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Catecholamine secretion from bullfrog adrenals in response to osmotic changes
Cbmp. Biochem.Phwiol.Vol. 94A. No. 3, pp. 539-548.1989 Printed in Great Br>tain
;
0300-9629/89 %3.00+0.00 1989 Pergamon Press plc
CATECHOLAMrNE SECRETION FROM BULLFROG ADRENALS IN RESPONSE TO OSMOTIC CHANGES HIROSHI KITA and EMIKO YASUGI-NAGAOKA Department
of Physiology,
Kawasaki
Medical
School,
Kurashiki,
Okayama
701-01,
Japan
(Receiced 24 April 1989) Abstract-l. High K +-induced catecholamine (CA) secretion from bullfrog adrenals was suppressed in hypertonic solutions made by adding sucrose or NaCl and enhanced in solutions made hypotonic with reduced [NaCI], 2. When the preparation was transferred from a solution made hypertonic with sucrose or NaCl to a normal saline solution. CA secretion was increased transitorily. 3. The magnitude of the transitory response depended on the duration of soaking in the hypertonic solution. 4. Higher tonicities produced larger transitory responses, irrespective of whether the solute added was sucrose or NaCI. 5. When the preparation was passed from normal saline to a hypotonic solution made by lowering [NaCl],. a similar increase in CA output was observed. 6. The transitory effect of the osmotic gradient was similarly observed in low Ca’+, Mg’+ EGTAcontaining solutions and in solutions whose sole divalent cation was Mg?+.
INTRODUCTION
Changes in the tonicity of the bathing solution markedly affect the release of transmitter at the vertebrate neuromuscular junction: hypertonic solutions increase the rate of spontaneous quanta1 release of acetylcholine recorded as miniature endplate potentials (MEPPs) and hypotonic solutions decrease it (Fatt and Katz, 1952; Furshpan, 1956; Kita and Van der Kloot, 1977). Strongly hypertonic solutions reduce the number of quanta released from stimulated nerve terminals, which is manifested by the amplitude of end-plate potentials (Kita and Van der Kloot, 1977; Thesleff, 1959). Although only a few papers have reported on the effect of tonicity on the secretion of catecholamine (CA) from adrenals, hypertonic solutions have also been shown to enhance spontaneous secretion and to inhibit secretion evoked by high K+ or nicotine (Ladona et al., 1984). CA secretion from isolated adrenal chromaf?in ceils has also been suppressed by hypertonic solutions (Holz and Senter, 1986: Knight and Baker, 1982: Pollard et al., 1984). Recently, it has been shown that the rate of fusion of phospholipid vesicles with planar phospholipid bilayer membranes is enhanced when the side containing the vesicles is hypertonic to the opposite side (Finkelstein et al., 1986). The number of fusion events induced by antidiuretic hormone in the toad urinary bladder decreases when the mucosal bathing medium of the bladder is made hypertonic (Kachadorian et al., 1981}. These reports suggest that the osmotic entry of water into intracellular vesicles which are in contact with the plasma membrane is essential for the exocytotic release of the vesicular contents and that the release is accomplished by vesicular swelling and subsequent rupture of vesicular and plasma membranes at the contact site.
We have studied the effects of osmotic pressure and its changes on CA secretion from bullfrog adrenals. By measuring concomitant secretion of dopamine-fihydroxylase, it has been shown that CA secretion from frog adrenals induced with high K’ or the ionophore X-537A involves exocytosis (Ricci et al., 1975). When the adrenal is transferred from hypertonic to isotonic, or from isotonic to hypotonic, there will be an interval during which the interior of the chromaffin cells is hypertonic to the extracellular fluid. This duplicates the circumstances that the above reports have predicted promote exocytosis. When the osmolarity of the extracellular solution was abruptly reduced, a period of enhanced CA secretion was observed. Some of the results have appeared in a short communication (Kita er al., 1982b).
MATERIALS AND METHODS A pair of adrenals with the attached kidneys were dissected from a pithed bullfrog, Runa cute.~heiana. Normal saline solution containing (in mM) I15 NaCI, 2 KC], 2.3 CaCl?. 4NaHC0, and IO glucose, with an osmolarity (mosmoljl) of 235 was used. Preparations were then stored overnight in oxygenated saline solution in a refrigerator to stabilize the spontaneous secretion rate. Some of the advantages of using isolated frog adrenals for studying CA secretion have been described elsewhere (Van der Kloot et al.. 1974). The next morning the adrenals were kept for 0.5-t hr in oxygenated saline solution at room temperature, after which they were passed at 5min intervals through a series of small beakers, each containing 3 ml of oxygenated solution. Before each transfer, solution adhering on the surface of the preparation was blotted off with filter paper. The osmolarity of the solution was changed by adding sucrose or NaCI, or by reducing NaCI. The CA released into the solution was determined by the hydroxyindole method (Laverty
and Taylor, 196X) using a throrescence spectrophotometer (HITACHI MPF-3). The details of the method have been described together with the detailed experimental procedure (Van der Kioot eit al.. 1974). Blood contains CA (Holzbauer and Sharman. 1972). Therefore, the inferior vena cava supplying the kidneys and adrenals was perfused with normal saline by a hypodermic needle inserted into the vessel before they were isolated.
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200
1 300
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RESXXTS High K’
-induced CA secretion irz hyprtonic
snhtiorts
When bullfrog adrenals are soaked in hypertonic solution, the ostnotic gradient across the secretory granule membrane works against granule swdling and therefore prevents exocytosis (Fink&stein et nl., 1986). Under these conditions it is expected that the stimulated secretion of CA with high K + will be suppressed. Xn Fig. IA, the tonicity was raised by adding 100,200 and 300 mM sucrose to the normal saline solution. CA secretion was stimulated with 40 mM KCI. fn solutions ~ontainjng 200 and 300 mM sucrose, the stimulated secretions were decreased su~stant~al~~, being 74 and 42% of the response in the normal saline, respectively. In other experiments in which solutions containing 500 mM sucrose were used, stimulation with 4OmM KC1 was ineftectivc. Raising of tonicity was also achieved by the addition of 50, 100 and 15OmM NaCl (Fig. 1B). The high K”-induced secretions were suppressed in 100 and 150mM NaCl solutions, being 69 and 25% of the control response, respectively. Therefore, the depressive effect of hypertonicity was independent of the chemical species producing it. It should be noted that the transition from 300 mM sucrose or 150mM NaCl to the normal saline produced a transitory increase in CA secretion, which is the main theme of the present work. On the ather hand, when the osmolarity was raised by the addition of 0, 100, 200 and 300 mM glycerol, CA secretion induced by 40 mM KC1 was not suppressed. In other experiments, CA secretions, in response to increasing ~on~~~trations of KCI (40, 60 and 80 mM). were examined in the presence and absence of atropine sulfate (10-’ g/ml) and hexameth~~nium chloride (5 x IO-’ g/ml)_ There was no discernible difference in the secretory response between the two kinds of solutions. Therefore, these drugs were omitted in the experiments using high K’
A ~y~otoni~ en~~~ronrne~t is considered to be favourable for vesicle swelling, which promotes exocytosis (Fink&&in ef al., 1986). Figure 2 shows that stimulation with 40mM KCI produced larger responses at lower tonicities, the responses in -25, -50 and - 75 mM NaCI solutions helng I .07, I.30 and 2.80 times that in the normal sahne solution, respectively. The reason for the delayed enhanced secretions after stjrnu~~tjon is unknown. Basal CA secretkm in j~~~@~t~~~~ s~~~~~#~~~ As shown in Fig. 3, the basal secretion of CA was affected slightly by hypertonic solutions with sucrose
Ttme Lmm) Fig, 1. The effects of hypertonic solutions on high K +induced CA secretion from isolated bulKrog adrenals. During the shaded intervals the solutions contained 40 mM KCI. In this and the following experiments in which 40 mM KC1 was used to stimulate secretion, the osmoiarity of the solutions without 40 mM KCI was adjusted to that with the hieh KS bv addine 80 mM sucrose. Tonic& was raised bv the addition of either sucrose (A) or NaCl (5). The figures across the top show the amount (in m&l) of added sucrose (A) or NaCI (B). The values for CA (in ~g~rnl~5rnjn) are averages from four (A) and three (B) experiments. The lines indicate the standard deviations of the measured values,
The broken line represents the average value before any application of high KC andior hypertonic solution.
below 50OmM, but the effect was not straightforward. In solutions containing 100 and 200mM sucrose, the secretion was suppressed stepwise. Then it was increased progressively as the concentration of added sucrose was raised from 300 to 500mM. the rest& being identical to that shown by the shaded bars in Fig. 6. Transition from SO0 to 0 mM sucrose produced a substantial increase in secretion as afready mentioned in Fig. l.
The bullfrog adrenal. is not as sensitive to changes in osmolarity as the frog neuromuscular junction (Fatt and Katz, 1952). The addition of 300 mM sucrose to a normal saline solution sometimes did not mcrease the secretion of CA. When 500 mM sucrose was added, the rate of CA secretion rose slowly during 15 min of exposure (Fig. 4). On return to the isotonic solution, the CA secretion substantially increased transitorily. Then the rate of secretion
541
Osmotic pressure and catecholamine secretion 0.25 -0 NaCl
j
-25
/
j
-50
-75
E
E 0.20 * & -a i y
0.15 -
9 z .$ 0.10 .E! % 5 0.05 -
0
I--Time
( min
h 150
100
0
)
Fig. 2. The effects on high K+-stimulated secretion of hypotonicity produced by reducing the concentration of NaCI. During the shaded intervals the solutions contained 40 mM KCI. The figures with a minus sign across the top show the amount (in mM) of NaCl removed from normal saline. The values for CA are the averages from three experiments.
declined gradually toward the initial level. The transfer was repeated four times and each time there was a transitory, substantial increase in secretion. The level of the basal secretion became progressively higher during the experiment. The magnitudes of the transient increases shown by the first clear bars following the shaded ones in Fig. 4 were, from the first bout to the fourth, 7.86, 9.00, 10.57 and 11.00 times the control value shown by the broken line. The reproducibility of the result indicates that the transitory response is not due to a breakdown of the chromaffin cells.
In experiments like that illustrated in Fig. 5, a pair of adrenals were soaked for 5, 10, 1.5 and 30 min in solutions containing 500 mM sucrose. After each soaking, the preparation was returned to the initial, isotonic saline. The CA output was enhanced depending upon the soaking duration, although there was no difference between 5 and 10 min soaking. The sizes of the increases were 4.64,4.57, 6.36 and 18.57 times the control value for 5, 10, 15 and 30 min soaking, respectively.
It is expected that until intracellular fluid equilibrates osmoticafly with hypertonic extracellular solution, the osmotic pressure gradient across the granule membrane produced on return to isotonic
An example of the dependence of response on osmotic pressure is shown transitions from solutions containing and 700 mM sucrose to normal saline
solution will be greater when the soaking duration of the preparation in the hypertonic solution is longer.
Time
the transitory in Fig. 6. The 100, 300, 500 enhanced the
( min)
Fig. 3. The effects of hypertonic solutions on basal secretion. The figures across the top show the amount (in mM) of sucrose added to a normal saline solution (0 mM) whose osmolarity was 235 mosmol/l. The values for CA are the averages from three experiments.
HIR~SMIKITA and EMIKOYASUGI-NAGAOKA
542
0
50
100 Time
Fig.
4. The effects of changes
during
which
the solutions
( mm)
in osmolarity on CA secretion. The shaded bars are for the time intervals contained 500mM sucrose. The values for CA are the averages from three experiments.
CA output to 1.82, 5.53, 7.53 and 14.88 times that of the control, respectively. Almost identical results were obtained when the solution was made hypertonic by adding NaCl rather than sucrose. The shifts from solutions containing 50, 150, 250 and 350 mM NaCl to normal saline solution increased the basal secretion 1.38-, 3.85, 5.96and 13.42-fold, respectively (averages from three experiments). The transitory responses are caused by osmotic pressure gradients, not by the chemical species. CA secretion in response to transition from isotonic to hypotonic The same circumstances as in the transition from hypertonic to isotonic are obtained when the preparation is transferred from isotonic to hypotonic
solution. In the experiment shown in Fig. 7, a pair of adrenals were first soaked in normal saline solution and then transferred to hypotonic solution (from open to stippled intervals). Except in a 90 mM NaCl solution (shown by -25 NaCl), the shifts from isotonic to hypotonic produced increases in CA secretion; 0.89-, 2.84- and 7.45fold increases for 90mM, 65 mM (-50) and 40mM NaCl (-7S), respectively. In the hypotonic solutions the increased CA secretion did not decrease. Rather it increased in 40mM NaCI. One possible explanation is that in hypotonic solutions water moves into the chromaffin cells, which helps the secretory granules to swell. This in turn accelerates the exocytotic release of CA (Finkelstein et al., 1986). Another possibility is that a fall in [NaCI], reduces the activity of the Na+-Ca* + exchange system of the chromaffin cell membrane
Fig. 5. The effects of varying the soaking duration in hypertonic solution. The durations of soaking the nreparation in solutions containing SO0 mM sucrose were 5, 10, 15 and 30 min as shown by the shaded bars. The values for CA are the averages from seven experiments
Osmotic
pressure
and catecholamine
secretion
543
Fig. 6. The effects of increasing tonicities on CA secretion. During the shaded intervals the solutions contained sucrose at the concentrations (in mM) indicated by the figures above the shaded bars. The values for CA are the averages from four experiments.
and consequently causes an accumulation of intracellular Ca2+ (Lastowecka and Trifaro, 1974); but this seems unlikely from the results shown in Fig. 8. On return to normal saline, the CA output decreased gradually toward the control level. Effect of restoration
of hypotonicity
on CA secretion
To examine whether sodium deficiency rather than an osmotic fall can increase CA output, the experiment illustrated in Fig. 8 was performed. The preparation was shifted from a normal saline solution to a NaCl-deficient solution that was osmotically restored to isotonic by adding an appropriate amount of sucrose. As shown in Fig. 8, shifts from normal to lower sodium concentrations did not cause transitory increases in CA secretion as long as the tonicity was kept constant. The CA output during the first 5 min
of the stippled intervals was 0.93, 0.93 and 1.18 times the control value for 90 mM NaCl + 50 mM sucrose, 65 + 100 and 40 + 150, respectively. Transitory
response in low Ca2+ solutions
The transitory response to osmotic changes was also observed in solutions with a low [Ca’+]. A pair of adrenals pretreated with low Ca2+ saline were subjected to transitions from hypertonic to isotonic in a low Ca2+, Mg2+ EGTA-containing solution (Fig. 9). The first and second transfers increased the control secretion 4.68 and 5.45 times, respectively. These results suggest that the transitory response was not due to stimulation of Cal+ influx. After the second transitory response, the secretion reached a plateau which was about four times the control. The cause of the increase is unclear.
Time
( min)
Fig. 7. The effects of decreasing tonicities on CA secretion. The stippled regions indicate the periods during which the concentration of NaCl was reduced by the amount (in mM) shown by the figures with a minus sign above the stippled bars. The values for CA are averages from three experiments.
HIROSHIKITA
544
and
EMIKO YASUGI-NAGAOKA
Timet min 1
Fig.
8. The effects
on CA secretion of reducing [NaClI, while keeping the tonicity isotonic. During the intervals the concentration of NaCl was reduced by the amount (in mM) shown by the figures wi% a minus sign above the stippled bars and the osmolarity of each solution was restored to normal by adding the appropriate amount of sucrose (in mM) as indicated by the figures with a plus sign. The values for CA are the averages from three experiments. stinnled
Trunsitory
response in Mg’+ solutions
Essentially the same results were obtained in solutions whose only divalent cation was Mg’+, again suggesting that the transitory stimulation of CA secretion was not due to stimulation of Cal+ influx. The preparation, which had been pretreated overnight in the refrigerator with 2.5 mM Mg’+ saline containing 1 mM EGTA and no Ca*+, was shifted from hypertonic (plus 500mM sucrose) to isotonic in the same solution. There was an interval of enhanced CA secretion following the transfer. The first and second transfers produced increases in secretion of 5.5 and 7.7 times the control value, respectively (average from three expe~ments). After the second transfer, the CA secretion remained
elevated, the level being about 5.5 times that of the control. The overall results obtained in Mg”+ salines were similar to those in low Ca2+ solutions.
The dependence of the transitory response on [Ca’+], was examined within the range of 0.1 to 1.0 mM (Fig. IO). The transition from 500 to 0 mM sucrose was performed at 0. I mM Ca* + and then the same transfer was repeated at 0.5 (Fig IOA), 0.7 (B) or l.OmM Ca2+ (C). In Fig. lOA, the transfer at 0.1 mM increased the control secretion (shown by the broken line) 12.52-fold and the same transfer at 0.5 mM produced an 8.75-fold increase. In Fig. 10B, the increase was 9.22fold at 0.1 mM and 6.96fold at 0.7 mM and in Fig. IOC, it was 7.91-and 4.72-fold at 0.1 and l.OmM, respectively. The values at higher Ca2+ concentrations were always smaller because of the elevated levels of control secretion, However, as shown in Fig. 10. the magnitudes of the transitory response were essentially the same at the two Ca” concentrations. It may generally be said that the transitory response is not enhanced by an increase in the extracellular concentration of Ca?” below 1 mM. This seems to be consistent with the results obtained in low Ca’+ solutions and in Mg2+ salines.
DISCUSSION 0
50
100
Ttme (msn 1
Fin. 9. The transitory response to an osmotic gradient in a Mg*+ EGTA-containing solution. The preparation was soaked overnight in a refrigerated low Ca*+ EGTA solution. The low Ca*+, EGTA solution was made without CaCl, but with 1 mM MaCl, and 1 mM EGTA (osmol=~240mosmol/l). D&g the intervals shown by the solid bars the low Ca*+, EGTA solution contained 500 mM sucrose. The values for CA are the averages from six experiments.
10-iCa zc,
Efect
of’ hypertonicit)
The osmotic hypothesis of Finkelstein et crf. (1986) predicts that secretion is suppressed in hypertoflic solutions. As the solution osmolarity is increased by adding sucrose (Fig. IA) or NaCl (Fig. 1Bf, CA secretion in response to 40mM K’ is progressively decreased. Depression of secretory activities by hypertonicity has also been reported in a wide variety of cells, including CA secretion from cat adrenals (Ladona et al., 1984). from isolated bovine adrenal
Osmotic
pressure
and catecholamine
secretion
545
B 0.06
c .E ‘E
0.04
Em $$& ‘u _” 0& 2E
IV
0.02
1
0
)
Time
( min ) 0.04
Time
,-. L
( min )
,
Time
( min )
Fig. IO. The influence of [Ca’+], on the transitory response. The [Ca’+], was increased from 0.1 mM to 0.5 (A), 0.7 (B) and 1.0 mM (C). The preparations were soaked overnight in refrigerated 0.1 mM Ca’+ saline solutions. During the shaded intervals solutions contained 500 mM sucrose. The values for CA are the averages from five (A), five (B) and six (C) experiments.
me&Nary
cells
rendered
leaky
by
intense
electric
fields (Knight and Baker, 1982) or by digitonin (Holz and Senter, 1986) and from cultured (Hampton and Holz, 1983) or dissociated (Pollard et al., 1984) chromaffin cells of bovine adrenal medulla. Our results and these examples seem to support the hypothesis. Though there is a possibility that the above-mentioned inhibition of secretion may not be caused by a direct effect of hypertonicity but by the elevation in ionic strength (Holz and Senter, 1986), we suggest the osmolarity itself, rather than ionic strength, is responsible for the decrease in CA secretion caused by hypertonicity in the present study, since CA secretion is suppressed to a comparable degree when osmolarity is increased to a similar extent with either sucrose or NaCl (Fig. 1). High K+-stimulated aldosterone secretion was also inhibited, to a similar extent, when the solution osmolarity was increased by the addition of NaCl, sucrose or mannitol (Schneider et al., 1985). When glycerol was used as the osmoticant, the inhibitory effect of hypertonicity on high K+stimulated secretion was no longer observed. At the frog neuromuscular junction, the frequency of MEPPs elevated by the addition of glycerol returned to the initial level within 15 min (Furshpan, 1956). The inability to depress stimulated CA secretion and the transient effect on the MEPP frequency of glycerol can be explained by assuming that glycerol molecules enter the cell interior much more easily than sucrose, reducing the osmotic gradient across the vesicle membrane. AS shown in Fig. 3 and during shaded intervals in
Fig. 6, hypertonic solutions with sucrose higher than 300 mM increase the basal CA secretion gradually, as has been shown previously (Ladona et al., 1984), though the critical concentration of sucrose is different. The basal level of norepinephrine secretion from pheochromocytoma cells (Englert and Perlman, 1981), the secretion of atria1 natriuretic factor from dispersed perifused myocytes of the rat (Gibbs, 1987) and parathyroid hormone secretion from dispersed bovine parathyroid cells (Chen et al., 1987) have been increased markedly by increased medium osmolality. It is known that hypertonic solutions produce marked increases in MEPP frequency at the frog neuromuscular junction, which is much more sensitive than frog adrenals to changes in osmolarity (Fatt and Katz, 1952; Furshpan, 1956; Kita and Van der Kloot. 1977). These phenomena seem to be a drawback to the osmotic hypothesis. However, the mechanism for the increase may be different from that for the transitory increase shown in Fig. 4. In one respect, the rise in secretion in hypertonic solutions is slow. During the interval in the hypertonic media, shrinkage of the chromaffin cells due to dehydration and a consequent elevation of the intracellular free Ca’+ concentration ([Ca’+],) will occur. The rise in [Ca*+], then enhances secretion, as suggested at the neuromuscular junction (Furshpan, 1956; Shimoni et al., 1977). It has also been reported that hyperosmoIarity accelerated 4sCa release from intracellular organelles into cytoplasm, which in turn enhanced amylase secretion (Case and Clausen, 1973). However, increasing osmolality by adding sucrose increased the secretion of parathyroid hormone with-
546
HIKOSHI KITA and EMIKO YASUGI-NAGAOKA
out any increase in cytosolic Ca” (Chen er u/., 1987), which suggests that the effect of high osmolality cannot be explained by cytosolic CaZ+ alone.
According to the hypothesis, hypotonic solutions are expected to increase secretion by accelerating water inflow into granules, thus causing them to swell. Figure 2 shows that high K+-induced secretion gradually increases as the tonicity of the bathing solution is reduced by decreasing [NaCl],. As shown in Fig. 7, basal secretion also increased in a hypotonic solution made by removing 75 mM NaCI. The facilitatory effect does not seem to be due to the reduction in [NaCl], , since the basal secretion did not change at the reduced [NaCI],, when the tonicity was restored by compensating for the decreased [NaCI],, with the addition of sucrose (Fig. 8). It has been reported that hyposmolar medium increased luteinizing hormone secretion from perifused, dispersed adenohypophyseal ceils of the rat (Greer er al., 1983). Hyposmotic reduction in fNaCl], has caused an increase in high K+-stimulated aldosterone secretion, but the increase did not occur with an equivalent isosmotic reduction in [NaCl], even when the reduction was large (Schneider et al., 198.5). Renin release from rat epithelioid cells has been stimulated by hypotonic solutions and this stimulatory effect may be related to the observed swelling of the secretory granules (Skartt and Taugner, 1987). These results are consistent with the hypothesis. but they are inconsistent with those obtained at the frog neuromuscular junction (Kita et al., 1982a). The cause of this difference is unknown. The transitory response CA secretion is enhanced transitorily when the andrenals are transferred from hypertonic to isotonic (Fig. 4) or from an isotonic to hypotonic solution (Fig. 7). At the time of transfer, the interior of the chromaffin cells and therefore the inside of the secretory vesicles within the cells transiently becomes hypertonic compared with the extracellular Ruid. This condition is considered to be precisely a duplicate of the circumstances that the results of the liposome-bilayer experiments by Finkelstein (it al. (1986) extrapolate. Under these circumstances, water penetrates into the vesicles causing them to swell. This, in turn, facilitates the fusion of the vesicular membrane with the plasma membrane and the fission of the fused membrane, resulting in exocytotic CA secretion. It has been proposed that the entry of Cl- or other permeant anions into the chromaffin granule is a prerequisite for exocytosis (Pazoles and Pollard, 1978). This chemiosmotic hypothesis has, however, been criticized by some workers (Baker and Knight, 1984; Englert and Perlman, 1981; Jones PI a/., 1987). Also, in this case, osmotic swelling of the cytoplasmic secretory granule is crucial to the exocytotic secretion of granule contents. Although the present results do not provide any direct evidence concerning vesicle swelling and whether it precedes or follows vesicle-plasma membrane fusion (Green, 1987), they are consistent with the idea that an osmotic gradient across the vesicle membrane plays a role in exocytosis.
This idea is further supported by the dependencies of the transitory response on the soaking duration in hypertonic solution (Fig. 5) and on the degree of hypertonicitv (Fig. 6). From our previous experiments with i”C-sucrosc (Van der Kloot et ul.. 1974). it is indicated that the extracellular compartment exchanges readily with incubation solutions. As the bullfrog adrenal is larger than the frog’s. the duration-dependency probably means that the longer soaking period simply allows the hypertonic solution to be better distributed throughout the adrenal. The transitory response caused by an abrupt fall in osmolarity has been demonstrated in other cells. It is known that frog muscles treated with glycerol lose contractility when they are returned to isotonic salines (Eisenberg et rd.. 1971). On return, the release of creatine kinase increases transitorily, suggesting that an osmotic gradient across the sarcolemma stimulates the release by an exocytosis-like process (Suarez-Kurtz. 1982). Return of sea urchin egg cortices from a hypertonic solution with calcium to an isotonic medium without calcium causes inlmediate exocytosis of cortical vesicles, which indicates a role of osmotic forces in exocytosis (Zimmerberg ~‘t al.. 1985; Zimmerberg and Whitaker, 1985). It has also been reported that prolactin release in rainbow trout is increased transitorily by transition to a lower osmolarity (Gonnet et al.. 1988).
It is widely accepted that an increase in [Ca’+ 1, triggers exocytosis, which results in the secretion of hormones and neurotransmitters (Douglas, 1978). According to Zimmerberg Pt al. (1980), the role of Ca” in fusion of phospholipid vesicles with a planar phospholipid bilayer membrane containing a Ca’ ’ binding protein is: (a) to increase the probability 01 vesicle-membrane contact and (b) to prolong the duration of the contact, allowing time for the osmotic swelling of the vesicles to occur. Therefore, Ca” alone does not complete exocytosis; release of the vesicular contents is accomplished solely by osmotic forces. Pollard et (I/.(I 980) have also suggested that the action of Ca’ ’ is to bind a Ca’+ -binding protein. synexin. which bridges the granule and the plasma membrane to fuse. Burgoyne et al. (1982), using the calmodulin antagonist, trifiuoperazine (TFP), separated the process of exocytosis into two stages: (1) transloc~~tion of secretory granules to the plasma which is TFP-insensitive or Ca-“ membrane, independent and (2) fusion of granules with the which is TFP-sensitive OI plasma membrane. Ca’ ’ -dependent. Stage (1) comprises positioning ol granules at the plasma membrane (De Lisle and Williams, 1986) and stage (2) includes both contact or fusion and fission (rupture) or release (Pollard et ~1.. 1980; Zimmerberg et al., 1980). Burgoyne et ul. (1982) have suggested that the fusion involves calmodulin. Although one of the important roles of a rise in [Ca’+ 1, is considered to be facilitation of translocation of granules by screening the negative surface charges on the granule and plasma membranes (cf. Van der Kloot and Kita, 1973), the view of Burgoyne et al. (1982) seems to be consistent with those 01 Zimmerberg et ~1. ( 1980) and Pollard et al. (1980) that
Osmotic pressure and catecholamine secretion Ca’+ acts on contact or fusion of secretory granules with the plasma membrane. Burgoyne et al. (1983) observed that in adrenal chromaffin cells calcium was localized at sites of granule-plasma membrane interaction. The final stage of exocytosis, fission or release of granule contents, is brought about by the driving force provided by granule swelling caused by an osmotic gradient across the granule membrane. Swelling of secretory granules prior to release that could contribute to membrane thinning and rupture has been reported in rat peritoneal mast cells (Breckenridge and Almers, 1987; Schmauder-Chock, 1987; Zimmerberg et al., 1987). The stimulatory effect of hypotonic solutions on renin release from rat epithelioid cells is possibly related to the observed swelling of the secretory granules (Skott and Taugner, 1987). The transitory response observed in the present experiments seems to reflect the final stage of exocytosis, fission (rupture) of the secretory granule which is fused with the plasma membrane. Therefore. the response is seen in low Ca’+ solutions (Fig. 9) as well as in Mg” salines and is insensitive to [Ca’+], in the range of O.lLl.OmM (Fig. 10). A possible
mechanism
The present experiments provide strong evidence for the involvement in exocytosis of the osmotic gradient between the inside and outside of the secretory granule, not only from the standpoint of the steady effect of tonicity changes, which usually have been carried out, but also from that of the effect of the abrupt fall in osmolarity, about which little has so far been reported. One of the favourable features of Finkelstein cr ul.‘s model is that the water permeability of the chromaffin granule membrane is close to that of phospholipid bilayers (Sharp and Sen, 1982). Under physiological conditions, no osmotic gradients across the plasma and granule membranes exist, since there is isosmolarity between cells and their environment. The best possibility for the physiological generation of an osmotic gradient rests in the contents of the granules themselves. The interior of the chromaffin granule in which CA and ATP are major osmotic constituents (Holz, 1986) would be hypertonic when the binding of CA with ATP is released. If there is a mechanism activated by a secretory stimulus to free the hormone from the ATP or to hydrolyse ATP within a granule whose membrane is fused with the plasma membrane at the attached site, this would make the granule interior hypertonic and trigger a sequence of events in exocytosis-water inflow, granule swelling and rupture of the granule resuhng in CA secretion. Acknolr/rd~emenl.F-We are grateful to Dr William Van der Kloot for reading and improving an earlier draft of this manuscript and to Miss Satoko Kumazawa for typing the manuscript. This work was supported in part by Project Research Grants from Kawasaki Medical School.
REFERENCES Baker P. F. and Knight D. E. (1984) Chemiosmotic hypotheSiS of exocytosis: a critique. B&r;. Rep. 4, 285-298.
547
Breckenridge L. J. and Almers W. (1987) Final steps in exocytosis observed in a cell with giant secretory granules. Proc. natn. Acad. Sci. USA 84, 1945-1949. Burgoyne R. D.. Geisow M. J. and Barron J. (1982) Dissection of stages in exocytosis in the adrenal chromaffin cell with use of trifluoperazine. Proc. Roy. Sot. Lond. (B) 216, 111-l 15. Burgoyne R. D.. Barron J. and Geisow M. J. (1983) Cytochemical localization of calcium binding sites in adrenal chromaffin cells and their relation to secretion. Cell Tiss. Res. 229. 207-2 1I. Case R. M. and Clausen T. (1973) The relationship between calcium exchange and enzyme secretion in the isolated rat pancreas. J. Physiol. 235, 75-102. Chen C. J., Anast C. S. and Brown E. M. (1987) High osmolality: a potent parathyroid hormone secretogogue in dispersed parathyroid cells. Endocrinology 121, 958-964. De Lisle R. C. and Williams J. A. (1986) Regulation of membrane fusion in secretory exocytosis. Ann. Ret’. Physiol. 48, 225-238. Douglas W. W. (1978) Stimulus-secretion coupling: variations on the theme of calcium-activated exocytosis involving cellular and extracellular sources of calcium. Ciba Foind. Symp. 54, 61-90. Eisenbera R. S.. Howell J. N. and Vauehan P. C. (1971) The maintenance of resting potentials ?n glycerol-treated muscle fibres. J. Physiol. 215, 95-102. Englert D. F. and Perlman R. L. (1981) Permeant anions are not required for norepinephrine secretion from pheochromocytoma cells. Biochim. biophys. Acta 674, 136143. Fatt P. and Katz B. (1952) Spontaneous subthreshold activity at motor nerve endings. J. Physiol. 117, 109%128. Finkelstein A., Zimmerberg J. and Cohen F. S. (1986) Osmotic swelling of vesicles: its role in the fusion of vesicles with planar phosphohpid bilayer membranes and its possible role in exocytosis. Ann. Rec. PhJsiol. 48, 163-174. Furshpan E. J. (1956) The effects of osmotic pressure changes on the spontaneous activity at motor nerve endings. J. Physiol. 134, 689-697. Gibbs D. M. (1987) Noncalcium-dependent modulation of in vitro atrial natriuretic factor release by extracellular osmolality. Endocrinology 120, 194197. Gonnet F., Prunet P., Tonon M. C., Dubourg P., Kah 0. and Vaudry H. (1988) Effect of osmotic pressure on prolactin release in rainbow trout: in vitro studies. Gen. camp. Endocrinol. 69, 252-261. Green D. P. L. (1987) Granule swelling and membrane fusion in exocytosis. J. Cell Sci. 88, 5477549. Greer M. A., Greer S. E., Opsahl Z., McCafferty L. and Maruta S. (1983) Hyposmolar stimulation of in ritro pituitary secretion of luteinizing hormone: a potential clue to the secretory process. Endocrinology 113, 1531-1533. Hampton R. Y. and Holz R. W. (1983) Effects of changes in osmolarity on the stability and function of cultured chromaffin cells and the possible role of osmotic forces in exocytosis. J. Ce// Biol. 96, 108221088. Hola R. W. (1986) The role of osmotic forces in exocytosis from adrenal chromaffin cells. Ann. Rev. PhFsiol. 48, 175-189. Holz R. W. and Senter R. A. (1986) Effects of osmolarity and ionic strength on secretion from adrenal chromaffin cells permeabilized with digitonin. J. Neurochem. 46, 1835-1842. Holzbauer M. and Sharman D. F. (1972) The distribution of catecholamines in invertebrates. In Handbook of Experimental Pharmacology (Edited by Blaschko H. and Muscholl E.). Vol. XXIII. DV. 110-185. &ringer. Berlin. Jones P. M., Keaney J. E: ‘and Howeli S.-L. (1987) Chemiosmotic lysis and insulin secretion: studies of isolated granules, intact and permeabilised rat islets of Langerhans. Biochim. biophys. Acta 929, 302Z310.
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HIROSHI KITA and
EMIKO YASUGI-NAGAOKA
Kachadorian W. A., Muller J. and Finkelstein A. (1981) Schmauder-Chock E. A. and Chock S. P. (1987) Mechanism Role of osmotic forces in exocytosis: studies of ADHof secretory granule exocytosis: can granule enlargement induced fusion in toad urinary bladder. J. CeN Biol. 91, precede pore formation? Histochem. J. 19, 413418. 584588. Schneider E. G., Radke K. J., Ulderich D. A. and Taylor Kita H. and Van der Kloot W. (1977) Time course and R. E. Jr (1985) Effect of osmolarity on aldosterone magnitude of effects of changes in tonicity on acetylsecretion. Endocrinology 116, 1621-1626. choline release at frog neuromuscular junction. J. NeuroSharp R. R. and Sen R. (1982) Water permeability of the physiol. 40, 2 12-224. chromaffin granule membrane. Biophys. J. 40, 17 25. Kita H., Narita K. and Van der Kloot, W. (1982a) The Shimoni Y., Alnaes E. and Rahamimoff R. (1977) Is relation between tonicity and impulse-evoked transmitter hypertonic neurosecretion from motor nerve endings a release in the frog. J. Physiol. 325, 213-222. calcium-dependent process? Nafure 267, 170-172. Kita H., Yasugi E. and Van der Kloot W. (1982b) TransiSkett 0. and Taugner R. (1987) Effects of extracellular tory effects of osmotic pressure gradients on acetylcholine osmolality on renin release and on the ultrastructure of the juxtaglomerular epithelioid cell granules. CeII Tixs. release at the neuromuscular junction and on catecholamine secretion at the adrenal gland of the frog. Res. 249, 325-329. Neurosci. Lett. 34, 171-176. Suarez-Kurtz G. (1982) Release of creatin kinase from frog muscle by osmotic changes. Am. J. Physiol. 242, Knight D. E. and Baker P. F. (1982) Calcium-dependence C398-C403. of catecholamine release from bovine adrenal medullary cells after exposure to intense electric fields. J. Membr. Thesleff S. (1959) Motor end-plate ‘desensitization’ by Biol. 68, 1077140. repetitive nerve stimuli. J. Physiol. 148, 659-664. Ladona M. G., Sanchez-Garcia P. and Garcia A. G. (1984) Van der Kloot W. and Kita H. (1973) The possible role of Effects of hypertonic solutions on catecholamine release fixed membrane surface charges in acetylcholine release at from cat adrenal glands. Neuroscience 12, 301-307. the frog neuromuscular junction. J. Membr. Biol. 14, Lastowecka A. and Trifaro J. M. (1974) The effect of 365-382. sodium and calcium ions on the release of catecholamines Van der Kloot W.. Kita H. and Kita K. (1974) Excitationfrom the adrenal medulla: sodium deprivation induces secretion coupling in the release of catecholamines from release by exocytosis in the absence of extracellular the in vitro frog adrenal: effects of K+, Ca2+, hypercalcium. J. Physiol. 236, 681-705. tonicity, NaZ+ and Ni’+. Comp. Biochem. Physiol. 47A, Laverty R. and Taylor K. M. (1968) The fluorometric assay 701-711. of catecholamines and related compounds: improvements Zimmerberg J. and Whitaker M. (1985) Irreversible swelling and extensions to the hydroxyindole technique. Analyr. of secretory granules during exocytosis caused by calcium. Biochem. 22, 269-279. Nature 315, 581-584. Pazoles C. J. and Pollard H. B. (1978) Evidence for stimuZimmerberg J., Cohen F. S. and Finkelstein A. (1980) lation of anion transport in ATP-evoked transmitter Micromolar Ca” stimulates fusion of lipid vesicles with release from isolated secretory vesicles. J. biol. Chem. 253, planar bilayers containing a calcium-binding protein. 3962-3969. Science 210, 906908. Pollard H. B., Pazoles C. J., Creutz C. E., Scott J. H., Zinder Zimmerberg J., Sardet C. and Epel D. (1985) Exocytosis of 0. and Hotchkiss A. (1984) An osmotic mechanism for sea urchin egg cortical vesicles in vitro is retarded by exocytosis from dissociated chromaffin cells. J. biol. hyperosmotic sucrose: kinetics of fusion monitored by Chem. 259, 11141121. quantitative light-scattering microscopy. J. Cell Biol. 101, Pollard H. B., Pazoles C. J., Creutz C. E. and Zinder 0. 239X-2410. (1980) Role of intracellular proteins in the regulation of Zimmerberg J., Curran M.. Cohen F. S. and Brodwick M. calcium action and transmitter release during exocytosis. (1987) Simultaneous electrical and optical measurements Monogr. Neural Sri. I, 106116. show that membrane fusion precedes secretory granule Ricci A. Jr, Sanders K. M., Portmore J. and Van der Kloot swelling during exocytosis of beige mouse mast cell. Proc. W. (1975) Effects of the ionophores, X-537A and A-23187 natn. Acad. Sci. USA 84, 1585-1589. on catecholamine release from the in c?tro frog adrenal. Life Sci. 16, 1777184.
;
0300-9629/89 %3.00+0.00 1989 Pergamon Press plc
CATECHOLAMrNE SECRETION FROM BULLFROG ADRENALS IN RESPONSE TO OSMOTIC CHANGES HIROSHI KITA and EMIKO YASUGI-NAGAOKA Department
of Physiology,
Kawasaki
Medical
School,
Kurashiki,
Okayama
701-01,
Japan
(Receiced 24 April 1989) Abstract-l. High K +-induced catecholamine (CA) secretion from bullfrog adrenals was suppressed in hypertonic solutions made by adding sucrose or NaCl and enhanced in solutions made hypotonic with reduced [NaCI], 2. When the preparation was transferred from a solution made hypertonic with sucrose or NaCl to a normal saline solution. CA secretion was increased transitorily. 3. The magnitude of the transitory response depended on the duration of soaking in the hypertonic solution. 4. Higher tonicities produced larger transitory responses, irrespective of whether the solute added was sucrose or NaCI. 5. When the preparation was passed from normal saline to a hypotonic solution made by lowering [NaCl],. a similar increase in CA output was observed. 6. The transitory effect of the osmotic gradient was similarly observed in low Ca’+, Mg’+ EGTAcontaining solutions and in solutions whose sole divalent cation was Mg?+.
INTRODUCTION
Changes in the tonicity of the bathing solution markedly affect the release of transmitter at the vertebrate neuromuscular junction: hypertonic solutions increase the rate of spontaneous quanta1 release of acetylcholine recorded as miniature endplate potentials (MEPPs) and hypotonic solutions decrease it (Fatt and Katz, 1952; Furshpan, 1956; Kita and Van der Kloot, 1977). Strongly hypertonic solutions reduce the number of quanta released from stimulated nerve terminals, which is manifested by the amplitude of end-plate potentials (Kita and Van der Kloot, 1977; Thesleff, 1959). Although only a few papers have reported on the effect of tonicity on the secretion of catecholamine (CA) from adrenals, hypertonic solutions have also been shown to enhance spontaneous secretion and to inhibit secretion evoked by high K+ or nicotine (Ladona et al., 1984). CA secretion from isolated adrenal chromaf?in ceils has also been suppressed by hypertonic solutions (Holz and Senter, 1986: Knight and Baker, 1982: Pollard et al., 1984). Recently, it has been shown that the rate of fusion of phospholipid vesicles with planar phospholipid bilayer membranes is enhanced when the side containing the vesicles is hypertonic to the opposite side (Finkelstein et al., 1986). The number of fusion events induced by antidiuretic hormone in the toad urinary bladder decreases when the mucosal bathing medium of the bladder is made hypertonic (Kachadorian et al., 1981}. These reports suggest that the osmotic entry of water into intracellular vesicles which are in contact with the plasma membrane is essential for the exocytotic release of the vesicular contents and that the release is accomplished by vesicular swelling and subsequent rupture of vesicular and plasma membranes at the contact site.
We have studied the effects of osmotic pressure and its changes on CA secretion from bullfrog adrenals. By measuring concomitant secretion of dopamine-fihydroxylase, it has been shown that CA secretion from frog adrenals induced with high K’ or the ionophore X-537A involves exocytosis (Ricci et al., 1975). When the adrenal is transferred from hypertonic to isotonic, or from isotonic to hypotonic, there will be an interval during which the interior of the chromaffin cells is hypertonic to the extracellular fluid. This duplicates the circumstances that the above reports have predicted promote exocytosis. When the osmolarity of the extracellular solution was abruptly reduced, a period of enhanced CA secretion was observed. Some of the results have appeared in a short communication (Kita er al., 1982b).
MATERIALS AND METHODS A pair of adrenals with the attached kidneys were dissected from a pithed bullfrog, Runa cute.~heiana. Normal saline solution containing (in mM) I15 NaCI, 2 KC], 2.3 CaCl?. 4NaHC0, and IO glucose, with an osmolarity (mosmoljl) of 235 was used. Preparations were then stored overnight in oxygenated saline solution in a refrigerator to stabilize the spontaneous secretion rate. Some of the advantages of using isolated frog adrenals for studying CA secretion have been described elsewhere (Van der Kloot et al.. 1974). The next morning the adrenals were kept for 0.5-t hr in oxygenated saline solution at room temperature, after which they were passed at 5min intervals through a series of small beakers, each containing 3 ml of oxygenated solution. Before each transfer, solution adhering on the surface of the preparation was blotted off with filter paper. The osmolarity of the solution was changed by adding sucrose or NaCI, or by reducing NaCI. The CA released into the solution was determined by the hydroxyindole method (Laverty
and Taylor, 196X) using a throrescence spectrophotometer (HITACHI MPF-3). The details of the method have been described together with the detailed experimental procedure (Van der Kioot eit al.. 1974). Blood contains CA (Holzbauer and Sharman. 1972). Therefore, the inferior vena cava supplying the kidneys and adrenals was perfused with normal saline by a hypodermic needle inserted into the vessel before they were isolated.
/
200
1 300
/
0
/
RESXXTS High K’
-induced CA secretion irz hyprtonic
snhtiorts
When bullfrog adrenals are soaked in hypertonic solution, the ostnotic gradient across the secretory granule membrane works against granule swdling and therefore prevents exocytosis (Fink&stein et nl., 1986). Under these conditions it is expected that the stimulated secretion of CA with high K + will be suppressed. Xn Fig. IA, the tonicity was raised by adding 100,200 and 300 mM sucrose to the normal saline solution. CA secretion was stimulated with 40 mM KCI. fn solutions ~ontainjng 200 and 300 mM sucrose, the stimulated secretions were decreased su~stant~al~~, being 74 and 42% of the response in the normal saline, respectively. In other experiments in which solutions containing 500 mM sucrose were used, stimulation with 4OmM KC1 was ineftectivc. Raising of tonicity was also achieved by the addition of 50, 100 and 15OmM NaCl (Fig. 1B). The high K”-induced secretions were suppressed in 100 and 150mM NaCl solutions, being 69 and 25% of the control response, respectively. Therefore, the depressive effect of hypertonicity was independent of the chemical species producing it. It should be noted that the transition from 300 mM sucrose or 150mM NaCl to the normal saline produced a transitory increase in CA secretion, which is the main theme of the present work. On the ather hand, when the osmolarity was raised by the addition of 0, 100, 200 and 300 mM glycerol, CA secretion induced by 40 mM KC1 was not suppressed. In other experiments, CA secretions, in response to increasing ~on~~~trations of KCI (40, 60 and 80 mM). were examined in the presence and absence of atropine sulfate (10-’ g/ml) and hexameth~~nium chloride (5 x IO-’ g/ml)_ There was no discernible difference in the secretory response between the two kinds of solutions. Therefore, these drugs were omitted in the experiments using high K’
A ~y~otoni~ en~~~ronrne~t is considered to be favourable for vesicle swelling, which promotes exocytosis (Fink&&in ef al., 1986). Figure 2 shows that stimulation with 40mM KCI produced larger responses at lower tonicities, the responses in -25, -50 and - 75 mM NaCI solutions helng I .07, I.30 and 2.80 times that in the normal sahne solution, respectively. The reason for the delayed enhanced secretions after stjrnu~~tjon is unknown. Basal CA secretkm in j~~~@~t~~~~ s~~~~~#~~~ As shown in Fig. 3, the basal secretion of CA was affected slightly by hypertonic solutions with sucrose
Ttme Lmm) Fig, 1. The effects of hypertonic solutions on high K +induced CA secretion from isolated bulKrog adrenals. During the shaded intervals the solutions contained 40 mM KCI. In this and the following experiments in which 40 mM KC1 was used to stimulate secretion, the osmoiarity of the solutions without 40 mM KCI was adjusted to that with the hieh KS bv addine 80 mM sucrose. Tonic& was raised bv the addition of either sucrose (A) or NaCl (5). The figures across the top show the amount (in m&l) of added sucrose (A) or NaCI (B). The values for CA (in ~g~rnl~5rnjn) are averages from four (A) and three (B) experiments. The lines indicate the standard deviations of the measured values,
The broken line represents the average value before any application of high KC andior hypertonic solution.
below 50OmM, but the effect was not straightforward. In solutions containing 100 and 200mM sucrose, the secretion was suppressed stepwise. Then it was increased progressively as the concentration of added sucrose was raised from 300 to 500mM. the rest& being identical to that shown by the shaded bars in Fig. 6. Transition from SO0 to 0 mM sucrose produced a substantial increase in secretion as afready mentioned in Fig. l.
The bullfrog adrenal. is not as sensitive to changes in osmolarity as the frog neuromuscular junction (Fatt and Katz, 1952). The addition of 300 mM sucrose to a normal saline solution sometimes did not mcrease the secretion of CA. When 500 mM sucrose was added, the rate of CA secretion rose slowly during 15 min of exposure (Fig. 4). On return to the isotonic solution, the CA secretion substantially increased transitorily. Then the rate of secretion
541
Osmotic pressure and catecholamine secretion 0.25 -0 NaCl
j
-25
/
j
-50
-75
E
E 0.20 * & -a i y
0.15 -
9 z .$ 0.10 .E! % 5 0.05 -
0
I--Time
( min
h 150
100
0
)
Fig. 2. The effects on high K+-stimulated secretion of hypotonicity produced by reducing the concentration of NaCI. During the shaded intervals the solutions contained 40 mM KCI. The figures with a minus sign across the top show the amount (in mM) of NaCl removed from normal saline. The values for CA are the averages from three experiments.
declined gradually toward the initial level. The transfer was repeated four times and each time there was a transitory, substantial increase in secretion. The level of the basal secretion became progressively higher during the experiment. The magnitudes of the transient increases shown by the first clear bars following the shaded ones in Fig. 4 were, from the first bout to the fourth, 7.86, 9.00, 10.57 and 11.00 times the control value shown by the broken line. The reproducibility of the result indicates that the transitory response is not due to a breakdown of the chromaffin cells.
In experiments like that illustrated in Fig. 5, a pair of adrenals were soaked for 5, 10, 1.5 and 30 min in solutions containing 500 mM sucrose. After each soaking, the preparation was returned to the initial, isotonic saline. The CA output was enhanced depending upon the soaking duration, although there was no difference between 5 and 10 min soaking. The sizes of the increases were 4.64,4.57, 6.36 and 18.57 times the control value for 5, 10, 15 and 30 min soaking, respectively.
It is expected that until intracellular fluid equilibrates osmoticafly with hypertonic extracellular solution, the osmotic pressure gradient across the granule membrane produced on return to isotonic
An example of the dependence of response on osmotic pressure is shown transitions from solutions containing and 700 mM sucrose to normal saline
solution will be greater when the soaking duration of the preparation in the hypertonic solution is longer.
Time
the transitory in Fig. 6. The 100, 300, 500 enhanced the
( min)
Fig. 3. The effects of hypertonic solutions on basal secretion. The figures across the top show the amount (in mM) of sucrose added to a normal saline solution (0 mM) whose osmolarity was 235 mosmol/l. The values for CA are the averages from three experiments.
HIR~SMIKITA and EMIKOYASUGI-NAGAOKA
542
0
50
100 Time
Fig.
4. The effects of changes
during
which
the solutions
( mm)
in osmolarity on CA secretion. The shaded bars are for the time intervals contained 500mM sucrose. The values for CA are the averages from three experiments.
CA output to 1.82, 5.53, 7.53 and 14.88 times that of the control, respectively. Almost identical results were obtained when the solution was made hypertonic by adding NaCl rather than sucrose. The shifts from solutions containing 50, 150, 250 and 350 mM NaCl to normal saline solution increased the basal secretion 1.38-, 3.85, 5.96and 13.42-fold, respectively (averages from three experiments). The transitory responses are caused by osmotic pressure gradients, not by the chemical species. CA secretion in response to transition from isotonic to hypotonic The same circumstances as in the transition from hypertonic to isotonic are obtained when the preparation is transferred from isotonic to hypotonic
solution. In the experiment shown in Fig. 7, a pair of adrenals were first soaked in normal saline solution and then transferred to hypotonic solution (from open to stippled intervals). Except in a 90 mM NaCl solution (shown by -25 NaCl), the shifts from isotonic to hypotonic produced increases in CA secretion; 0.89-, 2.84- and 7.45fold increases for 90mM, 65 mM (-50) and 40mM NaCl (-7S), respectively. In the hypotonic solutions the increased CA secretion did not decrease. Rather it increased in 40mM NaCI. One possible explanation is that in hypotonic solutions water moves into the chromaffin cells, which helps the secretory granules to swell. This in turn accelerates the exocytotic release of CA (Finkelstein et al., 1986). Another possibility is that a fall in [NaCI], reduces the activity of the Na+-Ca* + exchange system of the chromaffin cell membrane
Fig. 5. The effects of varying the soaking duration in hypertonic solution. The durations of soaking the nreparation in solutions containing SO0 mM sucrose were 5, 10, 15 and 30 min as shown by the shaded bars. The values for CA are the averages from seven experiments
Osmotic
pressure
and catecholamine
secretion
543
Fig. 6. The effects of increasing tonicities on CA secretion. During the shaded intervals the solutions contained sucrose at the concentrations (in mM) indicated by the figures above the shaded bars. The values for CA are the averages from four experiments.
and consequently causes an accumulation of intracellular Ca2+ (Lastowecka and Trifaro, 1974); but this seems unlikely from the results shown in Fig. 8. On return to normal saline, the CA output decreased gradually toward the control level. Effect of restoration
of hypotonicity
on CA secretion
To examine whether sodium deficiency rather than an osmotic fall can increase CA output, the experiment illustrated in Fig. 8 was performed. The preparation was shifted from a normal saline solution to a NaCl-deficient solution that was osmotically restored to isotonic by adding an appropriate amount of sucrose. As shown in Fig. 8, shifts from normal to lower sodium concentrations did not cause transitory increases in CA secretion as long as the tonicity was kept constant. The CA output during the first 5 min
of the stippled intervals was 0.93, 0.93 and 1.18 times the control value for 90 mM NaCl + 50 mM sucrose, 65 + 100 and 40 + 150, respectively. Transitory
response in low Ca2+ solutions
The transitory response to osmotic changes was also observed in solutions with a low [Ca’+]. A pair of adrenals pretreated with low Ca2+ saline were subjected to transitions from hypertonic to isotonic in a low Ca2+, Mg2+ EGTA-containing solution (Fig. 9). The first and second transfers increased the control secretion 4.68 and 5.45 times, respectively. These results suggest that the transitory response was not due to stimulation of Cal+ influx. After the second transitory response, the secretion reached a plateau which was about four times the control. The cause of the increase is unclear.
Time
( min)
Fig. 7. The effects of decreasing tonicities on CA secretion. The stippled regions indicate the periods during which the concentration of NaCl was reduced by the amount (in mM) shown by the figures with a minus sign above the stippled bars. The values for CA are averages from three experiments.
HIROSHIKITA
544
and
EMIKO YASUGI-NAGAOKA
Timet min 1
Fig.
8. The effects
on CA secretion of reducing [NaClI, while keeping the tonicity isotonic. During the intervals the concentration of NaCl was reduced by the amount (in mM) shown by the figures wi% a minus sign above the stippled bars and the osmolarity of each solution was restored to normal by adding the appropriate amount of sucrose (in mM) as indicated by the figures with a plus sign. The values for CA are the averages from three experiments. stinnled
Trunsitory
response in Mg’+ solutions
Essentially the same results were obtained in solutions whose only divalent cation was Mg’+, again suggesting that the transitory stimulation of CA secretion was not due to stimulation of Cal+ influx. The preparation, which had been pretreated overnight in the refrigerator with 2.5 mM Mg’+ saline containing 1 mM EGTA and no Ca*+, was shifted from hypertonic (plus 500mM sucrose) to isotonic in the same solution. There was an interval of enhanced CA secretion following the transfer. The first and second transfers produced increases in secretion of 5.5 and 7.7 times the control value, respectively (average from three expe~ments). After the second transfer, the CA secretion remained
elevated, the level being about 5.5 times that of the control. The overall results obtained in Mg”+ salines were similar to those in low Ca2+ solutions.
The dependence of the transitory response on [Ca’+], was examined within the range of 0.1 to 1.0 mM (Fig. IO). The transition from 500 to 0 mM sucrose was performed at 0. I mM Ca* + and then the same transfer was repeated at 0.5 (Fig IOA), 0.7 (B) or l.OmM Ca2+ (C). In Fig. lOA, the transfer at 0.1 mM increased the control secretion (shown by the broken line) 12.52-fold and the same transfer at 0.5 mM produced an 8.75-fold increase. In Fig. 10B, the increase was 9.22fold at 0.1 mM and 6.96fold at 0.7 mM and in Fig. IOC, it was 7.91-and 4.72-fold at 0.1 and l.OmM, respectively. The values at higher Ca2+ concentrations were always smaller because of the elevated levels of control secretion, However, as shown in Fig. 10. the magnitudes of the transitory response were essentially the same at the two Ca” concentrations. It may generally be said that the transitory response is not enhanced by an increase in the extracellular concentration of Ca?” below 1 mM. This seems to be consistent with the results obtained in low Ca’+ solutions and in Mg2+ salines.
DISCUSSION 0
50
100
Ttme (msn 1
Fin. 9. The transitory response to an osmotic gradient in a Mg*+ EGTA-containing solution. The preparation was soaked overnight in a refrigerated low Ca*+ EGTA solution. The low Ca*+, EGTA solution was made without CaCl, but with 1 mM MaCl, and 1 mM EGTA (osmol=~240mosmol/l). D&g the intervals shown by the solid bars the low Ca*+, EGTA solution contained 500 mM sucrose. The values for CA are the averages from six experiments.
10-iCa zc,
Efect
of’ hypertonicit)
The osmotic hypothesis of Finkelstein et crf. (1986) predicts that secretion is suppressed in hypertoflic solutions. As the solution osmolarity is increased by adding sucrose (Fig. IA) or NaCl (Fig. 1Bf, CA secretion in response to 40mM K’ is progressively decreased. Depression of secretory activities by hypertonicity has also been reported in a wide variety of cells, including CA secretion from cat adrenals (Ladona et al., 1984). from isolated bovine adrenal
Osmotic
pressure
and catecholamine
secretion
545
B 0.06
c .E ‘E
0.04
Em $$& ‘u _” 0& 2E
IV
0.02
1
0
)
Time
( min ) 0.04
Time
,-. L
( min )
,
Time
( min )
Fig. IO. The influence of [Ca’+], on the transitory response. The [Ca’+], was increased from 0.1 mM to 0.5 (A), 0.7 (B) and 1.0 mM (C). The preparations were soaked overnight in refrigerated 0.1 mM Ca’+ saline solutions. During the shaded intervals solutions contained 500 mM sucrose. The values for CA are the averages from five (A), five (B) and six (C) experiments.
me&Nary
cells
rendered
leaky
by
intense
electric
fields (Knight and Baker, 1982) or by digitonin (Holz and Senter, 1986) and from cultured (Hampton and Holz, 1983) or dissociated (Pollard et al., 1984) chromaffin cells of bovine adrenal medulla. Our results and these examples seem to support the hypothesis. Though there is a possibility that the above-mentioned inhibition of secretion may not be caused by a direct effect of hypertonicity but by the elevation in ionic strength (Holz and Senter, 1986), we suggest the osmolarity itself, rather than ionic strength, is responsible for the decrease in CA secretion caused by hypertonicity in the present study, since CA secretion is suppressed to a comparable degree when osmolarity is increased to a similar extent with either sucrose or NaCl (Fig. 1). High K+-stimulated aldosterone secretion was also inhibited, to a similar extent, when the solution osmolarity was increased by the addition of NaCl, sucrose or mannitol (Schneider et al., 1985). When glycerol was used as the osmoticant, the inhibitory effect of hypertonicity on high K+stimulated secretion was no longer observed. At the frog neuromuscular junction, the frequency of MEPPs elevated by the addition of glycerol returned to the initial level within 15 min (Furshpan, 1956). The inability to depress stimulated CA secretion and the transient effect on the MEPP frequency of glycerol can be explained by assuming that glycerol molecules enter the cell interior much more easily than sucrose, reducing the osmotic gradient across the vesicle membrane. AS shown in Fig. 3 and during shaded intervals in
Fig. 6, hypertonic solutions with sucrose higher than 300 mM increase the basal CA secretion gradually, as has been shown previously (Ladona et al., 1984), though the critical concentration of sucrose is different. The basal level of norepinephrine secretion from pheochromocytoma cells (Englert and Perlman, 1981), the secretion of atria1 natriuretic factor from dispersed perifused myocytes of the rat (Gibbs, 1987) and parathyroid hormone secretion from dispersed bovine parathyroid cells (Chen et al., 1987) have been increased markedly by increased medium osmolality. It is known that hypertonic solutions produce marked increases in MEPP frequency at the frog neuromuscular junction, which is much more sensitive than frog adrenals to changes in osmolarity (Fatt and Katz, 1952; Furshpan, 1956; Kita and Van der Kloot. 1977). These phenomena seem to be a drawback to the osmotic hypothesis. However, the mechanism for the increase may be different from that for the transitory increase shown in Fig. 4. In one respect, the rise in secretion in hypertonic solutions is slow. During the interval in the hypertonic media, shrinkage of the chromaffin cells due to dehydration and a consequent elevation of the intracellular free Ca’+ concentration ([Ca’+],) will occur. The rise in [Ca*+], then enhances secretion, as suggested at the neuromuscular junction (Furshpan, 1956; Shimoni et al., 1977). It has also been reported that hyperosmoIarity accelerated 4sCa release from intracellular organelles into cytoplasm, which in turn enhanced amylase secretion (Case and Clausen, 1973). However, increasing osmolality by adding sucrose increased the secretion of parathyroid hormone with-
546
HIKOSHI KITA and EMIKO YASUGI-NAGAOKA
out any increase in cytosolic Ca” (Chen er u/., 1987), which suggests that the effect of high osmolality cannot be explained by cytosolic CaZ+ alone.
According to the hypothesis, hypotonic solutions are expected to increase secretion by accelerating water inflow into granules, thus causing them to swell. Figure 2 shows that high K+-induced secretion gradually increases as the tonicity of the bathing solution is reduced by decreasing [NaCl],. As shown in Fig. 7, basal secretion also increased in a hypotonic solution made by removing 75 mM NaCI. The facilitatory effect does not seem to be due to the reduction in [NaCl], , since the basal secretion did not change at the reduced [NaCI],, when the tonicity was restored by compensating for the decreased [NaCI],, with the addition of sucrose (Fig. 8). It has been reported that hyposmolar medium increased luteinizing hormone secretion from perifused, dispersed adenohypophyseal ceils of the rat (Greer er al., 1983). Hyposmotic reduction in fNaCl], has caused an increase in high K+-stimulated aldosterone secretion, but the increase did not occur with an equivalent isosmotic reduction in [NaCl], even when the reduction was large (Schneider et al., 198.5). Renin release from rat epithelioid cells has been stimulated by hypotonic solutions and this stimulatory effect may be related to the observed swelling of the secretory granules (Skartt and Taugner, 1987). These results are consistent with the hypothesis. but they are inconsistent with those obtained at the frog neuromuscular junction (Kita et al., 1982a). The cause of this difference is unknown. The transitory response CA secretion is enhanced transitorily when the andrenals are transferred from hypertonic to isotonic (Fig. 4) or from an isotonic to hypotonic solution (Fig. 7). At the time of transfer, the interior of the chromaffin cells and therefore the inside of the secretory vesicles within the cells transiently becomes hypertonic compared with the extracellular Ruid. This condition is considered to be precisely a duplicate of the circumstances that the results of the liposome-bilayer experiments by Finkelstein (it al. (1986) extrapolate. Under these circumstances, water penetrates into the vesicles causing them to swell. This, in turn, facilitates the fusion of the vesicular membrane with the plasma membrane and the fission of the fused membrane, resulting in exocytotic CA secretion. It has been proposed that the entry of Cl- or other permeant anions into the chromaffin granule is a prerequisite for exocytosis (Pazoles and Pollard, 1978). This chemiosmotic hypothesis has, however, been criticized by some workers (Baker and Knight, 1984; Englert and Perlman, 1981; Jones PI a/., 1987). Also, in this case, osmotic swelling of the cytoplasmic secretory granule is crucial to the exocytotic secretion of granule contents. Although the present results do not provide any direct evidence concerning vesicle swelling and whether it precedes or follows vesicle-plasma membrane fusion (Green, 1987), they are consistent with the idea that an osmotic gradient across the vesicle membrane plays a role in exocytosis.
This idea is further supported by the dependencies of the transitory response on the soaking duration in hypertonic solution (Fig. 5) and on the degree of hypertonicitv (Fig. 6). From our previous experiments with i”C-sucrosc (Van der Kloot et ul.. 1974). it is indicated that the extracellular compartment exchanges readily with incubation solutions. As the bullfrog adrenal is larger than the frog’s. the duration-dependency probably means that the longer soaking period simply allows the hypertonic solution to be better distributed throughout the adrenal. The transitory response caused by an abrupt fall in osmolarity has been demonstrated in other cells. It is known that frog muscles treated with glycerol lose contractility when they are returned to isotonic salines (Eisenberg et rd.. 1971). On return, the release of creatine kinase increases transitorily, suggesting that an osmotic gradient across the sarcolemma stimulates the release by an exocytosis-like process (Suarez-Kurtz. 1982). Return of sea urchin egg cortices from a hypertonic solution with calcium to an isotonic medium without calcium causes inlmediate exocytosis of cortical vesicles, which indicates a role of osmotic forces in exocytosis (Zimmerberg ~‘t al.. 1985; Zimmerberg and Whitaker, 1985). It has also been reported that prolactin release in rainbow trout is increased transitorily by transition to a lower osmolarity (Gonnet et al.. 1988).
It is widely accepted that an increase in [Ca’+ 1, triggers exocytosis, which results in the secretion of hormones and neurotransmitters (Douglas, 1978). According to Zimmerberg Pt al. (1980), the role of Ca” in fusion of phospholipid vesicles with a planar phospholipid bilayer membrane containing a Ca’ ’ binding protein is: (a) to increase the probability 01 vesicle-membrane contact and (b) to prolong the duration of the contact, allowing time for the osmotic swelling of the vesicles to occur. Therefore, Ca” alone does not complete exocytosis; release of the vesicular contents is accomplished solely by osmotic forces. Pollard et (I/.(I 980) have also suggested that the action of Ca’ ’ is to bind a Ca’+ -binding protein. synexin. which bridges the granule and the plasma membrane to fuse. Burgoyne et al. (1982), using the calmodulin antagonist, trifiuoperazine (TFP), separated the process of exocytosis into two stages: (1) transloc~~tion of secretory granules to the plasma which is TFP-insensitive or Ca-“ membrane, independent and (2) fusion of granules with the which is TFP-sensitive OI plasma membrane. Ca’ ’ -dependent. Stage (1) comprises positioning ol granules at the plasma membrane (De Lisle and Williams, 1986) and stage (2) includes both contact or fusion and fission (rupture) or release (Pollard et ~1.. 1980; Zimmerberg et al., 1980). Burgoyne et ul. (1982) have suggested that the fusion involves calmodulin. Although one of the important roles of a rise in [Ca’+ 1, is considered to be facilitation of translocation of granules by screening the negative surface charges on the granule and plasma membranes (cf. Van der Kloot and Kita, 1973), the view of Burgoyne et al. (1982) seems to be consistent with those 01 Zimmerberg et ~1. ( 1980) and Pollard et al. (1980) that
Osmotic pressure and catecholamine secretion Ca’+ acts on contact or fusion of secretory granules with the plasma membrane. Burgoyne et al. (1983) observed that in adrenal chromaffin cells calcium was localized at sites of granule-plasma membrane interaction. The final stage of exocytosis, fission or release of granule contents, is brought about by the driving force provided by granule swelling caused by an osmotic gradient across the granule membrane. Swelling of secretory granules prior to release that could contribute to membrane thinning and rupture has been reported in rat peritoneal mast cells (Breckenridge and Almers, 1987; Schmauder-Chock, 1987; Zimmerberg et al., 1987). The stimulatory effect of hypotonic solutions on renin release from rat epithelioid cells is possibly related to the observed swelling of the secretory granules (Skott and Taugner, 1987). The transitory response observed in the present experiments seems to reflect the final stage of exocytosis, fission (rupture) of the secretory granule which is fused with the plasma membrane. Therefore. the response is seen in low Ca’+ solutions (Fig. 9) as well as in Mg” salines and is insensitive to [Ca’+], in the range of O.lLl.OmM (Fig. 10). A possible
mechanism
The present experiments provide strong evidence for the involvement in exocytosis of the osmotic gradient between the inside and outside of the secretory granule, not only from the standpoint of the steady effect of tonicity changes, which usually have been carried out, but also from that of the effect of the abrupt fall in osmolarity, about which little has so far been reported. One of the favourable features of Finkelstein cr ul.‘s model is that the water permeability of the chromaffin granule membrane is close to that of phospholipid bilayers (Sharp and Sen, 1982). Under physiological conditions, no osmotic gradients across the plasma and granule membranes exist, since there is isosmolarity between cells and their environment. The best possibility for the physiological generation of an osmotic gradient rests in the contents of the granules themselves. The interior of the chromaffin granule in which CA and ATP are major osmotic constituents (Holz, 1986) would be hypertonic when the binding of CA with ATP is released. If there is a mechanism activated by a secretory stimulus to free the hormone from the ATP or to hydrolyse ATP within a granule whose membrane is fused with the plasma membrane at the attached site, this would make the granule interior hypertonic and trigger a sequence of events in exocytosis-water inflow, granule swelling and rupture of the granule resuhng in CA secretion. Acknolr/rd~emenl.F-We are grateful to Dr William Van der Kloot for reading and improving an earlier draft of this manuscript and to Miss Satoko Kumazawa for typing the manuscript. This work was supported in part by Project Research Grants from Kawasaki Medical School.
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