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Measurement of prolactin release and cytosolic calcium in estradiol-primed lactotrophs
Life Sciences, Vol. 53, pp. 1605-1616 Printed in the USA
Pergamon Press
MEASUREMENT OF PROLACTIN RELEASE AND CYTOSOLIC CALCIUM IN ESTRADIOL-PRIMED LACTOTROPHS S.H. Shin, C. Soukup, S.C. Pang +, T.J. Kubiseski* and T.G. Flynn* Departments of Physiology, Anatomy + and Biochemistry* Queen's University Kingston, Ontario, Canada K7L 3N6
(Received in final form September 14, 1993)
Summary_
We have developed a perifusion system that can measure both changes of cytosolic free calcium concentration [Ca2+]i and prolactin release simultaneously from cultured lactotrophs. This model incorporated a commonly-used perifusion system to a spectrofluorometer. Indo-1 loaded cells were injected into Sephadex G-150 matrix in the cuvette at a site where the emitting light of the fluorometer projects. During perifusion periods, the perifusate was collected in a fraction collector, while optical density of the emitting light at 405 nm was recorded. The [Ca2+]i was calculated based on an ionomycin and Mn 2+ quenching technique. As expected, TRH (1 umol/1) stimulated prolactin release from cultured lactotrophs in this system. We further observed that prolactin releases as induced by TRH and ionomycin were not proportional with changes of the [Ca2+]i, suggesting that changes of [Ca2+]i is not the sole final pathway of intracellular transduction systems for prolactin release. In lactotrophs, changes in the cytosolic free calcium concentration ([Ca2+]i) are an important aspect of the subcellular transduction system of prolactin release (27, 12). Simultaneous measurement of levels of [Ca2+]~ and prolactin release from a specimen are necessary in correlating the relationship between alteration in [Ca2+]i and prolactin release. Our system is an improved version of previosly published work by Law et al. (13). In order to establish a model system of this type, it is essential first to obtain a homogeneous or near homogeneous population of lactotrophs. This can be achieved by raising blood concentrations of estradiol for a 10 to 13 week period through implantation
Address all correspondence to: Dr. S.H. Shin, Department of Physiology, Queen's University,Botterell Hall, Kingston, Ontario, Canada KTL 3N6 0024-3205/93 $6.00 + .00 Copyright © 1993Pergamon Press Ltd All rights reserved.
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of an estrogen capsule. Secondly, a perifusion system which is incorporated to a fluorometer is used to facilitate the continuous measurement of changes in [Ca2+]i. This was accomplished by several modifications of a commonly used perifusion system. In this study, we introduced a perifusion system which allowed us to simultaneously measure changes of [Ca2+]i and prolactin release, and have found that [Ca2+]i and prolactin release were not always proportionally related. This illustrates that altered [Ca2+]i levels are not the sole final regulators of the subcellular transduction systems of prolactin release. Materials and Methods Animal Male Sprague-Dawley rats (Charles River, CD, Canadian Breeding Farm and Laboratories, Montreal) were housed in a controlled environment with illumination for 14 h daily (06.00 - 20.00 h) at a temperature of 25 __. 1 "C. Purina Lab Chow and tap water were supplied ad libitum. 10 to 13 weeks before the experiments, rats received implants of Silastic tubing (Silastic medical grade tubing, 0.062 inches inner diameter, 0.125 inches outer diameter, 3 cm in length, Dow Corning Corp) containing estradiol-17/3 (60 mg). The tubings were sealed with a plug of Silastic medical grade elastomer. These estradiol-primed rats showed a mean plasma estradiol concentration of 1.6 ___0.4 nmol/l (19). Using indirect immunohistofluorescent techniques, the population of lactotrophs was found to increase to 82 _+ 3% of the total dispersed pituitary cell population after the 10-13 week priming period (unpublished observation) which is consistent with others (16, 18). Pituitary cells were dispersed as described in the following section and cultured for 2-4 days prior to perifusion experiments. Cell dispersion and culture The estradiol-primed male rats (300-330 g) were decapitated and their adenohypophyses were collected and dispersed with trypsin (bovine pancreas Type III, Sigma Chemical Co., St. Louis) under sterile conditions as previously described (22). Briefly, the adenohypophyses were cut to small pieces (< 1 mm 3) and placed in Spinner's minimum essential medium (S-MEM) (Gibco Labs., Grand Island, NY) containing 0.05% trypsin and 0.1% bovine serum albumin (BSA) (Cohn Fraction V, Sigma Chemical Co.) in a Spinner's flask. Following 1 h dispersion at 37"C, the cells were collected by centrifugation (750 x g for 10 min). The pellet was resuspended in SMEM containing 0.02% lima bean trypsin inhibitor (type II-L, Sigma Chemical Co.) and recentrifuged. The dispersed adenohypophysial cells were suspended in 4 ml of Dulbecco's modified Eagle's medium (DMEM) supplemented with 2.5% fetal calf serum and, 15% horse serum (Gibco Lab., Grand Island, N.Y.). Estradiol and penicillin (Sigma Chemical Co.) were added to make 1 nmol/1 and 50 IU/ml, respectively (DMEM culture medium). Each pituitary yielded approximately 10-12 million cells. The cell suspension was distributed to 2 Petri dishes (35x10 style Falcon Plastic, Oxnard, CA) (2 ml/dish; 5 - 6 million cells). The adenohypophysial cells were cultured in a water jacketed incubator at 37"C under a water saturated atmosphere of 5% CO 2 and 95% O z for 2-4 days. The cells were cultured in Petri dishes which were not treated for monolayer culture. Therefore, they did not attach to the dish, but formed cell clusters. These aggregations could easily be recovered without enzyme treatment or mechanical scraping. This allowed the use of undamaged cells for subsequent experiments. Emission spectra Acetoxymethyl Indo-1 (Indo-1, AM; Molecular Probes, Inc., Eugene, OR, USA) was hydrolyzed using an esterase present in pituitary ceils in order to
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Cytosolic[Ca2+]i and Prolactin Release
examine the emission spectra. Cultured adenohypophysial cells were collected by centrifugation (750 x g for 10 min) and were resuspended in 1 ml DMEM. 10 ~1 dimethylsulfoxide (DMSO, Baker Chemical Co., Phillipsburg, N J, USA) solution containing 10 nmole Indo-1, AM was added in the 1 ml DMEM of the cell suspension to make 10 t,mol/l Indo-1, AM. This suspension was incubated for 45 min at 37°C. The cells in the medium containing Indo-1 were left in room temperature for 1 h and were homogenized in a glass tube. The homogenate was left in room temperature for 1 h in order to further hydrolyze ester of Indo-1, AM, and removed cell debris by centrifugation (20,000 x g for 30 min). The supernatant was used to test emission spectra with different gel matrices (Sephadex G-25, G-150), and different perifusion media (DMEM with or without phenol red and 0.1% BSA).
Construction of cuvette for perifusion A semi-micro size disposable cuvette (Dynalon Disposable polystyrene cuvettes, 1.5 ml, 10x4x45 mm, Canlab Scientific Products, Toronto, Canada) was chosen for this study to reduce the dead space of the Sephadex gel matrix. The lower right corner of the cuvette face which receives excitation spectra, was drilled and a silicone tubing (1/23 inch inner diameter, 5/64 inch outer diameter) was inserted in the hole. The lower part of the cuvette was plugged with glass wool (Corning Glass Works, Corning, NY, USA) in order to prevent leakage of the gel matrix. The cuvette was packed with Sephadex gel matrix which was equilibrated with the following perifusion media: DMEM with or without phenol red and with or without 0.1% BSA (DMEM-BSA). The cuvette was capped with a 00 size rubber stopper which had been pierced by a 18 gauge steel tubing (syringe needle, Becton, Dickinson & Co.). The stopper was then connected to a medium reservoir with Tygon tubing (R-3603, 1/32 inch inner diameter, 3/32 outer diameter). It is necessary for a matrix to be present in the cuvette to attain a successful perifusion system. Commonly used matrices for perifusion are Sephadex gels or Bio-gels. We scanned the spectra of Sephadex G-25 and G-150 in order to find suitable gel for this study. The Sephadex G-25 gel blocked exciting/emitting light slightly more than the Sephadex G-100 gel. Sephadex G-150 is more translucent than Sephadex G-25 and thus, it was easier to locate the injected cells in the cuvette. Therefore, Sephadex G-150 was chosen for this study.
Loading cells into the cuvette The primary cultured cells were collected by centrifugation (750 x g for 10 min) from the culture medium and resuspended in DMEM. 10 ~l dimethylsulfoxide (DMSO) solution containing 10 nmole Indo-1, AM was added to the 10 ml DMEM to make 1 ~mol/1 Indo-1, AM and incubated for 30 rain at 37"C. The cells were recovered by centrifugation (750 x g for 10 rain) and resuspended in 10 ml DMEM-BSA. The suspension of Indo-1 loaded cells were left for 30 min at room temperature. The cells were recovered by centrifugation and resuspended in 50 ~1 DMEM-BSA. The cuvette containing 1 ml of Sephadex G-150 was equilibriated with perifusion medium (DMEM-BSA). 0.2 ml of DMEM-BSA was added on top of the Sephadex matrix. The cell suspension was then injected into the Sephadex G-150 matrix using a Pasteur pipette which had been previously equilibrated with the perifusion medium (DMEM-BSA). The injection of the Indo-1 loaded cells was aimed to a position between 9 and 18 mm from the bottom of the cuvette where the excitation light projects in LS-50 Spectrofluorometer (Perkin-Elmer Ltd., Buckinghamshire, UK). The cuvette, loaded with cells, was left for 30 minutes at room temperature. The Indo-1 loaded cells
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were left for 1 h or more at room temperature to allow time for the cells to hydrolyze the ester of Indo-1, AM.
Perifusion Two jacketed columns were used to warm up the medium and the mediumcontaining secretagogue to 37"C before perifusing the media through the cuvette. DMEM-BSA was pumped out (12000 Vario Perpex, LKB) from the cuvette at a rate of 0.4 ml/min for a period of 20 min before experiments were performed in order for cells to adjust to a perifusion environment at 37°C. During the experimental period, the flow rate was 0.4 ml/min and the perifusate was collected into disposable cups (24x14 mm, Sarstedt Canada inc., V-St. Laurent, Quebec) at the rate of one sample per minute. Unless otherwise specified, the cells were perifused with DMEM-BSA or DMEM-BSA containing an appropriate concentration of secretagogues. Dead spaces between the medium reservoir and the cuvette, and between the cuvette and the collection of perifused sample were 0.3 ml and 0.9 ml, respectively.
Fluorometry The temperature of the cuvette was maintained at 37"C by circulating water through a jacket surrounding the cuvette chamber in the spectrofluorometer. The excitation wavelength was 329 nm, and the emission wavelength was 405 nm. Excitation and emission slit widths were 5 nm and 10 nm, respectively (15). The relationship between intensity of fluorescence and [Ca2+]i was established using the ionomycin and Mn 2÷ quenching technique (2). [Ca2÷]i was then computed based on the method of Tsien et al (28) with a Ka = 250 nM (5,8). Frozen TRH (1 mmol/1, Sigma) stock solution was used to make a 1 ~mol/l solution. Ionomycin (Sigma) was dissolved in absolute alcohol (5 mmol/l). The ionomycin solution was diluted in DMEM or DMEM-13SA solution to make the appropriate concentrations. A stock solution of 1 mol/l manganese chloride (Mallinckrodt) was made in distilled water and diluted in DMEM or DMEM-BSA to make 0.1 mmol/1 Mn 2÷ in order to quench extracellular free Ca 2÷. Other quenching solutions made were 1 mmol/l Mn 2÷ plus 0.1% Triton X-100 (BDH Chemicals) or 1 mmol/1 Mn 2+ plus 10 ~mol/1 ionomycin to quench [Ca2+]i.
Radioimmunoassay Appropriate volumes of the perifused medium were assayed in triplicate using the radioimmunoassay kit for rat prolactin, which was kindly supplied by Drs. A.F. Parlow and S. Raiti through the Rat Pituitary Hormone Distribution Program. The quantity of prolactin was expressed in terms of NIAMDD rat prolactin RP-3. Coefficients of variation for inter- and intra-assay variability were 14.5% and 7.2% respectively. The sensitivity was 0.03 ng/tube. 50 ,1 of perifused medium were assayed in triplicate using the radioimmunoassay kit for rat TSH. The quantity of TSH was expressed in terms of NIAMDD rat TSH RP-2. The sensitivity of TSH was 0.03 ng/tube.
Measurement of area The area under the curves in the original graphs was measured using SigmaScan (Jandel Scientific, San Rafael, Calif. U.S.A.) which quantifies the area in square millimeters. The square millimeters of the areas were expressed as undefined units (U2). Mean values of prolactin concentration during the period of control are represented by mean __ SEM of 10 samples during a 10 min period.
Statistics Differences between 2 group means were compared using the Student t-test, with a p-value less than 0.05 or 0.01 considered to be significant or highly significant respectively. Data are presented as mean _ SEM (standard error of the mean).
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Results
Phenol red (15 mg/1) has been used to monitor pH changes in culture medium. Emission spectra of DMEM plus 10 ~,1 hydrolyzed Indo-1/ml with and without phenol red from excitation light at 329 nm were scanned in order to determine whether the phenol red interferes with fluorometry. Optical density of emission light was expressed in arbitrary units (OD). The difference between emission light at 405 nm of DMEM and DMEM containing the 10 ,1 digested Indo-1/ml was 160 OD (control), but the difference between DMEM plus the phenol red and DMEM plus the 15 mg/l phenol red and the Indo-1 was 104 OD (phenol red). The 15 mg/l phenol red reduced (control minus phenol red) optical density by 56 OD, which indicated that a substantial amount of emitting/exciting light was impeded by the phenol red. DMEM without phenol red was therefore used in this study. Protein has been included in the perifusion medium for experiments measuring prolactin release because basal prolactin release is very low and pituitary cells do not respond well to stimulation without protein in the medium. The most commonly used protein is 0.1% BSA in medium. The emission spectra of DMEM plus 10 ~1 digested Indo-1/ml with and without 0.1% BSA were scanned. When we compared the difference between DMEM plus digested Indo-1 with and without the 0.1% BSA, the 0.1% BSA reduced a small amount (15 OD) of the emitting light at 405 nm. The digested Indo-1 (10 ~,1)was injected into the Sephadex G-150 matrix between 9 and 18 mm from the bottom of the cuvette, and the rate of washout of Indo-1 was tested. The half-life of Indo-1 washout in the cuvette was 68 min (flow rate: 0.4 ml/min) indicating that Indo-1 was not easily sieved through the Sephadex G-150. The following experiments were repeated at least 3 times and they were reproducible. Typical results were shown. TRH was selected to test whether the perifusion system was performing properly. TRH (1 ,mol/1) stimulated prolactin release and elevated [Ca2+]i, confirming previous works (9, 1, 13) (Fig. 1). In order to calibrate the [Ca2+]i, ionomycin (5 umol/1) was perifused after the TRH experiment. Ionomycin elevated [Ca2+]i with a single peak, after which [Ca2+]~ was depressed below basal levels even though the ionomycin was perifused for a 10 rain period (Fig. 1). Prolactin was also released as a single peak. Both the peak [Ca2+]i and the relative amount of elevated [Ca2+]i induced by ionomycin were higher (peak, 32.5 +_ 7.30D; area under the curve, 42.3 _+ 14.9 U 2) than those (peak, 12.7 + 3 . 8 0 D ; area under the curve, 13.5 _+ 4.2 U 2) induced by TRH. However, peak prolactin concentration (peak concentration, 240 _+ 24 ng/ml) and total amount of released prolactin (area under the curve, 593.2 + 168.5 U 2) during the TRH perifusion period were higher than those (peak concentration, 165 -+ 22 ng/ml; area under the curve, 259.5 -+ 69.5 U 2) during the 5 ~,mol/1 ionomycin perifusion. The difference between effects of TRH and ionomycin on [Ca2+]i and prolactin release were highly significant (p < 0.01, n = 4). The pituitary cells other than lactotrophs will contribute to the elevation of [Ca2+]~ but it will be less than 20% of the elevated [Ca2+]i since purity of the lactrotrophs is 82%. After 20% discount of the elevated [Ca2+]i by ionomycin, [Ca2+]i induced by ionomycin is significantly higher than that of TRH (p < 0.01). These observations show that changes in [CaZ+]~ and prolactin release are not proportional.
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A dynamic penfuslon system for momtormg both changes of [Ca- )]j and prolactin release. Optical density of emission wavelen~gth at 405 nm (excitation wavelength = 329) was scanned during 90 mm experimental period (lower panel) while perifusate was collected throughout the experimental period (0.4 ml/min/fraction), and assayed prolactin concentration in the fraction (upper panel). Perifusion was performed in the following sequence: medium (10 min), TRH (1 ~mol]l) (10 min), medium (10 min), ionomycin (5 ~mol/)(10 min), medium (10 rain), Mnz+ (0.1 mmol/1)(20 min), Mn -~+ (1 retool/l) plus ionomycin (5 ,mol/1)(20 min). 0.1% BSA was included for the entire perifusion period. Prolactin concentration was measure by RIA. Each point represents the mean of triplicate measurements. The concentration of TSH was measured using triplicates of 50 ~1 out of 400 ,1 fractions. TSH was assayed in 3 different experiments and each radioimmunoassay resulted in a good standard curve, but basal or TRH-induced TSH concentrations were not detectable. This indicated that the number of thyrotrophs in the estradiol-primed adenohypophysis were insignificant. Even though it is known that a few umol/1 of ionomycin, a calcium ionophore (14) are sufficient to allow calcium entry into the cells (1), the [Ca2+]i elevation induced by the 5 ~mol/1 ionomycin only occurred for a brief period (Fig. 1). This observation was unexpected, but understandable since the ionomycin is a lipid soluble compound
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Cytosolic [Ca2+]i and Prolactin Release
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(14). The ionomycin will form hydrophobic binding to the BSA and thus availability of the ionomycin in the perifusion medium will be reduced. Therefore, ionomycin was dissolved in DMEM without BSA, and the experiments described above were repeated. The ionophore elevated [Ca2+]i with a sharp peak. The elevated [Ca2+]~ was then depressed below basal concentrations and was then elevated again. The second peak was lower than the first peak indicating that [Ca2+]i did not saturate the Indo-1 present in the cytosol during the 10 min perifusion period (Fig. 2). 1200
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Fig. 2 A dynamic perifusion system for monitoring both changes of [Ca2+)]i and prolactin release. Experimental design is essentially the same as one in Fig. 1 except that BSA was excluded in later part of experiment. Optical density scanned during 90 min experimental period (lower panel) while perifusate was collected throughout the experimental period (0.4 ml/min/fraction), and assayed prolactin concentration in the fraction (upper panel). Perifusion was performed in the following sequence: medium (10 min), TRH (1 ~mol/1)(10 min), medium (10 min), ionomycin (5 ~mol/) (10 min), medium (10 rain), Mn 2+ (0.1 mmol/l)(20 min), Mn 2+ (1 mmol/1) plus 0.1% Triton X-100 (20 min). 0.1% BSA was included for first 30 min perifusion period, but excluded for the rest of the experimental period (60 min) beginning with the ionomycin. Each point for prolactin concentration represents the mean value of triplicate measurements.
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We next perifused DMEM without BSA for a 10 min period after a 1 umol/1 TRH perifusion in order to wash out residual BSA in the system, and then 10 umol/1 ionomycin in DMEM without the BSA was perifused. [Ca2+]i was elevated from 610 OD to 680 OD and the elevated [Ca2+]i concentration was sustained (Fig. 3). The calcium complex of extracellular Indo-1 was then quenched with 0.2 mmol/l Mn 2+ in DMEM without BSA. The optical density of emission light decreased from 610 OD to 498 OD by Mn 2÷ quenching. The [Ca2+]~ complex of Indo-1 was quenched with I mmol/1 Mn 2÷ plus 10 mmol/l ionomycin in DMEM without BSA. The optical density of light emission was decreased from 498 OD to 470 OD by the Mn 2÷ plus ionomycin quenching (Fig. 3). We next tested the quenching of the [Ca2+]i complex of Indo-1 with 1 mmol/1 Mn 2÷ plus 0.1% Triton X-100. Quenching after destruction of the membrane with 0.1% Triton X-100 was the same as the quenching which occurred using 1 mmol/1 Mn 2÷ plus 10 retool/1 ionomycin. [CaZ÷]i was calculated as follows: [Ca2+]i = Kd x (FFmi,/Fmax-F) where Kd is 250 mM and F is the optical density. Basal [Ca2+]i = 250 x (498-470 OD/680-610 OD) -- 100 nM. Discussion
Cell homogeneity The number of lactotrophs present in the anterior pituitary is a small fraction of the total adenohypophyseal cell population. It has been difficult to study subcellular transduction systems such as adenylate cyclase, the phospholipase C system and changes in [Ca2+]i in lactotrophs due to the heterogeneous population of adenohypophyseal cells. Only somatotrophs have been purified and successfully studied with respect to their subcellular transduction systems (17). Many significant works have been performed with tumour cell lines (4, 7, 21), but these cell lines have characteristics which deviate from those of normal pituitary cells. Estradiol-priming of male rats for 1013 weeks increased the number of lactotrophs to 82 + 3% in the primary cultured adenohypophysial cells. This is not a new observation but confirms other works (16, 18). The dominant population of lactotrophs could then be used to study the subcellular transduction system without the application of further purifying techniques. When our primary cultured pituitary cells were perifused with a high concentration of TRH (1 ~,mol/l), no detectable amount of TSH was released which showed that insignifcant number of thyrotrophs were present in the culture. Estrogen-priming of male rats caused some changes in the characteristics of the lactotrophs. Unprimed male rat lactotrophs, for example, do not respond to somatostatin, but somatostatin effectively inhibits prolactin release from the estradiol-primed lactotrophs (3, 10). Primed lactotrophs are more sensitive to stimulating agents such as TRH (20).
Perifusion system Our perifusion system is the first practical design to directly correlate changes of [Ca 2+]i and prolactin release using primary cultured pituitary cells. Phenol red was not used as a pH indicator in the culture medium during these experiments because it interfered with fluorometry. BSA was added to the perifusion medium because basal release of prolactin is very low and pituitary cells do not respond well to a secretagogue without added protein in the medium. Fortunately, 0.1% BSA which is commonly used for perifusion experiments, did not seriously interfere with the fluorometry in our experimental conditions.
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The system used in these experiments was basically the same as commonly used perifusion systems with several modifications. We chose a semimicro disposable polystyrene cuvette as the perifusion chamber in order to reduce the size of dead space. 500
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Fig. 3 A dynamic perifusion system for monitoring both changes of [Ca2+)]i and prolactin release. Experimental design is the same as one in Fig. 1 except that BSA was excluded in later part of experiment. Optical density was scanned during 90 min experimental period (lower panel) while perifusate was collected throughout the experimental period (0.4 ml/min/fraction), and assayed prolactin concentration in the fraction (upper panel). Perifusion was performed in the following sequence; medium (10 min), TRH (1 umol/l) (10 min), medium (10 min), ionomycin (10 ~mol/)(20 rain), medium (10 min), Mn 2÷ (0.2 mmol/l)(20 min), Mn 2+ (1 retool/l) plus isonomycin (10 ~mol/1) (20 rain). 0.1% BSA was included for first 20 rain perifusion period, but excluded for the rest of the experimental period (80 rain) after TRH perifusion. Each point for prolactin concentration represents the mean value of triplicate measurements.
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This allowed less dilution of released hormone during perifusion and sharper resolution. This particular type of polystyrene cuvette has a circular instead of a square opening. As a result, it was easier to connect this cuvette to an inlet. It was also easier to inject Indo-1 loaded cells into the Sephadex gel matrix in this type of cuvette than in flow cell cuvette. To reduce the amount of Indo-1 outside of the cells when they are injected in the gel matrix, the cells recovered after incubation with Indo-1 were resuspended in 10 ml DMEM-BSA and left for 30 rain at room temperature to allow the cells to hydrolyze the Indo-1, AM. The cells were then be injected into the gel matrix. Water soluble compounds should be sieved through the Sephadex within 2 - 3 times the void volume of the matrix. Indo-1 had a very long half-life (68 rain) in the gel matrix with flow rate of 0.4 ml/min. It is probably mainly due to the aromatic retardation in Sephadex. Because of this long retardation period, the washing process of the Indo-1 loaded cells in the DMEM-BSA decreased background fluorescence.
Perifusion vs static incubations Most studies concerned with [Ca2+]i have been performed with cells suspended in a cuvette (11, 15) which is a static incubation system. In general, hormone secretion patterns of dynamic perifusion experiments are substantially different from the patterns of static incubation systems. For example, the effect of a secretagogue on prolactin release can be seen immediately with a perifusion system (24), but it requires almost a 30 min incubation period to make a respectable dose-response curve with a static monolayer incubation system (25). The discrepancy between a dynamic perifusion and a static incubation system can partially be explained by several features: (1) feedback effects: Pituitary hormones released into the medium can exert a feedback effect on the pituitary cells present in the culture. Thus, hormone release would be influenced by either negative or positive feedback. (2) downregulation: Long term exposure of pituitary cells to a secretagogue may exert receptor down regulation, and thus sensitivity to a secretagogue could be decreased. (3) cumulative response vs. dynamic changes: A large amount of hormone may he released at the moment when the preincubation medium is changed with fresh medium which is temperature and CO 2 equilibriated (26). Hormone already released in the medium may mask a brief burst of hormone release if the total amount of hormone released during the brief burst is small relative to total amount of the released hormone present in the medium.
Effect of ionomycin Ionomycin did not work as anticipated in presence of 0.1% BSA in DMEM. Normally a few ~,mol/1 of ionomycin are sufficient to make calcium permeable in cells (1) and the influxed calcium will saturate Indo-1 in the cytosol. We used 5 umol/l ionomycin for the purpose of calibration of [Ca2÷]i. However, the availability of ionomycin in 0.1% BSA solution was presumably so reduced that the cells were only partially permeable for a brief period of time since ionomycin is a hydrophobic 9+ . . . . compound (14), and reduced free [Ca- ]i below basal levels after the lnmal surge m 2+ .'7+ [Ca ]i, This depression of [Ca- ]i was most likely caused by an over-reaction of the calcium pump. Our observation indicated that ionomycin should be dissolved in a protein free medium when maximum [Ca2+]i is calibrated. No protein should also be present for the Mn z÷ quenching of cytosol. Thus, Mn 2÷ can diffuse into the cytosol which were induced by ionomycin. We, therefore, dissolved ionomycin in a protein free DMEM, but this still did not satisfactorily work for calibration of the maximum [Ca2+]~. Our interpretation was that a small amount of protein may have been left in the perifusion system even when protein free ionomycin solution was used. The residual protein in the
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perifusion system likely absorbed the ionomycin. Next, we used protein free DMEM during the 10 rain buffer perifusion after stimulation by a secretagogue in order to remove protein in the perifusion system, and increased the ionomycin concentration to 10 umol/1. In this experimental condition, [Ca2+]i was elevated and remained so illustrating that calcium was diffused into the cytosol by the ionomycin. During the period in which the calcium concentration in the cytosol is increased by ionomycin, a large amount of prolactin was released, but it would be difficult to compare the observations between the ionomycin-induced and TRH-induced prolactin release since their experimental conditions were different. Ionomycin was dissolved in the protein free DMEM while TRH was treated in DMEM containing 0.1% BSA.
Effects of TRH TRH is not the physiological PRF, but is a very powerful stimulating agent of prolactin release from estradiol primed lactotrophs (20, 23). TRH stimulates prolactin release in 2 phases in a perifusion system; an initial surge of prolactin and maintenance of elevated rate of release. TRH activates phospholipase C which hydrolyses phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-triphosphate (IP3) and 1,2-diacylglycerol (DAG). The initial surge is triggered by an increase in [Ca2+]i which is mobilized by the action of IP 3, and the elevated release is believed to be dependent on DAG which activates protein kinase C (6). Ionomycin, a calcium ionophore increases [Ca2+]i due to an influx of extracellular calcium. The hormone release is supposed to be proportional to the [Ca2÷]i. However, the increase in [Ca2+]~ induced by ionomycin is much higher than the one elevated by TRH, while the increase in prolactin release induced by TRH is much higher than that induced by ionomycin in the experiments which included 0.1% BSA in the perifusion medium throughout experiment (Fig. 1). The discrepancy between [CaZ÷]i and rate of prolactin release indicates that the elevation of [Ca2+]i is not the sole final common pathway of prolactin release. This conclusion agrees with other studies using freshly dispersed rat pituitary cells (13). A similar system was previously introduced (13) but substantially different from our system. The major differences between ours and the previous system are: (1) we used estradiol primed, primary cultured pituitary cells (82% lactotrophs) while Law et al. (13) used acutely dispersed female pituitary cells, and (2) Law et al (13) expressed their [Ca2÷]i with relative changes while our method introduced a calibration method of [Ca2+]i in a perifusion system. In conclusion, we have introduced a practical perifusion system which can simultaneously measure both [Ca2÷]~ and prolactin release. This dynamic perifusion system is an excellent tool for the study of the physiological role of [Ca2÷]~ changes on prolactin release.
Acknowledgements This work was supported by the Medical Research Council of Canada. The authors express their thanks to Drs. A.F. Parlow and S. Raiti for supplying the prolactin RIA kit through the National Hormone and Pituitary Program, NIDDKD, to Mr. M. Lim for excellent technical help and to Ms. Bonnie Stewart for excellent secretarial help.
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References
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
S.N. AKERMAN, Endocrinology. 129 475-488 (1991) A.M. CAPPONI, P.D. LEW, W. SCHLEGEL and T. POZZAN, Methods in Enzymology 124 116-135 (1986) G.R. COOPER and S.H. SHIN, Can. J. Physiol. Pharmacol. 59 1082-1088 (1981) J.J. ENYEAR, S.S. SHEU and P.M. HINKLE, J. Biol. Chem. 262 3154-3159 (1987) E.W. Gelfand, R.K. CHEUNG and S. GRINSTEIN, J. Biol. Chem. 261 11520 (1986) M.C. GERSHENGORN, Ann. Rev. Physiol. 48 515-526 (1986) M.C. GERSHENGORN and C. THAW, Endocrinology. 113 1522-1524 (1983) G. GRYNKIEWICZ, M. POENIE and R.Y. TSIEN, J. Biol. Chem, 26.__Q03440-3450 (1985) S. HANNA and S.H. SHIN, Neuroendocrinology. 55 591-599 (1992) N. KIMURA, C. HAYAFUGI, H. KONAGAYA and K. TAKAHASHI, Endocrinology. 119 1028-1036 (1986) B.D. KOCH, J.B. BLALOCKI and A. SCHONBUNN, J. Biol. Chem, 26___33216-225 (1988) S.W.J.LAMBERTS and R.M. MACLEOD, Physiol. Rev. 70 279-318 (1990) G.J. LAW, J.A. PACHTER and P.S. DANNIES, Mol. Endocrinol. 3 539-546 (1989) C. LIU and T.E. HERMAN, J. Biol. Chem. 253, 5892-5894 (1978) B.T. LUSSIER, M.B. FRENCH, B.C. MOOR and J. KRAICER, Endocrinology 128 570-582 (1991) G. MARTINEZ DE LA ESCALERA, T.F.J. MARTIN and R.I. WEINER, Neuroendocrinology. 46 545-548 (1987) N. NARAYANAN, B. LUSSIER, M. FRENCH and J. KRAICER, Endocrinology 124 484-495 (1989) H. NOGAMI, Cell Tissue Res. 237 195-202 (1984) S. PAPAS, S.H. SHIN and M.C. OBONSAWIN, Neuroendocrinology. 47 154-158 (1988) M. PIERCY and S.H. SHIN, Neuroendocrinology. 31 270-275 (1980) W. SCHLEGEL and C.B. WOLLHEIM, J. Cell Biol. 99 83-87 (1984) S.H. SHIN, Life Sci. 22 67-74 (1978a) S.H. SHIN, Life Sci. 23 1813-1818 (1978b) S.H. SHIN and S.F. HANNA, Can. J. Physiol. Pharmacol. 68 1226-1230 (1990) S.H. SHIN and R. STIRLING, J. Endocrinology. 118 287-294 (1988) R. STIRLING and S.H. SHIN, Mol Cell Endocrinol. 70 65-72 (1990) S.S. STOJILKOVIC, S. ISUMI and K.J. CATF, J. Biol. Chem. 263 13054-13061 (1988) R.Y. TSIEN, T. POZZAN and T.J. RINK, J. Cell Biol. 94 440-445 (1982)
Pergamon Press
MEASUREMENT OF PROLACTIN RELEASE AND CYTOSOLIC CALCIUM IN ESTRADIOL-PRIMED LACTOTROPHS S.H. Shin, C. Soukup, S.C. Pang +, T.J. Kubiseski* and T.G. Flynn* Departments of Physiology, Anatomy + and Biochemistry* Queen's University Kingston, Ontario, Canada K7L 3N6
(Received in final form September 14, 1993)
Summary_
We have developed a perifusion system that can measure both changes of cytosolic free calcium concentration [Ca2+]i and prolactin release simultaneously from cultured lactotrophs. This model incorporated a commonly-used perifusion system to a spectrofluorometer. Indo-1 loaded cells were injected into Sephadex G-150 matrix in the cuvette at a site where the emitting light of the fluorometer projects. During perifusion periods, the perifusate was collected in a fraction collector, while optical density of the emitting light at 405 nm was recorded. The [Ca2+]i was calculated based on an ionomycin and Mn 2+ quenching technique. As expected, TRH (1 umol/1) stimulated prolactin release from cultured lactotrophs in this system. We further observed that prolactin releases as induced by TRH and ionomycin were not proportional with changes of the [Ca2+]i, suggesting that changes of [Ca2+]i is not the sole final pathway of intracellular transduction systems for prolactin release. In lactotrophs, changes in the cytosolic free calcium concentration ([Ca2+]i) are an important aspect of the subcellular transduction system of prolactin release (27, 12). Simultaneous measurement of levels of [Ca2+]~ and prolactin release from a specimen are necessary in correlating the relationship between alteration in [Ca2+]i and prolactin release. Our system is an improved version of previosly published work by Law et al. (13). In order to establish a model system of this type, it is essential first to obtain a homogeneous or near homogeneous population of lactotrophs. This can be achieved by raising blood concentrations of estradiol for a 10 to 13 week period through implantation
Address all correspondence to: Dr. S.H. Shin, Department of Physiology, Queen's University,Botterell Hall, Kingston, Ontario, Canada KTL 3N6 0024-3205/93 $6.00 + .00 Copyright © 1993Pergamon Press Ltd All rights reserved.
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of an estrogen capsule. Secondly, a perifusion system which is incorporated to a fluorometer is used to facilitate the continuous measurement of changes in [Ca2+]i. This was accomplished by several modifications of a commonly used perifusion system. In this study, we introduced a perifusion system which allowed us to simultaneously measure changes of [Ca2+]i and prolactin release, and have found that [Ca2+]i and prolactin release were not always proportionally related. This illustrates that altered [Ca2+]i levels are not the sole final regulators of the subcellular transduction systems of prolactin release. Materials and Methods Animal Male Sprague-Dawley rats (Charles River, CD, Canadian Breeding Farm and Laboratories, Montreal) were housed in a controlled environment with illumination for 14 h daily (06.00 - 20.00 h) at a temperature of 25 __. 1 "C. Purina Lab Chow and tap water were supplied ad libitum. 10 to 13 weeks before the experiments, rats received implants of Silastic tubing (Silastic medical grade tubing, 0.062 inches inner diameter, 0.125 inches outer diameter, 3 cm in length, Dow Corning Corp) containing estradiol-17/3 (60 mg). The tubings were sealed with a plug of Silastic medical grade elastomer. These estradiol-primed rats showed a mean plasma estradiol concentration of 1.6 ___0.4 nmol/l (19). Using indirect immunohistofluorescent techniques, the population of lactotrophs was found to increase to 82 _+ 3% of the total dispersed pituitary cell population after the 10-13 week priming period (unpublished observation) which is consistent with others (16, 18). Pituitary cells were dispersed as described in the following section and cultured for 2-4 days prior to perifusion experiments. Cell dispersion and culture The estradiol-primed male rats (300-330 g) were decapitated and their adenohypophyses were collected and dispersed with trypsin (bovine pancreas Type III, Sigma Chemical Co., St. Louis) under sterile conditions as previously described (22). Briefly, the adenohypophyses were cut to small pieces (< 1 mm 3) and placed in Spinner's minimum essential medium (S-MEM) (Gibco Labs., Grand Island, NY) containing 0.05% trypsin and 0.1% bovine serum albumin (BSA) (Cohn Fraction V, Sigma Chemical Co.) in a Spinner's flask. Following 1 h dispersion at 37"C, the cells were collected by centrifugation (750 x g for 10 min). The pellet was resuspended in SMEM containing 0.02% lima bean trypsin inhibitor (type II-L, Sigma Chemical Co.) and recentrifuged. The dispersed adenohypophysial cells were suspended in 4 ml of Dulbecco's modified Eagle's medium (DMEM) supplemented with 2.5% fetal calf serum and, 15% horse serum (Gibco Lab., Grand Island, N.Y.). Estradiol and penicillin (Sigma Chemical Co.) were added to make 1 nmol/1 and 50 IU/ml, respectively (DMEM culture medium). Each pituitary yielded approximately 10-12 million cells. The cell suspension was distributed to 2 Petri dishes (35x10 style Falcon Plastic, Oxnard, CA) (2 ml/dish; 5 - 6 million cells). The adenohypophysial cells were cultured in a water jacketed incubator at 37"C under a water saturated atmosphere of 5% CO 2 and 95% O z for 2-4 days. The cells were cultured in Petri dishes which were not treated for monolayer culture. Therefore, they did not attach to the dish, but formed cell clusters. These aggregations could easily be recovered without enzyme treatment or mechanical scraping. This allowed the use of undamaged cells for subsequent experiments. Emission spectra Acetoxymethyl Indo-1 (Indo-1, AM; Molecular Probes, Inc., Eugene, OR, USA) was hydrolyzed using an esterase present in pituitary ceils in order to
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Cytosolic[Ca2+]i and Prolactin Release
examine the emission spectra. Cultured adenohypophysial cells were collected by centrifugation (750 x g for 10 min) and were resuspended in 1 ml DMEM. 10 ~1 dimethylsulfoxide (DMSO, Baker Chemical Co., Phillipsburg, N J, USA) solution containing 10 nmole Indo-1, AM was added in the 1 ml DMEM of the cell suspension to make 10 t,mol/l Indo-1, AM. This suspension was incubated for 45 min at 37°C. The cells in the medium containing Indo-1 were left in room temperature for 1 h and were homogenized in a glass tube. The homogenate was left in room temperature for 1 h in order to further hydrolyze ester of Indo-1, AM, and removed cell debris by centrifugation (20,000 x g for 30 min). The supernatant was used to test emission spectra with different gel matrices (Sephadex G-25, G-150), and different perifusion media (DMEM with or without phenol red and 0.1% BSA).
Construction of cuvette for perifusion A semi-micro size disposable cuvette (Dynalon Disposable polystyrene cuvettes, 1.5 ml, 10x4x45 mm, Canlab Scientific Products, Toronto, Canada) was chosen for this study to reduce the dead space of the Sephadex gel matrix. The lower right corner of the cuvette face which receives excitation spectra, was drilled and a silicone tubing (1/23 inch inner diameter, 5/64 inch outer diameter) was inserted in the hole. The lower part of the cuvette was plugged with glass wool (Corning Glass Works, Corning, NY, USA) in order to prevent leakage of the gel matrix. The cuvette was packed with Sephadex gel matrix which was equilibrated with the following perifusion media: DMEM with or without phenol red and with or without 0.1% BSA (DMEM-BSA). The cuvette was capped with a 00 size rubber stopper which had been pierced by a 18 gauge steel tubing (syringe needle, Becton, Dickinson & Co.). The stopper was then connected to a medium reservoir with Tygon tubing (R-3603, 1/32 inch inner diameter, 3/32 outer diameter). It is necessary for a matrix to be present in the cuvette to attain a successful perifusion system. Commonly used matrices for perifusion are Sephadex gels or Bio-gels. We scanned the spectra of Sephadex G-25 and G-150 in order to find suitable gel for this study. The Sephadex G-25 gel blocked exciting/emitting light slightly more than the Sephadex G-100 gel. Sephadex G-150 is more translucent than Sephadex G-25 and thus, it was easier to locate the injected cells in the cuvette. Therefore, Sephadex G-150 was chosen for this study.
Loading cells into the cuvette The primary cultured cells were collected by centrifugation (750 x g for 10 min) from the culture medium and resuspended in DMEM. 10 ~l dimethylsulfoxide (DMSO) solution containing 10 nmole Indo-1, AM was added to the 10 ml DMEM to make 1 ~mol/1 Indo-1, AM and incubated for 30 rain at 37"C. The cells were recovered by centrifugation (750 x g for 10 rain) and resuspended in 10 ml DMEM-BSA. The suspension of Indo-1 loaded cells were left for 30 min at room temperature. The cells were recovered by centrifugation and resuspended in 50 ~1 DMEM-BSA. The cuvette containing 1 ml of Sephadex G-150 was equilibriated with perifusion medium (DMEM-BSA). 0.2 ml of DMEM-BSA was added on top of the Sephadex matrix. The cell suspension was then injected into the Sephadex G-150 matrix using a Pasteur pipette which had been previously equilibrated with the perifusion medium (DMEM-BSA). The injection of the Indo-1 loaded cells was aimed to a position between 9 and 18 mm from the bottom of the cuvette where the excitation light projects in LS-50 Spectrofluorometer (Perkin-Elmer Ltd., Buckinghamshire, UK). The cuvette, loaded with cells, was left for 30 minutes at room temperature. The Indo-1 loaded cells
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were left for 1 h or more at room temperature to allow time for the cells to hydrolyze the ester of Indo-1, AM.
Perifusion Two jacketed columns were used to warm up the medium and the mediumcontaining secretagogue to 37"C before perifusing the media through the cuvette. DMEM-BSA was pumped out (12000 Vario Perpex, LKB) from the cuvette at a rate of 0.4 ml/min for a period of 20 min before experiments were performed in order for cells to adjust to a perifusion environment at 37°C. During the experimental period, the flow rate was 0.4 ml/min and the perifusate was collected into disposable cups (24x14 mm, Sarstedt Canada inc., V-St. Laurent, Quebec) at the rate of one sample per minute. Unless otherwise specified, the cells were perifused with DMEM-BSA or DMEM-BSA containing an appropriate concentration of secretagogues. Dead spaces between the medium reservoir and the cuvette, and between the cuvette and the collection of perifused sample were 0.3 ml and 0.9 ml, respectively.
Fluorometry The temperature of the cuvette was maintained at 37"C by circulating water through a jacket surrounding the cuvette chamber in the spectrofluorometer. The excitation wavelength was 329 nm, and the emission wavelength was 405 nm. Excitation and emission slit widths were 5 nm and 10 nm, respectively (15). The relationship between intensity of fluorescence and [Ca2+]i was established using the ionomycin and Mn 2÷ quenching technique (2). [Ca2÷]i was then computed based on the method of Tsien et al (28) with a Ka = 250 nM (5,8). Frozen TRH (1 mmol/1, Sigma) stock solution was used to make a 1 ~mol/l solution. Ionomycin (Sigma) was dissolved in absolute alcohol (5 mmol/l). The ionomycin solution was diluted in DMEM or DMEM-13SA solution to make the appropriate concentrations. A stock solution of 1 mol/l manganese chloride (Mallinckrodt) was made in distilled water and diluted in DMEM or DMEM-BSA to make 0.1 mmol/1 Mn 2÷ in order to quench extracellular free Ca 2÷. Other quenching solutions made were 1 mmol/l Mn 2÷ plus 0.1% Triton X-100 (BDH Chemicals) or 1 mmol/1 Mn 2+ plus 10 ~mol/1 ionomycin to quench [Ca2+]i.
Radioimmunoassay Appropriate volumes of the perifused medium were assayed in triplicate using the radioimmunoassay kit for rat prolactin, which was kindly supplied by Drs. A.F. Parlow and S. Raiti through the Rat Pituitary Hormone Distribution Program. The quantity of prolactin was expressed in terms of NIAMDD rat prolactin RP-3. Coefficients of variation for inter- and intra-assay variability were 14.5% and 7.2% respectively. The sensitivity was 0.03 ng/tube. 50 ,1 of perifused medium were assayed in triplicate using the radioimmunoassay kit for rat TSH. The quantity of TSH was expressed in terms of NIAMDD rat TSH RP-2. The sensitivity of TSH was 0.03 ng/tube.
Measurement of area The area under the curves in the original graphs was measured using SigmaScan (Jandel Scientific, San Rafael, Calif. U.S.A.) which quantifies the area in square millimeters. The square millimeters of the areas were expressed as undefined units (U2). Mean values of prolactin concentration during the period of control are represented by mean __ SEM of 10 samples during a 10 min period.
Statistics Differences between 2 group means were compared using the Student t-test, with a p-value less than 0.05 or 0.01 considered to be significant or highly significant respectively. Data are presented as mean _ SEM (standard error of the mean).
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Cytosolic [Ca2+]i and Prolactin Release
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Results
Phenol red (15 mg/1) has been used to monitor pH changes in culture medium. Emission spectra of DMEM plus 10 ~,1 hydrolyzed Indo-1/ml with and without phenol red from excitation light at 329 nm were scanned in order to determine whether the phenol red interferes with fluorometry. Optical density of emission light was expressed in arbitrary units (OD). The difference between emission light at 405 nm of DMEM and DMEM containing the 10 ,1 digested Indo-1/ml was 160 OD (control), but the difference between DMEM plus the phenol red and DMEM plus the 15 mg/l phenol red and the Indo-1 was 104 OD (phenol red). The 15 mg/l phenol red reduced (control minus phenol red) optical density by 56 OD, which indicated that a substantial amount of emitting/exciting light was impeded by the phenol red. DMEM without phenol red was therefore used in this study. Protein has been included in the perifusion medium for experiments measuring prolactin release because basal prolactin release is very low and pituitary cells do not respond well to stimulation without protein in the medium. The most commonly used protein is 0.1% BSA in medium. The emission spectra of DMEM plus 10 ~1 digested Indo-1/ml with and without 0.1% BSA were scanned. When we compared the difference between DMEM plus digested Indo-1 with and without the 0.1% BSA, the 0.1% BSA reduced a small amount (15 OD) of the emitting light at 405 nm. The digested Indo-1 (10 ~,1)was injected into the Sephadex G-150 matrix between 9 and 18 mm from the bottom of the cuvette, and the rate of washout of Indo-1 was tested. The half-life of Indo-1 washout in the cuvette was 68 min (flow rate: 0.4 ml/min) indicating that Indo-1 was not easily sieved through the Sephadex G-150. The following experiments were repeated at least 3 times and they were reproducible. Typical results were shown. TRH was selected to test whether the perifusion system was performing properly. TRH (1 ,mol/1) stimulated prolactin release and elevated [Ca2+]i, confirming previous works (9, 1, 13) (Fig. 1). In order to calibrate the [Ca2+]i, ionomycin (5 umol/1) was perifused after the TRH experiment. Ionomycin elevated [Ca2+]i with a single peak, after which [Ca2+]~ was depressed below basal levels even though the ionomycin was perifused for a 10 rain period (Fig. 1). Prolactin was also released as a single peak. Both the peak [Ca2+]i and the relative amount of elevated [Ca2+]i induced by ionomycin were higher (peak, 32.5 +_ 7.30D; area under the curve, 42.3 _+ 14.9 U 2) than those (peak, 12.7 + 3 . 8 0 D ; area under the curve, 13.5 _+ 4.2 U 2) induced by TRH. However, peak prolactin concentration (peak concentration, 240 _+ 24 ng/ml) and total amount of released prolactin (area under the curve, 593.2 + 168.5 U 2) during the TRH perifusion period were higher than those (peak concentration, 165 -+ 22 ng/ml; area under the curve, 259.5 -+ 69.5 U 2) during the 5 ~,mol/1 ionomycin perifusion. The difference between effects of TRH and ionomycin on [Ca2+]i and prolactin release were highly significant (p < 0.01, n = 4). The pituitary cells other than lactotrophs will contribute to the elevation of [Ca2+]~ but it will be less than 20% of the elevated [Ca2+]i since purity of the lactrotrophs is 82%. After 20% discount of the elevated [Ca2+]i by ionomycin, [Ca2+]i induced by ionomycin is significantly higher than that of TRH (p < 0.01). These observations show that changes in [CaZ+]~ and prolactin release are not proportional.
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Cytosolic [Ca2+]i and Prolactin Release
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A dynamic penfuslon system for momtormg both changes of [Ca- )]j and prolactin release. Optical density of emission wavelen~gth at 405 nm (excitation wavelength = 329) was scanned during 90 mm experimental period (lower panel) while perifusate was collected throughout the experimental period (0.4 ml/min/fraction), and assayed prolactin concentration in the fraction (upper panel). Perifusion was performed in the following sequence: medium (10 min), TRH (1 ~mol]l) (10 min), medium (10 min), ionomycin (5 ~mol/)(10 min), medium (10 rain), Mnz+ (0.1 mmol/1)(20 min), Mn -~+ (1 retool/l) plus ionomycin (5 ,mol/1)(20 min). 0.1% BSA was included for the entire perifusion period. Prolactin concentration was measure by RIA. Each point represents the mean of triplicate measurements. The concentration of TSH was measured using triplicates of 50 ~1 out of 400 ,1 fractions. TSH was assayed in 3 different experiments and each radioimmunoassay resulted in a good standard curve, but basal or TRH-induced TSH concentrations were not detectable. This indicated that the number of thyrotrophs in the estradiol-primed adenohypophysis were insignificant. Even though it is known that a few umol/1 of ionomycin, a calcium ionophore (14) are sufficient to allow calcium entry into the cells (1), the [Ca2+]i elevation induced by the 5 ~mol/1 ionomycin only occurred for a brief period (Fig. 1). This observation was unexpected, but understandable since the ionomycin is a lipid soluble compound
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Cytosolic [Ca2+]i and Prolactin Release
1611
(14). The ionomycin will form hydrophobic binding to the BSA and thus availability of the ionomycin in the perifusion medium will be reduced. Therefore, ionomycin was dissolved in DMEM without BSA, and the experiments described above were repeated. The ionophore elevated [Ca2+]i with a sharp peak. The elevated [Ca2+]~ was then depressed below basal concentrations and was then elevated again. The second peak was lower than the first peak indicating that [Ca2+]i did not saturate the Indo-1 present in the cytosol during the 10 min perifusion period (Fig. 2). 1200
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Fig. 2 A dynamic perifusion system for monitoring both changes of [Ca2+)]i and prolactin release. Experimental design is essentially the same as one in Fig. 1 except that BSA was excluded in later part of experiment. Optical density scanned during 90 min experimental period (lower panel) while perifusate was collected throughout the experimental period (0.4 ml/min/fraction), and assayed prolactin concentration in the fraction (upper panel). Perifusion was performed in the following sequence: medium (10 min), TRH (1 ~mol/1)(10 min), medium (10 min), ionomycin (5 ~mol/) (10 min), medium (10 rain), Mn 2+ (0.1 mmol/l)(20 min), Mn 2+ (1 mmol/1) plus 0.1% Triton X-100 (20 min). 0.1% BSA was included for first 30 min perifusion period, but excluded for the rest of the experimental period (60 min) beginning with the ionomycin. Each point for prolactin concentration represents the mean value of triplicate measurements.
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We next perifused DMEM without BSA for a 10 min period after a 1 umol/1 TRH perifusion in order to wash out residual BSA in the system, and then 10 umol/1 ionomycin in DMEM without the BSA was perifused. [Ca2+]i was elevated from 610 OD to 680 OD and the elevated [Ca2+]i concentration was sustained (Fig. 3). The calcium complex of extracellular Indo-1 was then quenched with 0.2 mmol/l Mn 2+ in DMEM without BSA. The optical density of emission light decreased from 610 OD to 498 OD by Mn 2÷ quenching. The [Ca2+]~ complex of Indo-1 was quenched with I mmol/1 Mn 2÷ plus 10 mmol/l ionomycin in DMEM without BSA. The optical density of light emission was decreased from 498 OD to 470 OD by the Mn 2÷ plus ionomycin quenching (Fig. 3). We next tested the quenching of the [Ca2+]i complex of Indo-1 with 1 mmol/1 Mn 2÷ plus 0.1% Triton X-100. Quenching after destruction of the membrane with 0.1% Triton X-100 was the same as the quenching which occurred using 1 mmol/1 Mn 2÷ plus 10 retool/1 ionomycin. [CaZ÷]i was calculated as follows: [Ca2+]i = Kd x (FFmi,/Fmax-F) where Kd is 250 mM and F is the optical density. Basal [Ca2+]i = 250 x (498-470 OD/680-610 OD) -- 100 nM. Discussion
Cell homogeneity The number of lactotrophs present in the anterior pituitary is a small fraction of the total adenohypophyseal cell population. It has been difficult to study subcellular transduction systems such as adenylate cyclase, the phospholipase C system and changes in [Ca2+]i in lactotrophs due to the heterogeneous population of adenohypophyseal cells. Only somatotrophs have been purified and successfully studied with respect to their subcellular transduction systems (17). Many significant works have been performed with tumour cell lines (4, 7, 21), but these cell lines have characteristics which deviate from those of normal pituitary cells. Estradiol-priming of male rats for 1013 weeks increased the number of lactotrophs to 82 + 3% in the primary cultured adenohypophysial cells. This is not a new observation but confirms other works (16, 18). The dominant population of lactotrophs could then be used to study the subcellular transduction system without the application of further purifying techniques. When our primary cultured pituitary cells were perifused with a high concentration of TRH (1 ~,mol/l), no detectable amount of TSH was released which showed that insignifcant number of thyrotrophs were present in the culture. Estrogen-priming of male rats caused some changes in the characteristics of the lactotrophs. Unprimed male rat lactotrophs, for example, do not respond to somatostatin, but somatostatin effectively inhibits prolactin release from the estradiol-primed lactotrophs (3, 10). Primed lactotrophs are more sensitive to stimulating agents such as TRH (20).
Perifusion system Our perifusion system is the first practical design to directly correlate changes of [Ca 2+]i and prolactin release using primary cultured pituitary cells. Phenol red was not used as a pH indicator in the culture medium during these experiments because it interfered with fluorometry. BSA was added to the perifusion medium because basal release of prolactin is very low and pituitary cells do not respond well to a secretagogue without added protein in the medium. Fortunately, 0.1% BSA which is commonly used for perifusion experiments, did not seriously interfere with the fluorometry in our experimental conditions.
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Cytosolie [Ca2+]i and Frolactin Release
1613
The system used in these experiments was basically the same as commonly used perifusion systems with several modifications. We chose a semimicro disposable polystyrene cuvette as the perifusion chamber in order to reduce the size of dead space. 500
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500
468.8
, 0
TIME (MINUTES)
Fig. 3 A dynamic perifusion system for monitoring both changes of [Ca2+)]i and prolactin release. Experimental design is the same as one in Fig. 1 except that BSA was excluded in later part of experiment. Optical density was scanned during 90 min experimental period (lower panel) while perifusate was collected throughout the experimental period (0.4 ml/min/fraction), and assayed prolactin concentration in the fraction (upper panel). Perifusion was performed in the following sequence; medium (10 min), TRH (1 umol/l) (10 min), medium (10 min), ionomycin (10 ~mol/)(20 rain), medium (10 min), Mn 2÷ (0.2 mmol/l)(20 min), Mn 2+ (1 retool/l) plus isonomycin (10 ~mol/1) (20 rain). 0.1% BSA was included for first 20 rain perifusion period, but excluded for the rest of the experimental period (80 rain) after TRH perifusion. Each point for prolactin concentration represents the mean value of triplicate measurements.
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This allowed less dilution of released hormone during perifusion and sharper resolution. This particular type of polystyrene cuvette has a circular instead of a square opening. As a result, it was easier to connect this cuvette to an inlet. It was also easier to inject Indo-1 loaded cells into the Sephadex gel matrix in this type of cuvette than in flow cell cuvette. To reduce the amount of Indo-1 outside of the cells when they are injected in the gel matrix, the cells recovered after incubation with Indo-1 were resuspended in 10 ml DMEM-BSA and left for 30 rain at room temperature to allow the cells to hydrolyze the Indo-1, AM. The cells were then be injected into the gel matrix. Water soluble compounds should be sieved through the Sephadex within 2 - 3 times the void volume of the matrix. Indo-1 had a very long half-life (68 rain) in the gel matrix with flow rate of 0.4 ml/min. It is probably mainly due to the aromatic retardation in Sephadex. Because of this long retardation period, the washing process of the Indo-1 loaded cells in the DMEM-BSA decreased background fluorescence.
Perifusion vs static incubations Most studies concerned with [Ca2+]i have been performed with cells suspended in a cuvette (11, 15) which is a static incubation system. In general, hormone secretion patterns of dynamic perifusion experiments are substantially different from the patterns of static incubation systems. For example, the effect of a secretagogue on prolactin release can be seen immediately with a perifusion system (24), but it requires almost a 30 min incubation period to make a respectable dose-response curve with a static monolayer incubation system (25). The discrepancy between a dynamic perifusion and a static incubation system can partially be explained by several features: (1) feedback effects: Pituitary hormones released into the medium can exert a feedback effect on the pituitary cells present in the culture. Thus, hormone release would be influenced by either negative or positive feedback. (2) downregulation: Long term exposure of pituitary cells to a secretagogue may exert receptor down regulation, and thus sensitivity to a secretagogue could be decreased. (3) cumulative response vs. dynamic changes: A large amount of hormone may he released at the moment when the preincubation medium is changed with fresh medium which is temperature and CO 2 equilibriated (26). Hormone already released in the medium may mask a brief burst of hormone release if the total amount of hormone released during the brief burst is small relative to total amount of the released hormone present in the medium.
Effect of ionomycin Ionomycin did not work as anticipated in presence of 0.1% BSA in DMEM. Normally a few ~,mol/1 of ionomycin are sufficient to make calcium permeable in cells (1) and the influxed calcium will saturate Indo-1 in the cytosol. We used 5 umol/l ionomycin for the purpose of calibration of [Ca2÷]i. However, the availability of ionomycin in 0.1% BSA solution was presumably so reduced that the cells were only partially permeable for a brief period of time since ionomycin is a hydrophobic 9+ . . . . compound (14), and reduced free [Ca- ]i below basal levels after the lnmal surge m 2+ .'7+ [Ca ]i, This depression of [Ca- ]i was most likely caused by an over-reaction of the calcium pump. Our observation indicated that ionomycin should be dissolved in a protein free medium when maximum [Ca2+]i is calibrated. No protein should also be present for the Mn z÷ quenching of cytosol. Thus, Mn 2÷ can diffuse into the cytosol which were induced by ionomycin. We, therefore, dissolved ionomycin in a protein free DMEM, but this still did not satisfactorily work for calibration of the maximum [Ca2+]~. Our interpretation was that a small amount of protein may have been left in the perifusion system even when protein free ionomycin solution was used. The residual protein in the
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perifusion system likely absorbed the ionomycin. Next, we used protein free DMEM during the 10 rain buffer perifusion after stimulation by a secretagogue in order to remove protein in the perifusion system, and increased the ionomycin concentration to 10 umol/1. In this experimental condition, [Ca2+]i was elevated and remained so illustrating that calcium was diffused into the cytosol by the ionomycin. During the period in which the calcium concentration in the cytosol is increased by ionomycin, a large amount of prolactin was released, but it would be difficult to compare the observations between the ionomycin-induced and TRH-induced prolactin release since their experimental conditions were different. Ionomycin was dissolved in the protein free DMEM while TRH was treated in DMEM containing 0.1% BSA.
Effects of TRH TRH is not the physiological PRF, but is a very powerful stimulating agent of prolactin release from estradiol primed lactotrophs (20, 23). TRH stimulates prolactin release in 2 phases in a perifusion system; an initial surge of prolactin and maintenance of elevated rate of release. TRH activates phospholipase C which hydrolyses phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-triphosphate (IP3) and 1,2-diacylglycerol (DAG). The initial surge is triggered by an increase in [Ca2+]i which is mobilized by the action of IP 3, and the elevated release is believed to be dependent on DAG which activates protein kinase C (6). Ionomycin, a calcium ionophore increases [Ca2+]i due to an influx of extracellular calcium. The hormone release is supposed to be proportional to the [Ca2÷]i. However, the increase in [Ca2+]~ induced by ionomycin is much higher than the one elevated by TRH, while the increase in prolactin release induced by TRH is much higher than that induced by ionomycin in the experiments which included 0.1% BSA in the perifusion medium throughout experiment (Fig. 1). The discrepancy between [CaZ÷]i and rate of prolactin release indicates that the elevation of [Ca2+]i is not the sole final common pathway of prolactin release. This conclusion agrees with other studies using freshly dispersed rat pituitary cells (13). A similar system was previously introduced (13) but substantially different from our system. The major differences between ours and the previous system are: (1) we used estradiol primed, primary cultured pituitary cells (82% lactotrophs) while Law et al. (13) used acutely dispersed female pituitary cells, and (2) Law et al (13) expressed their [Ca2÷]i with relative changes while our method introduced a calibration method of [Ca2+]i in a perifusion system. In conclusion, we have introduced a practical perifusion system which can simultaneously measure both [Ca2÷]~ and prolactin release. This dynamic perifusion system is an excellent tool for the study of the physiological role of [Ca2÷]~ changes on prolactin release.
Acknowledgements This work was supported by the Medical Research Council of Canada. The authors express their thanks to Drs. A.F. Parlow and S. Raiti for supplying the prolactin RIA kit through the National Hormone and Pituitary Program, NIDDKD, to Mr. M. Lim for excellent technical help and to Ms. Bonnie Stewart for excellent secretarial help.
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