evoked dopamine efflux was consistent with the regression analysis shown in the inset to Fig. 1, which predicted that a 71% suppression of protein kinase C activity would reduce dopamine efflux by 33%. The observed decrease was 32%. Conversely, staurosporine failed to suppress evoked dopamine efflux to the extent predicted by its suppression of protein kinase C. The 67% attenuation of protein kinase C activity was predicted to reduce evoked dopamine efflux by 31%, whereas the actual attenuation was only 12%.
fig.ommitteedw1w), 百拇医药
Figure 2. Effect of chelerythrine or staurosporine on either protein kinase C (PKC) activity or evoked dopamine efflux (Dop). Columns represent means, and bars indicate the SE. Asterisks indicate statistically significant differences from vehicle treatment (*, P < 0.05; **, P < 0.01). Vehicle-treated groups were combined, because the results were virtually identical with water and dimethylsulfoxide. The number of preparations in the cells examined for PKC activity was 6 in the vehicle group, 6 in the chelerythrine group, and 4 in the staurosporine group. The number of preparations in the dopamine efflux experiments was 20 in the vehicle group, 6 in the chelerythrine group, and 14 in the staurosporine group.w1w), 百拇医药
The neuromodulatory effect of C-type natriuretic peptide was abolished by either chelerythrine or staurosporine, as indicated in Fig. 3. The maximal suppression of evoked dopamine efflux was 25 ± 4% in the presence of the chelerythrine vehicle. In contrast, C-type natriuretic peptide failed to attenuate evoked dopamine efflux in the presence of chelerythrine, which had already lessened evoked dopamine efflux 32%, as indicated in Fig. 2. The control value for chelerythrine of 100% in this figure represents the dopamine efflux in the presence of chelerythrine alone (i.e. a lower control efflux than in the presence of the chelerythrine diluent). The curves differed significantly when compared by ANOVA for repeated measures (P = 0.0006). The results were similar, with staurosporine significantly blunting the neuromodulatory effects of C-type natriuretic peptide (P = 0.0004). Again, it should be noted that staurosporine alone diminished evoked dopamine efflux 12%, as shown in Fig. 2.
fig.ommitteedm+g62gz, 百拇医药
Figure 3. Effects of chelerythrine (10 µM) and staurosporine (10 nM) on the neuromodulatory effect of C-type natriuretic peptide (CNP). All symbols represent means, with bars indicating the SE. Asterisks indicate a statistical difference between the control and treated curves. The chelerythrine treatment curve differed from vehicle (P = 0.0006), as did the staurosporine treatment curve (P = 0.0004). The number of experiments per group is indicated in parentheses.m+g62gz, 百拇医药
The influence of C-type natriuretic peptide on protein kinase C activity and evoked dopamine efflux could be mediated by an activation of NPR-A, NPR-B, or NPR-C. The first two possibilities were tested by examining the influence of C-type natriuretic peptide on guanylyl cyclase activity. As shown in Fig. 4, C-type natriuretic peptide augmented guanylyl cyclase activity at concentrations of 10 and 100 nM, but not at lower concentrations. The stimulatory effect of C-type natriuretic peptide on guanylyl cyclase activity was both statistically significant (P < 0.0001 by ANOVA) and sustained in the presence of chelerythrine. The two curves were nearly identical (P = 0.99, by ANOVA). Virtually identical results were obtained in the presence of staurosporine (data not shown). A correlation of guanylyl cyclase activation with evoked dopamine efflux resulted in a statistically insignificant relationship, with a correlation coefficient of 0.14 (P > 0.20; shown in inset of Fig. 4). The guanylyl cyclase activation by C-type natriuretic peptide also correlated poorly with protein kinase C suppression, yielding a correlation coefficient of 0.67 (P > 0.10; correlation not shown).
fig.ommitteede, 百拇医药
Figure 4. Effect of chelerythrine on guanylyl cyclase activation by C-type natriuretic peptide (CNP). Symbols depict mean values, and bars indicate the SE. The number of preparations per group is indicated by N. The guanylyl cyclase stimulation by C-type natriuretic peptide was statistically significant (P = 0.0001), but not different between the curves (P = 0.99). The inset demonstrates the correlation between cGMP concentrations and evoked dopamine efflux at corresponding concentrations of C-type natriuretic peptide. The correlation value of 0.14 was not statistically significant (P > 0.20).e, 百拇医药
The possibility that C-type natriuretic peptide acts via the NPR-C to suppress protein kinase C activity and evoked dopamine efflux was explored using a peptide corresponding to the first 15 cytoplasmic amino acids of the NPR-C. The NPR-C peptide suppressed protein kinase C activity in a concentration-dependent manner from 0.1 to 10 pM, as indicated in Fig. 5. The EC50 averaged 0.30 ± 0.10 pM, and a maximal effect was achieved at a concentration of 1 pM. The 56 ± 10% maximal suppression of protein kinase C by the NPR-C peptide was virtually identical to the maximal effect of C-type natriuretic peptide shown in Fig. 1 (55 ± 8%). A scrambled version of the peptide failed to influence protein kinase C activity, and the two curves differed statistically (P = 0.0012; data not shown). The active NPR-C peptide also significantly suppressed evoked dopamine efflux from permeabilized cells at concentrations of 1 and 10 pM. The maximal suppression of evoked dopamine efflux was 40 ± 12%, a value slightly greater than that achieved with C-type natriuretic peptide in Fig. 1. The EC50 for the effect averaged 0.49 ± 0.20 pM. The inset to Fig. 5 indicates the correlation between the effects of the NPR-C peptide on protein kinase C activity and evoked dopamine efflux. The correlation predicts a 6.2% drop in evoked dopamine efflux for each 10% decline in protein kinase C activity. These data indicate that an intracellular region of the NPR-C reproduces the effect of C-type natriuretic peptide on both protein kinase C activity and evoked dopamine efflux, consistent with a mediatory role for the NPR-C.e, 百拇医药
fig.ommitteede, 百拇医药
Figure 5. Effect of a pentadecapeptide ([1–15]), representing the first 15 amino acids of the NPR-C protruding into the cytosol, on protein kinase C (PKC) activity and evoked dopamine efflux in permeabilized PC12 cells. The pentadecapeptide significantly reduced both parameters at 1 and 10 pM (*, P < 0.05; **, P < 0.01). The inset demonstrates the correlation between the two parameters at equivalent concentrations of the pentadecapeptide. The correlation was statistically significant (P < 0.001).(George J. Trachte)
The observation that natriuretic peptides and protein kinase C activators influence evoked neurotransmitter efflux by diametrically opposed mechanisms prompted an investigation of the influence of natriuretic peptides on protein kinase C activity and the potential involvement of this pathway in neuromodulatory responses to natriuretic peptides. C-Type natriuretic peptide attenuated both evoked dopamine efflux and protein kinase C activity in a concentration-dependent manner consistent with a 10% diminution in protein kinase C activity producing a 4.6–6.2% reduction in evoked dopamine efflux. The ability of C-type natriuretic peptide to suppress evoked dopamine efflux was abolished by treatment with the protein kinase C inhibitors chelerythrine (10 µM) and staurosporine (10 nM). Both chelerythrine and staurosporine attenuated protein kinase C activity at the concentrations used. The natriuretic peptide C receptor (NPR-C) appeared to mediate the attenuation of protein kinase C activity, because the effect was mimicked by a pentadecapeptide fragment of the NPR-C, and the effect of C-type natriuretic peptide was attenuated by an antibody generated against the same region of the receptor. These data suggest that C-type natriuretic peptide attenuates neurotransmitter efflux by a mechanism involving suppression of neuronal protein kinase C activity via an interaction with the NPR-C.
Introductionse}t, 百拇医药
NATRIURETIC PEPTIDES were discovered as water-soluble components of atria that induce diuretic and natriuretic responses (1). Natriuretic peptides subsequently were identified as cyclic peptides consisting of approximately 20–40 amino acids containing a 17-member ring (2). Four major forms have been identified, including atrial natriuretic peptide (2), B-type natriuretic peptide (3), C-type natriuretic peptide (4), and urodilatin (5). These peptides interact with at least three receptors, two of them particulate guanylyl cyclases termed natriuretic peptide A or B receptors (NPR-A or NPR-B) (6, 7). Both atrial and B-type natriuretic peptide interact preferentially with NPR-A, and C-type natriuretic peptide interacts preferentially with NPR-B (8, 9). All four naturally occurring natriuretic peptides are ligands for the third natriuretic peptide receptor, NPR-C, which is a truncated receptor lacking guanylyl cyclase activity and containing only a 37-amino-acid extension into the cytosol (10, 11). The guanylyl cyclase receptors are thought to mediate renal and vascular effects of natriuretic peptides. NPR-C has been implicated in divergent signaling pathways, such as a suppression of adenylyl cyclase activity (12), activation of phospholipase C (13) and attenuation of mitogen-activated protein kinase (14). This investigation centers on the signal transduction pathway accounting for neuromodulatory effects of natriuretic peptides, a pathway that has been attributed to both NPR-C activation (15) and guanylyl cyclase activation (16). This information is critical to defining the mechanisms accounting for the marked effect of the natriuretic peptide endocrine system on blood pressure.
Natriuretic peptides were observed to reduce evoked neurotransmitter efflux in adrenergic tissue (17) shortly after their discovery. The mechanism accounting for the inhibition involves the NPR-C, based on the fact that agents solely interacting with the NPR-C mimic the effect of naturally occurring natriuretic peptides (15). Furthermore, some endogenous natriuretic peptides neuromodulate in the absence of a detectable elevation of guanylyl cyclase activity (18). A knockdown of NPR-C levels in PC12 cells also eliminates neuromodulatory effects of natriuretic peptides (19). Additional evidence involves a pentadecapeptide fragment of the NPR-C reproducing the effects of applied natriuretic peptides in permeabilized PC12 cells (20). Finally, an antibody generated against the active pentadecapeptide region of the NPR-C prevents the effects of natriuretic peptides in permeabilized PC12 cells (20). These data provide a strong rationale for considering the NPR-C to be the neuromodulatory natriuretic peptide receptor, but the NPR-A also has been invoked as this receptor (16).
The postreceptor mechanism accounting for the neuromodulatory effect of natriuretic peptides in PC12 cells involves a desensitization of the exocytotic apparatus to calcium (21). The natriuretic peptides increase calcium influx induced by a depolarizing concentration of potassium in PC12 cells (21). This curious mechanism of action is remarkably opposite that of protein kinase C activators, which augment evoked neurotransmitter efflux by sensitizing neurotransmitter efflux to calcium while reducing calcium influx into PC12 cells (22, 23, 24). These diametrically opposed actions of natriuretic peptides and protein kinase C activators formed the rationale for an investigation of the influence of natriuretic peptides on protein kinase C activity and the involvement of protein kinase C in neuromodulatory effects of natriuretic peptides. The specific receptor involvement was explored with an active peptide fragment of the NPR-C and with an antibody generated against this peptide. The specific hypothesis tested in this manuscript is that natriuretic peptides activate the NPR-C to attenuate protein kinase C activity, leading to a reduction in evoked dopamine efflux.
Materials and Methods], http://www.100md.com
Cell culture], http://www.100md.com
Pheochromocytoma cells were grown in DMEM supplemented with 5% horse serum and 10% fetal calf serum. Cells were cultured on 25-cm2 culture flasks in a 5% carbon dioxide atmosphere. Cells were used for experiments at passages 18–38 at a cell number varying from 1–3 million. The cells were used 7–10 d after differentiation with 200 ng/ml 7S nerve growth factor (Sigma, St. Louis, MO) combined with reduction of serum content to 1% fetal bovine serum and elimination of horse serum. The differentiated cells were grown on collagen-coated flasks.], http://www.100md.com
Dopamine efflux experiments in intact cells], http://www.100md.com
Dopamine efflux was induced by exposing the cells to a depolarizing concentration of potassium chloride for 5 min. The constituents of the depolarizing buffer included the following: 76 mM sodium chloride, 40 mM potassium chloride, 25 mM sodium bicarbonate, 1.2 mM sodium phosphate monobasic, 0.5 mM magnesium chloride, 2.5 mM calcium chloride, and 10 mM glucose. After exposure to the high potassium buffer, the cell supernate was poured into tubes containing 60 mg alumina to extract catecholamines. Cells were suspended in 1 ml perchloric acid (0.05 N) and sonicated to release intracellular catecholamines. Cellular debris was pelleted by centrifuging at 5000 x g, and the supernate was incubated with 60 mg alumina after addition of 1 ml Tris buffer (3 M) to raise the pH. Both released and cellular catecholamines were exposed to the alumina for 10 min with agitation, followed by a 10-min incubation with water and 10 min with perchloric acid (0.2 N). The perchloric acid fraction was collected for injection into a Spectraphysics (Thermo Separation Products, San Jose, CA) SP20 high performance liquid chromatograph with electrochemical detection to determine the dopamine content of each fraction. Dihydroxybenzylamine (10 ng) was included as an internal standard to assess recovery. All samples were corrected for recovery. The total cellular dopamine was calculated by summing the amount released with the amount remaining in the cells after the experiment. The percent release was calculated by dividing the amount released by the total cellular dopamine. Norepinephrine was detected inconsistently; therefore, only results with dopamine are reported.
Experiments were conducted to examine the effect of C-type natriuretic peptide in the presence of chelerythrine (10 µM; Sigma), staurosporine (10 nM; Sigma), or their diluents. Chelerythrine was dissolved in water, and staurosporine was dissolved in dimethylsulfoxide.[]am, http://www.100md.com
Cellular permeabilization[]am, http://www.100md.com
Cells were permeabilized with 10 µM digitonin for 5 min. The digitonin was dissolved in an intracellular buffer consisting of the following: 157 mM potassium glutamate, 10 mM HEPES, 5 mM magnesium sulfate, and 5 mM ATP. This procedure was used in protein kinase C assays and in dopamine efflux experiments examining the effects of either intracellular regions of the NPR-C or antibodies generated against intracellular regions of the NPR-C. In the latter experiments both calcium chloride (4 mM) and EGTA (4 mM) were included in the intracellular buffer to yield a predicted free calcium concentration of 10 µM (25).[]am, http://www.100md.com
Protein kinase C assay
Protein kinase C activity was measured in permeabilized cells using the colorimetric PKC Spinzyme assay kit (Pierce Chemical Co., Rockford, IL) with glycogen synthase as the enzyme substrate. After permeabilization, the cells were scraped from 10 culture flasks, pelleted at 1000 x g, and suspended in intracellular buffer containing calcium and EGTA. Tetradecanoylphorbol ester (10 µM) was used as an additional stimulus for protein kinase C activity. Peptides, including C-type natriuretic peptide, and a pentadecapeptide representing an intracellular region of the NPR-C with neuromodulatory activity, were tested for their influence on protein kinase C activity by including them in the assay. The effect of an antibody reacting with the NPR-C also was ascertained. The antibody was included at a concentration of 10 µg/ml. The reaction was terminated after 20 min by placing the reaction mixture on Spinzyme separation units with the addition of phosphopeptide binding buffer, as recommended by the manufacturer. After two cycles of phosphopeptide binding buffer addition and two cycles of phosphopeptide elution buffer addition, the absorbance at 570 nm was determined to indicate the amount of protein kinase C activity. The difference between absorbance in reactions performed at 4 and 37 C was interpreted as the amount of protein kinase C activity, and results are reported as raw absorbance data.
Dopamine efflux from permeabilized cells{x, 百拇医药
Dopamine efflux from permeabilized cells was stimulated by inclusion of calcium (4 mM). The extraction procedure and measurement were identical to those described for intact cells. Control efflux of dopamine was designated as the difference between dopamine efflux in the presence of EGTA alone (4 mM) and efflux in the presence of both EGTA (4 mM) and calcium chloride (4 mM). The combination yields a predicted free calcium concentration of 10 µM (24).{x, 百拇医药
Guanylyl cyclase assay{x, 百拇医药
Guanylyl cyclase activity was measured indirectly by determining the cGMP concentrations of cells after exposure to C-type natriuretic peptide in the presence of protein kinase C inhibitors. The assay was conducted in depolarizing buffer supplemented with isobutylmethylxanthine (2.5 mM) to suppress phosphodiesterase activity. cGMP was extracted with 1 ml ethanol. Cellular debris was pelleted at 1000 x g, the ethanol supernatant was evaporated, and cyclic nucleotides were resuspended in assay buffer. cGMP levels were quantified by RIA using a TRK.500 kit (Amersham Pharmacia Biotech, Arlington Heights, IL).
Peptides and antibodies|vnv5, http://www.100md.com
C-Type natriuretic peptide was purchased from Peninsula Laboratories, Inc. (Belmont, CA). A peptide representing the active region of the NPR-C was purchased from Chiron Corp. (Raleigh, NC). This peptide consisted of the following amino acids, arranged in an amino to carboxyl terminus orientation: RKKYRITIERRNHQE. A scrambled peptide was used as a negative control. The scrambled peptide had the following sequence: TYNHERRIRKIQEKR. An antibody reacting with the active NPR-C pentadecapeptide was generated in rabbits by Research Genetics, Inc. (Huntsville, AL). The antibody titer increased to a maximum of 12,300, and the antibody was partially purified on a protein A-Superose column as previously reported (20). The peptides and antibody were used to test whether the effects of C-type natriuretic peptide on evoked dopamine efflux and protein kinase C activity were mediated by NPR-C.|vnv5, http://www.100md.com
Statistical comparisons|vnv5, http://www.100md.com
Curves were compared by ANOVA for repeated measures. Individual values were compared by paired t test, with appropriate adjustments for multiple comparisons. Linear regression analyses were performed using Cricket Graph. P 0.05 was considered statistically significant.*-!5n, http://www.100md.com
Results*-!5n, http://www.100md.com
C-Type natriuretic peptide both suppressed protein kinase C activity and evoked dopamine efflux in a concentration-dependent manner, as shown in Fig. 1. The suppression of protein kinase C activity exceeded 60%, with a clear maximum at a C-type natriuretic peptide concentration of 10 nM. Evoked dopamine efflux also was reduced maximally at 10 nM C-type natriuretic peptide, and the maximum effect was 26 ± 4% attenuation. The reductions in protein kinase C activity were statistically significant at peptide concentrations of 1, 10, and 100 nM. Evoked dopamine efflux was suppressed to a statistically significant level at C-type natriuretic peptide concentrations of 0.1, 1, and 10 nM. The concentration of peptide producing a half-maximal effect averaged 0.22 ± 0.08 nM for protein kinase C activity and 0.63 ± 0.28 nM for evoked dopamine efflux. The 50% effective concentrations (EC50) were not significantly different for the two responses (P = 0.21).
fig.ommitteed#1y$', http://www.100md.com
Figure 1. Effect of C-type natriuretic peptide (CNP) on protein kinase C (PKC) activity and evoked dopamine efflux. All symbols represent the mean ± SE. The number of preparations per group was 18 for evoked dopamine efflux and 9 for PKC activity, except for the value at 100 nM, where the numbers were 9 and 2, respectively. Asterisks indicate a statistically significant difference from control (**, P < 0.01; *, P < 0.05) determinted by paired t test with appropriate correction for multiple comparisons. The inset demonstrates the correlation between PKC activity and evoked dopamine efflux at the various concentrations of C-type natriuretic peptide. The correlation is statistically significant (P < 0.001).#1y$', http://www.100md.com
The relationship between C-type natriuretic peptide effects on protein kinase C activity and evoked dopamine efflux is depicted in the inset to Fig. 1. The regression analysis describing this relationship is statistically significant (P < 0.001) and predicts that a 100% reduction in protein kinase C activity would reduce evoked dopamine efflux to 10.0% of the total cell content. This represents a 46% reduction from the dopamine efflux induced by the depolarizing stimulus in the absence of natriuretic peptides (i.e. 18.6% of total cell dopamine content). Thus, a 10% reduction in protein kinase C activity correlates with a 4.6% reduction from control levels of evoked dopamine efflux.
Both chelerythrine and staurosporine were used to test the relevance of protein kinase C inhibition to neuromodulatory effects of C-type natriuretic peptide. As shown in Fig. 2, chelerythrine suppressed protein kinase C activity approximately 71% and attenuated evoked dopamine efflux 32%. Staurosporine produced a similar inhibitory effect on protein kinase C activity, reducing it 67%, but only marginally affected evoked dopamine efflux, reducing it approximately 12%. These data for protein kinase C activity show that the protein kinase C inhibitors used in this study were effective at the concentrations used. The chelerythrine effect on evoked dopamine efflux was consistent with the regression analysis shown in the inset to Fig. 1, which predicted that a 71% suppression of protein kinase C activity would reduce dopamine efflux by 33%. The observed decrease was 32%. Conversely, staurosporine failed to suppress evoked dopamine efflux to the extent predicted by its suppression of protein kinase C. The 67% attenuation of protein kinase C activity was predicted to reduce evoked dopamine efflux by 31%, whereas the actual attenuation was only 12%.
fig.ommitteedw1w), 百拇医药
Figure 2. Effect of chelerythrine or staurosporine on either protein kinase C (PKC) activity or evoked dopamine efflux (Dop). Columns represent means, and bars indicate the SE. Asterisks indicate statistically significant differences from vehicle treatment (*, P < 0.05; **, P < 0.01). Vehicle-treated groups were combined, because the results were virtually identical with water and dimethylsulfoxide. The number of preparations in the cells examined for PKC activity was 6 in the vehicle group, 6 in the chelerythrine group, and 4 in the staurosporine group. The number of preparations in the dopamine efflux experiments was 20 in the vehicle group, 6 in the chelerythrine group, and 14 in the staurosporine group.w1w), 百拇医药
The neuromodulatory effect of C-type natriuretic peptide was abolished by either chelerythrine or staurosporine, as indicated in Fig. 3. The maximal suppression of evoked dopamine efflux was 25 ± 4% in the presence of the chelerythrine vehicle. In contrast, C-type natriuretic peptide failed to attenuate evoked dopamine efflux in the presence of chelerythrine, which had already lessened evoked dopamine efflux 32%, as indicated in Fig. 2. The control value for chelerythrine of 100% in this figure represents the dopamine efflux in the presence of chelerythrine alone (i.e. a lower control efflux than in the presence of the chelerythrine diluent). The curves differed significantly when compared by ANOVA for repeated measures (P = 0.0006). The results were similar, with staurosporine significantly blunting the neuromodulatory effects of C-type natriuretic peptide (P = 0.0004). Again, it should be noted that staurosporine alone diminished evoked dopamine efflux 12%, as shown in Fig. 2.
fig.ommitteedm+g62gz, 百拇医药
Figure 3. Effects of chelerythrine (10 µM) and staurosporine (10 nM) on the neuromodulatory effect of C-type natriuretic peptide (CNP). All symbols represent means, with bars indicating the SE. Asterisks indicate a statistical difference between the control and treated curves. The chelerythrine treatment curve differed from vehicle (P = 0.0006), as did the staurosporine treatment curve (P = 0.0004). The number of experiments per group is indicated in parentheses.m+g62gz, 百拇医药
The influence of C-type natriuretic peptide on protein kinase C activity and evoked dopamine efflux could be mediated by an activation of NPR-A, NPR-B, or NPR-C. The first two possibilities were tested by examining the influence of C-type natriuretic peptide on guanylyl cyclase activity. As shown in Fig. 4, C-type natriuretic peptide augmented guanylyl cyclase activity at concentrations of 10 and 100 nM, but not at lower concentrations. The stimulatory effect of C-type natriuretic peptide on guanylyl cyclase activity was both statistically significant (P < 0.0001 by ANOVA) and sustained in the presence of chelerythrine. The two curves were nearly identical (P = 0.99, by ANOVA). Virtually identical results were obtained in the presence of staurosporine (data not shown). A correlation of guanylyl cyclase activation with evoked dopamine efflux resulted in a statistically insignificant relationship, with a correlation coefficient of 0.14 (P > 0.20; shown in inset of Fig. 4). The guanylyl cyclase activation by C-type natriuretic peptide also correlated poorly with protein kinase C suppression, yielding a correlation coefficient of 0.67 (P > 0.10; correlation not shown).
fig.ommitteede, 百拇医药
Figure 4. Effect of chelerythrine on guanylyl cyclase activation by C-type natriuretic peptide (CNP). Symbols depict mean values, and bars indicate the SE. The number of preparations per group is indicated by N. The guanylyl cyclase stimulation by C-type natriuretic peptide was statistically significant (P = 0.0001), but not different between the curves (P = 0.99). The inset demonstrates the correlation between cGMP concentrations and evoked dopamine efflux at corresponding concentrations of C-type natriuretic peptide. The correlation value of 0.14 was not statistically significant (P > 0.20).e, 百拇医药
The possibility that C-type natriuretic peptide acts via the NPR-C to suppress protein kinase C activity and evoked dopamine efflux was explored using a peptide corresponding to the first 15 cytoplasmic amino acids of the NPR-C. The NPR-C peptide suppressed protein kinase C activity in a concentration-dependent manner from 0.1 to 10 pM, as indicated in Fig. 5. The EC50 averaged 0.30 ± 0.10 pM, and a maximal effect was achieved at a concentration of 1 pM. The 56 ± 10% maximal suppression of protein kinase C by the NPR-C peptide was virtually identical to the maximal effect of C-type natriuretic peptide shown in Fig. 1 (55 ± 8%). A scrambled version of the peptide failed to influence protein kinase C activity, and the two curves differed statistically (P = 0.0012; data not shown). The active NPR-C peptide also significantly suppressed evoked dopamine efflux from permeabilized cells at concentrations of 1 and 10 pM. The maximal suppression of evoked dopamine efflux was 40 ± 12%, a value slightly greater than that achieved with C-type natriuretic peptide in Fig. 1. The EC50 for the effect averaged 0.49 ± 0.20 pM. The inset to Fig. 5 indicates the correlation between the effects of the NPR-C peptide on protein kinase C activity and evoked dopamine efflux. The correlation predicts a 6.2% drop in evoked dopamine efflux for each 10% decline in protein kinase C activity. These data indicate that an intracellular region of the NPR-C reproduces the effect of C-type natriuretic peptide on both protein kinase C activity and evoked dopamine efflux, consistent with a mediatory role for the NPR-C.e, 百拇医药
fig.ommitteede, 百拇医药
Figure 5. Effect of a pentadecapeptide ([1–15]), representing the first 15 amino acids of the NPR-C protruding into the cytosol, on protein kinase C (PKC) activity and evoked dopamine efflux in permeabilized PC12 cells. The pentadecapeptide significantly reduced both parameters at 1 and 10 pM (*, P < 0.05; **, P < 0.01). The inset demonstrates the correlation between the two parameters at equivalent concentrations of the pentadecapeptide. The correlation was statistically significant (P < 0.001).(George J. Trachte)