Increase of cyclic GMP induced in murine thymocytes by thymosin fraction 5

Using a radioimmunoassay (RIA) for the determination of adenosine 3'5' cyclic monophosphate (cAMP) and an acetylation-RIA procedure to measure guanosine 3'5' cyclic monophosphate (cGMP), we observed that cGMP levels, but not cAMP levels, were significantly elevated in murine thymocytes which had been incubated with preparations containing the thymic hormone, thymosin. Stimulation of intracellular cGMP levels was seen as early as 1 minute after incubation with thymosin fraction 5 and was maximal at approximately 10 minutes. Dose response studies indicated an optimum stimulation of cGMP with a thymosin concentration of 100 microg/ml. A control spleen fraction prepared by an identical procedure as fraction 5 did not affect the levels of either cyclic nucleotide.     

Regulation of intracellular cyclic GMP levels in olfactory sensory neurons

Cyclic AMP is the primary second messenger mediating odorant signal transduction in mammals. A number of studies indicate that cyclic GMP is also involved in a variety of other olfactory signal transduction processes, including adaptation, neuronal development, and long-term cellular responses in the setting of odorant stimulation. However, the mechanisms that control the production and degradation of cGMP in olfactory sensory neurons (OSNs) remain unclear. Here, we investigate these mechanisms using primary cultures of OSNs. We demonstrate that odorants increase cGMP levels in intact OSNs in vitro. Different from the rapid and transient cAMP responses to odorants, the cGMP elevation is both delayed and sustained. Inhibition of soluble guanylyl cyclase and heme oxygenase blocks these odorant-induced cGMP increases, whereas inhibition of cGMP PDEs (phosphodiesterases) increases this response. cGMP PDE activity is increased by odorant stimulation, and is sensitive to both ambient calcium and cAMP concentrations. Calcium stimulates cGMP PDE activity, whereas cAMP and protein kinase A appears to inhibit it. These data demonstrate a mechanism by which odorant stimulation may regulate cGMP levels through the modulation of cAMP and calcium level in OSNs. Such interactions between odorants and second messenger systems may be important to the integration of immediate and long-term responses in the setting odorant stimulation.     

Capsaicin-Induced Ion Fluxes Increase Cyclic GMP but Not Cyclic AMP Levels in Rat Sensory Neurones in Culture

Capsaicin, which induces fluxes of sodium, calcium, and potassium ions in a subset of both neonatal and adult rat dorsal root ganglion neurones, increased cyclic GMP (cGMP) levels by a factor of 20 (EC50 0.07 microM) to 10-20 pmol cGMP/mg protein in these cells. Cyclic AMP (cAMP) levels were unaffected. Nonneuronal cells derived from rat ganglia, and both neurones and nonneuronal cells from chick were unresponsive to capsaicin. Capsaicin-induced cGMP elevation in rat dorsal root ganglion (DRG) neurones was unaffected by pertussis toxin, lowered by compounds that block voltage-sensitive calcium channels, and was abolished by the removal of extracellular calcium. Calcium, guanidine, and rubidium fluxes were unaffected by treatment of DRG cells with sodium nitroprusside or dibutyryl cGMP. The cGMP response to capsaicin is thus a function of capsaicin-evoked calcium uptake through voltage-sensitive calcium channels. Elevated cGMP levels do not, however, contribute to capsaicin-evoked ion fluxes or to their desensitisation.     

Cyclic nucleotides and calcium in human lymphocytes induced to divide

Intracellular concentrations of cyclic adenosine 3'-5' monophosphate (cAMP) and cyclic guanosine 3'-5' monophosphate (cGMP) were measured in human lymphocytes induced to divide by the addition of lectins, 12-O-tetra-decanoylphorbol-13-acetate (TPA) and the calcium ionophore A 23187. cGMP levels rose within minutes without concomitant alterations in cAMP concentration. The cAMP and cGMP levels rose during the prereplicative and replicative phases respectively. Under calcium depleting conditions, both the fluctuations in cyclic nucleotide levels and the increase in [3H[ thymidine incorporation into DNA were abolished, suggesting a role for calcium ions in the regulation of lymphocyte proliferation.     

Calcium and cyclic nucleotide regulation in incubated mouse retinas.

When retinas from dark-adapted C57BL/6 mice were incubated in the dark for 5 min at 37 degrees C in Earle's medium, they contained 80-120 pmol/mg protein of cGMP and about 13 pmol/mg protein of cAMP. When the incubation in darkness was in calcium-deficient Earle's medium with 3 mM EGTA, a 10-20 fold increase occurred in the cGMP level, peaking at 2-3 min, but no change occurred in cAMP. This elevated level fell in 3 min to normal dark levels on return to normal Earle's medium, but was still about three times that of control levels after 15 min in EGTA-containing solution. Bright light after 2 min of dark incubation of dark-adapted retinas resulted in a 40-50% fall in cGMP, and bright light sharply reduced the elevated dark cGMP level of retinas in calcium-deficient media with 3 mM EDTA. However, no depression of normal dark levels of cGMP has thus far been obtained by increasing external calcium levels, even in the presence of the ionophore A23187. All the above phenomena involving dark cGMP levels and calcium are similar in Earle's medium with 100 mM of K+ substituted for Na+. Congenic rodless (rd/rd) mouse retinas have less than 5% of control cGMP and show only traces of calcium sensitivity. Thus, the above phenomena in controls are likely to be largely occurring in rods. The data suggest a dependency of the dark cGMP level on the calcium level, but that the light-induced fall in cGMP may largely be calcium insensitive.     

The role of cyclic nucleotides in the regulation of mitotic activity in SSPE virus-infected human brain tissue.

The intracellular level of cGMP was independent of the rate of cell division in cells derived from virally infected brain tissue. The phosphodiesterase inhibitor R07-2956 (4-dimethoxybenzyl-2-imidazolidinone) increased the intracellular level of cGMP in virally infected brain cells, but it did not effect the level of cAMP. There was no correction between the increase in cGMP levels following addition of R07-2956 and changes in mitotic activity in the brain cell cultures. Experimental manipulations which increased the cAMP level were accompanied by a decreased mitotic rate indicating there was a correlation between mitotic activity and the level of cAMP in the same cells. Raising the intracellular level of cAMP by exogenous db-cAMP or cAMP or the use of other phosphodiesterase inhibitors routinely increased the level of cGMP as well. Conversely increasing the intracellular cGMP level by adding the exogenous cGMP increased the level of both cGMP and cAMP.A tissue culture system was used with the cell line derived from viral infected human brain tissue originally obtained from a patient with subacute sclerosing panencephalitis (SSPE). The intracellular levels of cAMP and cGMP were monitored by radioimmunoassay following manipulation of the system by addition of exogenous cGMP (0.05 mM), addition of exogenous db-cAMP (0.5 mM), or cAMP (0.5 mM) and the use of phosphodiesterase inhibitors: theophylline (1.0 mM), papaverine (50 μg/ml), 4-3-butoxy-4-methoxy benzyl-2-imidozalidinone (R020-1724) and R07-2956. Cell division was monitored in treated and non-treated cultures at 24 h intervals by analyzing the cell number and mitotic index.High levels of cGMP were found in cells which were not actively dividing but high levels were just as apt to be present in dividing cells. There was an inverse relationship between cell division and the level of cAMP.     

Increased cyclic GMP levels lead to a stimulation of elastin production in ligament fibroblasts that is reversed by cyclic AMP

The effects of cyclic nucleotides on elastin synthesis were studied in ligamentum nuchae fibroblasts by adding exogenous cyclic nucleotide derivatives or beta-adrenergic agents to cell culture medium. Elastin synthesis was enhanced (approximately 80%) by dibutyryl cGMP (Bt2cGMP) in concentrations ranging from 0.01 to 100 nM. Two other cGMP derivatives, 8-bromoguanosine 3':5'-cyclic monophosphate (8-Br-cGMP) and 2'-deoxy-cGMP, were also potent stimulators of elastin synthesis. In the absence of calcium, basal elastin production was substantially decreased (40% of control) and cGMP analogs no longer stimulated elastin synthesis, suggesting a role for calcium in the cGMP response. Bt2cAMP had no demonstrable effect on elastin production except at high concentrations which produced a nonspecific decrease equivalent to the decrease in total protein synthesis. Similarly, elevation of endogenous cellular cAMP levels by beta-adrenergic stimulation produced no change in elastin production. When 8-Br-cGMP was added to cells together with Bt2cAMP, cGMP-dependent stimulation of elastin production was abolished by cAMP in a dose-dependent fashion. These results suggest a coordinated means by which elastin production is controlled in ligament cells, i.e. increased cGMP levels lead to a stimulation of elastin production that is reversed by cAMP.     

Inhibition of nitric oxide synthase enhances contractile response of ventricular myocytes from streptozotocin-diabetic rats

The contractile hyporesponsiveness of the streptozotocin diabetic rat heart in vitro to β-adrenergic agonists is eliminated when the heart is perfused with N^G-nitro-l-arginine methyl ester (l-NAME), a non-selective inhibitor of nitric oxide synthase (NOS). The following study evaluated the hypothesis that an increased production of NO/cGMP within the diabetic myocyte inhibits the β-adrenergic-stimulated increase in calcium current and contractile response. Male Sprague-Dawley rats were given an intravenous injection of streptozotocin (60 mg/kg). After 8 weeks, L-type calcium currents were recorded in ventricular myocytes using the whole cell voltage-clamp method. Shortening of isolated myocytes was determined using a video edge detection system. cAMP and cGMP were measured using radioimmunoassay. Nitric oxide production was determined using the Griess assay kit. Basal cGMP levels and nitric oxide production were elevated in diabetic myocytes. Shortening of the diabetic myocytes in response to isoproterenol (1 μM) was markedly diminished. However, there was no detectable difference in the isoproterenol-stimulated L-type calcium current or cAMP levels between control and diabetic myocytes. Acute superfusion of the diabetic myocyte with l-NAME (1 mM) decreased basal cGMP and markedly enhanced the shortening response to isoproterenol but did not alter isoproterenol-stimulated calcium current. These data suggest that increased production of NO/cGMP within the diabetic myocyte suppressed β-adrenergic stimulated shortening of the myocyte. However, NO/cGMP apparently does not suppress shortening of the myocyte by inhibition of the β-stimulated calcium current.     

Effects of ascorbic acid and sodium ascorbate on cyclic nucleotide metabolism in human lymphocytes.

L-ascorbic acid (LAA) augmented cGMP many-fold in highly purified human peripheral blood lymphocytes. The cGMP response occurred within 10 sec and persisted for at least 60 min. D-ascorbic acid (DAA) and dehydroascorbic acid (DHAA) were also equally active in enhancing cGMP concentrations but metabolic precursors of ascorbic acid and other inorganic acids did not increase cGMP levels. Determination of the amount of DHAA contaminating the LAA precluded the possibility that it was solely responsible for the enhanced cGMP levels. The sodium or calcium salts of ascorbic acid did not increase cGMP concentrations. If these neutralized preparations were acidified, increased cGMP concentrations were then noted. In broken cell preparations, LAA, DAA, and DHAA and to a lesser extent sodium ascorbate (NaA) enhanced guanylate cyclase activity while neither inhibited cAMP or cGMP phosphodiesterase (PDE) activity. The possible role of H2O2, fatty acid liberation, prostaglandin production, oxidizing-reducing agents, and free radical formation in mediating the effects of ascorbic acid on cGMP levels were evaluated, but none of these potential mechanisms were definitively proven to be a required intermediary for the cGMP enhancing activity of ascorbic acid. LAA, DHAA or NaA did not induce lymphocyte transformation or modulate lectin-induced mitogenesis.     

cAMP activates adenylate and guanylate cyclase of Dictyostelium discoideum cells by binding to different classes of cell-surface receptors. A study with extracellular Ca2+

cAMP induces a transient increase of cAMP and cGMP levels in Dictyostelium discoideum cells. Fast binding experiments reveal three types of cAMP-binding site (S, H and L), which have different off-rates (t0.5, 0.7-15 s) and different affinities (Kd, 15-450 nM). A time- and cAMP-concentration-dependent transition of H- to L-sites occurs during the binding reaction (Van Haastert, P.J.M. and De Wit, R.J.W. (1984) J. Biol. Chem. 13321-13328). Extracellular Ca2+ had multiple effects on cAMP-binding sites. (i) The number of H + L-sites increased 2.5-fold, while the number of S-sites was not strongly affected. (ii) The Kd of the S-sites was reduced from 16 nM to 5 nM (iii) The conversion of H-sites to L-sites was inhibited up to 80%. The kinetics of the cAMP-induced cAMP accumulation was not strongly altered by Ca2+, but the amount of cAMP produced was inhibited up to 80%. The kinetics of the cAMP-induced cGMP accumulation was strongly altered; maximal levels were obtained sooner, and the Ka was reduced from 15 to 3.5 nM cAMP. Ca2+, Mg2+ and Mn2+ increased the number of binding sites, all with EC50 = 0.5 mM. The S-sites and the cGMP response were modified by equal Ca2+ concentrations and by higher concentrations of Mg2+ and Mn2+ (EC50 are respectively 0.4 mM, 2.5 mM and about 25 mM). The conversion of H- to L-sites and the cAMP response were specifically inhibited by Ca2+ with EC50 = 20 microM. It is concluded that cAMP activates guanylate cyclase through the S-sites; adenylate cyclase is activated by the H + L-sites, in which the appearance of the L-sites during the binding reaction represents the coupling of occupied surface cAMP receptors to adenylate cyclase.     

Diverse directional changes of cGMP relative to cAMP in E. coli.

The relationships of the changes of cAMP and cGMP concentrations in $\text{E. coli}$ varied as a function of experimental conditions. (1) Cells starved for carbon source for a short time period had high cAMP and low cGMP concentrations. Addition of carbon source (succinate, glucose or α-methyl glucoside) led to a decrease in cAMP and an increase in cGMP (bi-directional change). (2) Washed cells starved for glucose for long time periods had low cAMP levels which did not change on glucose addition. Addition of succinate or glucose to such cells led to a transient increase in cGMP levels (uncoupled change). The cGMP concentration peaked at 15 minutes or 1 hour after glucose or succinate addition, respectively. (3) Sham shift-up experiments (addition of α-methyl glucoside to cultures growing in succinate) in $\text{E. coli}$ 1100 and CA 8000 showed decreases in cGMP levels in both strains; however, cAMP levels increased in the former (bi-directional change) and decreased in the latter (unidirectional change).     

Effects of N-Acetyl Aspartate, Aspartate, and Glutamate on cAMP and cGMP Levels in Developing Rat Cerebral Cortex

N-Acetyl-aspartate (N-Ac-Asp) incubated with minced cerebral cortex caused a dose-dependent increase in the levels of cAMP and cGMP. This effect was followed during postnatal development. N-Ac-Asp elicits the greatest increase in cAMP in 5-day-old and in cGMP in 40-day-old rats. The levels of cyclic AMP were always higher than those of cGMP. We also studied the effects of L-aspartate (Asp) and L-glutamate (Glu) on the levels of cyclic nucleotides in the cerebral cortex minces of rats different ages, and observed that both amino acids produced the maximum increase in cAMP at 10 days, whereas in the case of cGMP the maximal effect of Asp occurs earlier than 20 days and of Glu after 40 days. In the adult rat, the N-Ac-Asp effect on cAMP was greater than that produced by either Asp or Glu, whereas the levels of cGMP were similarly affected by all three. The data show a peak response of cAMP and cGMP to N-Ac-Asp, Asp, and Glu during cortical maturation. Because this response varies with postnatal time, N-Ac-Asp, and Glu may act upon different receptor sites.     

Significance of Ca2+, Rb+ fluxes, of cAMP and cGMP for the CCK8-modulated insulin release

In rat pancreatic islets the effects of cholecystokinin-8 (CCK8) on glucose-mediated insulin release, 45Ca2+ net uptake, 45Ca2+ efflux, 86Rb+ efflux, cAMP- and cGMP levels were studied. In the presence of a substimulatory glucose concentration (3 mM) CCK8 concentrations of up to 1 microM had no effect on insulin release, but CCK8 at 10 nM potentiated the stimulatory effect of glucose (11.1 mM). 10 nM CCK8 enhanced glucose-stimulated 45Ca2+ net uptake but was ineffective at substimulatory glucose levels. CCK8 had no effect on cAMP and cGMP levels in the presence of 11.1 mM glucose, CCK8 increased 86Rb+ (a measure of K+) in the presence of both 3 and 11.1 mM glucose. This effect was abolished when Ca2+ was omitted from the perifusion medium. CCK8 did not alter glucose (11.1 mM)-stimulated 45Ca2+ efflux rate. These data indicate that (1) CCK8 potentiates glucose-stimulated insulin secretion possibly via an effect on Ca2+ uptake, 2) by affecting Ca2+ uptake, CCK8 enhances K+ efflux, and 3) CCK8 does not mediate its effect via cAMP or cGMP. With respect to 86Rb+ efflux the mechanism of CCK8 action appears to be different from that of glucose. When the mechanism of CCK action on islets is compared with that on exocrine pancreas (data from others) there are similarities (importance of Ca2+ uptake and non-importance of cAMP and cGMP).     

Phosphatidic acid and the calcium-dependent actions of gonadotropin-releasing hormone in pituitary gonadotrophs

The stimulation of luteinizing hormone (LH) release and cyclic GMP (cGMP) production in rat anterior pituitary cells by gonadotropin-releasing hormone (GnRH) are receptor mediated and calcium dependent, and have been shown to be accompanied by increased phospholipid turnover and arachidonic acid release. The incorporation of 32Pi into the total phospholipid fraction of pituitary gonadotrophs was significantly elevated by 10(-8) M GnRH, with specific increases in the labeling of phosphatidylinositol and phosphatidic acid (PA). Since PA acts as a calcium ionophore in several cell types, its effects upon calcium-mediated gonadotroph responses were compared with those elicited by GnRH. In rat pituitary gonadotrophs prepared by centrifugal elutriation, PA stimulated LH release and cGMP production by 9-fold and 5-fold, respectively. The stimulation of LH release by 30 microM PA was biphasic in its dependence on extracellular calcium concentration, rising from zero in the absence of calcium to a maximum of 10-fold at 0.5 mM Ca2+ and declining at higher calcium concentrations. In dose-response experiments, PA was 3-fold more potent at 0.5 mM Ca2+ than at 1.2 mM Ca2+. The cGMP response to PA in cultured gonadotrophs was also calcium dependent, and was progressively enhanced by increasing Ca2+ concentrations up to 1.5 mM. The ability of PA to stimulate both LH release and cGMP formation in a calcium-dependent manner suggests that endogenous PA formed in response to GnRH receptor activation could function as a Ca2+ ionophore in pituitary gonadotrophs, and may participate in the stimulation of gonadotroph responses by GnRH and its agonist analogs.     

Antagonistic effect of cAMP and cGMP on the kinetic behaviour of phosphofructokinase from rat erythrocytes and reticulocytes

Fructose-6-phosphate (F6P)-saturation curves (up to 5 mM F6P) for phosphofructokinase (PFK) have been studied at physiological pH (7.1) and inhibitory (1.5 mM) or non-inhibitory (0.25 mM) ATP levels, in rat erythrocytes and reticulocytes. The addition of 300 microM cAMP to control samples activates the enzyme and displaces F6P-saturation curve towards the left, while the addition of cGMP inhibits the enzyme and shifts the curve to the right. The cAMP positive allosteric effect is more evident at inhibitory ATP levels, while the inhibitory effect of cGMP is very similar at both ATP levels. This antagonistic effect is exerted at the same regulatory site, since cAMP also activates the enzyme when cGMP is previously present in the reaction mixture. The physiological significance of this antagonism is not yet clear.     

Photoreceptor channel activation by nucleotide derivatives

Cyclic nucleotide activated sodium currents were recorded from photoreceptor outer segment membrane patches. The concentration of cGMP and structurally similar nucleotide derivatives was varied at the cytoplasmic membrane face; currents were generated at each concentration by the application of a voltage ramp. Nucleotide-activated currents were analyzed as a function of both concentration and membrane potential. For cGMP, the average K0.5 at 0 mV was 24 microM, and the activation was cooperative with an average Hill coefficient of 2.3. Of the nucleotide derivatives examined, only 8-[[(fluorescein-5-yl-carbamoyl)methyl]thio]-cGMP (8-Fl-cGMP) activated the channel at lower concentrations than cGMP with a K0.5 of 0.85 microM. The next most active derivative was 2-amino-6-mercaptopurine riboside 3',5'-monophosphate (6-SH-cGMP) which had a K0.5 of 81 microM. cIMP and cAMP had very high K0.5 values of approximately 1.2 mM and greater than 1.5 mM, respectively. All nucleotides displayed cooperativity in their response and were rapidly reversible. Maximal current for each derivative was compared to the current produced at 200 microM cGMP; only 8-Fl-cGMP produced an identical current. The partial agonists 6-SH-cGMP, cIMP, and cAMP activated currents which were approximately 90%, 80%, and 25% of the cGMP response, respectively. 5'-GMP, 2-aminopurine riboside 3',5'-monophosphate, and 2'-deoxy-cGMP produced no detectable current. The K0.5 values for cGMP activation, examined from -90 to +90 mV, displayed a weak voltage dependence of approximately 400 mV/e-fold; the index of cooperativity was independent of the applied field.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anti-diuresis in the blood-feeding insect Rhodnius prolixus Stål: antagonistic actions of cAMP and cGMP and the role of organic acid transport

Secretion of primary urine by upper Malpighian tubules of the blood-sucking insect Rhodnius prolixus has recently been shown to be inhibited by cyclic GMP (cGMP). In the present work, we have demonstrated that cGMP has effects antagonistic to those of cAMP in Rhodnius tubules and have further characterized the effects of cGMP on tubular secretion. Cyclic GMP inhibited secretion at all concentrations from 5x10(-6) to 10(-3)M, though this inhibition was partially or wholly reversed by large (2mM) doses of cAMP. While sub-maximal concentrations of cGMP did not significantly alter [K(+)] and [Na(+)] of secreted fluid, high external [cGMP] reduced secretion to minimal levels and caused [K(+)] and [Na(+)] to approach pre-stimulation levels. Cyclic GMP does not appear to affect the permeability of the lower Malpighian tubule to water. Both cAMP and cGMP likely enter tubule cells by way of an organic acid transporter whose activity is induced by feeding. Sensitivity of the tubules to exogenous cGMP and cAMP, which is assumed to be a function of transport activity, reaches a peak approximately 5 days after the blood meal and declines rapidly thereafter. Transport of anions into upper tubules involves at least two different transporters: one for acylamides (e.g., p-aminohippuric acid) and another for sulphonates (e.g., amaranth, phenol red). Amaranth and phenol red blocked the actions of both cGMP and cAMP, whereas p-aminohippuric acid was without effect. This suggests that cyclic nucleotides enter by way of the sulphonate transporter.     

Atrial natriuretic peptides stimulate renal gluconeogenesis

Atrial natriuretic peptide (5-28AA; ANP) and atrial extract (ANS) stimulated rat renal gluconeogenesis in cortical tubule suspension in a dose dependent fashion only from substrates that enter gluconeogenesis via phosphoenol-pyruvate carboxylase. The effects of ANP and ANS were significantly potentiated by cAMP and cGMP, whereas methoxamine showed no effect. Extracellular calcium revealed a key role for ANP and ANS response to gluconeogenesis: a concentration of calcium higher than 1 mM was essential. Isolated cells from cortex which lost cell membrane polarity by warming but responded solely to cAMP and cGMP showed no effect by ANP nor ANS. These data suggest that ANP or ANS may act mainly from the basolateral site in the proximal tubule cell and promote gluconeogenesis through cAMP and/or cGMP system.     

Cyclic nucleotides and lipolysis in rat fat cells. Interaction between calcium ionophore A23187 and FCCP,uncoupler of oxidative phosphorylation

The interaction between calcium ionophore A23187 and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) has been studied at the level of cyclic-AMP (cAMP), cyclic-GMP (cGMP) and lipolysis in isolated rat fat cells. Ionophore A23187 (1–10 μM) stimulated cGMP accumulation and glycerol release without affecting cAMP level. FCCP (1–100 μM) inhibited the effect of A23187 on cGMP level and glycerol release, but did not affect or increase cAMP. Thus a correlation exists between the changes of cGMP levels and lipolysis and a dissociation of lipolysis from cAMP.     

Effect of calcium on the binding in vitro of [3H]cAMP to synaptosomal membranes from rat brain

1. The binding of [3H]cAMP in vitro to synaptosomal membranes from rat brain was resolved in two components; one saturable at 20 nM cAMP with dissociation constant (KD) of 4.7 nM, and another nonsaturable within the 5-133 nM cAMP concentration range with an estimated KD value of 0.26 microM. 2. MgATP at concentration of 0.4 mM effected complete inhibition of the binding of [3H]cAMP to synaptosomal membranes throughout the used concentration range. This and the above finding indicate that the studied binding was focused on to the cAMP kinase on the membrane. 3. Calcium at concentrations of 0.1 and 10 mM stimulated a transient 20-30% increase of [3H]cAMP binding to the membranes which was influenced, as regards its time of appearance, by the concentration of cAMP. 4. The stimulation by calcium of the binding of [3H]cAMP to the membranes was inversely related to the phosphorylation of an Mr = 80,000 membrane protein, indicating stimulation of a negative effector function of cAMP--through cAMP-mediated phosphorylation--in the phosphorylation by calcium of this substrate. Moreover, the temporal displacement by cAMP of the peak of [3H]cAMP binding, produced similar temporal displacement of the inhibitory effect of cAMP on the Mr = 80,000 substrate phosphorylation. 5. These results suggest interaction in vitro of calcium and cAMP in modulation of the activity of cAMP kinase on the synaptosomal membranes.