On the role of extracellular Ca2+ for prolactin release and adenosine 3′:5′-monophosphate formation induced by thyroliberin in cultured rat pituitary cells

In cultured rat pituitary tumour cells (GH₃ cells) the absence of extracellular Ca⁺⁺ or addition of NaEGTA reduced spontaneous prolactin (PRL) release and abolished the stimulatory effect of thyroliberin (TRH). Readdition of CaCl₂, but not of equimolar concentrations of MgCl₂ increased spontaneous hormone release, and restored the effect of TRH. The calcium ionophore, A-23187, induced PRL release during normal calcium conditions, but not when an excess NaEGTA was present. TRH increased cyclic AMP accumulation in the presence and the absence of extracellular calcium. The effect of TRH on PRL release and cyclic AMP formation occured concomitantly with an increased efflux of ⁴⁵Ca²⁺. Intracellular electrophysiological recordings from the same single cells before and after TRH activation showed increased frequency and duration of the Ca²⁺ dependent action potentials. We conclude that TRH elevates the Ca²⁺ influx which depends on the depolarizing action current, and this effect is probably linked to formation of cyclic AMP and PRL release.     

Dissociation between Ca2+-ATPase and alkaline phosphatase activities in plasma membranes of rat duodenum

The presence of Ca²⁺-ATPase activities with high-affinity sites for Ca²⁺ in brush border as well as basolateral plasma membranes of rat duodenal epithelium has been reported previously (Ghijsen, W.E.J.M. and van Os, C.H. (1979) Nature 279, 802–803). Since both plasma membranes contain alkaline phosphatase (EC 3.1.3.1), which also can be stimulated by Ca²⁺, the substrate specificity of Ca²⁺-induced ATP-hydrolysis has been studied to determine whether or not alkaline phosphatase and Ca²⁺-ATPase are two distinct enzymes. In basolateral fragments, the rate of Ca²⁺-dependent ATP-hydrolysis was greater than that of ADP, AMP and $\text{p-}\text{nitrophenylphosphate}$ at Ca²⁺ concentrations below 25 μM. At 0.2 mM Ca²⁺ the rates of ATP, ADP, AMP and $\text{p-}\text{nitrophenylphosphate}$ hydrolysis were not significantly different. In brush border fragments the rates of ATP, ADP and AMP hydrolysis were identical at low Ca²⁺, but at 0.2 mM Ca²⁺, Ca²⁺-induced hydrolysis of ADP and AMP was greater than either ATP or $\text{p-}\text{nitrophenylphosphate}$. Alkaline phosphatase in brush border and basolateral membranes was inhibited by 75% after addition of 2.5 mM theophylline. Ca²⁺-stimulated ATP hydrolysis at 1 μM Ca²⁺ was not sensitive to theophylline in basolateral fragments while the same activity in brush border fragments was totally inhibited. At 0.2 mM Ca²⁺, Ca²⁺-induced ATP hydrolysis in both basolateral and brush border membranes was sensitive to theophylline. Oligomycin and azide had no effect on Ca²⁺-stimulated ATP hydrolysis, either at low or at high Ca²⁺ concentrations. Chlorpromazine fully inhibited Ca²⁺-stimulated ATP hydrolysis in basolateral fragments at 5 μM Ca²⁺, while it had no effect in brush border fragments. From these results we conclude that, (i) Ca²⁺-ATPase and alkaline phosphatase are two distinct enzymes, (ii) high-affinity Ca²⁺-ATPase is exclusively located in basolateral plasma membranes, (iii) alkaline phosphatase activity, present on both sides of duodenal epithelium, is stimulated slightly by low Ca²⁺ concentrations, but this Ca²⁺-induced activity is inhibited by theophylline and shows no specificity with respect to ATP, ADP or AMP.     

Control of Ca2+ efflux and cyclic AMP by 5-hydroxytryptamine and dopamine in abalone gill.

5-Hydroxytrptamine increased the rate of Ca²⁺ efflux and the concentration of endogenous cyclic AMP in abalone gill in both 10 mM and 50 mM CaCl₂ concentrations externally. Dopamine decreased the rate of Ca²⁺ efflux in 50 mM CaCl₂ but slightly increased the efflux rate in 10 mM CaCl₂. At both external Ca²⁺ concentrations, dopamine increased the endogenous cyclic AMP concentration in the gill. 5-Hydroxytryptamine but not dopamine was found to activate adenylate cyclase in broken cell preparations of abalone gill. Cyclic AMP-dependent protein kinase activity was also demonstrated in homogenate fractions of abalone gill. It is suggested that both Ca²⁺ and cyclic AMP act as second messengers in the response of abalone gill to 5-hydroxytryptamine and dopamine.     

Rearrangement of intramembranous particles and fusion promoted in chicken erythrocytes by intracellular Ca2+

Ca²⁺ was introduced into fresh and ATP-depleted chicken erythrocytes through the aid of the ionophore A-23187.Intracellular Ca²⁺ (10–40 mM) induced fusion in ATP-depleted cells after 30–60 min incubation at 37°C, but not in fresh cells. Fresh cells underwent a higher degree of haemolysis than ATP-depleted cells after accumulation of Ca²⁺. Uptake of Ca²⁺ was the same in these two systems.Intracellular Ca²⁺ induced rearrangement of intramembranous particles, as revealed by freeze-etching studies. The intramembranous particles in the protoplasmic face of fractured membranes obtained from fresh cells incubated with 1 mM of Ca²⁺ were more scattered and their density was lower than in control cells. Incubation with higher concentrations of Ca²⁺ (10–40 mM) induced transient changes in the intramembranous particles' density with the appearance of protrusions and depressions on the protoplasmic and exoplasmic faces of the fractured membranes, respectively. These effects were reversible upon removal of Ca²⁺ by washing the cells with ethyleneglycol $\text{bis}\text{(α-}\text{aminoethylether}\text{)-N,N′-}\text{tetraacetic}$ acid; rearrangement of intramembranous particles was less evident after accumulation of Ca²⁺ in ATP-depleted cells, whose fractured membranes did not contain any protrusions or depressions.Transferring Ca²⁺-loaded cells to the cold caused the formation of large smooth areas devoid of intramembranous particles in the protoplasmic face of the fractured membranes.Cells containing Ca²⁺ appeared spherical, and removal of Ca²⁺ restored the normal oval shape of chicken erythrocytes.     

Signal transduction and motility ofDictyostelium

This review is concerned with the roles of cyclic GMP and Ca²⁺ ions in signal transduction for chemotaxis ofDictyostelium. These molecules are involved in signalling between the cell surface cyclic AMP receptors and cytoskeletal myosin II involved in chemotactic cell movement. Evidence is presented for uptake and/or eflux of Ca²⁺ being regulated by cyclic GMP. The link between Ca²⁺, cyclic GMP and chemotactic cell movement has been explored using streamer F mutants whose primary defect is in the structural gene for the cyclic GMP-specific phosphodiesterase. This mutation causes the mutants to produce an abnormally prolonged peak of cyclic GMP accumulation in response to stimulation with the chemoattractant cyclic AMP. The production and relay of cyclic AMP signals is normal in these mutants, but certain events associated with movement are (like the cyclic GMP response) abnormally prolonged in the mutants. These events include Ca²⁺ uptake, myosin II association with the cytoskeleton and regulation of both myosin heavy and light chain phosphorylation. These changes can be correlated with changes in the shape of the amoebae after chemotactic stimulation. Other mutants in which the accumulation of cyclic GMP in response to cyclic AMP stimulation was absent produced no myosin II responses.A model is described in which cyclic GMP (directly or indirectly via Ca²⁺) regulates accumulation of myosin II on the cytoskeleton by regulating phosphorylation of the myosin heavy and light chain kinases.     

Lanthanum stimulates the accumulation of cyclic AMP and inhibits secretion and thromboxane B2 formation in human platelets

La³⁺ was found to inhibit the secretion of 5-hydroxytryptamine and the production of thromboxane B₂ by washed platelets exposed to collagen or thrombin. In addition, La³⁺ inhibited secretion in response to sodium arachidonate, although the conversion of arachidonate to thromboxane B₂ was not affected.La³⁺ was also found to enhance the accumulation of cyclic AMP under basal conditions and in response to prostaglandin E₁, in washed platelets. The inhibition of cyclic AMP accumulation by ADP was prevented by La³⁺, suggesting that the effect of ADP on cyclic AMP metabolism was dependent upon the presence or flux of calcium at the platelet membrane.La³⁺ inhibited the activity of adenylate cyclase in platelet lysates both in response to prostaglandin E₁ and to F^?, indicating a possible effect at the catalytic subunit of the enzyme. None of the observed effects of La³⁺ could be reversed by the addition of Ca²⁺ up to 10 mM. The stimulation of cyclic AMP production by La³⁺ may largely explain the inhibitory effect of La³⁺ upon platelet secretion and thromboxane B₂ production. These results also suggest that Ca²⁺ localised at the platelet plasma membrane may be important in the regulation of cyclic AMP metabolism.     

Hormone-induced changes in pyruvate kinase activity in isolated hepatocytes II. Relation to the hormonal regulation of gluconeogenesis gluconeogenesis

1. 1. Incubation of isolated hepatocytes with glucagon (10⁻⁶ M) or dibutyryl cyclic AMP (0.1 mM) causes a decrease in pyruvate kinase activity of 50%, measured at suboptimal substrate (phosphoenolpyruvate) concentrations and 1 mM Mg_free²⁺. The magnitude of the decrease in activity is not influenced by the applied extracellular concentrations of lactate (1 and 5 mM), glucose (5 and 30 mM) or fructose (10 and 25 mM). With all three substrates comparable inhibition percentages are induced by glucagon or dibutyryl cyclic AMP.

2. 2. The extent of inhibition of pyruvate kinase induced by incubation of hepatocytes with glucagon or dibutytyl cyclic AMP is not influenced by the extracellular Ca²⁺ concentration nor by the presence of 2 mM EGTA. The reactivation of pyruvate kinase seems to be inhibited by a high concentration of extracellular Ca²⁺ (2.6 mM) as compared to a low concentration of extracellular Ca²⁺ (0.26 mM).

3. 3. Incubation of hepatocytes in a Na⁺-free, high K⁺-concentration medium does not influence the magnitude of the pyruvate kinase inhibition induced by dibutyryl cyclic AMP. However, the reactivation reaction is stimulated under these incubation conditions.

4. 4. Incubation of hepatocytes with dibutyryl cyclic GMP (0.1 mM) leads to a 25% decrease in pyruvate kinase activity. The magnitude of the inhibition by dibutyryl cyclic (GMP) is not influenced by the presence of pyruvate (1 mM) or glucose (5 mM and 30 mM).

5. 5. The relative insensitivity of the pyruvate kinase inhibition induced by glucagon, dibutyryl cyclic AMP and dibutyryl cyclic GMP to the extracellular environment leads to the conclusion that the hormonal regulation of pyruvate kinase is not the only site of hormonal regulation of glycolysis and gluconeogenesis. It is concluded that hormonal regulation of pyruvate kinase activity is exerted by changes in the degree of (de)phosphorylation of the enzyme reflecting acute hormonal control as well as by changes in the concentration of the allosteric activator fructose 1,6-diphosphate. The latter depends at least in part on the hormonal control of the phosphofructokinase-fructose-1,6-phosphatase cycle.

Calcium and pancreatic β-cell function I. Stimulatory effects of pentobarbital on insulin release

Islets microdissected from ob/ob-mice were exposed to 3mM pentobarbital in media which were normal or deficient in Ca²⁺. This treatment resulted in a marked decrease of the islet content of cyclic AMP recorded in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Pentobarbital had a dual effect on insulin release. In addition to being a potent inhibitor of glucose-stimulated insulin release in media containing 2.56 mM Ca²⁺ it increased the amounts of insulin released in high glucose media deficient in Ca²⁺. There was a transient stimulation with ordinary concentrations of Ca²⁺ and 3 mM glucose when the media also contained 3-isobutyl-1-methylxanthine. The stimulatory effect of pentobarbital persisted after replacing part of the Ca²⁺ in the β-cell membrane with lanthanum ions and it could not be mimicked by lowering the oxygen tension of the incubation medium. It is suggested that pentobarbital stimulation of insulin release is the result of a specific action of the drug on the distribution of Ca²⁺ within the pancreatic β-cells.     

Protein kinase and cyclic AMP levels in differentiating myoblasts are altered by extracellular calcium

Protein kinase specific activities and cyclic AMP levels show a similar pattern of response, when the Ca²⁺ concentration is altered in the culture medium of differentiating chick skeletal muscle cells; an increase at intermediate Ca²⁺ concentrations (0.05–0.2mM), followed by a decrease at higher concentrations (2mM). Effects of Ca²⁺ on protein kinase appear to be on cyclic AMP-independent enzymes in both nucleus and cytoplasm, and are quite the reverse of Ca²⁺ effects on the muscle-specific enzyme, creatine kinase.     

On the role of calcium as second messenger in liver for the hormonally induced activation of glycogen phosphorylase

We have studied the mode of action of three hormones (angiotensin, vasopressin and phenylephrine, an α-adrenergic agent) which promote liver glycogenolysis in a cyclic AMP-independent way, in comparison with that of glucagon, which is known to act essentially via cyclic AMP. The following observations were made using isolated rat hepatocytes: (a) In the normal Krebs-Henseleit bicarbonate medium, the hormones activated glycogen phosphorylase (EC 2.4.1.1) to about the same degree. In contrast to glucagon, the cyclic AMP-independent hormones did not activate either protein kinase (EC 2.7.1.37) or phosphorylase $\text{b}$ kinase (EC 2.7.1.38). (b) The absence of Ca²⁺ from the incubation medium prevented the activation of glycogen phosphorylase by the cyclic AMP-independent agents and slowed down that induced by glucagon. (c) The ionophore A 23187 produced the same degree of activation of glycogen phosphorylase, provided that Ca²⁺ was present in the incubation medium (d) Glucagon, cyclic AMP and three cyclic AMP-independent hormones caused an enhanced uptake of ⁴⁵Ca; it was verified that concentrations of angiotensin and of vasopressin known to occur in haemorrhagic conditions were able to produce phosphorylase activation and stimulate ⁴⁵Ca uptake. (e) Appropriate antagonists (i.e. phentolamine against phenylephrine and an angiotensin analogue against angiotensin) prevented both the enhanced ⁴⁵Ca uptake and the phosphorylase activation.We interpret our data in favour of a role of calcium (1) as the second messenger in liver for the three cyclic AMP-independent glycogenolytic hormones and (2) as an additional messenger for glucagon which, via cyclic AMP, will make calcium available to the cytoplasm either from extracellular or from intracellular pools. The target enzyme for Ca²⁺ is most probably phosphorylase $\text{b}$ kinase.     

Calmodulin-like activity in the soluble fraction of Escherichia coli

A heat-stable factor with properties similar to those of calmodulin was found in the fraction containing Ca²⁺-dependent cyclic AMP phosphodiesterase of $\text{Escherichia}\text{coli}$. The factor activated such enzymes as cyclic nucleotide phosphodiesterase of bovine brain, (Ca²⁺,Mg²⁺)ATPase of human erythrocyte menbrane and myosin light chain kinase of rabbit myometrium in a Ca²⁺-dependent fashion with an apparent K_a of 5 × 10^?5$\text{M}$. The factor and brain calmodulin had no effect on the phosphodiesterase of $\text{E.}\text{coli}$. It may be concluded that calmodulin or a calmodulin-like protein occurs in prokaryotes.     

Calcium,cyclic GMP and the control of myosin II during chemotactic signal transduction ofDictyostelium

Evidence is presented for Ca²⁺ and cyclic GMP being involved in signal transduction between the cell surface cyclic AMP receptors and cytoskeletal myosin II involved in chemotactic cell movement. Ca²⁺ is shown to be required for chemotactic aggregation of amoebae. The evidence for uptake and/or eflux of this ion being regulated by the nucleotide cyclic GMP is discussed. The connection between Ca²⁺, cyclic GMP and chemotactic cell movement has been explored using “streamer F” mutants. The primary defect in these mutants is in the structural gene for the cyclic GMP-specific phosphodiesterase which results in the mutants producing an abnormally prolonged peak of accumulation of cyclic GMP in response to stimulation with the chernoattractant cyclic AMP. While events associated with production and relay of cyclic AMP signals are normal, certain events associated with movement are (like the cyclic GMP response) abnormally prolonged in the mutants. These events include Ca²⁺ uptake, myosin II association with the cytoskeleton and inhibition of myosin heavy and light chain phosphorylation. These changes can be correlated with the amoebae becoming elongated and transiently decreasing their locomotive speed after chemotactic stimulation. Other mutants studied in which the accumulation of cyclic GMP in response to cyclic AMP stimulation was absent produced no myosin II responses. Models are described in which cyclic GMP (directly or indirectly via Ca²⁺) regulates accumulation of myosin II on the cytoskeleton by inhibiting phosphorylation of the myosin heavy and light chain kinases.     

INFLUENCE OF DIVALENT CATIONS ON REGULATION OF CYCLIC GMP AND CYCLIC AMP LEVELS IN BRAIN TISSUE

—Depolarizing concentrations of K⁺ elevate levels of both adenosine 3′,5′monophosphate (cyclic AMP) and guanosine 3′,5′monophosphate (cyclic GMP) in incubated slices of mouse cerebellum. Calcium is an essential requirement for the K⁺ -induced accumulation of cyclic GMP. Barium and Sr²⁺, but not Mn²⁺ or Co²⁺, can substitute for Ca²⁺ in this process. Relatively high concentrations of Mg²⁺ inhibit the effect of Ca²⁺ on K⁺-induced accumulation of cyclic GMP. In contrast, depolarizing concentrations of K⁺ are capable of elevating cyclic AMP levels in brain slices suspended in media containing Mg²⁺ and no other divalent cations. High concentrations of Ca²⁺ (1 mm or greater) augment this Mg²⁺ -dependent, K⁺-induced accumulation of cyclic AMP, however. Strontium and Mn²⁺, but not Ba²⁺ or Co²⁺, can substitute for Ca²⁺ in this process, and high concentrations of Mg²⁺ are not inhibitory. The divalent cation ionophore, A-23187 (10 μm ), in the presence of extracellular Ca²⁺ elevates the level of cyclic GMP, but not cyclic AMP, in incubated mouse cerebellum slices. The results of this study indicate that intracellular Ca²⁺ concentration is a major factor regulating cyclic GMP levels in brain. In addition the present results suggest that, in brain tissue, depolarization-induced accumulation of cyclic GMP, but not cyclic AMP, is closely linked to some Ca²⁺-dependent mechanism(s) mediating release of intracellular substances.     

Amylase release from rat parotid glands II. Calcium kinetics

The kinetics of ⁴⁵Ca²⁺ uptake, efflux, and calcium potentiation of amylase release by slices of rat parotid glands were examined. Pretreatment of the tissue with 11.25 mM ⁴⁵Ca²⁺ medium increased the total tissue ⁴⁵calcium content. Lanthanum (1 mM) decreased tissue uptake, blocked the slow components of exchange and appeared to inhibit transcellular calcium movement. Neither dibutyryl cyclic AMP nor caffeine caused consistently significant effects on ⁴⁵Ca²⁺ kinetics, or total ⁴⁵calcium content. Carbamylcholine increased the initial rate of ⁴⁵Ca²⁺ uptake, but had no effect on total uptake.Elevation of the extracellular Ca²⁺ concentration to 11.25 mM during stimulation of amylase release resulted in an initial decrease in the rate of amylase release followed by a potentiation of release which developed slowly, requiring 40–50 min to reach the maximal response.The inability to detect release-related changes in either calcium influx or mobilization, and the lengthy times and high Ca²⁺ concentrations required to achieve calcium potentiation suggests that calcium does not couple amylase release.     

Adrenocorticotropic hormone and dibutyryl adenosine cyclic monophosphate-mediated Ca uptake by rat adrenal glands

The effect of adrenocorticotropic hormone and dibutyryl cyclic AMP on the uptake of⁴⁵Ca²⁺ by the rat adrenal gland has been investigated. After injection of ⁴⁵Ca²⁺ and adrenocorticotropic hormone into rats, the adrenal ⁴⁵Ca²⁺ concentration was significantly enhanced 90 to 180 min following hormone administration. The rise in adrenal ⁴⁵Ca²⁺ content was accompanied by a marked increase of the serum corticosterone levels. During incubation of rat adrenal glands in the presence of ⁴⁵Ca²⁺, adrenocorticotropic hormone and dibutyryl cyclic AMP caused significant accumulation of adrenal ⁴⁵Ca²⁺ and increased corticosterone synthesis. The degree of stimulation of both adrenal ⁴⁵Ca²⁺ uptake and corticosterone synthesis by adrenocorticotropic hormone or dibutyryl cyclic AMP was dependent upon the concentration of calcium in the incubation medium and upon the amount of adrenocorticotropic hormone or dibutyryl cyclic AMP added. Theophylline mimicked the stimulatory effect of adrenocorticotropic hormone and dibutyryl cyclic AMP and increased the uptake of ⁴⁵Ca²⁺ by rat adrenal glands in vitro. Determination of calcium by atomic absorption spectroscopy showed that the adrenocorticotropic hormone-mediated adrenal ⁴⁵Ca²⁺ uptake was due to a net accumulation of calcium in the tissue and not only to an increased rate of exchange of extracellular ⁴⁵Ca²⁺ with the intracellular calcium pool. Adrenocorticotropic hormone-stimulated adrenal ⁴⁵Ca²⁺ uptake was not observed when steroidogenesis was inhibited with elipten. Both adrenocorticotropic hormone-mediated corticosterone synthesis and adrenal ⁴⁵Ca²⁺ uptake were abolished after treatment of rats with cycloheximide but not after treatment with actinomycin D, indicating that adrenal ⁴⁵Ca²⁺ uptake and steroidogenesis have similar requirements for de novo protein synthesis, but not RNA synthesis.     

The activation by Ca2+ of platelet phospholipase A2: Effects of dibutyryl cyclic adenosine monophosphate and 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate

Thrombin-induced release of arachidonic acid from human platelet phosphatidylcholine is found to be more than 90% impaired by incubation of platelets with 1 mM dibutyryl cyclic adenosine monophosphate (Bt₂ cyclic AMP) or with 0.6 mM 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8), an intracellular calcium antagonist. Incorporation of arachidonic acid into platelet phospholipids is not enhanced by Bt₂ cyclic AMP. The addition of external Ca²⁺ to thrombin-treated platelets incubated with Bt₂ cyclic AMP or TMB-8 does not counteract the observed inhibition. However, when divalent cation ionophore A23187 is employed as an activating agent, much less inhibition is produced by Bt₂ cyclic AMP or TMB-8. The inhibition which does result can be overcome by added Ca²⁺. Inhibition of arachidonic acid liberation by Bt₂ cyclic AMP, but not by TMB-8, can be overcome by high concentrations of A23187. When Mg²⁺ is substituted for Ca²⁺, ionophore-induced release of arachidonic acid from phosphatidylcholine of inhibitor-free controls is depressed and inhibition by Bt₂ cyclic AMP is slightly enhanced. The phospholipase A₂ activity of platelet lysates is increased by the presence of added Ca²⁺, however, the addition of either A23187 or Bt₂ cyclic AMP is without effect on this activity. We suggest that Bt₂ cyclic AMP may promote a compartmentalization of Ca²⁺, thereby inhibiting phospholipase A activity. The compartmentalization may be overcome by ionophore. By contrast, TMB-8 may immobilize platelet Ca²⁺ stores in situ or restrict access of Ca²⁺ to phospholipase A in a manner not susceptible to reversal by high concentrations of ionophore.     

A quench-flow kinetic investigation of calcium ion accumulation by isolated cardiac sarcoplasmic reticulum. Dependence of initial velocity on free calcium ion concentration and influence of preincubation with a protein kinase,MgATP, and cyclic AMP

Ca²⁺ accumulation at pH 6.8 by isolated rabbit heart microsomes derived chiefly from sarcoplasmic reticulum was investigated by a quench-flow technique. The reaction was terminated at preset times by addition to the reaction mixture of an equal volume of 10 to 50 mM ethyleneglycol-bis-(β-aminoethyl ether)-N,N′-tetraacetic acid buffered at pH 6.0. The initial velocity of Ca²⁺ accumulation by microsomal preparations exhibiting a steady state Ca²⁺ accumulation of 25.6 nmol Ca²⁺/mg increased from 3.67 to 33.4 nmol Ca²⁺/mg · s as the free Ca²⁺ concentration was raised from 0.2 to 18.9 μM. Preincubation of the cardiac microsomes with a partly purified soluble cardiac cyclic AMP-dependent protein kinase, MgATP, and cyclic AMP lead to a significant increase in the initial Ca²⁺ accumulation rate. The amounts of Ca²⁺ that were found to accumulate in the first 200 ms of the reaction are comparable to the quantities of the ion that according to literature data need to be removed from the myofilaments and the myoplasm for induction of relaxation of the myocardial fibers.     

Adrenocorticotropic hormone and dibutyryl adenosine cyclic monophosphate-mediated Ca2+ uptake by rat adrenal glands

The effect of adrenocorticotropic hormone and dibutyryl cyclic AMP on the uptake of ⁴⁵Ca²⁺ by the rat adrenal gland has been investigated. After injection of ⁴⁵Ca²⁺ and adrenocorticotropic hormone into rats, the adrenal ⁴⁵Ca²⁺ concentration was significantly enhanced 90 to 180 min following hormone administration. The rise in adrenal ⁴⁵Ca²⁺ content was accompanied by a marked increase of the serum corticosterone levels. During incubation of rat adrenal glands in the presence of ⁴⁵Ca²⁺, adrenocorticotropic hormone and dibutyryl cyclic AMP caused significant accumulation of adrenal ⁴⁵Ca²⁺ and increased corticosterone synthesis. The degree of stimulation of both adrenal ⁴⁵Ca²⁺ uptake and corticosterone synthesis by adrenocorticotropic hormone or dibutyryl cyclic AMP was dependent upon the concentration of calcium in the incubation medium and upon the amount of adrenocorticotropic hormone or dibutyryl cyclic AMP added. Theophylline mimicked the stimulatory effect of adrenocorticotropic hormone and dibutyryl cyclic AMP and increased the uptake of ⁴⁵Ca²⁺ by rat adrenal glands in vitro. Determination of calcium by atomic absorption spectroscopy showed that the adrenocorticotropic hormone-mediated adrenal ⁴⁵Ca²⁺ uptake was due to a net accumulation of calcium in the tissue and not only to an increased rate of exchange of extracellular ⁴⁵Ca²⁺ with the intracellular calcium pool. Adrenocorticotropic hormone-stimulated adrenal ⁴⁵Ca²⁺ uptake was not observed when steroidogenesis was inhibited with elipten. Both adrenocorticotropic hormone-mediated corticosterone synthesis and adrenal ⁴⁵Ca²⁺ uptake were abolished after treatment of rats with cycloheximide but not after treatment with actinomycin D, indicating that adrenal ⁴⁵Ca²⁺ uptake and steroidogenesis have similar requirements for de novo protein synthesis, but not RNA synthesis.     

Cyclic AMP stimulation of membrane phosphorylation and Ca2+-activated,Mg2+-dependent ATPase in cardiac sarcoplasmic reticulum

Ca²⁺-activated ATPase (EC 3.6.1.15) in canine cardiac sarcoplasmic reticulum was stimulated 50–80% by cyclic adenosine 3′ : 5′-monophosphate. The relationship of this stimulation to cyclic AMP-dependent membrane phosphorylation with phosphoester bands was studied. Cyclic AMP stimulation of ATPase activity was specific for Ca²⁺-activated ATPase and was half-maximal at about 0.1 μM which is similar to the concentration required for half-maximal stimulation of membrane phosphorylation by endogenous cyclic AMP-stimulated protein kinase (EC 2.7.1.37). Cyclic AMP stimulation of Ca²⁺-activated ATPase was calcium dependent and maximal at calculated Ca²⁺ concentrations of 2.0 μM. Cyclic AMP-dependent Ca²⁺-activated ATPase correlated well with the cyclic AMP-dependent membrane phosphorylation of which 80% was 20 000 molecular weight protein identified by sodium dodecyl sulfate discontinuous polyacrylamide gel electrophoresis. In trypsin-treated microsomes, cyclic AMP did not stimulate Ca²⁺-activated ATPase or phosphorylation of the 20 000 molecular weight membrane protein. An endogenous calcium-stimulated protein kinase (probably phosphorylase b kinase) with an apparent K_m for ATP of 0.21–0.32 mM was present and appeared to be involved in the cyclic AMP-dependent phosphorylation of the 20 000 molecular weight protein which was calcium dependent. Cyclic guanosine 3′ : 5′-monophosphate did not inhibit any of the stimulatory effects of cyclic AMP. These data suggest that the cyclic AMP stimulation of Ca²⁺-activated ATPase in cardiac sarcoplasmic reticulum is mediated by the 20 000 molecular weight phosphoprotein product of a series of kinase reactions similar to those activating phosphorylase b.