Introduction

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The renin activity of the systemic circulating blood is determined primarily by the rate of its secretion from the JG cells (reviewed by Hackenthal et al., 1990). JG cells reside in the medial layer of the distal segment of the afferent arterioles of the kidney and undergo reversible metaplastic transformation between contractile vascular smooth muscle cells and secretory epithelioid cells depending on their renin secretory activity (Cantin et al., 1977; Taugner et al., 1987; Hackenthal et al., 1990). The rate of renin secretion from the JG cells is regulated by a wide variety of factors, among which inhibitory factors are generally vasoconstrictors and their inhibitory effects on renin secretion depends on the presence of extracellular Ca⁺⁺ (Vandongen and Peart, 1974; Hackenthal et al., 1990). We subsequently have found that not the extracellular Ca⁺⁺ per se but the intracellular Ca⁺⁺ inhibits renin secretion (Park and Malvin, 1978). Furthermore, we have demonstrated an inverse correlationship between the rate of renin secretion and estimated intracellular Ca⁺⁺ concentration of the JG cell: 10⁸ M or higher Ca⁺⁺ concentration causing inhibition of renin secretion (Park et al., 1986). Moreover, a wide variety of calmodulin antagonists were found to block the inhibitory effect of Ca⁺⁺ on renin secretion, which supports the possibility of the inhibitory effect of Ca⁺⁺ on renin secretion being through calmodulin mediation (Park et al., 1986; Hackenthal et al., 1990). However, the next biochemical event(s), how the Ca⁺⁺-calmodulin complex inhibits renin secretory process, remains unknown.

The reversible phosphorylation and dephosphorylation of target protein(s) by an activated specific protein kinase(s) or phosphatase(s), respectively, is thought to be among the major biochemical events in the signal transduction cascade elicited by the intracellular messengers into cellular responses (Cohen, 1992). In our recent studies, we have found that several putative inhibitors of the Ca⁺⁺-calmodulin-dependent MLCK such as ML-9 block the inhibitory effect of Ca⁺⁺ on renin secretion (Park et al., 1996). A major question raised about the finding was whether or not the stimulatory effect of ML-9 on renin secretion was through its specific inhibitory action on the MLCK. Because the method for preparation of pure JG cells is not available presently, the direct inhibitory action of ML-9 on MLC phosphorylation can not be determined biochemically in JG cells. Given the existing methodological difficulties, we elected a pharmacological approach to address the question with the use of ML-7 and calyculin A.

ML-7, a putative selective inhibitor MLCK, is a structural analog of ML-9 with 10-fold greater potency in the inhibition of purified smooth muscle MLCK (Saitoh et al., 1987). On the other hand, the phosphorylated smooth muscle MLC is dephosphorylated by MLC phosphatase, an isoform of PPase-1 (Alessi et al., 1992; Gong et al., 1992; Ishihara et al., 1989a; Mitsui et al., 1992). Calyculin A is a putative selective inhibitor equally potent for both PPase-1 and -2A (Ishihara et al., 1989a). The phosphorylated MLC by MLCK can be dephosphorylated by specific PPase-1 (Alessi et al., 1992; Gong et al., 1992). On the basis of these facts, it is expected that ML-7 would be more potent than ML-9 in stimulation of renin secretion as well as reversal of renin secretion preihibited by Ca⁺⁺. It also is expected that ML-7 and calyculin A would have apparent antagonistic effects on renin secretion through their opposite actions on the level of MLC phosphorylation. We reasoned that if experimental results are consistent with these predictions, the data from the present studies would provide additional support to our hypothesis that reversible phosphorylation and dephosphorylation of MLC is involved in the regulatory mechanism of renin secretion.

	Methods

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Experiments were conducted with renal cortical slices of Sprague-Dawley rats of either sex weighing 200 to 300g. Animals were fed a low-salt diet (HarlanTeklad, Madison, WI) for 2 to 4weeks before the experiments to maintain high renin secretory activity (Park et al., 1978). Renal cortical slices (5 × 5 × 0.5 mm) were prepared on ice with a Stadie-Riggs microtome as described previously (Park and Malvin, 1978; Park et al., 1978). Renal cortical slices from three to five rats were pooled and preincubated in ~100 ml of KRB solution for 60 to 90 min with two to three washings to remove tissue debris and to stabilize renin secretory activity.

After completion of the preincubation, one or two cortical slices (10-20 mg) per glass test tube (1.5 × 4.5 cm) containing 1 ml of incubation medium were incubated at 37°C for two to three periods of 1 h each. The incubation was conducted in a shaking Dubnoff metabolic incubator gassed continuously with 95%O₂-5%CO₂. The composition of the standard KRB was as follows (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 the K⁺-rich depolarizing KRB (K⁺-rich KRB) was identical with that of the standard KRB except that KCl concentration was raised from 5 to 90 mM by substitution of 85 mM KCl for NaCl of the standard KRB. Ca⁺⁺-free K⁺-rich KRB was prepared by omitting CaCl₂ and including 1.0 mM ethylene glycol-bis(-aminoethylether)-N,N,N',N'-tetraacetic acid.

The first 1-h incubation in the standard KRB or in K⁺-rich KRB under control conditions served as the control (C). The subsequent second and third periods of incubation were conducted under experimental conditions with testing agent(s) and/or Ca⁺⁺ in the K⁺-rich KRB (E). A group of slices was incubated in the same medium as in the control period throughout the incubation periods in parallel with other groups of slices under experimental conditions, and served as controls for the time-dependent spontaneous changes in the rate of renin secretion (time control). At the end of each incubation period, the incubation medium was collected. An aliquot of collected medium was incubated with the plasma of 48-h nephrectomized rabbits. Renin activity was determined by of radioimmunoassay the generated ANG I. The rate of renin secretion from slices under experimental conditions was corrected with respect to spontaneous time-dependent changes in the rate of the time control (see above), which was usually <10% each hour. The rate of renin secretion is presented as nanograms of ANG I per 100 milligrams wet weight per hour (ng ANG I · 100 mg¹ · h¹) or changes in the rate of secretion during the experimental periods relative to the changes of the control period (E/C). E/C of the experimental groups was divided by E/C of the time control, and the rate of renin secretion of the control was assigned a relative value 1.0. All data are presented as means ± S.E. Differences in values between periods within the group and between groups were compared by paired and unpaired Student's t test, respectively. A value of P < .05 was the accepted level of significance.

ML-7 was obtained either from the Biomol (Plymouth Meeting, PA) or from the Sigma (St. Louis, MO), A23187 from the Sigma (St. Louis, MO) and calyculin A from the LC Laboratories (Woburn, MA), respectively. These reagents were dissolved in dimethyl sulfoxide as concentrated stock solution and aliquot was added in the incubation medium, such that the final concentration of dimethyl sulfoxide was less than 1%. ¹²⁵I-ANG I and antibody to ANG I for radioimmunoassay of renin activity were provided generously by Prof. Kyung Woo Cho, Department of Physiology, Jeonbuk National University Medical School, Jeonju, Korea.

	Results

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Effect of ML-7 on renin secretion in the standard KRB. After the first hour control incubation in KRB (C), slices were incubated in the KRB containing varying concentrations of ML-7 during the second hour (E). Figure 1 shows concentration-dependent stimulation of "resting" renin secretion. ML-7 significantly stimulated renin secretion at its concentration as low as 1 × 10⁶ M, reaching a maximal stimulation of 2.47 ± 0.16 fold (P < .001) at 3 × 10⁵ M. The concentration of ML-7 required for the half-maximal stimulation (EC₅₀) calculated by nonlinear curve fitting was found to be about 9 × 10⁶ M. 

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Concentration-dependent stimulation of renin secretion by ML-7 under "resting" conditions. Each point is mean ± S.E. from six observations. Renal cortical slices were incubated in the standard (Na+-rich) KRB during the first period (C) followed by the same medium containing varying concentrations of ML-7 (E). Control tissues were incubated in the standard KRB without ML-7 (Control). Changes in the rate of renin secretion in the control were assumed to be time-related spontaneous changes and registered as a relative value of 1.0. E/C for experimental samples was corrected with respect to spontaneous changes of the Control. The solid curve is computer fit to the concentration-response data with ML-7 (correlation coefficient, r = 0.97). For comparison, the concentration-response with ML-9 (Park et al, 1996) is included (dashed line). The calculated concentration for the half-maximal stimulation of ML-7 and ML-9 was 9 × 106 M and 2.44 × 105 M, respectively.

Effects of ML-7 on renin secretion in the presence and absence of calyculin A in the standard KRB. As summarized in table 1, no significant changes occurred in the rate of renin secretion when slices were incubated in KRB for two periods, i.e., time control (group I, P > .05). ML-7 (3 × 10⁵ M) stimulated renin secretion about 2-fold (group II, P < .001). Calyculin A alone apparently did not have an effect on the "resting" renin secretion in the standard KRB (table1, period I of group IV) compared with other control groups. Addition of ML-7 simultaneously with calyculin A (3 × 10⁶ M; group III) or subsequent to preincubation with calyculin A (group IV) did not stimulate renin secretion. In fact, the rate of renin secretion was inhibited to the level significantly lower than that before the addition of ML-7 (groups III and IV, P < .001).

View this table: [in this window] [in a new window]   TABLE 1 Effects of ML-7 on renin secretion in the presence and absence of Calyculin A in the standard KRB Values are means ± S.E. from nine observations. Renal cortical slices were incubated in the standard KRB (KRB) for two periods, I and II, of 1 h each. One group of slices was incubated in KRB for two periods and served as the time control (group I). For other groups, after the incubation in the KRB during period I, ML-7 (3 × 105 M) alone (group II) or ML-7 plus Calyculin A (3 × 106 M) (group III) was included in the KRB during period II. For group IV, incubation was conducted in the presence of Calyculin A during period I followed by the simultaneous presence of both Calyculin A and ML-7 during period II.

Effect of ML-7 on renin secretion in the presence and absence of calyculin A in Ca⁺⁺-free K⁺-rich KRB. In Ca⁺⁺-free K⁺-rich KRB throughout incubation periods, which served as time control, high renin secretory activity was maintained for 2 h (table 2, group I). Addition of ML-7 (3 × 10⁵ M) during the second period II significantly stimulated renin secretion as compared with that during period I (group II, P < .001). However, when calyculin A (3 × 10⁶ M) was included in the presence of ML-7 during the second period II, the rate of renin secretion stimulated by ML-7 was inhibited by 30% (group IV, P < .001) to the level of the Ca⁺⁺-free control medium (group I). In the presence of ML-7 throughout incubation (group III), the increased rate of renin secretion was maintained and significantly greater in both periods (281 ± 17.4 vs. 193 ± 12.0, P < .001; 278 ± 18.1 vs. 182 ± 7.7 ng ANG I · 100 mg¹ · h¹, P < .001, by unpaired t test) than that of the corresponding periods of the control (group I). Thus, the inhibition of renin secretion in the presence of calyculin A during period II in group IV was not caused by time-related decrease in the stimulatory effect of ML-7, but was due to an inhibitory effect of calyculin A. 

View this table: [in this window] [in a new window]   TABLE 2 Effects of ML-7 on renin secretion in the presence and absence of Calyculin A Ca++-free K+-rich KRB Values are means ± S.E. from nine observations. Renal cortical slices were incubated in the Ca++-free K+-rich KRB (Ca++) for periods I and II. One group of slices was incubated throughout in the Ca++-free KRB and served as the time control (group I). ML-7 (3 × 105 M) was present only during period II (group II) or during both periods (groups III and IV). For group IV, ML-7 and Calyculin A (3 × 106 M) were present simultaneously during period II.

Effect of ML-7 on inhibition of renin secretion by Ca⁺⁺ in K⁺-rich KRB. Slices were incubated in Ca⁺⁺-free K⁺-rich KRB during the first control period and then in the same medium but containing 2 mM Ca⁺⁺ during the second period. The rationale for the incubation in high K⁺-rich KRB was to depolarize JG cell membrane and thereby facilitate Ca⁺⁺ influx into JG cells through the voltage-gated Ca⁺⁺ channel (Park and Malvin, 1978; Churchill, 1980; Park et al., 1981). Consequently, an increased intracellular Ca⁺⁺ concentration is expected to inhibit renin secretion as reported previously (Park and Malvin, 1978; Churchill, 1980; Park et al., 1986). In the absence of ML-7 in the incubation medium, the rate of renin secretion decreased from 132 ± 2.07 during the first period to 37.8 ± 2.07 ng ANG I · 100 mg¹ · h¹ during the second period; that is, 70 ± 2.24% inhibition (table 3, P < .001, n = 9). In the presence of ML-7 throughout the incubation periods, ML-7 at 1 × 10⁶ M or higher concentrations significantly reduced the magnitude of inhibition in K⁺-rich KRB in a concentration-dependent manner. In the presence of 3 × 10⁵ M ML-7, the inhibition was only 15 ± 6.0% (P = .05, n = 6).

View this table: [in this window] [in a new window]   TABLE 3 Concentration-dependent protective effect of ML-7 against Ca++-induced inhibition of renin secretion in K+-rich KRB Values are means ± S.E. After incubation in the Ca++-free K+-rich KRB (Ca++), slices were incubated in the Ca++ (2 mM) containing K+-rich KRB (+Ca++). Both (Ca++) and (+Ca++) KRB contained a given concentration of ML-7.

Reversible protection by ML-7 against inhibition of renin secretion by Ca⁺⁺ in K⁺-rich KRB. Slices were incubated in Ca⁺⁺-free K⁺-rich KRB with or without containing 3 × 10⁵ M ML-7 during the first control period. This was followed by the second and third period of incubation in Ca⁺⁺-containing K⁺-rich KRB either with or without the continuous presence of ML-7. In the absence of ML-7, the rate of renin secretion during the second (E/C = 0.34 ± 0.016, P < .001, n = 8) and third period (E/C = 0.10 ± 0.07, P < .001) was inhibited significantly compared with the rate of renin secretion during the control period (fig. 2). However, in the continuous presence of ML-7 throughout all three periods, the rate of renin secretion was not altered significantly. When ML-7 was present during the first two periods, the rate of renin secretion during the second period was not different from that during the first control period (E/C = 0.94 ± 0.06, P > .05). Subsequent incubation in fresh Ca⁺⁺-containing K⁺-rich KRB but without ML-7 during the third period resulted in a significant inhibition of renin secretion compared with that during the first (and second) period (E/C = 0.52 ± 0.03, P < .001). Thus ML-7 appears to produce reversible protection against the inhibition of renin secretion by an increased intracellular Ca⁺⁺ concentration of JG cells.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Protection by ML-7 against the inhibition of renin secretion by Ca++. Each point is the mean ± S.E. from eight observations. During the first incubation period (Control) slices were incubated in Ca++-free K+-rich KRB (-Ca++) and subsequently in Ca++ (2 mM) containing K+-rich KRB (+Ca++) for the second and third periods. One group of slices was incubated in the presence of ML-7 (3 × 105 M) during the control period as well as during the second and third period (+ML-7). Another group was incubated in the presence of ML-7 during the control and second periods but not during the third period. E/C values during incubation periods in the presence of ML-7 were not different from the E/C value 1.0 by paired t test. E/C values during the incubation periods in the Ca++-containing medium without ML-7 were significantly smaller than the ratio of 1.0 (P < .001)

5

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Protection by ML-7 against the inhibition of renin secretion by Ca++ plus A23187. Each point is mean ± S.E. from eight observations. Experimental procedures were identical with those described in the legend for figure 2 except that the Ca++-containing K+-rich KRB during the second and third periods contained A23187 (105 M) (+Ca++ + A23187). E/C values for the incubation periods with ML-7 (3 × 105 M) were significantly smaller than the ratio of 1.0 during the control period (P < .05) but were significantly greater than those of without ML-7 (P < .001).

Effect of ML-7 on the reversal of renin secretion preinhibited by Ca⁺⁺. We often observed that renin secretion preinhibited by Ca⁺⁺ in high K⁺-depolarizing medium did not return completely to the preinhibited level on subsequent incubation in Ca⁺⁺-free medium (C.S. Park, M.H. Kim, H.W. Kim, H.S. Kim, Y.S. Hong, C.H. Leem and Y.J. Jang, unpublished observations). With the use of ML-7, we therefore tested whether or not MLCK activity is related to the incomplete reversal. Slices were incubated sequentially in Ca⁺⁺-free K⁺-rich KRB, Ca⁺⁺-containing K⁺-rich KRB and then back in Ca⁺⁺-free K⁺-rich KRB. The rate of renin secretion in Ca⁺⁺-containing K⁺-rich KRB, as expected, was inhibited by about 50% as compared with the level in Ca⁺⁺-free medium (fig. 4, P < .001). On subsequent incubation in Ca⁺⁺-free medium, the rate of renin did not increase significantly as compared with the level in Ca⁺⁺-containing medium. When ML-7 (3 × 10⁵ M) was included in the Ca⁺⁺-free medium during the third period, the reversal was complete. If both ML-7 and calyculin A (3 × 10⁶ M) were included simultaneously during the third period, there was no reversal at all on the removal of Ca⁺⁺, and the rate of renin secretion was decreased further by 43% as compared with that in Ca⁺⁺-containing medium (P < .001).

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Effects of ML-7 and Calyculin A on the reversal of renin secretion preinhibited by Ca++. Each point is the mean ± S.E. from eight observations. Slices were incubated in Ca++-free K+-rich KRB during the first incubation period (-Ca++) followed by Ca++-containing K+-rich KRB during the second period (+Ca++) and then back in Ca++-free K+-rich KRB during the third period. During the third period, one group of slices was incubated in Ca++-free KRB, the second group in the presence of ML-7 (3 × 105 M) and the third group in the simultaneous presence of both ML-7 and Calyculin A (3 × 106 M). Incubation in Ca++-containing K+-rich KRB, the rate of renin secretion was decreased significantly to about 50% of the value in the Ca++-free KRB (P < .001). On reincubation in Ca++-free KRB, E/C value during the third period was not significantly different than that during the second period (P > .05). In the presence of ML-7 (3 × 105 M), the E/C value during the third period was significantly greater than that during second period (P < .001) and was not different from 1.0. In the simultaneous presence of both ML-7 and Calyculin A (3 × 106 M), the E/C value during the third period was significantly less than that during the second period (P < .001).

	Discussion

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The present results obtained with the use of putative inhibitors of MLCK and phosphatase provide supporting evidence that reversible phosphorylation and dephosphorylation of MLC is a pivotal biochemical step in the control of renin secretion in an analogous manner to contraction and relaxation of the vascular smooth muscle. In fact, JG cells are transformed vascular smooth muscles of the distal segments of afferent arterioles of the kidney (Cantin et al., 1977; Taugner et al., 1987). Renin secretion generally is inhibited by a wide variety vasoconstrictors, whereas it is stimulated by vasodilators (Hackenthal et al., 1990 for recent review). Contraction and relaxation of smooth muscle and nonmuscle cells are regulated primarily by phosphorylation and dephosphorylation of MLC by MLCK and phosphatases, respectively (Kamm and Stull, 1985; Tan et al., 1992; Somlyo and Somlyo, 1994). Both regulation of renin secretion and contraction of smooth muscles are Ca⁺⁺-calmodulin mediated (Hackenthal et al., 1990; Kamm and Stull, 1985; Somlyo and Somlyo, 1994). By inference from such functional relevancies, we explored a possible involvement of Ca⁺⁺-calmodulin-dependent MLCK in the inhibition of renin secretion by an increased intracellular Ca⁺⁺ concentration of the JG cell. Consistent with this speculation, ML-9, a putative specific inhibitor of MLCK (Saitoh et al., 1987), was found to reversibly block the Ca⁺⁺-calmodulin-mediated inhibition of renin secretion (Park et al., 1996). Given problems inherent to the specificity of pharmacological agents and use of renal cortical slices with heterogeneous cell populations among which the JG cells are less than 1%, it was questioned whether the effect of ML-9 on renin secretion is through its effect specifically on MLCK in JG cells. To address this question, we used ML-7 and calyculin A.

ML-7 is a structural analog of ML-9 and a putative much stronger inhibitor of smooth muscle MLCK than ML-9 (Saitoh et al., 1987). Much the same as with ML-9 in our previous studies (Park et al., 1996), ML-7 stimulated renin secretion under "resting" conditions in a concentration-dependent manner (fig. 1). ML-7 also reversibly protected against inhibition of renin secretion when slices were incubated in Ca⁺⁺-containing K⁺-rich KRB in both the absence and presence of the calcium ionophore A23187. Under these experimental conditions, the inhibition of renin secretion most likely is caused by increased intracellular Ca⁺⁺ influx through both voltage-gated Ca⁺⁺ channels and the A23187-mediated pathway (Churchill, 1980; Park and Malvin, 1978; Park et al., 1981, 1996). The protection was concentration dependent and reversible and makes it unlikely that the effect of ML-7 on renin secretion is through its nonspecific cytotoxic effects. Most importantly, however, the lowest concentration of ML-7 for significant stimulation of "resting" renin secretion was 10⁶ M (fig. 1), which is about 10 times lower than that of ML-9 (Park et al., 1996). The calculated concentration required for the half-maximal stimulation was 9 × 10⁶ M ML-7 compared with 2.4 × 10⁵ M ML-9 (fig.1). These results are consistent with the order of known potency of ML-7 and ML-9 in the inhibiting activity of pure MLCK (Saitoh et al., 1987). These results provide additional support that these effects of ML-7 as well as ML-9 are mediated via inhibition of Ca⁺⁺-calmodulin-dependent MLCK.

ML-7 stimulated renin secretion from renal cortical slices when incubated in nominally Ca⁺⁺-free K⁺-rich KRB (table 2) where the intracellular Ca⁺⁺ concentrations ought to be lower and, as a functional consequence, lower activity of MLCK than in normal Ca⁺⁺-containing KRB (table 1). The relative magnitude of stimulation of renin secretion by ML-7 in both KRB was similar, but the absolute magnitude of stimulation was greater in Ca⁺⁺-free K⁺-rich KRB than in normal KRB. These findings suggest that the intracellular Ca⁺⁺ concentrations in Ca⁺⁺-free K⁺-rich KRB may be still high enough to activate MLCK appreciably. Inhibition of this low but appreciable activity of MLCK in Ca⁺⁺-free K⁺-rich KRB might produce the stimulation. Whereas the incomplete reversal of renin secretion preinhibited by Ca⁺⁺ on subsequent incubation only in Ca⁺⁺-free K⁺-rich KRB, complete reversal in the presence of ML-7 also can be accounted for by the residual activity of MLCK (fig. 4). A low but appreciable activity of MLCK in Ca⁺⁺-free K⁺-rich KRB is conceivable because Ca⁺⁺ concentration for half-maximal phosphorylation of MLC was about ~170 nM under conditions of MLCK sensitized to Ca⁺⁺ (Kitazawa et al., 1991; Tansey et al., 1994). This possibility also is consistent with our previous finding that renin secretion was stimulated only when the intracellular Ca⁺⁺ concentration was lowered below 10⁷ M (Park et al., 1986).

The phosphorylated MLC by MLCK is dephosphorylated by myosin phosphatase which is believed to be a subfamily of PPase-1 (Alessi et al., 1992; Gong et al., 1992; Ishihara et al., 1989a; Mitsui et al., 1992). Calyculin A is a putative selective inhibitor of PPase-1 with a much greater potency than okadaic acid (Ishihara et al., 1989a). We indeed found that calyculin A and okadaic acid, each at 10⁶ M added to the standard KRB, significantly inhibited "resting" renin secretion by paired comparisons, but the former was more potent the latter (32 ± 4% vs. 19 ± 4%, n = 8 for each group; (C.S. Park, M.H. Kim, H.W. Kim, H.S. Kim, Y.S. Hong, C.H. Leem and Y.J. Jang, unpublished observations). In the present study, calyculin A (3 ×10⁶ M) added to the standard KRB apparently did not have an inhibitory effect on "renin" secretion as compared with renin secretion of other control groups in unpaired experiments (table 1). Such an apparent lack of inhibitory effect of calyculin A on "resting" renin secretion could be caused partly by the variability of "resting" renin secretion among different renal cortical slices from different animals. Another perhaps more important reason could be the result of low PPase-1 activity of the vascular smooth muscles, especially being about 1:30 relative to MLCK activity (Takai et al., 1987). The relative magnitude of effects of ML-7 and calyculin A on "resting" renin secretion perhaps reflects the relative activity of MLCK and PPase-1 of JG cells (table 1). The extent of MLC-phosphorylation is determined by the relative activity of MLCK and PPase-1. It therefore follows that under "resting" conditions where MLCK activity is predominant over that of PPase-1, inhibition of PPase-1 activity with calyculin A may not markedly increase the extent of MLC-phosphorylation. Therefore, calyculin A in "resting" conditions may or may not result in a significant inhibition of renin secretion. However, in the presence of MLCK inhibitors such as ML-9 or in the absence of extracellular Ca⁺⁺, the activity of MLCK was found to be very low and, as a functional consequence, the level of MLC-phosphorylation was also low (Ishihara et al., 1989b; Gong et al., 1992; Suzuki and Itoh, 1993). Under such conditions, the level of MLC-phosphorylation would be determined primarily by PPase-1 activity. Our finding that calyculin A had a greater inhibitory effect on renin secretion in the presence of ML-7 (table 1, group III and table 2, group IV) than its absence (table 1, group IV) can be explained by the lowered level of MLC-phosphorylation. Accordingly, our findings of inhibitory effects of calyculin A on both stimulation (tables 1 and 2) and reversal (fig. 4) of renin secretion induced by ML-7 can be explained easily by increasing the level of MLC-phosphorylation as a result of an inhibition of presumably PPase-1. However, alternative possibilities such as direct activation by calyculin A of myosin ATPase activity or of ML-7-insensitive and Ca⁺⁺-independent MLCK(s) cannot be excluded with our present results. In addition, whether the inhibition of renin secretion by calyculin A is through an inhibition of PPase-1 or PPase-2A is under investigation.

Effects of inhibitors of MLCK and PPase on secretion from other types of secretory cells where Ca⁺⁺ stimulates secretion seem noteworthy. Some investigators found that the MLCK inhibitors such as ML-7 inhibits secretion (Choi et al., 1994; Kitani et al., 1992; Ohara-Imaizumi et al., 1992; Reig et al., 1993) but other investigators failed to observe any inhibitory effects (Kumakura et al., 1994; Nakanishi et al., 1989). The intracellular Ca⁺⁺ concentration required for stimulation of secretion ranged from 10 µM to 100 µM (Neher and Zucker, 1993). At such high intracellular Ca⁺⁺ concentrations, multifunctional Ca⁺⁺-calmodulin-dependent protein kinase II (CaMK II), which is activated half-maximally at micromolar Ca⁺⁺ (Kuret and Schulman, 1985), are likely to be activated. The activated CaMK II is known to phosphorylate MLCK and inhibit MLCK activity leading to an inhibition of MLC phosphorylation (Tan et al., 1992; Tansey et al., 1994). Thus at such high intracellular Ca⁺⁺ concentrations during secretion, MLCK activity might be inhibited, rather than stimulated. Also in the rat mast cell line, RBL-2H3, about 40% of the total cellular MLC was phosphorylated by MLCK under resting conditions (Choi et al., 1994; Ludowyke et al., 1989, 1996). When stimulated to secrete, MLC was phosphorylated at different residues by protein kinase C. Most importantly, secretion was correlated best with MLC phosphorylation by protein kinase C but not by MLCK (Choi et al., 1994; Ludowyke et al., 1989, 1996). When MLC is dually phosphorylated by both kinases, it inhibits actin-activated myosin ATPase (i.e., contraction) and disassembles myosin filaments into myosin monomer (Tan et al., 1992). In view of these findings, it is unlikely that Ca⁺⁺ stimulates secretion by activating MLCK, thereby phosphorylating MLC. Furthermore, Ca⁺⁺-induced secretion was inhibited by calyculin A and less potently by okadaic acid (Gutierrez et al., 1995; McFerran and Guild, 1995; Meyer-Alber et al., 1994; Nishikawa et al., 1994; Wagner et al., 1992). These results also make it unlikely that Ca⁺⁺ stimulates secretion by phosphorylation of a target protein(s) such as MLC. Thus, dephosphorylation of target protein(s) by protein phosphatases is likely to stimulate secretion. Among the several candidates for target proteins, dephosphorylation of which was blocked, calyculin A had a molecular weight about 20,000 daltons (Nishikawa et al., 1994; Wagner et al., 1992), which corresponds closely to that of MLC. In view of these findings, it is possible that Ca⁺⁺-induced stimulation of secretion also may be mediated by dephosphorylation of MLC through an inhibition of MLCK at high intracellular Ca⁺⁺ concentration, thus sharing a common regulating mechanism(s) underlying secretory processes for both Ca⁺⁺-induced stimulation and inhibition.

To summarize, it is concluded that phosphorylation of MLC₂₀ by MLCK activated at a low range of micromolar intracellular Ca⁺⁺ concentrations leads to inhibition of renin secretion. Conversely, dephosphorylation of MLC₂₀ by PPase-1 leads to stimulation. Thus, reversible phosphorylation of MLC₂₀ apparently plays a pivotal role in the control of the secretion of renin. However, our present results with the pharmacological inhibitors of MLCK and PPase-1 with limited specificity cannot exclude conclusively possible involvement of other kinases and phosphatases in the Ca⁺⁺-dependent regulation of renin secretion. Thus this conclusion is tentative and must be confirmed by more direct biochemical evidence.