Role of membrane sulfhydryl groups in stimulation of renin secretion by sulfhydryl reagents

Kidney International, Vol. 39 (1991), pp. 867—873

Role of membrane sulfhydryl groups in stimulation of renin secretion by suithydryl reagents PHILIP S. DOH, CHEOL Joo LEE, PETER M. HWANG, KYUNG Woo CHO, THOMAS W. HONEYMAN, and CHUN SIK PARK Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts, USA

Role of membrane suiThydryl groups in stimulation of renin secretion by sulfbydryl reagents. The present study was designed to address the reactivity and accessibility of the particular class of sulthydryl groups involved in the regulatory process of renin secretion. Both mercurial (such as P-chloromercuriphenyl sulfonate [PCMPSI) and non-mercurial sulthydryl reagents (for example, 6,6-dithiodinicotinic acid [DTDN}), which very slowly penetrate the cell membrane of intact cells, stimu-

have shown that sulfhydryl reagents such as PCMPS and

strongly inhibited it at high concentrations. Several reagents which are known to primarily deplete cellular reduced glutathione were without effect on renin secretion. The stimulation of renin secretion by PCMPS was rapid in onset, and prevented and reversed by DTT and L-cysteine. Furthermore, the maximal stimulatory effect of PCMPS was not addititive to that by diuretics with sulfhydryl reactivity (such as, ethacrynic acid and mersalyl). The stimulatory effect of PCMPS was not affected by diuretics which lack sulthydryl reactivity (such as, bumetanide and furosemide). These results suggest that sulfflydryl reagents of both with and without diuretic activity stimulate renin secretion by reacting with specific class of sulthydryl groups which are readily accessible from the extracellular compartment. In addition, these results provide further support the possibility that a sulfhydryl-disulfide interchange in the membrane may play a regulatory role in the renin secretory process.

involvement of sulfliydryl groups in the stimulation of renin

P-chloromercuribenzoate (PCMB) stimulate secretion [5—11]. It

thus seemed possible that membrane sulfhydryl groups may play a fundamental role in the stimulus-secretion coupling.

The present study was to define more closely the relationship between the accessibility and reactivity of the suithydryl groups lated renin secretion. The membrane permeant sulfliydryl reagent that may be involved in renin secretion by comparing the effects N-ethylmaleimide had no effect on renin secretion but its membrane of PCMPS and other poorly permeant or impermeant sulfhydryl impermeant derivative, stilbene maleimide, strongly stimulated secretion. Furthermore, disulfide reducing agents such as dithiothreitol reagents to the effects of permeant reagents which are known to (DTT) had no effect on renin secretion at low concentrations, but primarily deplete intracellular reduced glutathione. The specific secretion by putative sulfhydryl reagents was studied by assessing the protective and reversal effects of exogenously provided thiols on the stimulatory effect of PCMPS on renin secretion. Furthermore, we have evaluated the effects of disulfide reducing agents on renin secretion to explore the possibility whether

or not the membrane sulfhydryl-disulfide interchange modulates the rate of renin secretion. The results are consistent with the hypothesis that membrane sulthydryl-disulfide interchange may be a step in renin secretion. Methods

Studies on renin secretion in vitro were conducted with renal cortical slices of rabbits fed a sodium deficient diet (Bio-Serve, Frenchtown, New Jersey, USA) for two to three weeks before

We have previously demonstrated that a number of sulfhydry! reagents stimulate renin secretion in vitro from rabbit renal cortical slices [11. Among those tested reagents P-chloromercuriphenylsulfonate (PCMPS) was found to be the most potent, although this reagent is known to only slowly permeate across the cell membrane [2]. These results suggested that PCMPS may stimulate secretion by reacting with sulfflydryl groups and that these groups are likely superficially located in the juxtaglomerular (JG) cell plasma membrane [1]. In subsequent recent

the experiments. Renal cortical slices —0.5 mm thick were prepared with a Stadie-Riggs tissue slicer as described previously [I]. Slices were incubated at 37°C for two to three periods of one hour each, unless otherwise indicated, in 2 ml medium which was continuously gassed with 100% 02. The composition

of the standard incubation medium was (in mM): NaCl, 145; KC1, 5.0; CaCI2, 2.0; MgCI2, 1.0; glucose, 10; N-2-hydroxyethylenepiperazine-N-2'-ethanesufonic acid (HEPES), 10. The pH

study, loop diuretics such as ethacrynic acid with known sulthydryl reactivity [3], were found to stimulate renin secretion in a manner similar to that by PCMPS, suggesting that specific membrane suithydryl groups may be involved in the regulation of renin secretion [4]. Studies on other types of secretory cells

Received for publication May 14, 1990 and in revised form November 19, 1990 Accepted for publication November 20, 1990

© 1991 by the International Society of Nephrology

was adjusted to 7.0. The first one hour incubation in the standard medium served as the control. During subsequent periods, slices were incubated in the medium containing various sulfhydryl reagents and

other testing agents. At the end of each incubation period, medium was collected and renin activity was determined by radioimmunoassay of angiotensin I [12]. The rate of renin secretion is expressed as nanograms of angiotensin I generated per 100 mg wet tissue weight per hour (ng AI/l00 mg/hr) or as the ratio of renin secretion during experimental period to that 867

868

Doh et a!: Sulfhydryl group and renin secretion 35

Table 1. Effect of various reagents depleting cellular reduced glutathione on renin secretion (ng AI/lOO mg/hr)

30

0w 0

Control 55.5

7.0

62.3

9.6

26.0

2.2

50.9

7.7

Diamide (1.0 mM) 51.5 4.6 t-Butylhyperoxide (2.0 mM) 56.8 7.5 2-Cyclohexene-l-one (3.0 mM) 21.7 3.8 Diethyl maleate (2.0 mM) 50.1 5.6

20

a) C)

a)

15

a)

10

5 0 Control

lx 10

lx 10

1 x10

1 x 10—2

0.95

0,08

0.93

0.05

0.82

0.10

1.06

0.12

Values are mean SE from 6 observations. Renal cortical slices were incubated for 60 mm each before (Control) and after addition of test reagents.

Concentration, M

140

Fig. 1. Concentration-stimulation relationship of p-chloromercuriphe-

nylsulfonate (PCMPS, 0), p-chloromercuribenzoate (PCMB, A), 5,5' dithiobis (2-nitrobenzoic acid) (DTNB, 0), and 6,6'-dithiodinicotinic acid (DTDN, O) on renin secretion. Following the control incubation (C), the tissues were incubated for another 60 mm in the presence of varying concentrations of sulfhydryl reagents for another 60 miii. Each point is the mean SE from 5 to 6 observations.

Ratio

Reagent

26

120

w C .2

100 80 60

during the control period (E/C). Differences between values were determined by paired or unpaired Student's t-test. P-chloromercuriphenylsulfonic acid (PCMPS), p-chloromercuribenzoic acid (PCMB), 6,6-dithiodinicotinic acid (DTDN), 5,5 '-dithio-bis-(2-nitrobenzoic acid) (DTNB), 2-niercaptoethanol (2-ME), 2,3-dimercaptopropanol (2,3-DMP), diamide [diazenedicarboxylic acid bis (N,N-dimethylamide)I, t-butylhyperoxide, diethyl maleate, N-ethylmalemide and furosemide were obtained from Sigma Chemicals (St. Louis, Missouri, USA), 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (stilbene

maleimide) from Molecular Probes, Inc., (Eugene, Oregon, USA), and 2-cyclohexene- 1-one from Aldrich Chemical Co., Inc., (Milwaukee, Wisconsin, USA). Bumetanide was generously provided by Dr. P.F. Sorter of Hoffman-LaRoche, Inc., (New Jersey, USA). All other chemicals were of analytical grade. Results Figure 1 shows the concentration-stimulation relationship of

four sulfhydryl reagents: 2 mercurial reagents, PCMPS and PCMB and 2 non-mercurial reagents, DTDN and DTNB. The lowest concentrations which significantly stimulated renin secretion were: PCMPS, i0 M (51 18% increase); PCMB, 10 M (44 19%); DTDN, l0— M (25 8%); DTNB, 3 x 10

J

40 20

0 0

5 10 15

30

60

Time, minutes

Fig. 2. Time course of stimulation of renin secretion by PCMPS. Tissues were incubated in the absence of PCMPS for 60 mm. The rate of renin secretion during this control period was 32.3 6.6 ng AI/100 mg/hr (N = 6). Following the control incubation, tissues were incubated in the presence of 5 x iO M PCMPS. The medium was changed at 5, 10, 15, 30 and 60 mm after the start of incubation. The rate of renin secretion during each period expressed as a per hr rate was compared to that during the control period (E/C).

(Table 1). It is of interest to note that these glutathione depleting reagents were found to inhibit secretion in other secretory cells [10, 16, 17]. PCMPS has a low lipid solubility owing to its sulfonic acid

group and penetrates the cell membrane very slowly. There-

fore, it is expected to react primarily with superficial membrane sulthydryl groups, forming readily reversible mercaptides [21. As Figure 2 shows, PCMPS indeed elicited prompt secretory M (21 5.8%) indicating that mercurial sulfhydryl reagents are response; it stimulated renin secretion over 20-fold within the far more effective in stimulation of renin secretion than non- first five minutes of incubation and produced its maximal effect within 30 minutes. mercurial sulfhydryl reagents. To investigate if the intracellular sulffiydryl groups of nonIf sulfhydryl reagents stimulate renin secretion by specifically protein molecujes are involved in renin secretion, the effects of reacting with sulihydryl groups on membrane proteins, exogefour reagents which primarily deplete cellular reduced glutathi- nously provided thiols should prevent and reverse the stimulaone were assessed. Diamide, an oxidant of reduced glutathione tion. As Figure 3 shows, PCMPS (5 x i0 M) alone stimulated [13], t-butylhyperoxide, a substrate of glutathione peroxidase renin secretion 94.5 10.2-fold (N = 6, P < 0.001). Subsequent

[13, 141 and 2-cyclohexene-l-one and diethyl maleate, sub- addition of dithiothreitol (5 x 10 M), a thiol protecting agent strates for the glutathione S-transferase [151, are known to [18], to the medium still containing PCMPS markedly but not decrease cytosolic reduced glutathione and thereby shift cyto- completely reversed stimulation by PCMPS (Fig. 3A). In a solic sulfhydrylldisulfide balance to the oxidized form. None of parallel experiments DTT (5 x iO M) was first added to these reagents had any significant effect on renin secretion incubation medium. Although DTF by itself had no significant

869

Doh et al: Sulfhydryl group and renin secretion

120

94.50

10.20

w

w

0 30 a

80

C

0 a

U

aa C C

a

4.30

+

40

0

100

0

43.30

50

A

C.,

a a

60

C C

20

a

10

40 9.69

0-

0.88

1.00

Control

x

Control

B 2

1.58

0.05

PCMPS

PCMPS

(5 x 10r) cysteine

cysteine (1:3)

PCMPS

(1:1)

PCMPS PCMPS (5x i0 M( (5x 10'C M( DTT(5x103 M(

0.46

3.82

1.00

Fig. 4. Effect of L-cysteine on renin secretion stimulated by PCMPS. Tissues were incubated for two consecutive 60 mm periods. Following the control period, tissues were incubated for the second period in the presence of PCMPS (5 x l0 M) alone or in the presence of L-cysteine.

The magnitude of stimulation by PCMPS was greatly reduced by cysteine in a manner dependent on the molar ratio of PCMPS to cysteine. Results are mean SE from 6 observations.

0 w

20

C

0 1 C.)

a a

C

C

a

15

0 w C

0

Control

DTT

(5x i0 M(

DTT

PCMPS (5x iO- M(

Fig. 3. Effect of DTT on the stimulation and reversal of renin secretion

by PCMPS. Tissues were incubated for three consecutive 60 mm periods. The rate of renin secretion during the control period was 74.3 17.3 ng AI/100 mg/hr. PCMPS (5 x i0 M) significantly stimulated renin secretion which was greatly reversed, but not completely, to the

control level by addition of DTT (5 x l0 M) (A). In the presence of

0 a 0 a a

10

C

C

a 5

DTT, PCMPS failed to stimulate renin secretion (B). Results are mean SE from 6 observations. 0

effect on renin secretion (Fig. 3B), it completely blocked the secretory response to subsequently added PCMPS (Fig. 3B). Similar results were obtained with 1-cysteine as shown in Figure

4. PCMPS (5 x

iO M) stimulated

renin secretion 43.3

4.3-fold over the control value. When tissues were incubated in the medium containing both PCMPS and cysteine, the magnitude of stimulation of renin secretion by PCMPS was greatly reduced in a manner dependent upon the molar ratio of PCMPS to cysteine. At a 1:1 or 3:1 molar ratio of cysteine to PCMPS, the stimulation was only 3.82 0.46 and 1.58 0.05-fold, respectively, as compared to 43.3 4.3-fold by PCMPS alone (Fig. 4, P < 0.001). These results strongly suggest that PCMPS stimulates renin secretion by reacting reversively and specifically with sulfhydryl groups. N-ethylmaleimide, unlike PCMPS, with high permeability to cell membrane irreversively alkylates sulfhydryl groups [19],

Control

Stilbene maleimide

Washout

Fig. 5. Irreversible stimulation of renin secretion by stilbene maleim-

ide. Tissues were incubated in the standard incubation medium for three consecutive one-hour periods. Following the control incubation, tissues were incubated in the medium containing 1.0 x lO M stilbene nialeimide and then fresh medium free of stilbene maleimide (washout). The rate of renin secretion during the control period was 56.6 5.4 ngAI/l00 mg/hr (N = 6). The rate of renin secretion in the presence of

stilbene maleimide and washout period as compared to the control value was significant (P < 0.001).

meant sulfhydryl alkylating agent stilbene maleimide [20], significantly stimulated renin secretion 19.3 1.9-fold (Fig. 5; N = 6, P < 0.001). When slices were transfered to fresh medium free

of stilbene maleimide during the third hour, the rate of renin secretion maintained at the stimulated level (14.7 2.3-fold but had no effect on renin secretion (line B of Fig. 6) in over the control), indicating that the sulfhydryl groups were consistent with our previous results [1j. By contrast, an imper- irreversively alkylated.

870

Doh et a!: Su!fhydryl group and renin secretion 35

Table 3. Stimulation of renin secretion by PCMPS in tissues with and without pretreatment with DTT

30

0Ui 0 a (C ID UI

C C

ID

Renin secretion (ng AI/100 mg/hr)

25

Control 42.7 4.0 Control 65.6 13.4 Control 71.6 20.0

20

15 10

DTr

8.04

Washout 19.5

1.78

Ratio 0.48

0.06

58.7

15.6

69.2

15.6

PCMPS

1.0

Control 76.7 17.7

3670 662 PCMPS

3590 509

Values are mean SE from 6 incubation flasks. Slices were incubated in the standard medium for three consecutive periods each for 60 mm. Tissues were incubated in the standard medium during the first period

5

for the control. During the second period, 2 groups of tissues were

0

incubated in the medium containing DTT (1.0 x 10-2) and 1 group in the

DTT-free medium (control). During the third period, tissues were

II

(Control)

Incubation period

Fig. 6. Effect of N-ethylmaleimide (NEM) on the stimulatory effect of

PCMPS on ran/n secretion. Tissues were incubated for three consecutive 60 mm. periods. Following the control incubation, renin secretion was stimulated by including PCMPS (5 X iO M) for two consecutive periods. Renin secretion was stimulated 30.2 2.55 fold during the first hour and slightly declined to 19.3 3.1-fold (A). Addition of NEM (1.0 mM) to the medium had no significant effect on renin secretion nor on the stimulatory effectof PCMPS (29 1.5; B). In the third series (C), renin secretion was first stimulated by PCMPS (26.0 2.5) and then by PCMPS and NEM during the third incubation period. NEM again had no effect on renin secretion stimulated by PCMPS. Each point is the

mean SE from 6 observations. Table 2. Effect of disulfide reducing reagents on renm secretion (ng AI/100 mg/hr)

Control 2-ME 2,3-DMP DTT

DTT 4.88 0.83

+ Reagent

98 115.0

18.3 23.1

68.5 28.2

98.9

18.1

11.7

18.6

4.88 1,75

Ratio 0.66 0.27 0.14

0.07 0.03 0.03

Values are mean SE from 6 incubation flasks for each group. Renal cortical slices were incubated in the standard medium for 60 mm each in the absence (Control) and then presence of 1.0 X 10—2 M 2-mercaptoethanol (2-ME), 2,3-dimercaptopropanol (2,3-DMP) or dithiothreitol (DiT). All these three ififreducing reagents significantly inhibited ream secretion (P < 0.001).

The third test of an involvement of specific sulfhydryl groups in the renin secretory process was explored by examining the interaction between PCMPS and NEM. As shown in Figure 6, suithydryl groups were irreversively alkylated with NEM before (line B of Fig. 6) and after (line C of Fig. 6) stimulation of renin secretion by PCMPS. Regardless of sequence of treatment with NEM, the stimulatory effect of PCMPS was not affected. The results indicate that the sulthydryl groups involved in the stimulatory action of PCMPS are either unreactive or inacessible to NEM. The possibility that sulfhydryl-disulfide interchange may play

a role in renin secretion was examined with use of three

disulfide reducing reagents (Table 2). DTT by itself up to 5 x iO M had no significant effects on renin secretion (Fig. 3B). However, the reducing reagent at 1.0 >( 10_2 M inhibited renin secretion by 86.0 3% (N = 6, P < 0.001) without further inhibition at 3.0 x 10_2 M (data not shown). The other two reducing reagents, 2-ME and 2,3-DMP, also significantly inhibited renin secretion (N = 6, P < 0.001). D'TT was most potent,

incubated back in the DTT-free medium (washout) or DTT-free medium containing PCMPS (5 x l0—). Ratio is the rate of renin secretion during the third period to that during the first period.

and the dithiol reducing reagents 2,3-DMP had twice greater inhibitory effect than that of its monothiol reducing congener 2-ME.

The results of the preceding series of experiments suggest that the cellular sull'hydryl-disulfide interchange may play an

important role in renin secretion. Thus, we compared the stimulatory effect of PCMPS on renin secretion from tissues with and without pretreatment with 1.0 x 10—2 M DTT. As summarized in Table 3, renin secretion inhibited DTT was only partially recovered following the removal of the reducing reagent, suggesting that the re-oxidation of reduced thiols by cellular process may be a rather slow or only partially reversible

process. However, the maximal stimulatory effect of PCMPS on renin secretion in terms of both absolute rate and relative change over the control was not affected by DTF (P > 0.05). It was reported that non-diuretic sulthydryl reagents such as PCMPS blocked and reversed natriuresis by diuretics with sulfhydryl reactivity such as ethacrynic acid and mersalyl [21, 221. To investigate if there is any interaction between PCMPS and diuretics in their stimulation of renin secretion, the stimulatory effect of PCMPS on renin secretion was evaluated in the absence and presence of diuretics that have or lack sulfhydryl reactivity. The presence of bumetanide or furosemide, which do not have sulfhydryl reactivity [3], did not affect the stimulatory effect of PCMPS either in terms of absolute or relative values (Table 4). On the contrary, the stimulation by submaximal stimulatory concentrations of PCMPS was additive to the stimulation by ethacrynic acid (Table 5) or by mersalyl (Table 6). However, the stimulation by maximal stimulatory concentration of PCMPS alone was comparable to that by the effects of combining PCMPS with either ethacrynic acid or mersalyl suggesting that their maximal stimulatory effects are not additive. Discussion

The results of the present study with the use of several sulfhydryl reagents that have differing degree of chemical reactivity and different permeability to the cell membrane suggest that certain specific membrane suithydryl groups are involved in the renin secretory process [11. The organic mercurials PCMB and PCMPS, which are known to have high specific

871

Doh et a!: Sulfliydryl group and renin secretion

Table 4. Stimulation of renin secretion (ng AI/l00 mglhr) by PCMPS in the absence and presence of loop diuretics

Table 5. Stimulation of renin secretion (ng AI/100 mglhr) by PCMPS alone and in combination with EA

Ratio

Control 71.8 12.0 Bumetanide 59.7 6.5 Furosemide 58.9 8.8

PCMPS 2391

367

2320 371

38.7

8.9

Bumetanide ÷ PCMPS

PCMPS Conc.

x l0

3 6 x l0— M 5

x 10

Control

51.1±4.6 52.8

PCMPS + EA

PCMPS 221 391

11.3

37.5±2.8

15

988

50 1149± 66

1061

85

1008±61

388

44.7

6.4

2328 499

39.9

4.5

Values are mean SE from 6 incubation flasks. Slices were incubated for three consecutive periods of 60 mm each in the standard medium

Values are mean SE from 6 incubation flasks. Slices were incubated

(Control), in the presence of varying concentration of PCMPS (PCMPS)

2783

393

Furosemide + PCMPS 2387

506

2723

in the absence (Control) and presence of 3 x l0— M bumetanide or

and then in the presence of both PCMPS and EA (3 x 10-2 M).

furosemide for the first 60 mm and then another 60 mm in the presence

of 5 x iO M PCMPS alone or in the presence of PCMPS and a loop diuretic.

reactivity with sulfhydryls and form readily reversible mercaptide bond [2, 191, were most potent among the tested reagents (Fig. 1). Two poorly permeating organic disulfides, 5,5'-dithiobis (2-nitrobenzoic acid) and 6,6'-dithiodinicotinic acid [5, 23], also stimulated renin secretion but to a much less extent than the two organomercurials did (Fig. 1). Furthermore, stilbene maleimide, a cell membrane impermeant alkylating agent [20],

Table 6. Stimulation of renin secretion (ng AIIl00 mg/hr) by PCMPS alone and in combination with mersalyl PCMPS Conc. 3

X iO M

6 x lO M 5 x lO— M

Control 37.6

41.2 40.6

6.2 4.9 3.5

PCMPS 173 265 1130

PCMPS + mersalyl

22 19 125

1850

132 177

1750

284

1539

Values are mean SE from 6 incubation flasks. Slices were incubated for three consecutive periods of 60 mm each in the standard medium, in

the presence of varying concentrations of PCMPS and then in the

effectively stimulated renin secretion (Fig. 5) but the cell presence of both PCMPS and mersalyl (3 x iO M). membrane permeant N-ethylmaleimide (NEM) had no effect [1]

(line B of Fig. 6). Diamide [13], t-butylhyperoxide [13, 14], 2-cyclohexene-1-one, and diethyl maleate [15], all of which are bond with PCMPS is readily reversible [19]. The stimulation by cell permeant agents known to rapidly decrease cellular re- stilbene maleimide was not reversible (Fig. 5), but the stimuladuced glutathione and other thiols, had no effect on renin tion of PCMPS was reversible upon the removal of sulihydryl secretion (Table 1). Such variable effectiveness of these sulfliy- reagents from incubation medium [1]. Furthermore, the stimudryl reagents on renin secretion most likely reflects their lation by PCMPS was prevented and reversed by dithiothreitol reactivity with and accessibility to pertinent sulfhydryl groups. and L-cysteine (Figs. 3 and 4). One simple explanation to The present results that only poorly permeant or impermeant account for these results is that exogenously provided sullhysulihydryl reagents stimulate renin secretion suggest that the dryl reagents block the formation of mercaptide bond between sulfliydryl groups involved in renin secretion are likely super- PCMPS and sulfhydryl groups on membrane proteins, and ficially located in the cell membrane. Also supportive for this remove PCMPS from the mercaptide complexes. Additional evidence for the specific involvement of sulfliypossibility is the finding that the stimulation of renin secretion by PCMPS was rapid without progressive increase with increasing incubation time (Fig. 2). Stimulation of renin secretion by stilbene maleimide (Fig. 5) but not by NEM (Fig. 6) suggests

dryl groups in renin secretion comes from the observations that

plasmic glutathione, its concentration in the membrane may not achieve high enough to alkylate pertinent suithydryl groups for stimulation of renin secretion. The lack of effects of NEM and reagents depleting intracellular reduced glutathione (see above) on renin secretion suggests that the redox state of the intracel-

disulfides leads to stimulation of secretion in some manner (Fig.

6,6-Dithiodinicotinic acid and 5,5'-dithiobis (2-nitrobenzoic acid) stimulated renin secretion. These reagents are extremely that the membrane sulfliydryl groups are accessible but not specific for free sulThydryl groups and form mixed disulfide highly reactive to NEM. Since rapid penetration of NEM into bridges between the reagent and specific membrane sulfhydryl cells and trapped therein by reacting with the abundant cyto- groups [24]. Thus, it is reasonable to assume that formation of 1). Consistent with this possibility is the present finding that disulfide reducing reagents markedly inhibited renin secretion (Table 2). The inhibition appears to be caused by stoichiometric reduction of disulfides in that dithiol reducing reagents (2,3lular glutathione has no direct modulatory role in the redox DMP and DTT) had a twice the inhibitory effect of monothiol state of the membrane suilbydryl groups involved in renin reagent (2-DME). This result points to the possibility that the secretion. These findings may point to the location of the interchange between particular subclass of membrane sullhysuiThydryl groups in the outer phase of the cell membrane dryl and disulfide groups may play a key regulatory role in renin where the sulihydryl-disulfide interchange is not affected by the secretory process. Furthermore, the finding that the stimulatory effect of PCMPS on renin secretion was little affected by redox state of intracellular glutathione. It is of particular importance to determine whether the pretreatment of tissues with DTT (Table 3) suggests that the stimulatory effects of sulffiydryl reagents are exclusively attrib- majority of this particular subclass of membrane thiols is in uted to their reactions with sulThydryl groups. One approach reduced state. Miller and Farah reported that in vivo natriuretic effects of often used to test a specific involvement of sulthydryl groups is to use agents which react with sulfhydryl groups in different mercurial diuretics such as mersalyl were blocked and reversed chemical reactions. It is well known that the thioalkylation by by non-diuretic sulthydryl reagents such as PCMB, PCMPS and maleimides is practically irreversible whereas the mercaptide 2,3-DMP (BAL) [21]. In subsequent in vitro studies with

872

Doh ef a!: Sail hydryl group and renin secretion

isolated perfused thick ascending loop of Henle from rabbits, PCMB added to the tubular lumen was found to reverse the natriuretic effect of mersalyl [221. The antagonism by nondiuretic sulfhydryl reagents against the natriuretic effect of mersalyl suggests that sulfhydryl groups may be involved in the natriuretic action of mersalyl. The non-diuretic reagents may compete with mersalyl for sulfhydryl groups related to tubular sodium transport. Although sulfhydryl groups may be involved in both renin secretion and tubular sodium transport, they must be functionally independent in view of the opposite effects of PCMPS on renin secretion (stimulatory) on one hand and on

unlikely for several reasons. The stimulatory effects of PCMPS were reversed when PCMPS was removed [Fig. 3 of Ref. 1; 5, 7] or when exogenous thiols were added (Figs. 3 and 4) [8, 9]. Although high concentrations of PCMPS (>0.1 mM) have been demonstrated to increase cell membrane permeability, but only to small molecules such as monovalent cations and sucrose, but such changes only occurred after 30 minutes [2, 7]. By that time the maximal secretory effects of PCMPS are already expressed (Fig. 2) [5, 7—10]. Permeabilization of cell membrane by detergents sufficient enough for cytosolic proteins to leak out does not necessarily stimulate secretion [34—36]. Furthermore, pannatriuresis (inhibitory) on the other hand. Furthermore, no creatic 13-cells treated with PCMPS (0.1 m for 40 to 60 mm) additive effect on renin secretion of maximal stimulatory con- secreted insulin in response to glucose [6, 7], and no significant centrations of PCMPS to that of two sulfhydryl reactive diuretic morphological changes were noted [7]. Finally, we have found ethacrynic acid (Table 5) and mersalyl (Table 6) suggests that that incubation of slices in lyotropic salt solutions such as both non-diuretic and diuretic sulthydryl reagents stimulate KSCN almost completely inhibits, rather than stimulate, renin renin secretion through their reactions with the same subclass secretion (unpublished observation) as observed in other types of secretory cells [34, 35, 37]. Thus, it is unlikely that the effects of membrane sullhydryl groups.

Ethacrynic acid, mersalyl, bumetanide and furosemide, of PCMPS and other suithydryl reagents solely result from which are collectively called loop diuretics, are among the most "non-specific" lytic actions. We therefore favor the possibility

powerful pharmacological stimuli for renin secretion in vivo that sulthydryl reagents stimulate secretion by reversively [reviewed in 25, 26]. Since this group of diuretics is well activating some membrane event(s) of exocytosis by reaction established to inhibit the Na/K/2Cl cotransport in the thick with membrane sull'hydryl groups. ascending loop of Henle [22, 27, 281, the inhibition of the cotransport at the macula densa has been postulated to be Acknowledgments directly responsible for the diuretic-induced stimulation of renin We thank Carol A. Enterline for her help in the preparation of this secretion [25, 26]. From recent in vitro studies direct evidence manuscript. Peter M. Hwang was a summer research student fellow

for the presence of furosemide-sensitive Na/K/2Cl cotrans- from Cornell University. This research was supported by the Biomedport at the macula densa was presented [29—31]. However, ical Research Support Grant. Present address of-K.-W. Cho: Departunlike the powerful stimulatory effect of bumetanide and furo-

ment of Physiology, Jeonbug National University Medical School,

semide on renin secretion in vivo, we (Table 4) and other Jeonju, Korea.

investigators [32, 33] failed to observe any consistent stimulaReprint requests to Chun Sik Park, M.D., Ph.D., Department of tory effect in vitro in renal cortical slices preparations even at Physiology, University of Massachusetts Medical School, 55 Lake

millimolar concentrations at which they should completely Avenue North, Worcester, Massachusetts 01655, USA. inhibit the cotransport [27, 28]. Furthermore, in the presence of

millimolar concentrations of bumetanide and furosemide, PCMPS (Table 4) and ethacrynic acid [4] were able to stimulate renin secretion equally well as in their absence. The foregoing data suggest that sulfhydryl reagents of both with and without

diuretic activity stimulate renin secretion through their reactions with same sulfhydryl groups which have no functional role

in the Na/K/2Cl cotransport. These findings raise the possibility that loop diuretics with and without sulfhydryl reactivity may stimulate renin secretion by multiple cellular mechanisms of actions rather than by a single cellular mecha-

nism of solely inhibiting Na/K/2Cl cotransport. The potential importance of sulfhydryl groups in the secretory process was first noted by Douglas, Ishida and Poisner in 1965 [8] who found a marked reversible stimulation of vasopressin release from rat neurohypophyses in vitro by PCMB and other sulfhydryl reagents. In the intervening years, organic mercurials and disulfides were found to stimulate secretion of various hormones in a variety of other secretory cells [5—7, 9—11]. These findings coupled with our present findings that poorly permeant or impermeant suiThydryl reagents stimulate renin secretion point to the possibility that an exchange between sullhydryl-disulphide groups on the plasma membrane proteins promote secretion. Renin secretion might result from "non-specific" effects of sulfhydryl reagents leading to cell membrane lysis. This is

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