The role of subcellular organelles in hormone secretion

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Experimental

THE

ROLE IN

OF

Cell Research

93 (1975) 388-394

SUBCELLULAR

HORMONE

ORGANELLES

SECRETION

The Interactions of Calcium, Vitamin A, Vinblastine, and Cytochalasin B in PTH Secretion B. S. CHERTOW, G. A. WILLIAMS, GEORGIANN R. BAKER, R. D. SURBAUGH and G. K. HARGIS Departments

of Endocrinology

and Medicine,

of Illinois College of Medicine,

VA West Side Hospital and University Chicago,

IL 60612, USA

SUMMARY To determine the role of subcelhdar organelles in hormone secretion, we studied the interaction of low calcium concentration (low Ca), retinol (vitamin A, vit A), vinblastine (VB), and cytochalasin B (CB) in parathyroid hormone (PTH) secretion. Bovine parathyroid tissue pieces were incubated in media containing the above agents. Vit A stimulated PTH release to a mean of 170 % of control. This effect of vit A was diminished when tissues were simultaneouslv stimulated with low Ca and. furthermore, absent when tissues were pre-incubated in low Ca. VB had no effect on low Ca-stimulated secretion, but did inhibit vit A-induced secretion in the presence of low Ca. CB stimulated PTH secretion to a mean of 150% of control during the second and third hours of incubation. CB had at least an additive effect with low Ca in stimulating PTH secretion, with a more prompt and greater response than seen in normal calcium. VB did not inhibit the acute effect of CB-on sdcretion in normai calcium media, but did inhibit CB-induced secretion during the third hour of incubation. None of the agents stimulated the release of lysosomal cathepsin D, and vit A and CB did not stimulate the release of LDH. Our results suggest that; (1) vitA and low Ca stimulate PTH secretion through a common pathway involving the cell membrane; (2) CB stimulates PTH secretion through a separate effect on the cell membrane or submembrane microfilaments, which normally retards secretion of PTH; and (3) microtubular proteins may facilitate basal secretion of PTH, but are not involved in low Ca-stimulated secretion of PTH.

Microtubules, microfilaments, and lysosomes are involved in many cellular biological processes [9, 24, 311, including hormone secretion [13, 20, 25, 29, 331. Of particular interest to us has been the role of and interrelationship of these subcellular

Address reprint requests to: B. S. Chertow, Endocrinology Section (M.P. 172), Va West Side Hospital, P.O. Box 8195, Chicago, IL 60680, USA. Exptl Cell Res 93 (1975)

organelles and membrane-active agents in the release of hormone. In previous preliminary studies of parathyroid hormone (PTH) secretion from bovine parathyroid tissue pieces [5, 71, we showed that (1) retinol (vitamin A, vit A), a membrane surface-active agent [3], increased PTH secretion; (2) cytochalasin B (CB), an agent that disrupts microfilaments [31] but also modifies membrane topography [ 12,281 and

Subcellular organelles and hormone secretion function [27], increased PTH secretion; and (3) vinblastine (VB), a microtubule disrupter [24], inhibited secretion. Our ultrastructural study of parathyroid tissues [6] showed that low calcium-, vit A-, and CBstimulated secretion may be mediated through changes in the cell membrane, secretion granule membrane and/or phagolysosomes, and that microtubules may facilitate PTH secretion. The present study of the effects and interactions of low calcium, vit A, CB, and VB in PTH, cathepsin D, and LDH release were undertaken to relate the release of PTH to membrane and other subcellular organelle functions,

MATERIALS

AND METHODS

The details of parathyroid tissue incubation and parathyroid hormone immunoassay have been described previously [6, 7, 321. In brief, bovine parathyroid tissue pieces are incubated in Eagle’s media with 10% calf serum in 1.5 mM calcium. in 95 % 0, and 5 % CO,. at 37’C. The first 2 h of incubation-served as an equilibration period. Basal or zero time secretion was determined by measuring PTH in media collected durina the second hour of incubation. Then the media composition was changed to contain normal calcium, 1.5 mM, or low calcium, 0.75 mM, and the following constituents alone or in combination: vit A, 0.35 &ml; VB, 8.0 &ml; or CB. 5.0 m/ml. In one exneriment .on the interrelation between-the effects of low calcium and vit A, tissues were incubated in 0.75 mM calcium during the equilibration period (low calcium preincubation) and test period. Secretion rates of PTH were determined half-hourly or hourly using a modification of the mdioimmunoassay method of Amaud [2]. Matched controls were run throughout incubation in every experiment. Hourly secretion rates of control and test tissues were expressed as a per cent of the zero time base line. Then, the difference in secretion between control and test tissues was expressed as a per cent of the time-matched control. Statistical significance was determined by the paired r test or analysis of variance [l 11. Previous studies of PTH secretion using this model showed that these tissues respond appropriately to suppression and stimulation by high and low calcium concentration, respectively [32]. Detailed ultrastructure studies show intact cells and subcellular organelles responsive to physiologic stimuli and test agents

161.

Lysosomal cathepsm D activity was measured by the microassay of Hille et al. [16] using SH-labeled

389

hemoglobin as a substrate. The activity is determined by measuring the release of trichloracetic acid-soluble radioactivity. This microassay is approx. 300 times more sensitive than the method of Anson [1] and permits the measurement of low levels of cathepsin D release from parathyroid tissue into incubation media. To eliminate the possibility that increases in hormone release were related to nonspecific leakage secondary to membrane disruption, we monitored the release of lactate dehydrogenase (LDH) in the presence of vit A and CB. LDH was assayed by the LDH-P method (Calbiochem, La Jolla, Calif.), by determining the rate of oxidation of NADH to NAD+ by measuring the change in EM0 during the conversion of pyruvate to lactate. LDH was assayed on the same day of experiments since enzymic activity decreased with storage.

RESULTS Interactions between vitA and low calcium in PTH secretion Confirming previous studies, vitA in 1.5 mM calcium significantly stimulated PTH secretion throughout incubation to 170% of its 1.5 mM calcium control (~~0.001). In the presence of 0.75 mM calcium concentration, vit A stimulated mean PTH secretion throughout incubation to 130% of its low calcium control Q~0.01). Thus, the stimulating effect of vit A in low calcium concentration was less than that seen when vit A was incubated in media containing normal calcium concentration. When tissues were preincubated for 2 h in media containing a low calcium concentration, zero time PTH secretion increased approx. 3to 4-fold, and subsequent addition of vit A had no further significant stimulating effect (table 1 A, 1-3, and fig. 1). Interactions of VB and vit A on PTH secretion in low calcium media VB did not inhibit low calcium-stimulated secretion. However, VB did diminish the further stimulating effect of vit A in low calcium-stimulated tissue at 60 min, and completely inhibited the vit A effect at 120 min of incubation (table 1B, 1-2, and fig. 1). Exptl Cell Res 93 (1975)

390

Chertow et al.

Table 1. The interactions of vitamin A, calcium and vinblastine in PTH secretion Time (min) Expt

0

A. Effect of low calcium stimulation on vitd-induced (1) Control-l.5 mM Ca 512+ 41” vitA+l.S mM Ca 483f35 NS (12) (2) Control-O.75 mM Ca 571+17 vit A+0.75 mM Ca 486+41 NS (14) (3) Control-O.75 mM Ca 1 432k226 vit A+0.75 mM Ca 1834+284 NS V-3

30

60

120

180

125+ 9b 218fl8 ***

112+11 1751tl3 **

127+14 187+16 *

-

207rt20 268+29 **

242f26 291+27 *

-

136+26 121+26 NS

112f33 107+30 NS

231+26 274f27 NS 86+28 82+29 NS

292flO 274?rl4 NS 290+ 6 NS

266+15 248523 NS 280f 19 NS

set -

B. Effect of VB on low calcium-stimulated and vitd-induced secretion (1) Control-O.75 mM Ca 472+ 27 244+34 307f33 VBk0.75 mM Ca 453+ 36 306f35 242+37 NS NS NS (6) (2) VB+vit A+O.75 mM Ca 495+ 24 295f45 355f35 NS NS NS (6)

a Zero time values represent a 60 min collection of media prior to the addition of test agents and are expressed in pg/mg wet wt/hfS.E.M. During this period, tissues were incubated in 1.5 mM Ca with the exception of expt A-3 in which tissues were preincubated in 0.75 mM calcium before the addition of test agents. b The PTH secretion rates after zero time baseline are for the 30 or 60 min period preceding the indicated time and are expressed as a per cent of baselinefS.E.M. Significance levels for the difference between the indicated control and test experiments are indicated by: *p
Interactions between CB and VB in PTH secretion in normal calcium CB stimulated mean secretion to 137% of control (pcO.01). VB did not affect CBstimulated secretion during the first 120 min of incubation; however, at 180 min, VB-inhibited CB-induced secretion to 77 % of its CB alone control (table 2A, l-2, and fig. 1). Interactions between CB and low calcium in PTH secretion CB in media containing low calcium concentration stimulated the mean secretion to 138% of low calcium alone (p
containing a low concentration of calcium, in comparison with the effect of CB incubated in media containing a normal concentration of calcium (table 2 A, 3, and fig. 1). Effects of vitA, CB, and VB on cathepsin D release into media Control tissues released small amounts of cathepsin D into media. Vit A, CB and VB had no effect on the release of cathepsin D (table 3). Effects of vitA and CB on LDH release into media Vit A and CB had no significant effect on LDH release. This indicated that the in-

Subcellular organelles and hormone secretion 15mM

0.75mM

Co

Ca

I

140 Ix) loo 00 60 40 20 0

v1t A

VB

VB + Vat A

CB

vtt A

VB

VB + ‘At A

CB

Fig. 1. Abscissa: agent; ordinate: PTH release expressed as a per cent of control. Effects of low calcium (0.75 mM) on vitamin A- (0.35 &ml) and cytochalasin B- (5.0 pg/ml) induced and vinblastine- (8.0 pg/ml) inhibited mean PTH secretion rates. Cumulative secretion over 3 h of incubation was determined and the mean hourly secretion rate calculated and expressed as a per cent of its matched 1.5 mM or 0.75 mM calcium control. Experiments in which tissues were incubated in low calcium media are shown in the right panel. In one experiment, indicated by the hatched bar, tissues were preincubated in 0.75 t&I calcium during the zero time collection prior to the addition of test agents. For comparison, experiments in which tissues were incubated in normal calcium media (1.5 mM calcium) and previous studies with vinblastine [7] are shown in the left panel. Half-bracket indicates +1 S.E.M. * ~~0.05; ** ~~0.01; *** p~O.001; NS, not significant; ()=no. of expts.

391

[6]. An alternative explanation is that vit A and low calcium both modify the cell and/or secretion granule membrane, and in so doing, facilitate fusion of the cell and secretion granule membrane and release of hormone; initial changes of the membrane in response to low calcium may supersede the membrane effects of vit A. Supporting this possibility are recent studies showing that (1) vit A and calcium ion directly interact with membranes [3, 15, 193,and modify the stability of membrane monolayer-s[22], and (2) ultrastructurally [6], low calcium and vit A produce an increase in the cellular membrane convolutions and microvillous processes, respectively. Further support for the concept that vit A is indeed acting through an interaction with the cell and/or secretion membrane, are findings that show that HC or vit E, agents that antagonize the effect of vit A on membranes [19, 301, also diminish vit A-induced PTH secretion

PI. In previous studies of tissues incubated creasein PTH secretion observed with vit A and CB was not the result of nonspecific in 1.5 mM calcium media, VB inhibited PTH secretion [7] and decreased the number of leakage of hormone (table 4). microtubules [6]. These findings suggested that microtubules facilitated basal PTH DISCUSSION release. In the present study, VB has no Vit A clearly stimulated PTH secretion in inhibitory effect on tissues incubated in low vitro. On incubation and preincubation of calcium concentration; therefore, microtissues in low calcium media, this stimulat- tubules do not appear to have a role in low ing effect of vit A was diminished and ab- calcium-stimulated PTH secretion. These sent, respectively. These findings suggest findings are consistent with other studies that low calcium and vit A may act through [4] showing that at a calcium ion concentraa common pathway. One possibility is that tion that stimulates PTH secretion, microvit A increases the release of secretion tubule formation is actually inhibited and granules, and thus, depletion of granules reversed. Since vit A acts at the membrane level, after exposure to a low calcium stimulus does not allow for further effect of vit A. and VB inhibits the effect of vit A in normal However, this possibility is not supported or low calcium concentration, the effect of by our ultrastructural studies, which indeed vit A may be mediated through microshow an increase in granules in the pres- tubular proteins, perhaps in the cell memence of vit A or low calcium concentration brane. Other studies with VB or colchicine Exptl Cell Res 93 (1975)

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Chertow et al.

Table 2. The effects of calcium and vinblastine on cytochalasin B-induced secretion Time (min) Expt A. (1) Control-l.5 mM Ca CB+l.S mM Ca

(12) (2) Control-CB+1.5 mM Ca VB+CB+l.S mM Ca

(6) (3) Control-O.75 mM Ca CB+0.75 mM Ca

(8)

0

30

60

120

180

498+ 92” 507f 88 NS 708+133 675+147 NS 678k109 548f 49 NS

151+11 157fll NS 149222 135f 7 NS 139413 184+16 *

140+10 181+17 *

104~10 181+17 **

104+ 5 145+10 **

154+29 166+16 NS 175+12 262+10 ***

134f 16 145f 14 NS 179f22 224+ 16 NS

164f16 114f 5 ** 174+20 205f13 NS

a Zero time values represent a 60 min collection of media prior to the addition of test agents and are expressed in pdmg wet wt/hfS.E.M. During this period all tissues were incubated in 1.5 mM Ca. CB, cytochalasin B, 5.0 j&ml; VB, vinblastine, 8 pg/ml. For other explanations, cf table 1.

have also suggested that microtubular or VB-sensitive proteins exist in the cell membrane [23,28] and that VB may stabilize the cell membrane [26]. CB in low calcium concentration, in contrast to our findings with vit A in low calcium concentration, had at least an additive effect with low calcium in stimulating PTH secretion. These findings suggest that low calcium and CB act through different

and perhaps interacting subcellular mechanisms involving the membrane and/or submembrane microfilaments. CB is also known to inhibit hexose transport across the cell membranes, but this effect appears paradoxical to its stimulating effect on secretion [2 11. The inhibition of CB-stimulated PTH release by VB later during incubation may not be related to disruption of cytoplasmic

Table 3. The effect of vitamin A, cytochalasin B, and vinblastine on cathepsin D release from parathyroid tissue Time (min) 30b

0” base

Expt

line (dpmlmg wet wt/h)

A. Control Vitamin A B. Control Cytochalasin B C. Control Vinblastine

11 48O’kl 12 040 fl 24 080*+2 29 960 +3 24 840 +2 23 720 k2

760 880 680 560 120 040

(percentage of 119+11 119*19 104rt 9 98f 3

6ob base

120

180

96f 6 87f 5 1OOf 8 108f 6 88+12 74f 7

83+5 84+9 81f9 82f6 73k4 79+9

line)

108+ 8 112+13 98+ 6 lOO+ 8 86+ 8 98f30

a Zero time or base line represents a 60 min collection of media prior to the addition of test agents and is expressed as dpm released/mg wet wtlh. b Secretion rates after base line are for the 30 or 60 min period preceding the indicated time. c Values represent the mean+S.E.M. for 20 experiments in A, 7 experiments in B, and 4 experiments in C. * Base line values in experiments B and C are greater than A because the total 3H-hemoglobin substrate in the assay incubation mixture was increased in experiments B and C. Exptr CeIlRes

93 (1975)

Subcellular

Table 4. The effect of vitamin A and cytochalasin B on LDH release into mediaa LDH (mU/mg wet wt) Control Cytochalasin B Vitamin A

21.Ok3.2” 16.5k7.2 15.7f2.0

(1 Total enzymic activity per mg wet wt of tissue released into media during 180 min of incubation. * Values represent the meanfl S.E.M. of 4 experiments.

organelles

and hormone secretion

393

clarify the role of lysosomal activity in hormone secretion, our negative findings suggest that lysosomal enzymes, if directly involved in hormone secretion, are not released by exocytosis from lysosomes or from the secretion granule itself. We thank Mrs Anna B. Baker and Miss Joanne C. Vaccaro for their secretarial assistance.

REFERENCES microtubules by VB. In ultrastructural studies [6] we found that VB, besides decreasing the number of microtubules, produced stacking of endoplasmic reticulum. Therefore, the late effect of VB on cytochalasin B-induced secretion may be indirectly related to changes in the synthesis of PTH or transfer of prohormone from the endoplasmic reticulum to the Golgi for packaging into the secretion granule [14]. The release of PTH may be related to lysosomal activity. An increase in lysosomal activity has been observed ultrastructurally in newt parathyroid tissues on stimulation of PTH secretion by low calcium [17]. Since vit A and CB stimulate lysosomal enzyme release [S, lo], and, in our ultrastructural studies [6], increased phagolysosomal activity, the effects of these agents on PTH secretion may be mediated through lysosomal enzymes. Since a catheptic enzyme must convert precursor to secreted forms of PTH and a catheptic enzyme may act on basement membrane [18] to facilitate release of hormone, we studied the release of lysosomal cathepsin D into media. However, neither vit A, CB, or VI3 affected the release of cathepsin D. Although studies on tissue levels of cathepsin D as well as specific catheptic enzymes at the biochemical and ultrastructural level are in progress to

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