0013-7227/79/1055-1122$02.00/0 Endocrinology Copyright © 1979 by The Endocrine Society
Vol. 105, No. 5 Printed in U.S.A.
Alteration of the Intracellular Calcium Level Stimulates Gonadotropin Release from Cultured Rat Anterior Pituitary Cells* P. MICHAEL CONN,t DELORIS C. ROGERS, AND FATEJEET S. SANDHU Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 27710
ABSTRACT. We demonstrated that stimulation of LH release from pituitary cells by gonadotropin-releasing hormone (GnRH) requires extracellular CaJ+ (optimally, 1 mM). Mg2+ (up to 10 mM) did not substitute for this requirement. Stimulated, but not basal, LH release was blocked by La'i+ which competes for cellular calcium-binding sites. Antagonists of Ca2+ flux (D-600, methoxyverapamil) and Ca2+ transport (Ruthenium red) also inhibited GnRH stimulation of LH release. In the present work, we incubated pituitary cells with compounds which alter the intracellular level of Ca2+ and examined the effect on LH release. In separate studies, cultured cells were incubated with ionophores A23187 (Lilly) or X537A (Roche) or with Ca2+- and Mg2+-bearing lipid vesicles (liposomes). Ionophore A23187, which inserts Ca2+ channels into the plasma membrane, stimulated LH release from the cells in the presence of extracellular Ca2+. When A23187 and GnRH were both present in saturating amounts, the cells did not release more LH than with either secretogogue alone. Ionophore X537A, a less
T
HE SPECIFIC mechanism by which gonadotropin hormone-releasing hormone (GnRH) stimulates gonadotropin release from the pituitary is not well understood. Some reports implicated cAMP in this process (1, 2), although there has been some difficulty by other laboratories (3-8) both in reproducing the initial observation and in pursuing logical extrapolations of this finding (such as adenylate cyclase stimulation). As a result, there is some confusion about the precise mechanism of GnRH action. In particular, using the cultured dispersed pituitary cell system (9) it was possible to show that 1) intracellular
Received February 23, 1979 Address all correspondence and requests for reprints to: Dr. P. Michael Conn, Department of Pharmacology, P.O. Box 3813, Duke University Medical Center, Durham, North Carolina 27710. * In past publications, we have used the term pituicytes to describe cultured anterior pituitary cells. To avoid possible confusion with other references to posterior pituitary cells, we are avoiding the use of pituicyte in reference to anterior pituitary-derived cells. This work was supported by Grants from the NIH and the Commonwealth Fund (to P.M.C.) f Fellow of the Duke Center for Aging and Human Development.
specific ionophore which may enter cells, did not show a marked dependency on either extracellular Ca2+ or Mg2+ for stimulation of LH release. Thus, intracellular ion pools appeared sufficient to support maximal stimulation of LH release even though extracellular Ca2+ was required for GnRH stimulation. Ion-bearing (Ca2+ or Mg2+) liposomes were used to insert particular ions into the gonadotropes in order to establish the specific ion(s) which stimulated LH release. Introduction of Ca2+-bearing, but not Mg2+-bearing or control, liposomes stimulated LH release above the basal level. The data in the present studies show that artificial introduction of Ca2+ into cultured pituitary cells resulted in the release of LH from the gonadotrope in the absence of GnRH. By sequential and concomitant GnRH and ionophore incubations, these secretogogues appeared to act on the same releasable pool of LH. The data provide additional support for a role of Ca2+ in LH release from cultured rat pituitary cells. (Endocrinology 105: 1122, 1979)
and extracellular cAMP and the occupancy of protein kinase with cAMP pools did not change over a wide range of GnRH concentrations or times; 2) compounds which activate cAMP production in most cells, such as cholera toxin and prostaglandin Ei, also activated pitutiary cell cAMP production but failed to evoke LH release comparably to GnRH; and 3) neither dibutyryl cAMP itself nor methyl isobutyl xanthine stimulated LH release or potentiated the effect of GnRH. Thus, in the cultured pituitary cell system, cAMP could be reliably uncoupled from gonadotropin release. Recently (10), cyclic nucleotide fluctuations, especially a decline in cGMP, were noted to occur after treatment of short term (22 h) cultures of pituitary cells with GnRH. The authors did not propose that cGMP fulfilled the requirements of an intracellular messenger. We (11) showed that extracellular Ca2+ is an absolute and specific requirement for GnRH-stimulated LH release and that drugs which blocked Ca2+ flux also blocked GnRH-stimulated LH release. In the present work, we extended these observations by showing that introduction of Ca2+ into gonadotropes results in LH release in the absence of the releasing hormone.
1122
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 16:29 For personal use only. No other uses without permission. . All rights reserved.
Ca STIMULATES PITUITARY CELL LH RELEASE
Materials and Methods Preparation of cells Primary cultures of rat pituitary cells were prepared by collagenase-hyaluronidase dispersion of 25- to 40-day-old fe*, male rats (Zivic-Miller), as previously (9) reported, and were then incubated 2 days at 37 C in Multi-Well culture plates (Falcon Plastics, Los Angeles, CA). After the culture period, they were washed free of the media and debris and then incubated as indicated below. Pituitary cell incubations Cells were incubated in 1 ml for 3 h (or as indicated for time studies) in the presence of the various secretogogues or in media alone. Medium 199 containing 0.3% bovine serum albumin (M199/BSA; fraction V, Miles Laboratories, Inc., Elkhart, IN) and Dulbecco's phosphate-buffered saline (dPBS, Microbiological Associates, Bethesda, MD) containing 0.3% BSA (dPBS/ BSA) were used interchangeably for cell incubations throughout and were found to produce identical basal and stimulated (10~° M GnRH) levels of LH release from pituitary cells. In i> studies where the Ca2+ level was altered, dPBS was prepared without Ca2+ and adjusted to the desired concentration. When ionophores were used, a final concentration of 1% dimethylsulfo. oxide was added to all assay wells. Assays were performed in triplicate. Reagents Stock solutons of ionophore A23187 (kindly donated by Dr. Robert Hamill, Eli Lilly Research Laboratories, Indianapolis, IN) and R02-2985/1, the sodium salt of ionophore X537A (kindly donated by Dr. W. E. Scott, Hoffman-La Roche, Inc., " Nutley, NJ), were stored at —40 C in dimethylsulfoxide for up to 60 days before use. GnRH was obtained from the Hormone Distribution Program of the NIAMDD, NIH, and the National _ Pituitary Agency. RIA ofLH *
Rat LH (rLH) antisera (LH S-4), highly purified rLH for labeling (LH 1-4), and rLH standard (LH-RP-1) were made available by a grant from the NIAMDD Hormone Distribution ^ officer and were prepared by Dr. A. Parlow (Torrance, CA). RIAs were performed as described by the kit instructions using second (precipitating) antibody to separate bound and free , radioactivities. Radioiodination was performed by the chloramine-T procedure (12) and purified by affinity chromatography on Concanavalin A-Sepharose (Pharmacia Fine Chemicals, Piscataway, NJ).
Preparation of liposomes —^
•
Liposomes were prepared from a suspension of phosphatidyl serine, phosphatidyl choline, and cholesterol in modified Locke's media according to the method of Poste et al. (13) and loaded with ions present in the original suspension, as previously described (14). Liposomes were purified by gel filtration on Sephadex G-50 (coarse) using a 10 X 280-mm column and
1123
eluted with PBS-BSA. Either 45Ca2+ (approximately 10 juCi; New England Nuclear Corp., Boston, MA) or I25I (approximately 1 juCi; New England Nuclear) was used to locate the included and void volumes by adding either isotope to the original lipid suspension before the preparation of the liposomes. In general, 125I was more convenient because radioactivity could be determined nondestructively by y-emission spectroscopy and also because this isotope could be used to locate liposomes prepared without added Ca2+. Electron micrographs of the liposome fraction were prepared by negative staining with saturated uranyl acetate in 2 mM collidine, pH 6.5 (15). Preparations were viewed and photographed in a JEOL transmission electron microscope (JEOL, Inc., Peabody, MA) at 25,000- to 30,000-fold magnification. General Bioassays of LH were performed using collagenase-dispersed interstitial cells (16). LH release and testosterone production are expressed as the average of triplicate determinations showing the SEM. The biological activity of LH was measured after the removal of contaminating ionophores and concentration by affinity chromatography on Concanavalin A (Con A)-Sepharose (Pharmacia Fine Chemicals, Piscataway, NJ). Released LH was extensively dialyzed against dPBS and loaded onto a 3-ml column of Con A-Sepharose (packed gel volume). The LH was competitively removed from the column by 0.2 M a-methyl mannoside, dialyzed, and assayed. Recoveries [82 ± 4% (SEM); n = 32) were calculated using [125I]LH as an internal standard. The amount of 125I not attached to (recovery standard) LH was determined at the time of the purification of (released) LH by gel filtration on Sephadex G-75. Antibody for the testosterone RIA was kindly provided by Dr. Guy Abraham. Pituitary cells were quantitated by DNA analysis, as previously described (9).
Results Figure 1 shows LH release resulting from incubating pituitary cells in various concentrations of ionophores A23187 and X537A. For comparison, both basal and GnRH-stimulated (10~6 M) LH release are shown. A23187 and X537A exhibited ED50 doses of approximately 10~5 M and 25 jug/ml, respectively, for LH release, and the dose-response curves were particularly sharp in contrast to GnRH, which varied over about 3 orders of magnitude in this preparation (9). Maximal LH release observed with either ionophore was statistically indistinguishable from that seen with maximal GnRH stimulation. Figure 2 shows the time courses of A23187 and X537A stimulation of LH release. Both appear to require about 3 h to produce maximum LH release, which is similar to the time course observed for maximal GnRH stimulation (9) and suggests that Ca2+ flux itself is not rate limiting in the stimulation of LH release. Figure 3 shows the Ca2+ dependence of basal LH release and release stimulated by GnRH, A23187, or X537A. GnRH and A23187 showed marked dependency
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 16:29 For personal use only. No other uses without permission. . All rights reserved.
CONN, ROGERS, AND SANDHU
1124
[A23I87] , log M (••••)
Vol. 105, No. 5 Printed in U.S.A.
Alteration of the Intracellular Calcium Level Stimulates Gonadotropin Release from Cultured Rat Anterior Pituitary Cells* P. MICHAEL CONN,t DELORIS C. ROGERS, AND FATEJEET S. SANDHU Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 27710
ABSTRACT. We demonstrated that stimulation of LH release from pituitary cells by gonadotropin-releasing hormone (GnRH) requires extracellular CaJ+ (optimally, 1 mM). Mg2+ (up to 10 mM) did not substitute for this requirement. Stimulated, but not basal, LH release was blocked by La'i+ which competes for cellular calcium-binding sites. Antagonists of Ca2+ flux (D-600, methoxyverapamil) and Ca2+ transport (Ruthenium red) also inhibited GnRH stimulation of LH release. In the present work, we incubated pituitary cells with compounds which alter the intracellular level of Ca2+ and examined the effect on LH release. In separate studies, cultured cells were incubated with ionophores A23187 (Lilly) or X537A (Roche) or with Ca2+- and Mg2+-bearing lipid vesicles (liposomes). Ionophore A23187, which inserts Ca2+ channels into the plasma membrane, stimulated LH release from the cells in the presence of extracellular Ca2+. When A23187 and GnRH were both present in saturating amounts, the cells did not release more LH than with either secretogogue alone. Ionophore X537A, a less
T
HE SPECIFIC mechanism by which gonadotropin hormone-releasing hormone (GnRH) stimulates gonadotropin release from the pituitary is not well understood. Some reports implicated cAMP in this process (1, 2), although there has been some difficulty by other laboratories (3-8) both in reproducing the initial observation and in pursuing logical extrapolations of this finding (such as adenylate cyclase stimulation). As a result, there is some confusion about the precise mechanism of GnRH action. In particular, using the cultured dispersed pituitary cell system (9) it was possible to show that 1) intracellular
Received February 23, 1979 Address all correspondence and requests for reprints to: Dr. P. Michael Conn, Department of Pharmacology, P.O. Box 3813, Duke University Medical Center, Durham, North Carolina 27710. * In past publications, we have used the term pituicytes to describe cultured anterior pituitary cells. To avoid possible confusion with other references to posterior pituitary cells, we are avoiding the use of pituicyte in reference to anterior pituitary-derived cells. This work was supported by Grants from the NIH and the Commonwealth Fund (to P.M.C.) f Fellow of the Duke Center for Aging and Human Development.
specific ionophore which may enter cells, did not show a marked dependency on either extracellular Ca2+ or Mg2+ for stimulation of LH release. Thus, intracellular ion pools appeared sufficient to support maximal stimulation of LH release even though extracellular Ca2+ was required for GnRH stimulation. Ion-bearing (Ca2+ or Mg2+) liposomes were used to insert particular ions into the gonadotropes in order to establish the specific ion(s) which stimulated LH release. Introduction of Ca2+-bearing, but not Mg2+-bearing or control, liposomes stimulated LH release above the basal level. The data in the present studies show that artificial introduction of Ca2+ into cultured pituitary cells resulted in the release of LH from the gonadotrope in the absence of GnRH. By sequential and concomitant GnRH and ionophore incubations, these secretogogues appeared to act on the same releasable pool of LH. The data provide additional support for a role of Ca2+ in LH release from cultured rat pituitary cells. (Endocrinology 105: 1122, 1979)
and extracellular cAMP and the occupancy of protein kinase with cAMP pools did not change over a wide range of GnRH concentrations or times; 2) compounds which activate cAMP production in most cells, such as cholera toxin and prostaglandin Ei, also activated pitutiary cell cAMP production but failed to evoke LH release comparably to GnRH; and 3) neither dibutyryl cAMP itself nor methyl isobutyl xanthine stimulated LH release or potentiated the effect of GnRH. Thus, in the cultured pituitary cell system, cAMP could be reliably uncoupled from gonadotropin release. Recently (10), cyclic nucleotide fluctuations, especially a decline in cGMP, were noted to occur after treatment of short term (22 h) cultures of pituitary cells with GnRH. The authors did not propose that cGMP fulfilled the requirements of an intracellular messenger. We (11) showed that extracellular Ca2+ is an absolute and specific requirement for GnRH-stimulated LH release and that drugs which blocked Ca2+ flux also blocked GnRH-stimulated LH release. In the present work, we extended these observations by showing that introduction of Ca2+ into gonadotropes results in LH release in the absence of the releasing hormone.
1122
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 16:29 For personal use only. No other uses without permission. . All rights reserved.
Ca STIMULATES PITUITARY CELL LH RELEASE
Materials and Methods Preparation of cells Primary cultures of rat pituitary cells were prepared by collagenase-hyaluronidase dispersion of 25- to 40-day-old fe*, male rats (Zivic-Miller), as previously (9) reported, and were then incubated 2 days at 37 C in Multi-Well culture plates (Falcon Plastics, Los Angeles, CA). After the culture period, they were washed free of the media and debris and then incubated as indicated below. Pituitary cell incubations Cells were incubated in 1 ml for 3 h (or as indicated for time studies) in the presence of the various secretogogues or in media alone. Medium 199 containing 0.3% bovine serum albumin (M199/BSA; fraction V, Miles Laboratories, Inc., Elkhart, IN) and Dulbecco's phosphate-buffered saline (dPBS, Microbiological Associates, Bethesda, MD) containing 0.3% BSA (dPBS/ BSA) were used interchangeably for cell incubations throughout and were found to produce identical basal and stimulated (10~° M GnRH) levels of LH release from pituitary cells. In i> studies where the Ca2+ level was altered, dPBS was prepared without Ca2+ and adjusted to the desired concentration. When ionophores were used, a final concentration of 1% dimethylsulfo. oxide was added to all assay wells. Assays were performed in triplicate. Reagents Stock solutons of ionophore A23187 (kindly donated by Dr. Robert Hamill, Eli Lilly Research Laboratories, Indianapolis, IN) and R02-2985/1, the sodium salt of ionophore X537A (kindly donated by Dr. W. E. Scott, Hoffman-La Roche, Inc., " Nutley, NJ), were stored at —40 C in dimethylsulfoxide for up to 60 days before use. GnRH was obtained from the Hormone Distribution Program of the NIAMDD, NIH, and the National _ Pituitary Agency. RIA ofLH *
Rat LH (rLH) antisera (LH S-4), highly purified rLH for labeling (LH 1-4), and rLH standard (LH-RP-1) were made available by a grant from the NIAMDD Hormone Distribution ^ officer and were prepared by Dr. A. Parlow (Torrance, CA). RIAs were performed as described by the kit instructions using second (precipitating) antibody to separate bound and free , radioactivities. Radioiodination was performed by the chloramine-T procedure (12) and purified by affinity chromatography on Concanavalin A-Sepharose (Pharmacia Fine Chemicals, Piscataway, NJ).
Preparation of liposomes —^
•
Liposomes were prepared from a suspension of phosphatidyl serine, phosphatidyl choline, and cholesterol in modified Locke's media according to the method of Poste et al. (13) and loaded with ions present in the original suspension, as previously described (14). Liposomes were purified by gel filtration on Sephadex G-50 (coarse) using a 10 X 280-mm column and
1123
eluted with PBS-BSA. Either 45Ca2+ (approximately 10 juCi; New England Nuclear Corp., Boston, MA) or I25I (approximately 1 juCi; New England Nuclear) was used to locate the included and void volumes by adding either isotope to the original lipid suspension before the preparation of the liposomes. In general, 125I was more convenient because radioactivity could be determined nondestructively by y-emission spectroscopy and also because this isotope could be used to locate liposomes prepared without added Ca2+. Electron micrographs of the liposome fraction were prepared by negative staining with saturated uranyl acetate in 2 mM collidine, pH 6.5 (15). Preparations were viewed and photographed in a JEOL transmission electron microscope (JEOL, Inc., Peabody, MA) at 25,000- to 30,000-fold magnification. General Bioassays of LH were performed using collagenase-dispersed interstitial cells (16). LH release and testosterone production are expressed as the average of triplicate determinations showing the SEM. The biological activity of LH was measured after the removal of contaminating ionophores and concentration by affinity chromatography on Concanavalin A (Con A)-Sepharose (Pharmacia Fine Chemicals, Piscataway, NJ). Released LH was extensively dialyzed against dPBS and loaded onto a 3-ml column of Con A-Sepharose (packed gel volume). The LH was competitively removed from the column by 0.2 M a-methyl mannoside, dialyzed, and assayed. Recoveries [82 ± 4% (SEM); n = 32) were calculated using [125I]LH as an internal standard. The amount of 125I not attached to (recovery standard) LH was determined at the time of the purification of (released) LH by gel filtration on Sephadex G-75. Antibody for the testosterone RIA was kindly provided by Dr. Guy Abraham. Pituitary cells were quantitated by DNA analysis, as previously described (9).
Results Figure 1 shows LH release resulting from incubating pituitary cells in various concentrations of ionophores A23187 and X537A. For comparison, both basal and GnRH-stimulated (10~6 M) LH release are shown. A23187 and X537A exhibited ED50 doses of approximately 10~5 M and 25 jug/ml, respectively, for LH release, and the dose-response curves were particularly sharp in contrast to GnRH, which varied over about 3 orders of magnitude in this preparation (9). Maximal LH release observed with either ionophore was statistically indistinguishable from that seen with maximal GnRH stimulation. Figure 2 shows the time courses of A23187 and X537A stimulation of LH release. Both appear to require about 3 h to produce maximum LH release, which is similar to the time course observed for maximal GnRH stimulation (9) and suggests that Ca2+ flux itself is not rate limiting in the stimulation of LH release. Figure 3 shows the Ca2+ dependence of basal LH release and release stimulated by GnRH, A23187, or X537A. GnRH and A23187 showed marked dependency
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 16:29 For personal use only. No other uses without permission. . All rights reserved.
CONN, ROGERS, AND SANDHU
1124
[A23I87] , log M (••••)
