0013-7227/90/1274-1948$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 127, No. 4 Printed in U.S.A.
Interactions of Dopamine and Neurotensin on Calcium Fluxes and Prolactin Release in Normal Rat Pituitary Cells* IVAN S. LOGIN, SON I. KUAN, ALLAN M. JUDD, AND ROBERT M. M A C L E O D Departments of Neurology (I.S.L.) and Internal Medicine (S.I.K., A.M.J., R.M.M.) and the Center for Cancer Research, University of Virginia Health Sciences Center, Charlottesuille, Virginia 22908
ABSTRACT. We studied the role of calcium in dopaminergic
control of the neuroendocrine effects of neurotensin. In primary cultures of dispersed normal female rat anterior pituitary cells the interactions of dopamine and neurotensin were examined with reference to the rate of PRL release, the magnitude of 45 Ca2+ uptake, the rate of fractional 45Ca2+ efflux, and the dynamic response of the intracellular calcium concentration (Ca;) monitored with the fluorescent dye, Indo-1. Neurotensin stimulated calcium uptake and also mobilized a pool of intracellular calcium to increase Cai in a sustained plateau-like pattern. The response of PRL release and fractional efflux to neurotensin, however, each displayed typical spike and plateau profiles. In
I
T IS ESTABLISHED that among several intracellular signalling systems the regulation of hormone secretion is critically influenced by the cytoplasmic concentration of unbound calcium ions (Caj). Demonstration of a change in Ca;, however, reveals little about the mechanism(s) by which such a change occurred. For example, an increase in Ca; could result from a combination of increased calcium uptake from the extracellular space, enhanced mobilization of calcium bound or stored within cytoplasmic constituents, or a reduction in the active removal of cytoplasmic calcium by ATP-dependent Ca2,Mg2+-ATPase located in plasma membrane or cytoplasmic organelle membranes (1). Further, individual secretagogues may access unique combinations of these paths to change Ca;. Investigation of isotopic calcium fluxes in association with measurement of Caj are complementary techniques providing insight to these complex mechanisms.
the presence of dopamine the stimulation of PRL release and calcium uptake due to neurotensin were abolished, and the rise in Ca; was barely detectable, but neurotensin-stimulated fractional efflux persisted almost unchanged. These data suggest that dopamine may modulate Caj by inhibiting calcium uptake and possibly also by enhancing cellular calcium extrusion under stimulated conditions. Further, the increased inositol trisphosphate production reportedly stimulated by neurotensin apparently does not generate a spike-like response of intracellular calcium, and stimulated hormone release may display a spike and plateau pattern solely with a plateau Ca; profile. (Endocrinology 127: 1948-1955, 1990)
part to inhibition of the basal rate of calcium uptake (5, 6). The present work was designed to extend this knowledge by examining dopaminergic control of calcium fluxes modulated by neurotensin (7,8), the calcium channel activator maitotoxin (MTX) (9), and the dihydropyridine BAY K8644 (10-12), each thought to act by stimulating calcium uptake.
Materials and Methods Cell culture
Dopaminergic control of PRL release is associated with a reduction in Ca; (2-4), which appears to be due in Received May 4, 1990. Address requests for reprints to: Ivan S. Login, M.D., Department of Neurology, Box 394, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908. * This work was supported in part by Biomedical Research Grant 5-507-RR-05431 (to I.S.L.) and NIH Grants CA-38228 (to I.S.L.) and CA-07535-28 (to R.M.M.) from the NCI.
Anterior pituitary glands were removed quickly after decapitation of female Sprague-Dawley rats (200-250 g; Dominion Laboratories, Dublin, VA), and the glands were dispersed mechanically and enzymatically, as previously described (9). Dispersed cells were maintained in primary culture in 75-cm2 flasks (Corning, Medfield, MA) at a density of 1 X 107 cells/ flask in RPMI-1640 medium containing 2.5% fetal bovine and 7.5% horse sera for 3-4 days. The cells were harvested on the day of experimentation with a brief exposure to 0.05% trypsin and washing using an isotonic defined perifusate buffer (MBSS; 129 mM NaCl, 5.9 mM KC1, 1.5 mM CaCl2, 1.2 mM MgCl2, 11.8 mM glucose, 18 mM HEPES, and 0.2% BSA, pH 7.35). About 70% of the initial cell yield was recovered, and the viability was greater than 90% by trypan blue exclusion. Dopamine and neurotensin were purchased from Sigma (St. Louis, MO), BAY K8644 was a gift from Miles, Inc. (West
1948
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 05:15 For personal use only. No other uses without permission. . All rights reserved.
DOPAMINE AND NEUROTENSIN
1949
Haven, CT), and MTX was prepared and supplied to us by Dr. T. Yasumoto, Tohuko University (Sendai, Japan).
dispersed suspended cells were preincubated with test agents for 10 min before adding 45Ca2+ for 60 sec. Calcium uptake was then estimated with a vacuum filtration method (9).
Experimental design
Simultaneous description of fractional calcium efflux and rate of PRL release during perifusion. After harvesting, cells were labeled with 45Ca2+ (5 MCi/ml; 2.5 h; 1.5 mM CaCl2), mixed with an inert supporting matrix Bio-Gel, and loaded into a perifusion chamber at 37 C, as described previously (9). Perifusate of the first 45 min was discarded, after which 1-min fractions were collected to monitor simultaneously fractional calcium efflux and PRL release (9). In each experiment, the area under the response curve was integrated for both fractional calcium efflux and PRL release to analyze the results statistically.
The cultured cells were used to assess four cellular functions. Dynamic estimation of Cai. Indo-1, acetoxymethylester (AM), is trapped intracellularly by esterase cleavage to form Indo-1, a calcium-sensitive fluorescent dye in a manner similar to the related compound, fura. After excitation at 339 nm, Indo-1 changes its emission spectra upon calcium binding, with calcium enhancing the 400nm signal and reducing the 480-nm signal. The ratio of Indo-1 emission spectra (400 nm/480 nm) simultaneously monitored in an SLM 8000 Spectrofluorometer (SLM Instruments, Inc., Urbana, IL), thus, estimates intracellular calcium levels independently of the intracellular Indo-1 concentration (13). Cells (1.5 million/ml) were exposed to 2.5 nM Indo-1, AM (Molecular Probes, Eugene, OR), at 37 C for 30 min. All manipulations with Indo were performed in subdued light, and the cells were shaken constantly during the incubation. The cells were then washed twice and brought to the original density in MBSS. The cells accumulated about 40% of the Indo-1, AM, in agreement with other reports (14). There was no change in viability by trypan blue exclusion after Indo labeling. Labeled cells (2.5 ml) were distributed to individual disposable UV cuvettes, wrapped in aluminum foil, and held on ice to retard dye leakage. When ready for use, each cuvette was warmed to 37 C for 30 min to complete dye hydrolysis. Cell clumping during the recording was minimized by manual trituration immediately before cuvette positioning in the fluorometer and with continuous magnetic stirring during the recording. A water-jacketed cuvette holder maintained the desired temperature. Baseline recording lasted 30-60 sec, after which, without interruption, test agents were injected by Hamilton syringe (Reno, NV) into the stirred cell suspension, and recording was continued as necessary, with spectral data provided every second. Dye leakage from loaded cells into the buffer could contribute to the recorded signal. In control suspensions, therefore, Indolabeled cells were centrifuged, and the supernatant (containing any Indo-1 that leaked from the cells) was scanned for its 400and 480-nm signals. These values were subtracted from the sample spectra as a background correction. The signals were also corrected for cellular and buffer autofluorescence. None of the agents tested fluoresced at 400 or 480 nm. Labeled cells were scanned without drug treatments to define baseline conditions. The free Indo-1 signal was calibrated (13) with a series of Ca-EGTA standards so that the individual ratio values at each second could be converted by computer software to an equivalent concentration for Ca^ Data from replicate experiments were combined to generate a dynamic recording of mean (±SE) values for Cai. Calcium uptake into unlabeled cells. After harvesting, the cells were washed and resuspended in a balanced salt solution (BSS) differing from MBSS only in the absence of BSA (9). Briefly,
PRL RIA PRL concentrations were determined by standard RIA procedures, using reagents provided by the NIDDK Rat Pituitary Hormone Distribution Program. Results are expressed in terms of NIDDK rat PRL RP-3 standard (intraassay variability,
Vol. 127, No. 4 Printed in U.S.A.
Interactions of Dopamine and Neurotensin on Calcium Fluxes and Prolactin Release in Normal Rat Pituitary Cells* IVAN S. LOGIN, SON I. KUAN, ALLAN M. JUDD, AND ROBERT M. M A C L E O D Departments of Neurology (I.S.L.) and Internal Medicine (S.I.K., A.M.J., R.M.M.) and the Center for Cancer Research, University of Virginia Health Sciences Center, Charlottesuille, Virginia 22908
ABSTRACT. We studied the role of calcium in dopaminergic
control of the neuroendocrine effects of neurotensin. In primary cultures of dispersed normal female rat anterior pituitary cells the interactions of dopamine and neurotensin were examined with reference to the rate of PRL release, the magnitude of 45 Ca2+ uptake, the rate of fractional 45Ca2+ efflux, and the dynamic response of the intracellular calcium concentration (Ca;) monitored with the fluorescent dye, Indo-1. Neurotensin stimulated calcium uptake and also mobilized a pool of intracellular calcium to increase Cai in a sustained plateau-like pattern. The response of PRL release and fractional efflux to neurotensin, however, each displayed typical spike and plateau profiles. In
I
T IS ESTABLISHED that among several intracellular signalling systems the regulation of hormone secretion is critically influenced by the cytoplasmic concentration of unbound calcium ions (Caj). Demonstration of a change in Ca;, however, reveals little about the mechanism(s) by which such a change occurred. For example, an increase in Ca; could result from a combination of increased calcium uptake from the extracellular space, enhanced mobilization of calcium bound or stored within cytoplasmic constituents, or a reduction in the active removal of cytoplasmic calcium by ATP-dependent Ca2,Mg2+-ATPase located in plasma membrane or cytoplasmic organelle membranes (1). Further, individual secretagogues may access unique combinations of these paths to change Ca;. Investigation of isotopic calcium fluxes in association with measurement of Caj are complementary techniques providing insight to these complex mechanisms.
the presence of dopamine the stimulation of PRL release and calcium uptake due to neurotensin were abolished, and the rise in Ca; was barely detectable, but neurotensin-stimulated fractional efflux persisted almost unchanged. These data suggest that dopamine may modulate Caj by inhibiting calcium uptake and possibly also by enhancing cellular calcium extrusion under stimulated conditions. Further, the increased inositol trisphosphate production reportedly stimulated by neurotensin apparently does not generate a spike-like response of intracellular calcium, and stimulated hormone release may display a spike and plateau pattern solely with a plateau Ca; profile. (Endocrinology 127: 1948-1955, 1990)
part to inhibition of the basal rate of calcium uptake (5, 6). The present work was designed to extend this knowledge by examining dopaminergic control of calcium fluxes modulated by neurotensin (7,8), the calcium channel activator maitotoxin (MTX) (9), and the dihydropyridine BAY K8644 (10-12), each thought to act by stimulating calcium uptake.
Materials and Methods Cell culture
Dopaminergic control of PRL release is associated with a reduction in Ca; (2-4), which appears to be due in Received May 4, 1990. Address requests for reprints to: Ivan S. Login, M.D., Department of Neurology, Box 394, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908. * This work was supported in part by Biomedical Research Grant 5-507-RR-05431 (to I.S.L.) and NIH Grants CA-38228 (to I.S.L.) and CA-07535-28 (to R.M.M.) from the NCI.
Anterior pituitary glands were removed quickly after decapitation of female Sprague-Dawley rats (200-250 g; Dominion Laboratories, Dublin, VA), and the glands were dispersed mechanically and enzymatically, as previously described (9). Dispersed cells were maintained in primary culture in 75-cm2 flasks (Corning, Medfield, MA) at a density of 1 X 107 cells/ flask in RPMI-1640 medium containing 2.5% fetal bovine and 7.5% horse sera for 3-4 days. The cells were harvested on the day of experimentation with a brief exposure to 0.05% trypsin and washing using an isotonic defined perifusate buffer (MBSS; 129 mM NaCl, 5.9 mM KC1, 1.5 mM CaCl2, 1.2 mM MgCl2, 11.8 mM glucose, 18 mM HEPES, and 0.2% BSA, pH 7.35). About 70% of the initial cell yield was recovered, and the viability was greater than 90% by trypan blue exclusion. Dopamine and neurotensin were purchased from Sigma (St. Louis, MO), BAY K8644 was a gift from Miles, Inc. (West
1948
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 05:15 For personal use only. No other uses without permission. . All rights reserved.
DOPAMINE AND NEUROTENSIN
1949
Haven, CT), and MTX was prepared and supplied to us by Dr. T. Yasumoto, Tohuko University (Sendai, Japan).
dispersed suspended cells were preincubated with test agents for 10 min before adding 45Ca2+ for 60 sec. Calcium uptake was then estimated with a vacuum filtration method (9).
Experimental design
Simultaneous description of fractional calcium efflux and rate of PRL release during perifusion. After harvesting, cells were labeled with 45Ca2+ (5 MCi/ml; 2.5 h; 1.5 mM CaCl2), mixed with an inert supporting matrix Bio-Gel, and loaded into a perifusion chamber at 37 C, as described previously (9). Perifusate of the first 45 min was discarded, after which 1-min fractions were collected to monitor simultaneously fractional calcium efflux and PRL release (9). In each experiment, the area under the response curve was integrated for both fractional calcium efflux and PRL release to analyze the results statistically.
The cultured cells were used to assess four cellular functions. Dynamic estimation of Cai. Indo-1, acetoxymethylester (AM), is trapped intracellularly by esterase cleavage to form Indo-1, a calcium-sensitive fluorescent dye in a manner similar to the related compound, fura. After excitation at 339 nm, Indo-1 changes its emission spectra upon calcium binding, with calcium enhancing the 400nm signal and reducing the 480-nm signal. The ratio of Indo-1 emission spectra (400 nm/480 nm) simultaneously monitored in an SLM 8000 Spectrofluorometer (SLM Instruments, Inc., Urbana, IL), thus, estimates intracellular calcium levels independently of the intracellular Indo-1 concentration (13). Cells (1.5 million/ml) were exposed to 2.5 nM Indo-1, AM (Molecular Probes, Eugene, OR), at 37 C for 30 min. All manipulations with Indo were performed in subdued light, and the cells were shaken constantly during the incubation. The cells were then washed twice and brought to the original density in MBSS. The cells accumulated about 40% of the Indo-1, AM, in agreement with other reports (14). There was no change in viability by trypan blue exclusion after Indo labeling. Labeled cells (2.5 ml) were distributed to individual disposable UV cuvettes, wrapped in aluminum foil, and held on ice to retard dye leakage. When ready for use, each cuvette was warmed to 37 C for 30 min to complete dye hydrolysis. Cell clumping during the recording was minimized by manual trituration immediately before cuvette positioning in the fluorometer and with continuous magnetic stirring during the recording. A water-jacketed cuvette holder maintained the desired temperature. Baseline recording lasted 30-60 sec, after which, without interruption, test agents were injected by Hamilton syringe (Reno, NV) into the stirred cell suspension, and recording was continued as necessary, with spectral data provided every second. Dye leakage from loaded cells into the buffer could contribute to the recorded signal. In control suspensions, therefore, Indolabeled cells were centrifuged, and the supernatant (containing any Indo-1 that leaked from the cells) was scanned for its 400and 480-nm signals. These values were subtracted from the sample spectra as a background correction. The signals were also corrected for cellular and buffer autofluorescence. None of the agents tested fluoresced at 400 or 480 nm. Labeled cells were scanned without drug treatments to define baseline conditions. The free Indo-1 signal was calibrated (13) with a series of Ca-EGTA standards so that the individual ratio values at each second could be converted by computer software to an equivalent concentration for Ca^ Data from replicate experiments were combined to generate a dynamic recording of mean (±SE) values for Cai. Calcium uptake into unlabeled cells. After harvesting, the cells were washed and resuspended in a balanced salt solution (BSS) differing from MBSS only in the absence of BSA (9). Briefly,
PRL RIA PRL concentrations were determined by standard RIA procedures, using reagents provided by the NIDDK Rat Pituitary Hormone Distribution Program. Results are expressed in terms of NIDDK rat PRL RP-3 standard (intraassay variability,
