Volume 27, number 3
MOLECULAR & CELLULARBIOCHEMISTRY
November 1, 1979
THE R E G U L A T I O N OF CELL PROLIFERATION BY CALCIUM A N D CYCLIC AMP
J. F. W H I T F I E L D , A. L. BOYNTON, J. P. MACMANUS, M. SIKORSKA*, and B. K. TSANG
Animal and Cell Physiology Group, Division of Biological Sciences, National Research Council of Canada, Ottawa, Canada K I A O R 6 (Received June 27, 1979)
Introduction
Many, if not most, cellular responses to different stimuli are wholly or partly mediated by changes in either Na+/K ÷ or Ca 2÷ fluxes. In fact, in many cases the final response is the culmination of sequential Na+/K+-mediated and Ca 2÷mediated processes. It is now known that cell proliferation is one of these ion-mediated responses, and that calcium ions and the hormones (calcitonin, l a , 25(OH)z vitamin D3, parathyroid hormone) which maintain their level in the blood are major regulators of D N A synthesis and mitotic activity in the bone marrow, liver and thymus of the rat 1-15. Calcium also positively controls the proliferation of non-tumorigenic epithelial and mesenchymally derived bovine, human and rodent cells in vitro 17-31. On the other hand, calcium has little or no influence on the proliferation of the corresponding tumorigenic cells 17-2°'22-24"25'27-3°, a very important fact which some day may prove to be the key to understanding neoplastic transformation. In this brief review there will be no detailed discussion of the inconclusive information on the still crudely localized cellular calcium fluxes related to cell proliferation. Instead, we will concentrate on the mounting evidence that calcium normally has nothing to do with the signal to start proliferative development, but does cooperate with that increasingly popular, * On Leave-of-Absencefrom the Medical Research Center, Polish Academy of Sciences,Warsaw. N.R.C.C. No. 17786.
many-talented trio of cyclic AMP (adenosine 3',5'-monophosphate), calmodulin (or CDR, a calcium-dependent regulatory protein) and protein kinase(s), to generate the later signal to initiate D N A synthesis. We will also speculate (heuristically, we hope!) on the reasons for calcium's mysterious inability to influence the proliferation of cancer cells.
Calcium's physiological uniqueness The calcium ion is certainly not an obvious choice for a regulator of such a complicated and important process as cell proliferation, because it is characterized only by its charge, coordination number, and unhydrated radius. However, it is just these properties which enable it, and only it, to activate certain ubiquitous multipurpose, calcium-binding proteins (e.g., calmodulin (CDR), troponin C) which regulate many different cellular functions 32"33. The importance of calcium in processes such as cell motility34, egg activation 35, muscle contraction a6, neurotransmission and secretion 37"38 has promted the evolutionary assignment of an entire hormone system and the expenditure of considerable amounts of energy on intracellular sequestration processes to regulate the extracellular and intracellular calcium ion concentrations. However, calcium must have even more regulatory functions because all cells work unceasingly to maintain a very steep electrochemical calcium gradient (from 10-3~ outside to 10 -7 or 10-SM in the cytosol) using appropriately sited CaZ+-transport ATPases to pump calcium ions out of the cell or into
Dr. I41..Junk b.v. Publishers - The Hague, The Netherlands
155
sequestration sites such as the cisternae of the endoplasmic reticulum 39-42. Because of this extraordinary steep gradient many different agents, the only common property of which is an ability to react with things on or in the cell membrane, cause calcium ions to squirt into the cell where they combine with the multipurpose calcium-dependent regulatory proteins. However, the nature of the response to this common calcium surge differs according to the cell's enzyme complement (i.e., its functional specialty). Calcium's special status as a regulator is emphasized by the fact that no hormones have been designed to regulate the blood level of magnesium, the other major divalent cation. Moreover, no cells struggle to produce and maintain a steep electrochemical gradient of magnesium ions 43, nor are there any multipurpose magnesium-dependent counterparts of the calcium-dependent calmodulin (CDR) 33. Nevertheless, as might be guessed from its constantly high intracellular level, magnesium is normally a permissive or function-maintaining (as opposed to function-initiating) factor needed by many enzymes including adenylate cyclase, Ca2+-transport ATPases and various protein kinases, which are three of the principal targets of the activating Ca-calmodulin (CDR) complexes produced during an intracellular calcium surge. Different though they may be, the functions of calcium and magnesium are obviously inextricably interwined because magnesium is needed by some enzymes after activation by calcium and some key magnesium-dependent enzymes do not function until activated by calcium. Therefore, magnesium deprivation can reduce or block various responses to an intracellular calcium surge.
The cell cycle Some cells, such as hepatocytes in the normal adult liver, neurones, adult parotid gland acinar cells and small peripheral blood lymphocytes contain reversibly or irreversibly repressed proliferogenic genes (i.e., certain regulatory genes and the structural genes coding for components of the proliferative machinery) and are engaged only in non-proliferative activities 12"44. We avoid a cell cycle (i.e., "G") designation such as Go for these cells simply because they are neither involved in a growth-division cycle nor enzymi156
cally equipped to do so. Their proliferative status must not be confused with that of other proliferatively quiescent cells such as serum- or calcium-deprived cells in vitro and certain stem cells in vitro, with still activated proliferogenic genes which are enzymically equipped to cycle and therefore merit the cycle designation "Go ''12'44. We believe that the stimulatory processes causing Go cells to resume cycling are different from the activating processes causing proliferatively inactivated cells to start cycling 1e'44, but some agents can trigger both of them. For example, the plant lectin concanavalin A proliferatively activates small peripheral blood lymphocytes and proliferatively stimulates already activated, but proliferatively quiescent, calcium-deprived late prereplicative R R thymic lymphocytes (vide infra). Proliferative activators somehow generate the first signal to start the production of the messenger R N A species responsible for the completely mysterious transitional processes 3°'44 which prepare the cell for entry into the prolfferatively committed G1 phase during which a different set of parallel and sequential reactions take over and bring the cell to a point where another signal is generated to start their entry into the S phase, the most vivid, but by no means the only, features of which are D N A synthesis and chromosome replication [Fig. 1]. After replicating its chromosomes, the cell enters a premitotic, G2, phase after which it progresses through the four stages of mitosis and finally divides [Fig. 1]. A daughter cell may become proliferatively inactivated and turn to other things, it may move directly to the threshold of the G1 phase (bypassing the now unnecessary transitional phase) and start another cycle, or it may lapse into a proliferatively quiescent Go state [Fig. 1] 12"3°'44. The composition of the cell's enzyme complement and surface components constantly changes as it progresses through the growthdivision cycle. Therefore, an intracellular surge of a regulator such as cyclic AMP at different points in the cell cycle will elicit entirely different responses. For example, a cyclic AMP surge in the G1 phase of the rat hepatocyte triggers D N A synthesis, but an earlier surge in the transitional phase cannot do so simply because the cell has not yet begun to make its DNA-synthetic enzymes (vide infra ).
RR (rapidly responding) thymic lyrnphocytes
Fig. 1. A highly symbolic depiction of the constantly changing (and mostly unknown) interdependent processes of the different phases of proliferative development. These phases are defined by DNA-synthetic and mitotic signposts and the inconspicuousness of the events in the gaps (G1, G2) between them. Everything starts when P, a proliferogenic agent or procedure (e.g., isoproterenol for salivary gland acinar cells, partial hepatectomy for hepatocytes, plant lectins for small lymphocytes) proliferatively activates the cell, thereby causing it to enter the transitional phase (Trans.)at point 1. Many things, such as proliferatively meaningless calcium and cyclic AMP surges, possibly proliferatively important changes in Na+/K + fluxes, and definitely proliferatively important increases in mRNA, ribosomal RNA and polyamine syntheses, happen during this transitonal phase of prereplicative development. The transitional processes bring the cell to point 2, the threshold of the G1 phase of prereplicative development. The G1 phase is very important for our story, because it includes the critical, DNA-synthetic process which (as indicated) is triggered by the combined efforts of calcium and a second, or Ol, cyclic AMP surge. The cell then replicates its chromosomes (as well as other more mysterious things) in the S phase. In the G2 phase it prepares itself for various mitotic tasks, such as chromosome condensation and massive microtubule redistribution, and it finally undergoes mitosis and cytokinesis. Each daughter cell may repress its proliferogenic genes to become proliferatively inactive; become proliferatively quiescent (Go) without being proliferatively inactivated; or immediately initiate another cycle from point 2 without having to go through another transitional phase. If any one of the interlinked processes of the G1 kaleidoscope should fail, the cell enters a O 0 state without inactivating its proliferogenic genes. The longer the cell stays in the Go state, the more it loses the accumulated components and systems needed for proliferation, and the longer it will take to reach the S phase when stimulated. In other words, the proliferative status of the cell regresses to that of a freshly activated cell at point 1.
T h e large l y m p h o c y t e p o p u l a t i o n in the t h y m u s gland of a 200 g male, S p r a g u e - D a w l e y rat is maintained by the rapid proliferation (total cycle times of 5 to 7 hours) of stem cells (lymphoblasts) w h o s e p r o g e n y b e c o m e smaller with each division b e c a u s e of an increasing repression of their nuclear activity (seen as c h r o m a t i n condensation) and a c o n s e q u e n t failure of their intermitotic growth to k e e p pace with their rapid multiplication 3'4s. In o t h e r words, these cells have a division cycle rather than a growth-division cycle. T h e e n d - p r o d u c t s of this i m m e n s e proliferative activity are the small proliferatively inactivated l y m p h o c y t e s which o c c u p y the bulk of the t h y m u s gland 45. A b o u t 10 to 15% of the cells in large, freshly isolated thymic l y m p h o c y t e populations susp e n d e d at a c o n c e n t r a t i o n of a b o u t 107 cells/ml of a serum-free, low (0.02 mM)-calcium tissue culture m e d i u m are actively cycling large and m e d i u m lymphoblasts as, and a b o u t 75% are proliferatively inactivated small lymphocytes. T h e remaining 10 to 15% (i.e., a b o u t 106 cells/ml) are proliferatively activated R R l y m p h o c y t e s which c a n n o t e n d o g e n o u s l y initiate D N A synthesis, but are enzymically e q u i p p e d to do so p r o m p t l y after exposure to any of several agents, one of which is the calcium ion. Thus, briefly o v e r w h e l m i n g the cells' calcium h o m e o static systems b y suddenly raising the ionic calcium concentration to 1.5 mM (and then leaving it elevated for as little as 5 minutes or as long as 5 hours) caused the small s u b p o p u l a tion of R R cells to initiate D N A synthesis (as indicated by an increase in nuclear-labelling with [3H]-thymidine or [6-3H]-deoxyuridine) between 45 and 60 minutes later [Fig. 2A], and t h e n to start entering mitosis 2 hours afterwards 46-s0. W e do not k n o w w h e t h e r the R R cells were b l o c k e d in the late G I phase or in the D N A synthetic S phase, but this in no w a y detracts f r o m the i m p o r t a n c e of the fact that calcium can trigger D N A synthesis. Exactly the same inv o l v e m e n t of calcium in D N A synthesis has b e e n o b s e r v e d in the equally rapidly r e s p o n d i n g C F U - S spleen c o l o n y - f o r m i n g cells in suspensions of m o u s e b o n e m a r r o w cells 51-53. Needless to say, the p r e c e n d e n t for all of this is calcium's triggering (i.e., activation) of processes in the fertilized egg which lead to the initiation of 157
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0 30 60 120 0 ~0 60 MINUTES AFTER INCREASINGTHE C~LCIUM CONCENTRATIONFROM 0.02 rnbl TO 1.5mM_
Fig. 2. Stimulations of cyclic AMP and DNA syntheses in freshly prepared 14's° thymic lymphocyte suspensions by raising the extracellular calcium concentration from 0.02 to 1.5 raM. A. The stimulation of cyclic AMP synthesis as indicated by increased production of [3HI-cyclic AMP from a pre-labeled intracellular [3H]-ATP pool was chromatographically determined according to MAcMANuS and WHrrFmLD46. There was no change in the level of [aH]-ATP during the period of observation which could have been a more trivial reason for the [3H]-cyclic A M P surge46. DNA-synthefic activity was autoradiographicaily determined by pulse (15-min.)-labeling of cell nuclei with [3H]thymidines°. B. The stimultaneous calcium-induced depression of the incorporation of radioactivity from [3H]thymidine into the cold acid-insoluble fraction (i.e., DNA) 7~. Each point is the mean ± S.E.M. of the values from 8 to 12 cell suspensions. Note: CPM means counts per min. in this and all other Figures.
[Fig. 3]. However, pretreatment with parathyroid hormone (which did not by itself stimulate thymic lymphocyte adenylate cyclase in broken cell preparations) enabled such small increases in the extracellular calcium concentration to stimulate both cyclic AMP synthesis and RR cell proliferation48. The calcium-induced cyclic AMP surge in the thymic lymphocyte suspensions was undetectable with other methods such as the competitive radioligand-binding assays which do not have the radiometric assay's ability to distinguish newly synthesized (i.e., labeled) cyclic AMP from "old" (i.e., unlabeled) cyclic AMP. However, as will be seen below, the binding assays easily detected the similar cyclic AMP surge which preceded the calcium-induced initiation of DNA synthesis in much more homogeneous populations of calcium-deprived rat liver cells [Figs 14 and 16]. Nevertheless, small though it was, the calcium-induced cyclic AMP surge in the thymic lymphocyte suspensions was necessary because its prevention or facilitation (by respectively stimulating or inhibiting cyclic 3',5'-
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MOLECULAR & CELLULARBIOCHEMISTRY
November 1, 1979
THE R E G U L A T I O N OF CELL PROLIFERATION BY CALCIUM A N D CYCLIC AMP
J. F. W H I T F I E L D , A. L. BOYNTON, J. P. MACMANUS, M. SIKORSKA*, and B. K. TSANG
Animal and Cell Physiology Group, Division of Biological Sciences, National Research Council of Canada, Ottawa, Canada K I A O R 6 (Received June 27, 1979)
Introduction
Many, if not most, cellular responses to different stimuli are wholly or partly mediated by changes in either Na+/K ÷ or Ca 2÷ fluxes. In fact, in many cases the final response is the culmination of sequential Na+/K+-mediated and Ca 2÷mediated processes. It is now known that cell proliferation is one of these ion-mediated responses, and that calcium ions and the hormones (calcitonin, l a , 25(OH)z vitamin D3, parathyroid hormone) which maintain their level in the blood are major regulators of D N A synthesis and mitotic activity in the bone marrow, liver and thymus of the rat 1-15. Calcium also positively controls the proliferation of non-tumorigenic epithelial and mesenchymally derived bovine, human and rodent cells in vitro 17-31. On the other hand, calcium has little or no influence on the proliferation of the corresponding tumorigenic cells 17-2°'22-24"25'27-3°, a very important fact which some day may prove to be the key to understanding neoplastic transformation. In this brief review there will be no detailed discussion of the inconclusive information on the still crudely localized cellular calcium fluxes related to cell proliferation. Instead, we will concentrate on the mounting evidence that calcium normally has nothing to do with the signal to start proliferative development, but does cooperate with that increasingly popular, * On Leave-of-Absencefrom the Medical Research Center, Polish Academy of Sciences,Warsaw. N.R.C.C. No. 17786.
many-talented trio of cyclic AMP (adenosine 3',5'-monophosphate), calmodulin (or CDR, a calcium-dependent regulatory protein) and protein kinase(s), to generate the later signal to initiate D N A synthesis. We will also speculate (heuristically, we hope!) on the reasons for calcium's mysterious inability to influence the proliferation of cancer cells.
Calcium's physiological uniqueness The calcium ion is certainly not an obvious choice for a regulator of such a complicated and important process as cell proliferation, because it is characterized only by its charge, coordination number, and unhydrated radius. However, it is just these properties which enable it, and only it, to activate certain ubiquitous multipurpose, calcium-binding proteins (e.g., calmodulin (CDR), troponin C) which regulate many different cellular functions 32"33. The importance of calcium in processes such as cell motility34, egg activation 35, muscle contraction a6, neurotransmission and secretion 37"38 has promted the evolutionary assignment of an entire hormone system and the expenditure of considerable amounts of energy on intracellular sequestration processes to regulate the extracellular and intracellular calcium ion concentrations. However, calcium must have even more regulatory functions because all cells work unceasingly to maintain a very steep electrochemical calcium gradient (from 10-3~ outside to 10 -7 or 10-SM in the cytosol) using appropriately sited CaZ+-transport ATPases to pump calcium ions out of the cell or into
Dr. I41..Junk b.v. Publishers - The Hague, The Netherlands
155
sequestration sites such as the cisternae of the endoplasmic reticulum 39-42. Because of this extraordinary steep gradient many different agents, the only common property of which is an ability to react with things on or in the cell membrane, cause calcium ions to squirt into the cell where they combine with the multipurpose calcium-dependent regulatory proteins. However, the nature of the response to this common calcium surge differs according to the cell's enzyme complement (i.e., its functional specialty). Calcium's special status as a regulator is emphasized by the fact that no hormones have been designed to regulate the blood level of magnesium, the other major divalent cation. Moreover, no cells struggle to produce and maintain a steep electrochemical gradient of magnesium ions 43, nor are there any multipurpose magnesium-dependent counterparts of the calcium-dependent calmodulin (CDR) 33. Nevertheless, as might be guessed from its constantly high intracellular level, magnesium is normally a permissive or function-maintaining (as opposed to function-initiating) factor needed by many enzymes including adenylate cyclase, Ca2+-transport ATPases and various protein kinases, which are three of the principal targets of the activating Ca-calmodulin (CDR) complexes produced during an intracellular calcium surge. Different though they may be, the functions of calcium and magnesium are obviously inextricably interwined because magnesium is needed by some enzymes after activation by calcium and some key magnesium-dependent enzymes do not function until activated by calcium. Therefore, magnesium deprivation can reduce or block various responses to an intracellular calcium surge.
The cell cycle Some cells, such as hepatocytes in the normal adult liver, neurones, adult parotid gland acinar cells and small peripheral blood lymphocytes contain reversibly or irreversibly repressed proliferogenic genes (i.e., certain regulatory genes and the structural genes coding for components of the proliferative machinery) and are engaged only in non-proliferative activities 12"44. We avoid a cell cycle (i.e., "G") designation such as Go for these cells simply because they are neither involved in a growth-division cycle nor enzymi156
cally equipped to do so. Their proliferative status must not be confused with that of other proliferatively quiescent cells such as serum- or calcium-deprived cells in vitro and certain stem cells in vitro, with still activated proliferogenic genes which are enzymically equipped to cycle and therefore merit the cycle designation "Go ''12'44. We believe that the stimulatory processes causing Go cells to resume cycling are different from the activating processes causing proliferatively inactivated cells to start cycling 1e'44, but some agents can trigger both of them. For example, the plant lectin concanavalin A proliferatively activates small peripheral blood lymphocytes and proliferatively stimulates already activated, but proliferatively quiescent, calcium-deprived late prereplicative R R thymic lymphocytes (vide infra). Proliferative activators somehow generate the first signal to start the production of the messenger R N A species responsible for the completely mysterious transitional processes 3°'44 which prepare the cell for entry into the prolfferatively committed G1 phase during which a different set of parallel and sequential reactions take over and bring the cell to a point where another signal is generated to start their entry into the S phase, the most vivid, but by no means the only, features of which are D N A synthesis and chromosome replication [Fig. 1]. After replicating its chromosomes, the cell enters a premitotic, G2, phase after which it progresses through the four stages of mitosis and finally divides [Fig. 1]. A daughter cell may become proliferatively inactivated and turn to other things, it may move directly to the threshold of the G1 phase (bypassing the now unnecessary transitional phase) and start another cycle, or it may lapse into a proliferatively quiescent Go state [Fig. 1] 12"3°'44. The composition of the cell's enzyme complement and surface components constantly changes as it progresses through the growthdivision cycle. Therefore, an intracellular surge of a regulator such as cyclic AMP at different points in the cell cycle will elicit entirely different responses. For example, a cyclic AMP surge in the G1 phase of the rat hepatocyte triggers D N A synthesis, but an earlier surge in the transitional phase cannot do so simply because the cell has not yet begun to make its DNA-synthetic enzymes (vide infra ).
RR (rapidly responding) thymic lyrnphocytes
Fig. 1. A highly symbolic depiction of the constantly changing (and mostly unknown) interdependent processes of the different phases of proliferative development. These phases are defined by DNA-synthetic and mitotic signposts and the inconspicuousness of the events in the gaps (G1, G2) between them. Everything starts when P, a proliferogenic agent or procedure (e.g., isoproterenol for salivary gland acinar cells, partial hepatectomy for hepatocytes, plant lectins for small lymphocytes) proliferatively activates the cell, thereby causing it to enter the transitional phase (Trans.)at point 1. Many things, such as proliferatively meaningless calcium and cyclic AMP surges, possibly proliferatively important changes in Na+/K + fluxes, and definitely proliferatively important increases in mRNA, ribosomal RNA and polyamine syntheses, happen during this transitonal phase of prereplicative development. The transitional processes bring the cell to point 2, the threshold of the G1 phase of prereplicative development. The G1 phase is very important for our story, because it includes the critical, DNA-synthetic process which (as indicated) is triggered by the combined efforts of calcium and a second, or Ol, cyclic AMP surge. The cell then replicates its chromosomes (as well as other more mysterious things) in the S phase. In the G2 phase it prepares itself for various mitotic tasks, such as chromosome condensation and massive microtubule redistribution, and it finally undergoes mitosis and cytokinesis. Each daughter cell may repress its proliferogenic genes to become proliferatively inactive; become proliferatively quiescent (Go) without being proliferatively inactivated; or immediately initiate another cycle from point 2 without having to go through another transitional phase. If any one of the interlinked processes of the G1 kaleidoscope should fail, the cell enters a O 0 state without inactivating its proliferogenic genes. The longer the cell stays in the Go state, the more it loses the accumulated components and systems needed for proliferation, and the longer it will take to reach the S phase when stimulated. In other words, the proliferative status of the cell regresses to that of a freshly activated cell at point 1.
T h e large l y m p h o c y t e p o p u l a t i o n in the t h y m u s gland of a 200 g male, S p r a g u e - D a w l e y rat is maintained by the rapid proliferation (total cycle times of 5 to 7 hours) of stem cells (lymphoblasts) w h o s e p r o g e n y b e c o m e smaller with each division b e c a u s e of an increasing repression of their nuclear activity (seen as c h r o m a t i n condensation) and a c o n s e q u e n t failure of their intermitotic growth to k e e p pace with their rapid multiplication 3'4s. In o t h e r words, these cells have a division cycle rather than a growth-division cycle. T h e e n d - p r o d u c t s of this i m m e n s e proliferative activity are the small proliferatively inactivated l y m p h o c y t e s which o c c u p y the bulk of the t h y m u s gland 45. A b o u t 10 to 15% of the cells in large, freshly isolated thymic l y m p h o c y t e populations susp e n d e d at a c o n c e n t r a t i o n of a b o u t 107 cells/ml of a serum-free, low (0.02 mM)-calcium tissue culture m e d i u m are actively cycling large and m e d i u m lymphoblasts as, and a b o u t 75% are proliferatively inactivated small lymphocytes. T h e remaining 10 to 15% (i.e., a b o u t 106 cells/ml) are proliferatively activated R R l y m p h o c y t e s which c a n n o t e n d o g e n o u s l y initiate D N A synthesis, but are enzymically e q u i p p e d to do so p r o m p t l y after exposure to any of several agents, one of which is the calcium ion. Thus, briefly o v e r w h e l m i n g the cells' calcium h o m e o static systems b y suddenly raising the ionic calcium concentration to 1.5 mM (and then leaving it elevated for as little as 5 minutes or as long as 5 hours) caused the small s u b p o p u l a tion of R R cells to initiate D N A synthesis (as indicated by an increase in nuclear-labelling with [3H]-thymidine or [6-3H]-deoxyuridine) between 45 and 60 minutes later [Fig. 2A], and t h e n to start entering mitosis 2 hours afterwards 46-s0. W e do not k n o w w h e t h e r the R R cells were b l o c k e d in the late G I phase or in the D N A synthetic S phase, but this in no w a y detracts f r o m the i m p o r t a n c e of the fact that calcium can trigger D N A synthesis. Exactly the same inv o l v e m e n t of calcium in D N A synthesis has b e e n o b s e r v e d in the equally rapidly r e s p o n d i n g C F U - S spleen c o l o n y - f o r m i n g cells in suspensions of m o u s e b o n e m a r r o w cells 51-53. Needless to say, the p r e c e n d e n t for all of this is calcium's triggering (i.e., activation) of processes in the fertilized egg which lead to the initiation of 157
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I$1 I II ~2+ 016=
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0 30 60 120 0 ~0 60 MINUTES AFTER INCREASINGTHE C~LCIUM CONCENTRATIONFROM 0.02 rnbl TO 1.5mM_
Fig. 2. Stimulations of cyclic AMP and DNA syntheses in freshly prepared 14's° thymic lymphocyte suspensions by raising the extracellular calcium concentration from 0.02 to 1.5 raM. A. The stimulation of cyclic AMP synthesis as indicated by increased production of [3HI-cyclic AMP from a pre-labeled intracellular [3H]-ATP pool was chromatographically determined according to MAcMANuS and WHrrFmLD46. There was no change in the level of [aH]-ATP during the period of observation which could have been a more trivial reason for the [3H]-cyclic A M P surge46. DNA-synthefic activity was autoradiographicaily determined by pulse (15-min.)-labeling of cell nuclei with [3H]thymidines°. B. The stimultaneous calcium-induced depression of the incorporation of radioactivity from [3H]thymidine into the cold acid-insoluble fraction (i.e., DNA) 7~. Each point is the mean ± S.E.M. of the values from 8 to 12 cell suspensions. Note: CPM means counts per min. in this and all other Figures.
[Fig. 3]. However, pretreatment with parathyroid hormone (which did not by itself stimulate thymic lymphocyte adenylate cyclase in broken cell preparations) enabled such small increases in the extracellular calcium concentration to stimulate both cyclic AMP synthesis and RR cell proliferation48. The calcium-induced cyclic AMP surge in the thymic lymphocyte suspensions was undetectable with other methods such as the competitive radioligand-binding assays which do not have the radiometric assay's ability to distinguish newly synthesized (i.e., labeled) cyclic AMP from "old" (i.e., unlabeled) cyclic AMP. However, as will be seen below, the binding assays easily detected the similar cyclic AMP surge which preceded the calcium-induced initiation of DNA synthesis in much more homogeneous populations of calcium-deprived rat liver cells [Figs 14 and 16]. Nevertheless, small though it was, the calcium-induced cyclic AMP surge in the thymic lymphocyte suspensions was necessary because its prevention or facilitation (by respectively stimulating or inhibiting cyclic 3',5'-
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