INTRODUCTION

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RENIN IS SYNTHESIZED, stored, and secreted from the juxtaglomerular (JG) cells in response to a wide variety of extracellular stimuli. Stimuli that are thought to raise the intracellular Ca²⁺ concentrations of the JG cells were found to inhibit renin secretion (see Ref. 10 for recent review). Pharmacological evidence suggests that Ca²⁺ binds to calmodulin and that the Ca²⁺-calmodulin complex inhibits renin secretion (10, 22). Our recent studies, using several agents known to selectively inhibit Ca²⁺-calmodulin-activated protein kinases, have demonstrated that putative myosin light chain kinase (MLCK) inhibitors, such as ML-9 [1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine], not only block but also reverse the Ca²⁺-induced inhibition of renin secretion (21). These results led us to postulate that Ca²⁺-calmodulin-activated MLCK phosphorylates the 20-kDa regulatory myosin light chain (MLC₂₀) (21).

Reversible phosphorylation and dephosphorylation of effector proteins by specific protein kinases and phosphatases, regulated by intracellular messengers such Ca²⁺ and cAMP, are generally believed to be the principal mechanisms by which extracellular stimuli are transduced into cellular responses (6, 31). In the smooth muscle, contraction and relaxation are regulated by phosphorylation and dephosphorylation of MLC₂₀ by MLCK and myosin light chain phosphatase (MLCP), respectively (1; see Ref. 26 for recent review). The JG cells are granulated epithelioid cells transformed from the vascular smooth muscle cells (10). Thus, analogous to the regulation of contraction and relaxation of smooth muscle by intracellular Ca²⁺, inhibition of renin secretion by Ca²⁺ through phosphorylation of MLC₂₀ by MLCK might be reversed by dephosphorylation of MLC₂₀ by MLCP.

The aim of this study was to identify the types of protein phosphatase (PP) involved in the regulation of renin secretion, perhaps through dephosphorylation of MLC₂₀, using various types of specific inhibitors of PP. Of the inhibitors we examined, calyculin A, a potent putative selective inhibitor of protein phosphatase type 1 (PP1) and type 2A (PP2A) (11), inhibited renin secretion under various experimental conditions consistent with the possibility.

	MATERIALS AND METHODS

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Materials. ML-9 and okadaic acid were obtained from either Biomol (Plymouth Meeting, PA) or Sigma (St. Louis, MO), calyculin A was obtained from Kamiya Biomedical (Thousand Oaks, CA), and cyclosporin A was obtained from Research Biochemicals International (Natick, MA). ML-9 was dissolved in ethanol and other drugs in dimethyl sulfoxide to make 100- to 1,000-fold concentrated stock solution. The same amount of solvent was added to the incubation of the control tissues.

Preparation of renal cortical slices and incubation. Experiments were conducted with renal cortical slices of Sprague-Dawley rats, either male or female, weighing 200-300 g. Animals were fed a low-salt diet (Harlan Teklad, Madison, WI) for 2-4 wk before the experiments to maintain high renin secretory activity (24). Renal cortical slices (5 × 5 × 0.5 mm) were prepared on ice using a Stadie-Riggs microtome, as previously described (23, 24). Renal cortical slices from 3-5 rats were pooled and preincubated in ~100 ml of Krebs-Ringer bicarbonate (KRB) solution and continuously gassed with 95% O₂-5% CO₂ at 37°C for 60-90 min, with 2-3 washings to remove tissue debris and to stabilize renin secretory activity. The standard Na⁺-rich KRB solution had the following composition (in mM): 120 NaCl, 5.0 KCl, 2.0 CaCl₂, 1.0 MgCl₂, 24 NaHCO₃, 1 Na₂HPO₄, and 10 glucose, pH 7.4. The composition of K⁺-rich KRB was identical to that of the Na⁺-rich KRB except that K⁺ concentration was raised from 5 to 90 mM by substitution of 85 mM KCl for equimolar NaCl in the Na⁺-rich KRB.

Radioimmunoassay of renin activity. At the end of each incubation period, the incubation medium was collected. Aliquots of collected medium were incubated with the plasma of 48-h nephrectomized rabbits. Renin activity was determined by measuring the generated ANG I using an angiotensin radioimmunoassay kit from New England Nuclear (Boston, MA).

Statistics. The rate of renin secretion from tissues under the experimental conditions was corrected with respect to the rate of the time control, which was usually <10% per hour. The rate of renin secretion is expressed as nanograms of ANG I per 100 milligrams wet weight per hour. Alternatively, some results are expressed as relative changes in the rate of renin secretion during the second- and third-hour periods (E) to that of the first-hour control period (C). The rate of renin secretion, in terms of both absolute and relative change during experimental periods, was divided by the E-to-C ratio of the time control to correct for nonspecific time-related changes in renin secretion. All data are expressed as means ± SE. Differences in values between groups and between periods within groups were compared by unpaired and paired Student's t-test, respectively. P < 0.05 was the accepted level of significance.

	RESULTS

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Effects of various PP inhibitors on renin secretion, under resting state, in Na⁺-rich KRB. After the first 1-h incubation without inhibitors (control), slices were incubated either with or without (control) a given phosphatase inhibitor during the second hour. Each inhibitor concentration used was at least 100× higher than that for the half-maximal inhibition (IC₅₀) reported in the literature, to ensure detection of the effect of each inhibitor on renin secretion. In the absence of an inhibitor (control), the rate of renin secretion during two consecutive incubation periods did not significantly change (Table 1). Inclusion of calyculin A (10⁶ M) or okadaic acid (10⁶-10⁵ M) in the medium significantly inhibited renin secretion (Table 1; P < 0.005, n = 8). On the other hand, cyclosporin A (10⁶ M), a well-known inhibitor of the Ca²⁺-calmodulin-dependent protein phosphatase 2B (PP2B) (calcineurin) with an IC₅₀ in the nanomolar range (14), was without effect (Table 1).

Effect of PP inhibitors on the reversal of renin secretion preinhibited by Ca²⁺. Incubation of slices in Ca²⁺-containing, K⁺-rich KRB caused a marked inhibition of renin secretion, as reported in our previous studies (21, 22). In our recent studies, this Ca²⁺-induced inhibition was found to be blocked by the calmodulin antagonist calmidazolium, as well as by putative selective inhibitors of MLCK such as ML-9 (21). These data led us to postulate that raised intracellular Ca²⁺, in Ca²⁺-containing, K⁺-rich KRB, binds to calmodulin and that the Ca²⁺-calmodulin complex then activates MLCK, phosphorylating MLC₂₀ and causing an inhibition of renin secretion (21). Subsequent incubation of slices in Ca²⁺-free, K⁺-rich KRB would deactivate MLCK as the intracellular Ca²⁺ concentration declines back to a low level. Subsequently, the phosphorylated MLC₂₀ would be dephosphorylated by MLCP and the inhibited renin secretion would be reversed.

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Concentration dependence of inhibitory effect of calyculin A on reversal of renin secretion. Slices were incubated in Ca²⁺-containing, K⁺-rich KRB and then in Ca²⁺-free, K⁺-rich KRB. The magnitude of the reversal of the inhibited secretion was normalized by assuming the reversal without calyculin A to be 100%. Figure 1 shows a concentration-dependent inhibition of the reversal. Calyculin A at 3 × 10⁸ M, the lowest concentration used, produced a significant inhibition of the reversal with a maximal inhibition of 71.4 ± 1.39% at 3 × 10⁶ M (P < 0.001, n = 8).

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Concentration-dependent inhibitory effect of calyculin A on reversal of renin secretion preinhibited by raised intracellular Ca2+. Each point represents mean ± SE from 8 observations. Slices were incubated in Ca2+-containing, K+-rich Krebs-Ringer bicarbonate (KRB) with or without calyculin A at 3 × 108, 3 × 107, and 3 × 106 M during first 1-h incubation. Second 1-h incubation followed, using Ca2+-free, K+-rich KRB in continuous presence of calyculin A (in same concentrations as in Ca2+-containing KRB). For control without calyculin A, rate of renin secretion significantly increased to 5.15 ± 0.31-fold (from 13.0 ± 1.29 to 64.3 ± 4.38 ng ANG I · 100 mg1 · h1; P < 0.001); this value (to 5.15-fold increase) was considered 100% reversal. Percent reversal in presence of calyculin A was normalized to this value. Calyculin A significantly inhibited reversal at all tested concentrations (P < 0.001).

Effect of calyculin A on renin secretion at normal, low, or high intracellular Ca²⁺concentrations. Slices were incubated in standard Na⁺-rich KRB; nominally Ca²⁺-free, K⁺-rich KRB; or Ca²⁺-containing, K⁺-rich KRB during the first control period in an attempt to vary intracellular Ca²⁺ concentrations. Calyculin A (10⁶ M) was added to the same KRB during the second incubation period. Calyculin A added to the standard KRB significantly inhibited secretion (Table 3; P < 0.001, n = 8). Note that the magnitude of the inhibition was the same as in the preceding series of experiments (Table 1). When slices were incubated in Ca²⁺-containing, K⁺-rich KRB, the rate of renin secretion was low, as expected. Calyculin A significantly further inhibited renin secretion (Table 3; P < 0.001, n = 8). Conversely, when slices were incubated in Ca²⁺-free, K⁺-rich KRB, the rate of renin secretion was high. Calyculin A again produced a significant inhibition (Table 3; P < 0.001, n = 9). Interestingly, the absolute magnitude of renin secretion inhibited by calyculin A was apparently proportional to the rate of renin secretion during the control period under varying incubation conditions, but the fractional inhibition was relatively constant (~30%) regardless of incubation conditions (Table 3).

View this table: [in this window] [in a new window]   Table 3.   Inhibitory effect of calyculin A (106 M) on renin secretion at normal, low, and high extracellular Ca2+ concentrations

Effect of calyculin A on renin secretion in presence and absence of ML-9 in Ca²⁺-free and Ca²⁺-containing K⁺-rich KRB. In our previous studies, ML-9, a putative selective inhibitor of MLCK (25), was found to stimulate renin secretion in the standard Na⁺-rich KRB (21). Note that in Ca²⁺-free, K⁺-rich KRB, the rate of renin secretion was approximately twofold greater in the presence of ML-9 (10⁴ M) than in its absence. Calyculin A inhibited renin secretion, on average, by 83.2 ± 12.0 and 43.8 ± 6.2 ng ANG I · 100 mg¹ · h¹ in the presence and absence of ML-9, respectively (Fig.2, P < 0.001, n = 9). In Ca²⁺-containing, K⁺-rich KRB, the rate of renin secretion was very low, as observed in the preceding series of experiments (Table 3). Calyculin A significantly inhibited renin secretion in both the presence and absence of ML-9 (Fig. 2; P < 0.001, n = 7). Calyculin A produced a similar magnitude of fractional inhibition (~30%) regardless of the presence of ML-9 and Ca²⁺ (Fig. 2).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Inhibitory effects of calyculin A on renin secretion in absence and presence of Ca2+ and ML-9 (104 M). Slices were incubated in Ca2+-free, K+-rich KRB (, ; n = 9) or Ca2+-containing, K+-rich KRB (,  n = 7) in absence (, ) and presence (, ) of ML-9 (104 M) during first 1-h incubation. Calyculin A (106 M) was included (, ) during second 1-h incubation. SE bars are not shown when bar is smaller than symbol size. Rate of renin secretion in presence of ML-9 was significantly greater than that in its absence in Ca2+-free, K+-rich KRB (P < 0.001) but not in Ca2+-containing, K+-rich KRB. Calyculin A significantly inhibited renin secretion under all 4 different incubation conditions (P < 0.001).

Effect of calyculin A on the stimulatory action of ML-9. Slices were incubated in Ca²⁺-free or Ca²⁺-containing, K⁺-rich KRB in the presence and absence of calyculin A (10⁶ M) during the control period. As expected on the basis of the preceding series of experiments (Table 3), the rate of renin secretion was high in Ca²⁺-free, K⁺-rich KRB and low in Ca²⁺-containing, K⁺-rich KRB (Fig. 3). The rate of renin secretion was lower in the presence of calyculin A than in its absence in Ca²⁺-free, K⁺-rich KRB but not in Ca²⁺-containing KRB. In the absence of calyculin A, ML-9 (10⁴ M) significantly stimulated renin secretion in both Ca²⁺-free KRB (Fig. 3; P < 0.001, n = 8) and Ca²⁺containing KRB (Fig. 3; P < 0.005, n = 8). In the presence of calyculin A, ML-9 did not stimulate secretion.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Stimulatory effects of ML-9 on renin secretion in absence and presence of Ca2+ and calyculin A (106 M). Each point represents mean ± SE from 8 observations. SE bars are not shown when bar is smaller than symbol size. Slices were incubated in Ca2+-free (, ) or Ca2+-containing (, ), K+-rich KRB in absence (, ) or presence (, ) of calyculin A (106 M) during 2 incubation periods. ML-9 (104 M) was included in same incubation medium during second incubation period. Rate of renin secretion was significantly lower in presence of calyculin A than in its absence in Ca2+-free, K+-rich KRB. In absence of calyculin A, ML-9 significantly stimulated secretion in both Ca2+-free and Ca2+-containing KRB (P < 0.05). In presence of calyculin A, ML-9 was without effect.

	DISCUSSION

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The reversible phosphorylation and dephosphorylation of a target protein by a specific protein kinase or phosphatase, respectively, are known to be among the principal biochemical mechanisms in the signal transduction cascade elicited by the intracellular messengers into cellular responses (6, 31). In the present study, we used several putative inhibitors of protein phosphatases as pharmacological probes to evaluate a specific PP and its substrate, which are involved in the regulation of renin secretion from JG cells. The serine/threonine-specific protein phosphatases are classified into four types, termed PP1, PP2A, PP2B (calcineurin), and PP2C, according to their substrate specificity, sensitivity to inhibitors, and dependency of enzyme activities on divalent cations (6, 31).

PP2B is a Ca²⁺-calmodulin-dependent enzyme with a few specific substrates and is sensitive to cyclosporin A (14). Cyclosporin A at 10⁶ M is expected to completely inhibit PP2B activity in view of its known nanomolar concentration range for 50% inhibition of the enzymatic activity (14). Cyclosporin A did not affect renin secretion under resting state, and did not affect the reversal of renin secretion preinhibited by raised intracellular Ca²⁺ concentrations (Tables 1 and 2). Cyclosporin A did not affect Ca²⁺-dependent inhibition of renin secretion in our recent study (21). Thus the PP2B is unlikely to be involved in the control of renin secretion.

PP1 and PP2A are the same gene products, with broad and overlapping substrate specificity (6, 31) and sensitivity to both calyculin A and okadaic acid (11). A significant inhibition of renin secretion by these two inhibitors indicates that PP1, PP2A, or both may play a role in the control of renin secretion. In terms of relative potency, calyculin A was more potent than okadaic acid (Tables 1 and 2). It is known that calyculin A and okadaic acid have similar inhibitory potencies for PP2A, but the former is 10- to 100-fold more potent for PP1 than the latter (11). Accordingly, our results point to the possibility that PP1 may be involved in the regulation of renin secretion. The relative potency of the two inhibitors, however, could depend on their relative membrane permeability and the intracellular concentrations of PP1 and PP2A. Therefore, our results should be considered as presumptive evidence.

In our recent study, Ca²⁺-induced inhibition of renin secretion was found to be blocked by the calmodulin antagonist calmidazolium, as well as by several putative inhibitors of MLCK such as ML-9 (21). These findings led us to postulate that raised intracellular Ca²⁺ concentrations activate MLCK through calmodulin mediation and, consequently, phosphorylate MLC₂₀, leading to the inhibition of renin secretion (21). In the smooth muscle, the MLC₂₀ phosphorylated by MLCK is known to be dephosphorylated by PP1 (2, 8, 11, 19) with the pharmacological characteristic of being very sensitive to calyculin A but much less so to okadaic acid (8, 12, 27). According to these findings, the inhibitory effect of calyculin A on the reversal of renin secretion preinhibited in Ca²⁺-containing, K⁺-rich KRB is likely to be a result of the inhibition of the dephosphorylation of MLC₂₀ via inhibition of PP1. Furthermore, in our previous study, the maximal protection by the MLCK inhibitor ML-9 of Ca²⁺-induced inhibition was ~70% (21), which, incidentally, is similar in magnitude to the maximal inhibition by calyculin A of the reversal of renin secretion preinhibited by Ca²⁺ (Figs. 1 and 2). These findings suggest that all the inhibition of renin secretion by Ca²⁺, through MLC₂₀ phosphorylation, can be reversed through its dephosphorylation by MLCP, a type 1 PP (2, 8, 11, 19).

According to the idea that phosphorylated MLC₂₀ inhibits renin secretion, the rate of renin secretion under various experimental conditions ought to be determined by the dynamic balance between dephosphorylated and phosphorylated MLC₂₀, which would be determined by the activities of MLCP and MLCK under given conditions. When the inhibitory effect of calyculin A on renin secretion was compared in all series of experiments (i.e., in the normal KRB; Ca²⁺-free, K⁺-rich KRB; or Ca²⁺-containing, K⁺-rich KRB, in the presence and absence of ML-9), the absolute magnitude of its inhibitory effect was proportional to the control secretory rate, but the fractional inhibition was constant at ~30% (Fig. 4). Thus the mode of action of calyculin A displays first-order kinetics: the rate of product formation is directly linearly proportional to the substrate concentration. But our results seem contrary to this expectation. In Ca²⁺-free, K⁺-rich KRB, the intracellular Ca²⁺ concentration of JG cells is likely to be lower than that in standard KRB and Ca²⁺-containing, K⁺-rich KRB (Table 3). Therefore, the activity of MLCK and, as a result, the concentration of phosphorylated MLC₂₀ would be low in Ca²⁺-free, K⁺-rich KRB. At low substrate concentrations of phosphorylated MLC₂₀, the activity of MLCP and the magnitude of the inhibitory effect of calyculin A on the enzyme are expected to be low in Ca²⁺-free, K⁺-rich KRB. Accordingly, the inhibitory effect of calyculin A on renin secretion in Ca²⁺-free, K⁺-rich KRB should be small, but our results were contrary. This apparent contradiction can be, in part, explained by the following. The activity of MLCP is reported to be low, especially relative to that of MLCK in smooth muscles (28). The present findings of the maximal effects of ~30% inhibition by calyculin A, compared with ~200% stimulation by ML-9, might reflect the low activity ratio of MLCP to that of MLCK in JG cells. In standard KRB and Ca²⁺-containing, K⁺-rich KRB, the high activity of MLCK and, consequently, the greater extent of phosphorylated MLC₂₀ could be primary determining factors in the control of renin secretion. Under such conditions, the ratio of MLCP activity to its substrate (phosphorylated MLC₂₀) level is expected to be low, and, therefore, the effect of calyculin A on the rate of renin secretion would be small. Conversely, in Ca²⁺-free, K⁺-rich KRB, the activity of MLCK and the level of phosphorylated MLC₂₀ are expected to be low (8, 12, 27), and the MLCP activity would be a primary determining factor in the rate of secretion. Accordingly, the inhibitory effect of calyculin A on renin secretion becomes prominent. Even under this condition, because of the high ratio of MLCP activity to its substrate concentration, the MLCP activity appeared to be capable of dephosphorylating only 30% of the phosphorylated MLC₂₀ (Fig. 4).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Inhibitory effect of calyculin A on renin secretion as a function of renin secretory rate. Rate of secretion was varied in range, from 24.6 to 269 ng ANG I · 100 mg1 · h1, by incubating slices in standard Na+-rich KRB or Ca2+-free or Ca2+-containing K+-rich KRB, with or without ML-9 (104 M). Control incubation was followed by 1-h incubation in presence of calyculin A (106 M). Linear regression equation from 7 groups was calculated by least-squares method. Correlation coefficient was 0.98.

As alluded to previously, ~70% of total renin secretion appears to be regulated in a Ca²⁺-dependent manner. The remaining 30% of renin secretion appears to be Ca²⁺ insensitive. In smooth muscles, calyculin A was found to induce contraction concomitantly with the phosphorylation of MLC₂₀ in a Ca²⁺-independent manner (8, 12, 27). As suggested by those studies, 30% of the total MLC₂₀ of the JG cells may be phosphorylated by a Ca²⁺-independent protein kinase of an as yet unknown nature. Calyculin A may block dephosphorylation of this portion of MLC₂₀, causing an inhibition of renin secretion. For example, in Ca²⁺-free, K⁺-rich KRB with ML-9, MLC₂₀ would not be phosphorylated by MLCK but by a Ca²⁺-independent kinase. By inhibition of the dephosphorylation of this MLC₂₀ by MLCP, calyculin A could produce a large inhibitory effect on renin secretion. In this scenario, the inhibition of renin secretion by calyculin A, in an apparent first-order kinetics, could reflect its effect on the dephosphorylation of MLC₂₀, which is phosphorylated by a Ca²⁺-insensitive kinase. The rate of MLC₂₀ phosphorylation could be proportional to the level of unphosphorylated MLC₂₀, perhaps in the first-order kinetics (as previously discussed). Findings in support of this scenario are that ML-9 significantly stimulated renin secretion in both Ca²⁺-free and Ca²⁺-containing, K⁺-rich KRB in the absence of calyculin A, but not in its presence (Fig. 3). Calyculin A could shift the dynamic balance of MLC₂₀ from the unphosphorylated form to the phosphorylated form by inhibiting dephosphorylation of MLC₂₀, which was largely phosphorylated either by a Ca²⁺-independent kinase in the Ca²⁺-free KRB or by Ca²⁺-calmodulin-dependent MLCK in Ca²⁺-containing KRB. As a result, the concentration of unphosphorylated MLC₂₀ available for MLCK already would be limited, so that an inhibition of MLCK activity by ML-9 might not lead to an appreciable magnitude of stimulation of renin secretion. These two alternative possibilities need to be tested in biochemical studies.

In other secretory cell types where Ca²⁺ is known to stimulate secretion, in contrast to the JG cell, calyculin A and okadaic acid were reported to inhibit secretion (17, 18, 20, 29). Thus as in the JG cells, dephosphorylation of a target protein by protein phosphatases, most likely PP1, appears to trigger secretion. MLC₂₀ is among the most frequently observed target proteins and the phosphorylation of MLC₂₀ was markedly increased in association with inhibition of secretion (5, 9, 15, 16, 20, 30). The findings most relevant to our discussion are that, in rat basophilic leukemia mast cells (RBL-2H3), 40% of the total MLC₂₀ was found to be phosphorylated at the MLCK site at Ser¹⁹ under resting conditions (i.e., when no secretion is occurring). Stimulation of RBL-2H3 cells with antigen, phorbol esters, or calcium ionophore A-23187 was found to phosphorylate MLC₂₀ at protein kinase C sites (Ser¹ and Ser²) in close correlation with secretion (5, 15, 16). As was also the case in platelets, secretion was found to be closely correlated with phosphorylation of MLC₂₀, at both sites, by MLCK and protein kinase C (30). An important finding resulting from these studies was that calcium ionophore-induced secretion was best correlated with MLC₂₀ phosphorylation at the protein kinase C site, not with MLCK sites (16, 30). Dual phosphorylation of MLC₂₀ at the MLCK site and protein kinase C site, in vitro, is known to decrease actin-activated ATPase activity of myosin that already has been phosphorylated by MLCK (3, 20). Thus in both secretory cell types in which Ca²⁺ stimulates secretion (as in the RBL-2H3 and platelets) or, conversely, in JG cells in which Ca²⁺ inhibits secretion, secretion may be elicited by inhibiting myosin ATPase through additional phosphorylation of MLC₂₀ at the protein kinase C sites in the former or through dephosphorylation of MLC₂₀ at MLCK sites in the latter. Despite the apparent Ca²⁺ paradox, secretion in both secretory cell types may be elicited by inhibition of myosin ATPase activity and, therefore, of actomyosin contraction in a manner recently postulated (23).

As schematically shown in Fig. 5, we postulate that phosphorylation of myosin light chain by Ca²⁺-dependent MLCK (and by Ca²⁺-independent kinases of unknown nature) inhibits renin secretion. Dephosphorylation of myosin light chain by MLCP, the serine/threonine-specific PP1, leads to stimulation of renin secretion. This hypothesis needs further biochemical evidence for a causal relationship to be determined between phosphorylation/dephosphorylation of MLC₂₀ and the rate of renin secretion. Currently available methods can enrich JG cells to 70-80% purity (7). Because every eukaryotic cell contains myosin (4) and renal vascular smooth muscles, in particular, they have a much greater myosin content than JG cells (10). Determination of MLC₂₀ in enriched JG cells with 20-30% contaminated non-JG cells would always give false-positive results to our hypothesis. To exclude such ambiguity, we employed cloned JG cells in culture and found that Ca²⁺ increased MLC₂₀ phosphorylation. This phosphorylation was decreased by ML-9 and increased by calyculin A (13). Although our preliminary results are consistent with our hypothesis, the secretory characteristics of these cloned JG cells are unknown and currently under investigation in our laboratory.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Schematic diagram of regulation of renin secretion by reversible phosphorylation of myosin light chain. MLCK, myosin light chain kinase; MLCP, myosin light chain phosphatase; PP1, protein phosphatase type 1.