Calcium dependence of hormone-stimulated cAMP accumulation in intact glial tumor cells.

The Ca2+ content of glial tumor (C6) cells was reduced approximately 5-fold by repeated treatment with media containing ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) without loss of cellular viability. The ability of the cells to accumulate cAMP in response to beta-adrenergic agonists was reduced 60 to 70% following Ca2+ depletion. Ca2+ did not affect the apparent KACT for norepinephrine, nor did it change the concentration of propranolol required to produce 50% inhibition of the maximal norepinephrine response. Phentolamine did not alter the Ca2+ dependence of the response. The binding of dihydroalprenolol by intact C6 cells was not influenced by Ca2+. Furthermore, pretreatment with norepinephrine did not affect the Ca2+ dependence of cAMP accumulation. The effects of Ca2+, therefore, appeared to be exerted on components of the adenylate cyclase system other than the catecholamine receptor. Micromolar free Ca2+ concentration in the extracellular medium were sufficient to restore a maximal norepinephrine response to Ca2+-depeleted cells. The effect of Ca2+ on cAMP accumulation in response to hormone was immediate and was rapidly reversible upon the addition of EGTA in excess of the cation. Cells in media containing Ca2+ exhibited a characteristic biphasic time course of cAMP accumulation; with Ca2+-depleted cells cAMP was accumulated more slowly and the subsequent decline in cAMP content was also reduced. Verapamil, an inhibitor of plasmalemmal Ca2+ influx, decreased the Ca2+-dependent component of the cAMP accumulation when added prior to the cation. The effect of Ca2+ on cAMP accumulation was reduced more extensively by pretreatment of cells at 45 degrees C under Ca2+-depleted (80% loss) than under Ca2+-restored (30% loss) conditions. Trifluoperazine at micromolar concentrations decreased the Ca2+-dependent increment in accumulation of cAMP in Ca2+-restored cells. This inhibition was not overcome by increasing concentrations of norepinephrine or of extracellular Ca2+.

The Ca2+ content of glial tumor (C6) cells was reduced approximately B-fold by repeated treatment with media containing ethylene glycol bis(P-aminoethyl ether) iV,N'-tetraacetic acid (EGTA) without loss of cellular viability.
The ability of the cells to accumulate CAMP in response to /I-adrenergic agonists was reduced 60 to 70% following Ca2+ depletion. Ca2+ did not affect the apparent KACT for norepinephrine, nor did it change the concentration of propranolol required to produce 50% inhibition of the maximal norepinephrine response. Phentolamine did not alter the Ca2+ dependence of the response.
The binding of dihydroalprenolol by intact C6 cells was not influenced by Ca2+. Furthermore, pretreatment with norepinephrine did not affect the Ca2+ dependence of CAMP accumulation. The effects of Ca2+, therefore, appeared to be exerted on components of the adenylate cyclase system other than the catecholamine receptor.
Micromolar free Ca2+ concentrations in the extracellular medium were sufficient to restore a maximal norepinephrine response to Ca2'-depleted cells. The effect of Ca2+ on CAMP accumulation in response to hormone was immediate and was rapidly reversible upon the addition of EGTA in excess of the cation. Cells in media containing Ca2+ exhibited a characteristic biphasic time course of CAMP accumulation; with Ca2+-depleted cells CAMP was accumulated more slowly and the subsequent decline in CAMP content was also reduced.
Verapamil, an inhibitor of plasmalemmal Ca2+ influx, decreased the Ca2+-dependent component of the CAMP accumulation when added prior to the cation. The effect of Ca2' on CAMP accumulation was reduced more extensively by pretreatment of cells at 45°C under Ca2+depleted (80% loss) than under Ca2+-restored (30% loss) conditions. Trifluoperazine at micromolar concentrations decreased the Ca2+-dependent increment in accumulation of CAMP in Ca2+-restored cells. This inhibition was not overcome by increasing concentrations of norepinephrine or of extracellular Ca2+.
Calcium-dependent regulation of brain adenylate cyclase is now recognized to be mediated through a specific Ca'+-binding protein. The Ca2'-dependent regulator protein (CDR)' has * This work was supported by United States Public Health Service Grant NS 10975. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' The abbreviations used are: CDK, calcium-dependent. regulator; EGTA, ethylene glycol bis (P-aminoethyl ether) N,N,N',N'-tetraacetic acid; Tes, N-[Tris(hydroxymethyl)methyl-2-amino]ethanesulfonic acid. been shown to activate the enzyme from detergent-dispersed (1,2) as well as from particulate (3,4) preparations of cerebral cortex. The adenylate cyclase activity of washed, particulate preparations from brain is comprised of two contributing components, one of which requires CDR, the CDR-dependent activity, like other adenylate cyclase activities, is enhanced by NaF (3) and by GTP (5). Furthermore, choleragen activation of the detergent-dispersed brain enzyme requires CDR (6). However, the role of CDR in hormone receptor-adenylate cyclase coupling in neural tissue has not been defined.
Ca2+ has been implicated in the control of responses of intact cells to hormones. The rate of accumulation of CAMP in response to norepinephrine in rat cerebral cortex slices or in response to histamine in guinea pig cerebral cortex slices was reduced when extracellular Ca*' concentrations were lowered with EGTA (7). In rat brain slices, accumulation of CAMP elicited by a-adrenergic agonists depended completely on the presence of extracellular Ca2' whereas accumulation of CAMP with /3-adrenergic agents was influenced minimally by extracellular Ca'+ (8). Formation of CAMP in response to isoproterenol in rat parotid slices (9, lo), to epinephrine in adipocytes (ll), and to adrenocorticotropin in perfused adrenal glands (12) was reduced when these tissues were incubated in Ca"'-depleted media. In contrast, hepatocytes depleted of Ca2+ exhibited an enhanced response to a-adrenergic agonists (13), a result compatible with the hypothesis that Ca2+ exerts an inhibitory action on cu-adrenergic receptor-stimulated adenylate cyclase activity in this cell.
The C6 glial tumor cell responds to P-adrenergic agonists with loo-fold increases in CAMP (14) and has been used as a model for the study of the P-adrenergic receptor-adenylate cyclase system. /3-Adrenergic receptor binding sites have been characterized in this cell line (15)(16)(17). C6 cells have been shown to contain CDR (18), and the catecholamine-stimulated adenylate cyclase activity of particulate preparations washed with Ca" chelators was observed to be enhanced approximately 40% by Ca* ' and CDR (19). CDR was found to lower the Ca2+ concentration required for maximal stimulation of the enzyme, but an absolute requirement of the hormonedependent activity of cell-free preparations for Ca*' and CDR was not demonstrated.
Furthermore, evidence that Ca2+ or CDR regulates adenylate cyclase activity in uiuo is lacking. The present study was undertaken to assess the role of CaZC in the regulation of CAMP synthesis in response to norepinephrine in intact C6 cells. The results obtained are consistent with the hypothesis that intracellular Ca2+ is required for padrenergic receptor-stimulated CAMP formation in these cells. CDR is proposed to be the intracellular mediator of this Ca*'dependent process. ride, histamine dihydrochloride, 5-hydroxytryptamine hydrochloride,   (20). Experimental cultures were grown in glass roller bottles, 23 cm in length and 11 cm in diameter.
Cultures were seeded at a density of 3 X lo7 cells/bottle and were grown for 7 days to a density of 3 x 10" cells/bottle. Medium (140 ml) was replaced three times weekly and consisted of Hams F-10 with 10% fetal bovine serum, penicillin (25 units EGTA were Ca"+-depleted, the Ca2+ content of cells incubated in medium containing EGTA was compared with that of cells incubated in medium containing Ca"' in excess of EGTA. Cells were separated from their extracellular media by centrifugation and were analyzed for Ca"' content by atomic absorption spectrophotometry (Table IA). The contribution of extracellular Ca2' was estimated from the inulin space of the pellet and subtracted. When two independent preparations of Ca'+-depleted and Ca"-restored cells were examined, it was observed that the Ca2+ content of the Ca'+-depleted cells was 21% of that present in Ca'+-restored cells. It was presumed that extracellular bound Ca2' did not contribute significantly to these measurements since pretreatment with LaC13 (26) did not alter the values obtained.
Ca2'-depleted and Ca"+-restored cells were examined initially for CAMP and ATP content in the absence and presence of 10 pM norepinephrine (Table IB). The ATP content of the or Ca*' -restored cells were incubated in polypropylene tubes for 30 min. Cells were separated from their extracellular media by centrifugation at 600 x g for 3 min. After allowing the tubes to drain, the insides of the tube were wiped dry with tissue paper and the pellet of cells was suspended in Ca*'-free water. Cells were analyzed for Ca'+ content by atomic absorption spectrophotometry. To determine the contribution of extracellular bound Ca2+ to total Ca*+ content, 5 mM LaClR (26) was added to portions of each preparation of cells and the suspensions incubated an additional 15 min. The Ca*' content of the cells was not found to be altered by the LaCla treatment.
The Ca*' content of the extracelhrlar media was also determined by atomic absorption spectrophotometry and was found to be 3 mM for Ca'+-restored cells and 30 PM for Ca"-depleted cells. The contribution of extracellular Ca"+ was estimated from the inulin space of each cell pellet and subtracted.
B Ca'+ + norepinephrine 2940 f 220 21.0 + 0.8 cells was not altered by Ca2+-depletion or by hormone treatment. The CAMP content of unstimulated, Ca'+-depleted cells was not significantly different from that of unstimulated cells to which Ca" was restored. However, following a 20-min incubation with norepinephrine differences in the amounts of CAMP accumulated were obtained; the CAMP content of Ca'"+-restored cells was 2.5-fold greater than that of Ca"depleted cells. Control experiments were performed with cells in monolayer culture in Hams F-10 medium containing methylisobutylxanthine (not shown). The CAMP accumulated in these cells in response to 10 pM norepinephrine for 20 min was 4.5 f 0.5 nmol/mg of protein; ATP content was 22 f 2 nmol/ mg of protein regardless of the presence of norepinephrine.
Ca2+-dependent CAMP Accumulation in Response to Hormone-The C6 clone of glial tumor cells has been characterized as possessing a classical P-adrenergic receptor which is coupled to the adenylate cyclase of cells in Ca2+-containing medium (14). In Table II the abilities of various agonists to stimulate CAMP accumulation in Ca'+-depleted and Ca'+restored cells are compared. At a concentration of 10 pM, norepinephrine and isoproterenol were more effective agonists than phenylephrine or dopamine. The effects of phenylephrine and dopamine are presumed to be mediated by a fladrenergic receptor since they were fully reversed by 10 PM propranolol but not by 10 PM phentolamine (data not shown). CAMP was increased by these agents both in Ca"-depleted and Ca'+-restored cells, but the extent of CAMP accumulation in Ca'+-restored cells was generally 2.5-fold greater. Histamine and serotonin were ineffective. In agreement with previous observations (14, 27), prostaglandin E1 produced a small increase in CAMP content; this increase was not affected by Ca2+ depletion.  2B) were examined. A rapid increase in the CAMP content of Ca2+-restored cells was apparent during the fist 20 min of incubation with hormone; this increase was followed by a decline in CAMP content which continued throughout the remaining 2% h of incubation. Ca'+-depleted cells accumulated CAMP more slowly during the first 30 min of incubation, but the decline in CAMP content seen in Ca'+-restored cells was not as apparent for Ca'+-depleted cells. In fact, 3 h after hormone addition the CAMP content of Ca*'-depleted cells was statistically equal to or greater than that of Ca'+-restored cells. In contrast to the CAMP content of cells, the increase in CAMP content of the extracellular medium was slow, but linear with time for at least 2 h following norepinephrine treatment. Thus, at earlier time points, the CAMP content of cell suspensions is due primarily to the cyclic nucleotide inside the cells whereas at later time points the CAMP content of cell suspensions comes primarily from the extracellular medium. The CAMP content of medium from Ca'+-restored cells was greater than that of medium from Ca'+-depleted cells throughout the 3-h period following hormone addition. (Y-and P-adrenergic blocking agents have been reported to have opposing effects on CAMP formed in response to norepinephrine in adipocytes (28) and in cultured cells of dissociated perinatal mouse brain (29); /3-adrenergic blockers inhibit while a-adrenergic blockers potentiate the hormonal response. The a-receptor is postulated to be linked to a Ca2+-dependent process inhibitory to CAMP synthesis (13). Therefore adrenergic blocking agents were tested for their ability to influence CAMP accumulation in response to norepinephrine in Ca'+depleted and Ca'+-restored cells (Table III) (16), was measured in intact Ca'+-depleted and Ca2+-restored cells. As shown in Fig.  3, specific dihydroalprenolol binding was a saturable process as a function of ligand concentration while nonspecific binding increased linearly. At limiting dihydroalprenolol concentrations specific binding was the predominant form whereas nonspecific binding predominanted at saturating ligand concentrations. A dissociation constant for specific binding of 4 nM was obtained using either Ca2+-depleted or Ca*+-restored cells. Furthermore, specific binding at saturating dihydroalprenolol was not changed by Ca*+ depletion, and 1.8 x 10" receptors/cell were calculated to be present in both preparations. This value is in close agreement with that obtained by Lucas and Bockaert (16) for washed membrane preparations of C6 cells (1.0 x lo4 receptors/cell).
Nonspecific binding was similarly unaffected by Ca2+ depletion. The results therefore do not support the assertion that cells depleted of Ca2+ have fewer P-receptors.
C6 cells are known to exhibit a decreased responsiveness to catecholamines when the cells are pretreated for several hours with these hormones (31, 32). The development of tachyphylaxis results, at least in part, from a decrease in catecholaminestimulated adenylate cyclase activity (33). It was of interest to determine whether cells which had become subsensitive to norepinephrine retained a Ca2+ requirement for CAMP accumulation in response to hormone. One monolayer culture of cells was pretreated with norepinephrine for 3 h in Hams F-10 medium while a second monolayer culture was pretreated in medium alone. Following the pretreatment, suspensions of Ca2+-depleted and Ca*+-restored cells were prepared from each monolayer culture and amounts of CAMP accumulated in response to fresh norepinephrine were determined (Fig. 4) The time required for Ca2+ to enhance accumulation of CAMP when added to Ca"+-depleted cells was investigated (Fig. 6A). A suspension of Ca2+-depleted cells was divided in half and each half was treated with hormone. One-half was treated with Ca2+ at 4% min after addition of hormone. In the absence of external Ca2', CAMP was accumulated in a nonlinear fashion during the 16 min following hormone addition. Ca2+ produced an immediate increase in CAMP accumulation which was linear with time for approximately 10 min after Ca2+ addition. The ability of EGTA to reduce the Ca2'-dependent component of CAMP accumulation was also examined (Fig. 6B). A suspension of Ca2+-restored cells was divided into equal portions and each portion treated with norepinephrine. One portion of cells was treated with EGTA in excess of Ca2' at 4% min after norepinephrine, and CAMP accumulation in the two suspensions of cells was compared. Accumulation of the nucleotide in the presence of Ca2+ was linear with respect to time for at least 12 min following hormone addition. When EGTA was added in excess of Ca2', the extent of CAMP accumulation was immediately reduced to that of the Ca2+depleted cells.
It was desired to ascertain whether the Ca2+ necessary for CAMP accumulation in response to hormone was extracellular or intracellular.
Preliminary evidence was consistent with an intracellular Ca2+ requirement for the hormonal response. First, repeated washing of the cells in monolayer culture with EGTA-containing medium was necessary to obtain a cell preparation exhibiting a decreased rate of CAMP accumulation in response to norepinephrine.
Second, micromolar free --k-r CoCI, ( Ca2' concentrations, which are believed to exist in intracellular fluids (34), were suffkient for maximal accumulation of CAMP (Fig. 5). On the other hand EGTA rapidly abolished the Ca2+-dependent component of CAMP accumulation when added to suspensions of Ca'+-restored cells (Fig. 6B), an observation consistent either with an extracellular Ca" requirement or a requirement for an intracellular Ca2+ pool in rapid equilibrium with extracellular Ca2+. Further evidence for an intracellular Cazc requirement is presented in Fig. 7. C6 cells were treated with verapamil, an agent which inhibits Ca" influx into cells (35), either prior to or following the addition of various Ca2+ concentrations, and accumulation of CAMP was compared with that in untreated controls. Cells treated with verapamil after restoration with Ca2+ accumulated CAMP in response to norepinephrine to the same extent as did control cells which were not treated with drug. However, when verapamil was added prior to Ca2', accumulation of the nucleotide was decreased. This effect of verapamil was competitive with respect to Ca2+ concentration; at 10 mM CaC12 the inhibition was almost fully overcome. Verapamil had no effect on CAMP accumulation in the absence of Ca'+. The adenylate cyclase activity of cerebral cortex homogenates exists in two forms, one of which is stabilized by Ca2+ to thermal denaturation (36). When intact C6 cells were pretreated at 45°C for varying lengths of time in the absence or in the presence of Ca'+, two components of hormone-stimulated CAMP accumulation could also be distinguished (Fig. 8) one-half was made 3 mM in CaCL while the other half was untreated.
Samples of Ca'+-restored cells were also divided into equal portions; one half was treated with 5 mM EGTA while the other half was untreated.
All samples of cells were then restored to 37"C, incubated for 20 min, and challenged with 10 j&M norepinephrine.
Suspensions were incubated with the hormone for 20 min and CAMP content determined.
Cells pretreated at 45°C without Ca*+ (0); cells pretreated at 45°C with Ca*' (0). Panel A, cells treated with hormone at 37"C, EGTA in excess of Ca*+; Panel B, cells treated with hormone at 37"C, Ca" in excess of EGTA. by guest on March 24, 2020 http://www.jbc.org/ Downloaded from component accounted for 80% of the total. Thus, exposing the cells to high temperature with Ca2+ increased the Ca" dependency of the hormonal response by selectively inactivating the component of the process which did not depend on Ca2+. In contrast, thermal pretreatment in the absence of Ca2+ inactivated both components of the process equally.
Effects of Trifluoperazine on Norepinephrine-stimulated CAMP Accumulation in C6 Cells-Phenothiazines competitively inhibit the stimulation by CDR of brain adenylate cyclase (5). Calcium-dependent binding sites for these drugs on CDR have been described and evidence provided that the phenothiazine Ca'+. CDR complex is not an activating species (37). It was therefore of interest to determine whether phenothiazines inhibit Ca2+-dependent CAMP accumulation in intact cells. The effect of trifluoperazine concentration on the CAMP content of nonstimulated cells and of norepinephrinetreated cells is presented in Fig. 9. At concentrations up to 30 PM, trifluoperazine had no effect on the CAMP content of Ca2+-depleted or Ca2+-restored cells in the absence of hormone (Panel A) or on the CAMP content of Ca2+-depleted cells exposed to hormone (Panel B) . However, hormone-stimulated CAMP accumulation in Ca2+-restored cells was progressively inhibited by increasing drug concentrations; at 30 pM trifluoperazine, CAMP accumulation in these cells was reduced to a value similar to that of Ca2+-depleted cells. Ca2+-dependent CAMP accumulation was reduced 50% by 15 pM drug. Other workers have observed that phenothiazines inhibit dopamine-sensitive (38) or norepinephrine-sensitive (39) adenylate cyclase of brain in a manner competitive with catecholamine.
To ascertain that the trifluoperazine effect described above was not due to occupation of the fi-adrenergic receptor by the drug, the norepinephrine concentration dependence of CAMP accumulation was determined in the absence or presence of 15 pM trifluoperazine (Fig. 10) with hormone for its receptor and Ca2+-dependent inhibition of CAMP accumulation at limiting or at saturating hormone concentrations.
The two inhibitory actions of trifluoperazine in C6 cells are summarized and contrasted in Table IV   FIG. 11. Inhibition by trifluoperazine of hormone-dependent CAMP accumulation in C6 cells as a function of calcium concentration. Cells were incubated in medium containing the indicated Ca'+ concentrations for 30 min. The cells were then treated with saline (O), 15 PM trifluoperazine (A), or 30 pM trifluoperazine (a) for an additional 10 min. Norepinephrine (10 pM) was added to all cells, and CAMP content measured after 20 min of incubation.
accumulation was virtually abolished but the capacity of norepinephrine to saturate the receptor was unaffected. Increasing concentrations of Ca2+ were ineffective in reversing the inhibition of Ca'+-dependent CAMP accumulation by 15 or by 30 pM trifluoperazine (Fig. 11). Both concentrations of drug decreased hormone-sensitive CAMP accumulation at all Ca2+ concentrations tested with the exception of no added Ca2+.

DISCUSSION
The results presented in this report demonstrate that Ca" depletion alters the capacity of C6 cells to accumulate CAMP in response to /3-adrenergic agonists. The data are consistent with the concept that intracellular, as opposed to extracellular, Ca2+ is a requirement for maximal CAMP formation in these cells in response to hormone. For example, simple replacement of the extracellular growth medium with medium containing EGTA had no effect on the ability of cells in monolayer culture to accumulate CAMP. Rather, repeated washing of the cells in EGTA-containing medium was necessary to observe a decrease in the accumulation of CAMP. Paradoxically, when Ca2+-depleted cells were subsequently restored with Cazc, EGTA effectively removed the Ca2+-dependent component of CAMP accumulation suggesting an extracellular Ca2+ requirement (Fig. 6). It is possible, however, that cells which have been re-exposed to the cation are not fully Ca2+-restored, but have only certain Ca2+ pools repleted. If the pool of intracellular Ca2+ which is needed for CAMP accumulation were in rapid equilibrium with the pool of extracellular Ca2', then EGTA addition would be expected to deplete both of these Ca2+ pools. The finding that cells in monolayer culture with normal stores of Ca2+ require more extensive EGTA treatment to inhibit CAMP accumulation may reflect a sequential mobilization of a strongly sequestered intracellular pool(s) of Ca" to a free Ca2' pool which can then be depleted by EGTA. Without a knowledge of the Ca2+ contents of these intracellular pools for either cell preparation, it is difficult to substantiate or disprove such an argument. A second observation implying the involvement of intracellular Ca2+ was the restoration of full hormonal responsiveness at equimolar concen-trations of Ca2+ and EGTA (Fig. 5). If the KD for Ca2+ .EGTA at pH 7.5 is assumed to be 0.1 pM (40) Lastly, Ca2+-dependent CAMP accumulation could be inhibited by verapamil, an agent which reduces Ca2+ influx, when the drug was added prior to Ca2+ restoration (Fig. 7). The only recognized intracellular Ca2+ receptor which has a stimulatory effect on the synthesis of CAMP is CDR. Although the experiments presented here do not demonstrate a CDR involvement in Ca2'-dependent CAMP accumulation in intact C6 cells, several observations favor a role for CDR in this process. First, Ca2+ did not affect the concentration of the substrate for adenylate cyclase in these cells. Although an obligatory relationship is known to exist between Ca2+ accumulation and energy utilization at the level of the mitochondrial respiratory chain (41), the ATP contents of Ca2'-depleted and Ca'+-restored cells were similar (Table I). Second, no effects of Ca2+ were noted on the interaction of agonist or antagonist with the adrenergic receptor; Ca2+ altered neither the KAc~ for norepinephrine ( Fig. 1) nor the extent of inhibition by propranolol of the response to norepinephrine. Dihydroalprenolol binding was unaffected by the absence or presence of Ca2+ (Fig. 3). Furthermore the development of preceptor subsensitivity did not change the Ca2+ requirement for CAMP accumulation (Fig. 4). Third, CAMP accumulation in the presence of Ca2+ was inhibited by the drug trifluoperazine. This observation directly implicates CDR since phenothiazines have been demonstrated (a) to inhibit competitively the CDR-dependent activation of brain adenylate cyclase (5) and cyclic nucleotide phosphodiesterase (42,43) and (b) to bind in Ca2+-dependent manner to specific sites on CDR (37) creating an apparently ineffective species. The specific effect of the drug on Ca2+-dependent CAMP accumulation was not attributed to competition with norepinephrine for its receptor because inhibitions by the drug of the Ca2'-dependent component of the activity were obtained at saturating concentrations of hormone (Fig. 10, Table IV). Concentrations of trifluoperazine which inhibited Ca'+-dependent CAMP accumulation were in the micromolar range ( Fig. 9), in agreement with those reported to be required for binding to CDR in vitro (37). Increasing concentrations of Ca2+ did not overcome the trifluoperazine inhibition of Ca2+-dependent CAMP accumulation (Fig. 11). Similarly, inhibition of CDR-dependent phosphodiesterase (43) or adenylate cyclase3 by phenothiazines in vitro is not overcome by Ca'+; the inhibition is reversed solely by CDR. A fourth observation consistent with a CDR involvement was the relative stability of the Ca2+dependent process of CAMP accumulation when cells were exposed to elevated temperature in the presence as compared to the absence of Ca2+ (Fig. 8). A similar observation has been made in brain homogenates; Ca2+ selectively stabilized to thermal denaturation the component of adenylate cyclase which depended on Ca2+ for activity (5). The stabilizing factor was identified to be CDR. The Ca2+-dependent form of brain adenylate cyclase in washed, particulate preparations from which CDR had been removed was protected by Ca2++CDR, but not by Ca2+ alone (3). A final piece of evidence supportive of a CDR involvement is that adenylate cyclase of cell-free preparations of C6 cells is stimulated to some extent by Ca2+ and CDR (19). Although CDR was not fully removed from and was not demonstrated to be a requirement of the enzyme, the addition of CDR served to increase the sensitivity of the enzyme to Ca2+. As was seen for CAMP accumulation in intact C6 cells, the adenylate cyclase activity of particulate fractions of these cells was influenced by micromolar free Ca" concentrations. The KAcT for norepinephrine was unaltered by Ca2+ in either system or by CDR itself in the cell-free system.
Investigations of the adenylate cyclase activity of homogenates of rat cerebral cortex revealed two contributing components, only one of which required CDR for activity (3). Although the CDR-requiring component from cerebral cortex is not known to be activated by hormones, it has several properties in common with Ca"-dependent CAMP accumulation in response to hormone in intact C6 cells. Both processes require micromolar free Ca*+ concentrations for optimal activity, are more stable to elevated temperatures in the presence of Ca2+, are inhibited by phenothiazines, and represent 65 to 80% of the total activity of the system. The basal adenylate cyclase activity of brain homogenates which did not depend on Ca'+ shared several features with CAMP accumulation in Ca"-depleted C6 cells. Each of these activities represented 20 to 35% of the total activity, was inactivated equally well at elevated temperatures in the absence or presence of Cap+, and was unaffected by phenothiazines at concentrations which inhibit the Ca'+-dependent processes. While it is clear that two components of the process of CAMP synthesis can be recognized in vivo as well as in vitro, the relationships between the Ca'+-dependent and Ca2+-independent processes are at this time unclear.
The CAMP content of Ca2+-restored cells was observed to increase rapidly during the first 20 min after norepinephrine addition and then to decline at a constant rate for approximately 90 min. The decline phase, which was noted for the CAMP content of cells but not for the CAMP content of the extracellular medium, was almost completely eliminated by Ca"+ depletion (Fig. 2). Since C6 cells are known to contain a Ca"+-dependent phosphodiesterase activity (18), it is attractive to speculate that this enzyme may be responsible for the decline in CAMP content of cells seen during longer incubations with hormone and that depletion of cellular Cast interferes with this activity. Such an interpretation should be viewed cautiously, however, in light of the complexity of the known adaptive changes in CAMP metabolism in C6 cells following prolonged hormonal exposure (31,33).
Participation of Ca" in the hormonal responses of other intact cell systems has been reported but important differences exist between the requirements of these systems and those of the C6 cell system. For example, formation of CAMP in brain slices in response to norepinephrine or histamine is dependent on Ca2+ (7). However, the source of the Ca2+ required for these responses is believed to be extracellular since EGTA added immediately before hormone to minimize disturbances of the intracellular Ca2' pools was found to reduce the rate of CAMP accumulation.
Furthermore, millimolar concentrations of Ca2+ as are ordinarily found in extracellular fluids were required to achieve maximal rates of CAMP synthesis. Norepinephrine-stimulated, Ca2+-dependent CAMP accumulation in brain slices was also shown to require the presence of adenosine. Yet neither adenosine nor high concentrations of adenosine deaminase had any effect on &?-dependent CAMP accumulation in C6 cells? Another major difference between the two intact cell systems is that the Ca"+ requirement for norepinephrine-stimulated CAMP formation in brain slices was mediated primarily by an (Yadrenergic receptor; Ca2+ effects on ,&receptor-stimulated CAMP accumulation were marginal (8). Since the C6 cell is a glial tumor cell, it will be of interest to determine whether the glial or neuronal elements of the brain slice preparation possess the Ca'+-dependent response. Rat parotid slices (10) and adipocytes (11) have been shown to accumulate more CAMP in response to catecholamines when Ca2+ is present than when it is absent from extracellular fluids. In the parotid system the Ca2+ effect was shown to be mediated by a /3-adrenergic receptor but the results with both parotid slices and adipocytes were consistent with an extracellular rather than an intracellular Ca2+ requirement. A third system in which Ca2' depletion has been clearly demonstrated to alter hormonestimulated CAMP accumulation is that of isolated hepatocytes. In contrast to the systems discussed above, Ca2'-depletion increases CAMP accumulation when these cells are treated with epinephrine or phenylephrine (13). The enhanced CAMP accumulation in Ca'+-depleted cells was blocked by several a-adrenergic antagonists but not by propranolol indicating that it was mediated by an cr-adrenergic receptor. Micromolar free Ca2+ concentrations were shown to inhibit CAMP accumulation in response to epinephrine suggesting that intracellular Ca'+ is responsible for the inhibitory control mechanism.
In view of the results presented in this report with adrenergic agonists and antagonists (Tables II and III), the receptor which mediates CAMP accumulation in Ca2+-depleted or Ca2'restored C6 cells is a PI-adrenergic receptor. No evidence has yet been provided by this or other laboratories that a-adrenergic receptors are linked in any manner to cyclic nucleotide metabolism in this cell line. On the other hand, cy-adrenergic antagonists facilitate CAMP accumulation in response to /Iadrenergic agonists in adipocytes (28) and cultured mouse brain cells (29) suggesting that the two types of adrenergic receptors may exert opposing regulatory controls in these systems. Since intracellular Ca2+ inhibits a-receptor-mediated CAMP accumulation in hepatocytes and facilitates P-receptormediated CAMP accumulation in C6 cells, it will be of considerable importance to an understanding of the relationships between Ca"+ and cyclic nucleotide metabolism to determine whether both Ca'+-dependent effects can be operative within the same cell or within different cells of a single tissue. In isolated hepatocytes phenylephrine and epinephrine, but not isoproterenol, produced a Ca2+ efflux of considerable magnitude (44) suggesting that a-adrenergic agonists promote increases in free intracellular Ca2' concentrations through mobilization of intracellular Ca2' pools resulting in inhibition of adenylate cyclase activity. Although the adenylate cyclase activity of C6 cell homogenates is stimulated by micromolar but inhibited by higher Ca"+ concentrations (19), no such biphasic effect of Ca2+ concentration was observed on /3-receptor-mediated CAMP accumulation in intact cells. It will be critical to determine whether a /I-adrenergic agonist can produce alterations in Ca2+ compartments within cells such as C6 thereby controlling rates of CAMP synthesis or whether Ca2+ is simply permissive for CAMP formation in response to this hormone.