J. Physiol. (1976), 260, pp. 105-115 With 6 text-figure8 Printed in Great Britain
105
STIMULATION OF 45Ca2+ EFFLUX FROM RAT PITUITARY BY LUTEINIZING HORMONE-RELEASING HORMONE AND OTHER PITUITARY STIMULANTS
BY J. A. WILLIAMS From the Department of Physiology, University of California, San Francisco, California 94143, U.S.A.
(Received 29 December 1975) SUMMARY
1. Rat anterior pituitaries were, incubated in medium containing 45Ca2+, superfused with non-radioactive medium and the efflux of 45Ca2+ studied. 2. When pituitaries from overiectomized rats were used the addition of LH-RH to the medium at a time when slow exponential efflux of Ca2+ was occurring produced a transient increase in 45Ca2+ efflux simultaneous with or before an increased release of LH. The stimulation of both 45Ca2+ efflux and LH release showed a similar concentration dependence on LHRH. 3. Increasing the K+ concentration of the medium tenfold also stimulated 45Ca2+ efflux and LH release. The response to LH-RH of both parameters was reduced by superfusion with calcium-free medium. 4. LH-RH induced only a small increase in pituitary 45Ca2+ efflux when intact or thyroidectomized male rats were used. TRH increased 45Ca2+ efflux from thyroidectomized rat pituitaries but had only a small effect when pituitaries from intact or ovariectomized rats were studied. 5. Pretreatment of ovariectomized rats with oestradiol inhibited the subsequent increase in 45Ca2+ efflux induced by LH-RH. 6. It is concluded that the secretagogue induced increase in 45Ca2+ efflux results from an increase in cellular Ca2+ activity which is presumably active in stimulus-secretion coupling. INTRODUCTION
Stimulus-secretion coupling of luteinizing hormone (LH) release in response to luteinizing hormone-releasing hormone (LH-RH) is believed to involve Ca2+ in a manner similar to that utilized in the release of secretory products from a number of tissues (Douglas, 1968; Rubin, 1970).
106 J. A. WILLIAMS Evidence for this scheme, in which receptor activation triggers Ca2+ entry, depolarization and hormone release, includes the finding that LH release is stimulated by an elevated concentration of K+ in the medium (Samli & Geschwind, 1968; Wakabayashi, Kamberi & McCann, 1969; Wakabayashi, Date & Tamaoki, 1973). An increase in K+ should depolarize the gonadotrophs (Williams, 1970) and does increase 45Ca2+ uptake into the whole pituitary (Milligan & Kraicer, 1971). Furthermore, LH release in response to both K+ and hypothalamic extract is reduced by incubation in Ca-free medium (Samli & Geschwind, 1968). Recently, however, evidence has been presented that hypothalamic releasing factors may not promote a specific calcium influx (Milligan & Kraicer, 1974; Eto, Wood, Hutchins & Fleischer, 1974). Since methods have not yet been developed to measure Ca2+ activity within secretary cells, there is little data showing how, or even whether a rise in intracellular free Ca2+ occurs; and if it does, whether Ca2+ directly promotes secretion. In the exocrine and endocrine pancreas, neurohypophysis and parotid glands, stimulators of granule release, are known to increase 45Ca2+ efflux from slowly exchanging compartments (Douglas & Poisner, 1964; Nielsen & Petersen, 1972; Case & Clausen, 1973; Matthews, Petersen & Williams, 1973; Malaisse, Brisson & Biard, 1973). We therefore decided to test whether LH-RH would analogously increase 45Ca2+ efflux from the anterior pituitary. We used pituitaries from ovariectomized rats since an increase in LH release can be easily demonstrated in this preparation (Osland, Gallo & Williams, 1975). The present results demonstrate a transient, concentration dependent increase in 45Ca2+ efflux in response to LH-RH and other pituitary stimulants. The possible relation to stimulussecretion coupling is discussed. METHODS
All studies were carried out using adult Sprague-Dawley rats. Ovariectomies were performed on 150-200 g female rats 3-5 weeks before use. Thyroidectomies were performed by injection of 1 mc l31J into 90-100 g rats and pituitaries were studied 6-8 weeks later. At the time of use the thyroidectomized rats weighed 200-250 g as compared to intact litter mates which weighed 350-400 g. Intact male rats weighed 300-400 g. Anterior pituitaries were removed, cut into 8 pieces and incubated 1 hr in Krebs-Henseleit bicarbonate (KHB) (Matthews et al. 1973) containing 45Ca2+ 15 ,uc/ml. (New England Nuclear) in a shaking water-bath. The medium was equilibrated with 95 % 02-5 % CO2 (pH 7.35) and was maintained at 370 C in all experiments. The pituitary fragments were subsequently transferred to a 0 5 ml. volume superfusion apparatus through which pre-warmed KHB was passed at 2 chamber volumes/min (Osland et al. 1975). Effluent was collected into plastic scintillation vials during timed intervals (2 min unless otherwise specified). Scintillation fluid (Toluene: Triton X-100, 2:1, butyl PBD, 4 g/l.) was added and radioactivity determined by liquid scintillation counting. The efflux of 45Ca2+ is shown
LH-RH AND PITUITARY 45Ca2+ EFFLUX
107
either as a desaturation curve or expressed as the fractional efflux (rate constant) defined as the fraction of '5Ca2+ leaving the tissue per minute (Matthews et al. 1973). Pituitaries were dried overnight at 800 C and Ca2+ extracted with 0-1 N-HNO3 for 48 hr at room temperature and 45Ca2+ content determined. Stable Ca2+ content was determined by atomic absorption spectrophotometry of diluted extracts containing La3+ and HCl to prevent Ca2+ complex formation. Quench correction of radioactive samples was by the sample channels ratio technique. In some cases aliquots of the superfusate were removed and processed in duplicate for LH content by radioimmunoassay (Osland et al. 1975). The anti-ovine LH serum was provided generously by Dr G. D. Niswender (Colorado State University). LH values are expressed in terms of the NIAMDD rat LH-RP- 1 reference preparation. Synthetic LH-RH and crude rat hypothalamic extract were obtained from NIAMDD. Synthetic TRH was obtained from Dr R. Guillemin (Salk Institute). All were dissolved directly in KHB or else dissolved in H20 with aliquots added to KHB. When the concentration of K+ in the superfusing medium was increased tenfold, Na+ was reduced to maintain osmolarity. Ca-free KHB was prepared by omitting CaCl2 and had a Ca2+ content of approximately 5 /M as determined by atomic absorption spectrophotometry. All figures showing representative experiments are representative of three or more experiments. RESULTS
Preliminary experiments established that 45Ca2+ slowly equilibrated with total pituitary Ca2+ over several hours as reported by Milligan & Kraicer (1971). After 1 hr of incubation the total 45Ca2+ pool size was 500, ° 300 x E
&
ci. c 0 U
100 0
+0
20
00
0
20
40
60 80 100 Time (min)
120
140
Fig. 1. Efflux of 45Ca2+ from anterior pituitary fragments obtained from an intact male rat incubated 1 hr in 45Ca2+ and superfused at 1 ml./min (i.e. 2 chamber volumelmin). Each point represents counts remaining in the pituitary at the start of a 4 min collection period.
J. A. WILLIAMS 4-75 + 0-19 (mean + s.E.) n-mole/mg wet wt. while the total Ca2+ content was 5-37 + 007 n-mole/mg (n = 6). The 1 hr loading time was chosen since it was not feasible to load to equilibrium and then carry out a prolonged superfusion. Furthermore, in previous studies on the pancreas,
108
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160
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Fig. 2. Simultaneous determination of the effect of LH-RH on 4(Caz+ fractional efflux and LH release by ovariectomized rat pituitary fragments. LH-RH was present at a concentration of 10-6 M as indicated by the bar. Each point represents the midpoint of a 2 min collection period. A-A, 45Ca2+ fractional efflux; 0-0, LH release. A, superfusion with KHB. B, superfusion with O-Ca2+ KHB.
loading time did not greatly influence the 45Ca2+ efflux curve (Matthews et al. 1973; unpublished data). Fig. 1 shows the pattern of 45Ca2+ efflux after loading a normal rat pituitary 1 hr with 45Ca2+. Note the multiphasic nature of the washout pattern with a slow exponential release seen by 90 min. No difference in this pattern was seen between pituitaries from intact male or ovari-
LH-RH AND PITUITARY 45Ca2+ EFFLUX 109 ectomized female rats. When pituitaries from ovariectomized rats were stimulated with LH-RH at 120 min, the LH content of the superfusate was increased (Fig. 2A) as described previously (Osland et al. 1975). Simultaneous determination of 45Ca2+ efflux shows a transient increase in 45Ca2+ fractional efflux which then returned to the original value even 300
4sCa2+ efflux 1|LH
@200 200
C
/
0100 01
0'? .10-'° 10-9 10-11. 10-7 10-11 LH-RH (M)
10-S
Fig. 3. Effect of LH-RH on anterior pituitary 45Ca2+ efflux and LH release. Average maximum increase in 45Ca2+ efflux (@-@), as compared to fractional efflux immediately prior to LH-RH is plotted as a function of the concentration of LH-RH. Data for sustained LH released (0-0) measured in the same apparatus is from Osland, Gallo & Williams (1975). All 4VCa2+ points are the mean ± S.E. of five to eight experiments. TABLE 1. Effect of estradiol pre-treatment on basal and LH-RH-stimulated 45Ca efflux from rat pituitary glands 45Ca2+ fractional efflux Rats Ovariectomized
Basal 0-0041
LH-RH stimulated 0-0136
% increase 235 +33 62 +12
+0±0004 +0±0021 Ovariectomized 0-0046 0-0074 + oestradiol ±0-0004 ±0-0007 All rats were ovariectomized 3-4 weeks before use. Oestradiol benzoate (50 jug) was administered s.c. 2 hr before decapitation. Pituitaries were then incubated with 45Ca2+ 1 hr and superfused with KHB. Two min samples were collected from 110-140 min and LH-RH (2 x 10-7 M) was added after 120 min superfusion. Basal fractional efflux was determined from 116 to 120 min while LH-RH-stimulated fractional efflux is the maximum value reached after addition of LH-RH. All values are the mean ± s.E. for six rats.
110 J. A. WILLIAMS though LH release was maintained. The maximal increase in 45Ca2+ efflux as a function of the concentration of LH-RH is shown in Fig. 3. Also shown for comparison is the maintained increase in LH release (measured 30-60 min after LH-RH addition) previously measured under similar conditions (Osland et al. 1975). As shown in Table 1, pre-treatment of rats with a large dose of oestradiol known to inhibit LH-RH-stimulated LH release (Vilchez-Martinez, 0-015 200 _ 0010
1
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~~~~~~~~~~~~~ ~~~~C
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'. 0
0
100
120
140 Time (min)
160
180
Fig. 4. Simultaneous determination of the effect of an elevated concentration of K+ in the medium on 45Ca2+ fractional efflux and LH release. The concentration of K+ was increased tenfold (to 47 mM) as indicated by the bar. A-A, 45C+2 faractional efflux; *--, LH release.
Arimura, Debeljuk & Schally, 1974) reduced the 45Ca2+ efflux in response to this dose of LH-RH. Basal 45Ca2+ fractional efflux was not significantly changed. Since removal of Ca2+ from the medium is known to reduce LH release, the LH and 45Ca2+ release patterns were determined concurrently after superfusion with Ca2+ free medium and the results of a typical experiment shown in Fig. 2 B. Stimulation of both LH release and 45Ca2+ fractional efflux were reduced by about 60-70%. In four experiments using Ca2+ free medium 45Ca2+ fractional efflux was increased 76 + 14% by 10-6 MLH-RH as compared to 242+23% when superfused with KHB plus LH-RH. Elevation of the concentration of K+ in the medium to 10 times normal increased both 45Ca2+ efflux and LH release in a manner similar to that of
111 LH-RH AND PITUITAR Y 45Ca2+ EFFLUX LH-RH as shown in Fig. 4. Addition of theophylline (5 mmx) or dibutyryl cyclic AMP (1-2 mM) separately (three experiments each) or together had no effect on either LH release or 4OCa2+ fractional efflux. Fig. 5 shows the failure of dibutyryl cyclic AMP plus theophylline to affect either basal values or the basic pattern in response to LH-RH stimulation. The effect of pituitary stimulants was also examined in pituitaries from normal and thyroidectomized male rats. LH-RH induced only a 400
300
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120
180
200
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Fig. 5. Effect of dibutyryl cyclic AMP plus theophyline followed by LHRH on 45Ca2+ fractional efflux and LH release by pituitary fraglrments from an ovariectomized rat. Each point represents the mid point of a 2 min collection period. A-At 45Ca2+ fractional efflux; *-*, LH release. TABLE 2. Effects of various stimulators
on
4SCa2+ efflux from rat anterior pituitary 45Ca2+
fractional efflux % increase)
(max
Ovariectomized Thyroidectomized Normal male Stimulator 26 ± 9 (5) 242 + 23 (6) 34 + 6 (6) LH-RH (10-6M) 263 + 28 (7) 24 + 5 (6) 26 + 8 (5) TRH (2*8 x 10-7 M) 57 ± 11 (6) 192 + 39 (6) Hypothalamic extract (1 hypothalamus/ml.) 122 ± 13 (4) 119 ± 20 (5) K+ (54 mM) All values are the mean ± S.E. of the number of experiments shown in parentheses. All stimulators were applied at 120 min.
J. A. WILLIAMS slight increase in 45Ca2+ release when pituitaries from normal or thyroidectomized male rats were used, while TRH caused a large increase in 45Ca2+ efflux only when pituitaries from thyroidectomized rats were used (Table 2). That LH-RH and TRH increase 45Ca2+ release from pituitaries 112
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Fig. 6. Effect of LH-RH and TRH on 45Ca2+ fractional efflux by pituitary glands from ovariectomized (A) and thyroidectomized (B) rats. LH-RH was present at a concentration of 106 M while TRH was present at 2-8 x 1O-7 M when indicated.
previously stimulated by the other releasing hormone is shown in Fig. 6. Hypothalamic extract induced a larger release from ovariectomized rat pituitaries than from pituitaries from intact rats, whereas an elevated concentration of K+ in the medium increased 45Ca2+ efflux equally (Table 2).
LH-RH AND PITUITARY 45Ca2+ EFFLUX
113
DISCUSSION
In the present work LH-RH, TRH, hypothalamic extract and K+ all stimulated 45Ca2+ efflux from rat pituitaries. The action of LH-RH and TRH should be specific to the gonadotrophs and thyrotrophs respectively and this is consistent with the much greater effect of the appropriate releasing factor on pituitaries from ovariectomized or thyroidectomized animals compared to pituitaries from intact rats. The findings that LHRH and TRH still produce their characteristic increase in 45Ca2+ efflux in the presence of the other releasing factor (Fig. 6) confirms that each releasing hormone is acting on a separate cell population. Rat hypothalamic extract which should stimulate all types did produce a significantly greater effect than LH-RH when tested on pituitaries from intact rats but the effect was small when compared to the action of LH-RH in pituitaries from ovariectomized rats. There are several possible explanations for this result: hypothalamic extract may contain a mixture of stimulator and inhibitory factors; only certain pituitary cell types may show a stimulated 45Ca2+ release; or the effect of hypothalamic extract may be augmented in chronically stimulated cells. In contrast to LH-RH, K+ stimulated 45Ca2+ efflux similarly from both ovariectomized and intact rat pituitaries. Increased 45Ca2+ efflux and hormone release were tightly associated in response to three different stimuli. Efflux increases simultaneously with, or before the release of LH in response to LH-RH when both were measured. Furthermore, the magnitude of stimulated 4&Ca2+ efflux shows a similar dependence on the concentration of LH-RH as does LH release. Removal of extracellular Ca2+ reduced both LH release and stimulated 45Ca2+ efflux in response to LH-RH. Acute pre-treatment of ovariectomized rats with oestradiol also inhibited the LH-RH induced increase in 45Ca2+ efflux (Table 1). Other stimulators known to affect pituitary secretion also stimulated 45Ca2+ release in a manner similar to LH-RH. Thus, stimulation of 45Ca2+ release is a consistent accompaniment to release of at least LH and TSH and probably other adenohypophyseal hormones. While the underlying event leading to increased 45Ca2+ efflux cannot be uniquely determined at present, the finding of an increased Ca2+ efflux in response to pituitary stimulants clearly identifies an effect on cellular Ca2+. Ca2+ active in stimulus secretion coupling might enter the cytosol from the extracellular fluid or be released intracellularly from Ca2+ stored in organelles or bound to membranes (Borle, 1973). The stimulated 45Ca2+ efflux described here could result from a direct release of intracellular 45Ca2+ or from a rise in intracellular 4OCa2+ with displacement of 45Ca2+
114 J. A. WILLIAMS but is in either case consistent with a rise in the concentration of free intracellular Ca 2+. The cellular site of origin of the stimulated Ca2+ efflux is not known at present. It seems unlikely that the increased Ca2+ efflux comes from Ca2+ bound to secretary granules as the increased 45Ca2+ efflux is transient compared to hormone release. Ca2+ is present in most cells primarily in mitochondria (Borle, 1973) and is bound to plasma and intracellular membranes (Clemente & Meldolesi, 1975). While further work is necessary to establish the site and function of intracellular Ca2+ pools, the present work provides a new approach to the study of the role of Ca2+ in adenohypophyseal secretion. I thank B. Moffat, M. Lee and A. deCarlo for skilled technical assistance and Dr Urban Rosenqvist for providing the thyroidectomized rats. This work was supported by USPHS grants GM 19998 and AM 06704 from the National Institutes of Health and a Rockefeller Foundation Center grant.
REFERENCES
BORLE, A. B. (1973). Calcium metabolism at the cellular level. Fedn Proc. 32, 1944-1950. CASE, R. M. & CLAUSEN, T. (1973). The relationship between calcium exchange and enzyme secretion in the isolated rat pancreas. J. Physiol. 235, 75-102. CLEMENTE, F. & MELDOLESI, J. (1975). Calcium and pancreatic secretion. I. Subcellular distribution of calcium and magnesium in the exocrine pancreas of the guinea pig. J. cell Biol. 65, 88-102. DOUGLAS, W. W. (1968). Stimulus-secretion coupling: the concept and clues from chromaffin and other cells. Br. J. Pharmac. Chemother. 34, 451-474. DOUGLAS, W. W. & POISNER, A. M. (1964). Calcium movement in the neurohypophysis of the rat and its relation to the release of vasopressin. J. Physiol. 172, 19-30. ETO, S., WOOD, J. M., HUTCHINS, M. & FLEISCHER, N. (1974). Pituitary 45Ca++ uptake and release of ACTH, GH, and TSH: effect of verapamil. Am. J. Phyeiol. 226, 1315-1320. MALAISSE, W. J., BRISSON, G. R. & BAIRD, L. E. (1973). Stimulus-secretion coupling of glucose-induced insulin release. X. Effect of glucose on 45Ca efflux from perfused islets. Am. J. Physiol. 224, 389-394. MATTHEWS, E. K., PETERSEN, 0. H. & WILLIAMS, J. A. (1973). Pancreatic acinar cells: acetylcholine-induced membrane depolarization, calcium efflux and amylase
release. J. Physiol. 234, 689-701. MILLIGAN, J. V. & KRAICER, J. (1971). 45Ca uptake during the in vitro release of hormones from the rat adenohypophysis. Endocrinology 89, 766-773. MILLIGAN, J. V. & KRAICER, J. (1974). Physical characteristics of the Ca++ compartments associated with in vitro ACTH release. Endocrinology 94, 435-443. NIELSEN, S. P. & PETERSEN, 0. H. (1972). Transport of calcium in the perfused submandibular gland of the cat. J. Physiol. 223, 685-697. OSLAND, R. B., GALLO, R. V. & WILLIAMS, J. A. (1975). In vitro release of luteinizing hormone from anterior pituitary fragments superfused with constant or pulsatile ainounts of luteinizing hormone releasing factor. Endocrinology 96, 1210-1216.
LH-RH AND PITUITARY 45Ca2+- EFFLUX
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RUBIN, R. P. (1970). The role of calcium in the release of neurotransmitter substances and hormones. Pharmac. Rev. 22, 389-428. SAMLI, M. H. & GESCHWIND, I. I. (1968). Some effects of energy-transfer inhibitors and of Ca++-free or K-enhanced media on the release of luteinizing hormone (LH) from the rat pituitary gland in vitro. Endocrinology 82, 225-231. VILCHEZ-MARTINEZ, J. A., ARIMURA, A., DEBELJUK, L. & SCHALLY, A. V. (1974). Biphasic effect of estradiol benzoate on the pituitary responsiveness to LH-RH. Endocrinology 94, 1300-1303. WAKABAYASHI, K., DATE, Y. & TAMAOKI, B. (1973). On the mechanism of action of luteinizing hormone-releasing factor and prolactin release inhibiting factor. Endocrinology 92, 698-704. WAKABAYASHI, K., KAMBERI, I. A. & MCCANN, S. M. (1969). In vitro response of rat pituitary to gonadotrophic releasing factors and to ions. Endocrinology 85, 1046-1056. WILLIAMS, J. A. (1970). Origin of transmembrane potentials in non-excitable cells. J. theor. Biol. 28, 287-296.
105
STIMULATION OF 45Ca2+ EFFLUX FROM RAT PITUITARY BY LUTEINIZING HORMONE-RELEASING HORMONE AND OTHER PITUITARY STIMULANTS
BY J. A. WILLIAMS From the Department of Physiology, University of California, San Francisco, California 94143, U.S.A.
(Received 29 December 1975) SUMMARY
1. Rat anterior pituitaries were, incubated in medium containing 45Ca2+, superfused with non-radioactive medium and the efflux of 45Ca2+ studied. 2. When pituitaries from overiectomized rats were used the addition of LH-RH to the medium at a time when slow exponential efflux of Ca2+ was occurring produced a transient increase in 45Ca2+ efflux simultaneous with or before an increased release of LH. The stimulation of both 45Ca2+ efflux and LH release showed a similar concentration dependence on LHRH. 3. Increasing the K+ concentration of the medium tenfold also stimulated 45Ca2+ efflux and LH release. The response to LH-RH of both parameters was reduced by superfusion with calcium-free medium. 4. LH-RH induced only a small increase in pituitary 45Ca2+ efflux when intact or thyroidectomized male rats were used. TRH increased 45Ca2+ efflux from thyroidectomized rat pituitaries but had only a small effect when pituitaries from intact or ovariectomized rats were studied. 5. Pretreatment of ovariectomized rats with oestradiol inhibited the subsequent increase in 45Ca2+ efflux induced by LH-RH. 6. It is concluded that the secretagogue induced increase in 45Ca2+ efflux results from an increase in cellular Ca2+ activity which is presumably active in stimulus-secretion coupling. INTRODUCTION
Stimulus-secretion coupling of luteinizing hormone (LH) release in response to luteinizing hormone-releasing hormone (LH-RH) is believed to involve Ca2+ in a manner similar to that utilized in the release of secretory products from a number of tissues (Douglas, 1968; Rubin, 1970).
106 J. A. WILLIAMS Evidence for this scheme, in which receptor activation triggers Ca2+ entry, depolarization and hormone release, includes the finding that LH release is stimulated by an elevated concentration of K+ in the medium (Samli & Geschwind, 1968; Wakabayashi, Kamberi & McCann, 1969; Wakabayashi, Date & Tamaoki, 1973). An increase in K+ should depolarize the gonadotrophs (Williams, 1970) and does increase 45Ca2+ uptake into the whole pituitary (Milligan & Kraicer, 1971). Furthermore, LH release in response to both K+ and hypothalamic extract is reduced by incubation in Ca-free medium (Samli & Geschwind, 1968). Recently, however, evidence has been presented that hypothalamic releasing factors may not promote a specific calcium influx (Milligan & Kraicer, 1974; Eto, Wood, Hutchins & Fleischer, 1974). Since methods have not yet been developed to measure Ca2+ activity within secretary cells, there is little data showing how, or even whether a rise in intracellular free Ca2+ occurs; and if it does, whether Ca2+ directly promotes secretion. In the exocrine and endocrine pancreas, neurohypophysis and parotid glands, stimulators of granule release, are known to increase 45Ca2+ efflux from slowly exchanging compartments (Douglas & Poisner, 1964; Nielsen & Petersen, 1972; Case & Clausen, 1973; Matthews, Petersen & Williams, 1973; Malaisse, Brisson & Biard, 1973). We therefore decided to test whether LH-RH would analogously increase 45Ca2+ efflux from the anterior pituitary. We used pituitaries from ovariectomized rats since an increase in LH release can be easily demonstrated in this preparation (Osland, Gallo & Williams, 1975). The present results demonstrate a transient, concentration dependent increase in 45Ca2+ efflux in response to LH-RH and other pituitary stimulants. The possible relation to stimulussecretion coupling is discussed. METHODS
All studies were carried out using adult Sprague-Dawley rats. Ovariectomies were performed on 150-200 g female rats 3-5 weeks before use. Thyroidectomies were performed by injection of 1 mc l31J into 90-100 g rats and pituitaries were studied 6-8 weeks later. At the time of use the thyroidectomized rats weighed 200-250 g as compared to intact litter mates which weighed 350-400 g. Intact male rats weighed 300-400 g. Anterior pituitaries were removed, cut into 8 pieces and incubated 1 hr in Krebs-Henseleit bicarbonate (KHB) (Matthews et al. 1973) containing 45Ca2+ 15 ,uc/ml. (New England Nuclear) in a shaking water-bath. The medium was equilibrated with 95 % 02-5 % CO2 (pH 7.35) and was maintained at 370 C in all experiments. The pituitary fragments were subsequently transferred to a 0 5 ml. volume superfusion apparatus through which pre-warmed KHB was passed at 2 chamber volumes/min (Osland et al. 1975). Effluent was collected into plastic scintillation vials during timed intervals (2 min unless otherwise specified). Scintillation fluid (Toluene: Triton X-100, 2:1, butyl PBD, 4 g/l.) was added and radioactivity determined by liquid scintillation counting. The efflux of 45Ca2+ is shown
LH-RH AND PITUITARY 45Ca2+ EFFLUX
107
either as a desaturation curve or expressed as the fractional efflux (rate constant) defined as the fraction of '5Ca2+ leaving the tissue per minute (Matthews et al. 1973). Pituitaries were dried overnight at 800 C and Ca2+ extracted with 0-1 N-HNO3 for 48 hr at room temperature and 45Ca2+ content determined. Stable Ca2+ content was determined by atomic absorption spectrophotometry of diluted extracts containing La3+ and HCl to prevent Ca2+ complex formation. Quench correction of radioactive samples was by the sample channels ratio technique. In some cases aliquots of the superfusate were removed and processed in duplicate for LH content by radioimmunoassay (Osland et al. 1975). The anti-ovine LH serum was provided generously by Dr G. D. Niswender (Colorado State University). LH values are expressed in terms of the NIAMDD rat LH-RP- 1 reference preparation. Synthetic LH-RH and crude rat hypothalamic extract were obtained from NIAMDD. Synthetic TRH was obtained from Dr R. Guillemin (Salk Institute). All were dissolved directly in KHB or else dissolved in H20 with aliquots added to KHB. When the concentration of K+ in the superfusing medium was increased tenfold, Na+ was reduced to maintain osmolarity. Ca-free KHB was prepared by omitting CaCl2 and had a Ca2+ content of approximately 5 /M as determined by atomic absorption spectrophotometry. All figures showing representative experiments are representative of three or more experiments. RESULTS
Preliminary experiments established that 45Ca2+ slowly equilibrated with total pituitary Ca2+ over several hours as reported by Milligan & Kraicer (1971). After 1 hr of incubation the total 45Ca2+ pool size was 500, ° 300 x E
&
ci. c 0 U
100 0
+0
20
00
0
20
40
60 80 100 Time (min)
120
140
Fig. 1. Efflux of 45Ca2+ from anterior pituitary fragments obtained from an intact male rat incubated 1 hr in 45Ca2+ and superfused at 1 ml./min (i.e. 2 chamber volumelmin). Each point represents counts remaining in the pituitary at the start of a 4 min collection period.
J. A. WILLIAMS 4-75 + 0-19 (mean + s.E.) n-mole/mg wet wt. while the total Ca2+ content was 5-37 + 007 n-mole/mg (n = 6). The 1 hr loading time was chosen since it was not feasible to load to equilibrium and then carry out a prolonged superfusion. Furthermore, in previous studies on the pancreas,
108
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160
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0
Fig. 2. Simultaneous determination of the effect of LH-RH on 4(Caz+ fractional efflux and LH release by ovariectomized rat pituitary fragments. LH-RH was present at a concentration of 10-6 M as indicated by the bar. Each point represents the midpoint of a 2 min collection period. A-A, 45Ca2+ fractional efflux; 0-0, LH release. A, superfusion with KHB. B, superfusion with O-Ca2+ KHB.
loading time did not greatly influence the 45Ca2+ efflux curve (Matthews et al. 1973; unpublished data). Fig. 1 shows the pattern of 45Ca2+ efflux after loading a normal rat pituitary 1 hr with 45Ca2+. Note the multiphasic nature of the washout pattern with a slow exponential release seen by 90 min. No difference in this pattern was seen between pituitaries from intact male or ovari-
LH-RH AND PITUITARY 45Ca2+ EFFLUX 109 ectomized female rats. When pituitaries from ovariectomized rats were stimulated with LH-RH at 120 min, the LH content of the superfusate was increased (Fig. 2A) as described previously (Osland et al. 1975). Simultaneous determination of 45Ca2+ efflux shows a transient increase in 45Ca2+ fractional efflux which then returned to the original value even 300
4sCa2+ efflux 1|LH
@200 200
C
/
0100 01
0'? .10-'° 10-9 10-11. 10-7 10-11 LH-RH (M)
10-S
Fig. 3. Effect of LH-RH on anterior pituitary 45Ca2+ efflux and LH release. Average maximum increase in 45Ca2+ efflux (@-@), as compared to fractional efflux immediately prior to LH-RH is plotted as a function of the concentration of LH-RH. Data for sustained LH released (0-0) measured in the same apparatus is from Osland, Gallo & Williams (1975). All 4VCa2+ points are the mean ± S.E. of five to eight experiments. TABLE 1. Effect of estradiol pre-treatment on basal and LH-RH-stimulated 45Ca efflux from rat pituitary glands 45Ca2+ fractional efflux Rats Ovariectomized
Basal 0-0041
LH-RH stimulated 0-0136
% increase 235 +33 62 +12
+0±0004 +0±0021 Ovariectomized 0-0046 0-0074 + oestradiol ±0-0004 ±0-0007 All rats were ovariectomized 3-4 weeks before use. Oestradiol benzoate (50 jug) was administered s.c. 2 hr before decapitation. Pituitaries were then incubated with 45Ca2+ 1 hr and superfused with KHB. Two min samples were collected from 110-140 min and LH-RH (2 x 10-7 M) was added after 120 min superfusion. Basal fractional efflux was determined from 116 to 120 min while LH-RH-stimulated fractional efflux is the maximum value reached after addition of LH-RH. All values are the mean ± s.E. for six rats.
110 J. A. WILLIAMS though LH release was maintained. The maximal increase in 45Ca2+ efflux as a function of the concentration of LH-RH is shown in Fig. 3. Also shown for comparison is the maintained increase in LH release (measured 30-60 min after LH-RH addition) previously measured under similar conditions (Osland et al. 1975). As shown in Table 1, pre-treatment of rats with a large dose of oestradiol known to inhibit LH-RH-stimulated LH release (Vilchez-Martinez, 0-015 200 _ 0010
1
E
0~~~~~~~~~~~~~~~~~
u
~~~~~~~~~~~~~ ~~~~C
4
'. 0
0
100
120
140 Time (min)
160
180
Fig. 4. Simultaneous determination of the effect of an elevated concentration of K+ in the medium on 45Ca2+ fractional efflux and LH release. The concentration of K+ was increased tenfold (to 47 mM) as indicated by the bar. A-A, 45C+2 faractional efflux; *--, LH release.
Arimura, Debeljuk & Schally, 1974) reduced the 45Ca2+ efflux in response to this dose of LH-RH. Basal 45Ca2+ fractional efflux was not significantly changed. Since removal of Ca2+ from the medium is known to reduce LH release, the LH and 45Ca2+ release patterns were determined concurrently after superfusion with Ca2+ free medium and the results of a typical experiment shown in Fig. 2 B. Stimulation of both LH release and 45Ca2+ fractional efflux were reduced by about 60-70%. In four experiments using Ca2+ free medium 45Ca2+ fractional efflux was increased 76 + 14% by 10-6 MLH-RH as compared to 242+23% when superfused with KHB plus LH-RH. Elevation of the concentration of K+ in the medium to 10 times normal increased both 45Ca2+ efflux and LH release in a manner similar to that of
111 LH-RH AND PITUITAR Y 45Ca2+ EFFLUX LH-RH as shown in Fig. 4. Addition of theophylline (5 mmx) or dibutyryl cyclic AMP (1-2 mM) separately (three experiments each) or together had no effect on either LH release or 4OCa2+ fractional efflux. Fig. 5 shows the failure of dibutyryl cyclic AMP plus theophylline to affect either basal values or the basic pattern in response to LH-RH stimulation. The effect of pituitary stimulants was also examined in pituitaries from normal and thyroidectomized male rats. LH-RH induced only a 400
300
0*03
1-1
0
.12 x
I.-
200 ^ E
002
bo
-a=1 -J
0-1 ff00~0
100
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id
U
"A-W
-. _a _a
I
2 mM-DBc-AMP+
j 140 160 Time (min)
[5 mMtheophylline 0
100
120
180
200
0
Fig. 5. Effect of dibutyryl cyclic AMP plus theophyline followed by LHRH on 45Ca2+ fractional efflux and LH release by pituitary fraglrments from an ovariectomized rat. Each point represents the mid point of a 2 min collection period. A-At 45Ca2+ fractional efflux; *-*, LH release. TABLE 2. Effects of various stimulators
on
4SCa2+ efflux from rat anterior pituitary 45Ca2+
fractional efflux % increase)
(max
Ovariectomized Thyroidectomized Normal male Stimulator 26 ± 9 (5) 242 + 23 (6) 34 + 6 (6) LH-RH (10-6M) 263 + 28 (7) 24 + 5 (6) 26 + 8 (5) TRH (2*8 x 10-7 M) 57 ± 11 (6) 192 + 39 (6) Hypothalamic extract (1 hypothalamus/ml.) 122 ± 13 (4) 119 ± 20 (5) K+ (54 mM) All values are the mean ± S.E. of the number of experiments shown in parentheses. All stimulators were applied at 120 min.
J. A. WILLIAMS slight increase in 45Ca2+ release when pituitaries from normal or thyroidectomized male rats were used, while TRH caused a large increase in 45Ca2+ efflux only when pituitaries from thyroidectomized rats were used (Table 2). That LH-RH and TRH increase 45Ca2+ release from pituitaries 112
0-020 r A
0015 0010 ,
4
0'005
£ I.-
.N I
4 '4
110
0'020 rl
I
I
120
130
140
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140
150
B
,j 0020 IF_ tor I
oun r
- ---
o0oio 1 0*005;
_
110
_ ..iiiiiiiiii.LH-RH..
120
130 Time (min)
Fig. 6. Effect of LH-RH and TRH on 45Ca2+ fractional efflux by pituitary glands from ovariectomized (A) and thyroidectomized (B) rats. LH-RH was present at a concentration of 106 M while TRH was present at 2-8 x 1O-7 M when indicated.
previously stimulated by the other releasing hormone is shown in Fig. 6. Hypothalamic extract induced a larger release from ovariectomized rat pituitaries than from pituitaries from intact rats, whereas an elevated concentration of K+ in the medium increased 45Ca2+ efflux equally (Table 2).
LH-RH AND PITUITARY 45Ca2+ EFFLUX
113
DISCUSSION
In the present work LH-RH, TRH, hypothalamic extract and K+ all stimulated 45Ca2+ efflux from rat pituitaries. The action of LH-RH and TRH should be specific to the gonadotrophs and thyrotrophs respectively and this is consistent with the much greater effect of the appropriate releasing factor on pituitaries from ovariectomized or thyroidectomized animals compared to pituitaries from intact rats. The findings that LHRH and TRH still produce their characteristic increase in 45Ca2+ efflux in the presence of the other releasing factor (Fig. 6) confirms that each releasing hormone is acting on a separate cell population. Rat hypothalamic extract which should stimulate all types did produce a significantly greater effect than LH-RH when tested on pituitaries from intact rats but the effect was small when compared to the action of LH-RH in pituitaries from ovariectomized rats. There are several possible explanations for this result: hypothalamic extract may contain a mixture of stimulator and inhibitory factors; only certain pituitary cell types may show a stimulated 45Ca2+ release; or the effect of hypothalamic extract may be augmented in chronically stimulated cells. In contrast to LH-RH, K+ stimulated 45Ca2+ efflux similarly from both ovariectomized and intact rat pituitaries. Increased 45Ca2+ efflux and hormone release were tightly associated in response to three different stimuli. Efflux increases simultaneously with, or before the release of LH in response to LH-RH when both were measured. Furthermore, the magnitude of stimulated 4&Ca2+ efflux shows a similar dependence on the concentration of LH-RH as does LH release. Removal of extracellular Ca2+ reduced both LH release and stimulated 45Ca2+ efflux in response to LH-RH. Acute pre-treatment of ovariectomized rats with oestradiol also inhibited the LH-RH induced increase in 45Ca2+ efflux (Table 1). Other stimulators known to affect pituitary secretion also stimulated 45Ca2+ release in a manner similar to LH-RH. Thus, stimulation of 45Ca2+ release is a consistent accompaniment to release of at least LH and TSH and probably other adenohypophyseal hormones. While the underlying event leading to increased 45Ca2+ efflux cannot be uniquely determined at present, the finding of an increased Ca2+ efflux in response to pituitary stimulants clearly identifies an effect on cellular Ca2+. Ca2+ active in stimulus secretion coupling might enter the cytosol from the extracellular fluid or be released intracellularly from Ca2+ stored in organelles or bound to membranes (Borle, 1973). The stimulated 45Ca2+ efflux described here could result from a direct release of intracellular 45Ca2+ or from a rise in intracellular 4OCa2+ with displacement of 45Ca2+
114 J. A. WILLIAMS but is in either case consistent with a rise in the concentration of free intracellular Ca 2+. The cellular site of origin of the stimulated Ca2+ efflux is not known at present. It seems unlikely that the increased Ca2+ efflux comes from Ca2+ bound to secretary granules as the increased 45Ca2+ efflux is transient compared to hormone release. Ca2+ is present in most cells primarily in mitochondria (Borle, 1973) and is bound to plasma and intracellular membranes (Clemente & Meldolesi, 1975). While further work is necessary to establish the site and function of intracellular Ca2+ pools, the present work provides a new approach to the study of the role of Ca2+ in adenohypophyseal secretion. I thank B. Moffat, M. Lee and A. deCarlo for skilled technical assistance and Dr Urban Rosenqvist for providing the thyroidectomized rats. This work was supported by USPHS grants GM 19998 and AM 06704 from the National Institutes of Health and a Rockefeller Foundation Center grant.
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RUBIN, R. P. (1970). The role of calcium in the release of neurotransmitter substances and hormones. Pharmac. Rev. 22, 389-428. SAMLI, M. H. & GESCHWIND, I. I. (1968). Some effects of energy-transfer inhibitors and of Ca++-free or K-enhanced media on the release of luteinizing hormone (LH) from the rat pituitary gland in vitro. Endocrinology 82, 225-231. VILCHEZ-MARTINEZ, J. A., ARIMURA, A., DEBELJUK, L. & SCHALLY, A. V. (1974). Biphasic effect of estradiol benzoate on the pituitary responsiveness to LH-RH. Endocrinology 94, 1300-1303. WAKABAYASHI, K., DATE, Y. & TAMAOKI, B. (1973). On the mechanism of action of luteinizing hormone-releasing factor and prolactin release inhibiting factor. Endocrinology 92, 698-704. WAKABAYASHI, K., KAMBERI, I. A. & MCCANN, S. M. (1969). In vitro response of rat pituitary to gonadotrophic releasing factors and to ions. Endocrinology 85, 1046-1056. WILLIAMS, J. A. (1970). Origin of transmembrane potentials in non-excitable cells. J. theor. Biol. 28, 287-296.
