Biphasic Ca2+ response of adenylate cyclase. The role of calmodulin in its activation by Ca2+ ions

The Ca2+-dependent regulation of human platelet membrane adenylate cyclase has been studied. This enzyme exhibited a biphasic response to Ca2+ within a narrow range of Ca2+ concentrations (0.1-1.0 microM). At low Ca2+ (0.08-0.3 microM) adenylate cyclase was stimulated (Ka = 0.10 microM), whereas at higher Ca2+ (greater than 0.3 microM) the enzyme was inhibited to 70-80% control (Ki = 0.8 microM). Membrane fractions, prepared by washing in the presence of LaCl3 to remove endogenous calmodulin (approximately equal to 70-80% depletion), exhibited no stimulation of adenylate cyclase by Ca2+ but did show the inhibitory phase (Ki = 0.4 microM). The activation phase could be restored to La3+-washed membranes by addition of calmodulin (Ka = 3.0 nM). Under these conditions it was apparent that calmodulin reduced the sensitivity of adenylate cyclase to Ca2+ (Ki = 0.8 microM). Prostaglandin E1 (PGE1) did not alter Ki or Ka values for Ca2+. Calmodulin did not alter the EC50 for PGE1 stimulation of adenylate cyclase but increased the Vmax (1.5-fold). The calmodulin antagonist trifluoperazine potently inhibited adenylate cyclase in native membranes (80%) and to a much lesser extent in La3+-washed membranes (15%). This inhibition was due to interaction of trifluoperazine with endogenous calmodulin since trifluoperazine competitively antagonized the stimulatory effect of calmodulin on adenylate cyclase in La3+-washed membranes. We propose that biphasic Ca2+ regulation of platelet adenylate cyclase functions to both dampen (low Ca2+) and facilitate (high Ca2+) the haemostatic function of platelets.     

Ca2+-dependent inhibition of smooth muscle adenylate cyclase activity

We have examined the inhibitory regulation by Ca2+ of the adenylate cyclase activity associated with microsomes isolated from bovine aorta smooth muscle. In the presence of 2 mM MgCl2, Ca2+ (0.8-100 microM) inhibited in a noncompetitive manner activation of the enzyme by GTP, Gpp[NH]p, or forskolin. In all instances the value for half-maximal inhibition was between 2 and 3 microM. In contrast, Ca2+ inhibited the activation by MgCl2 (2-50 mM), alone or in the presence of GTP, in a competitive manner. The inhibition of adenylate cyclase by 10 microM Ca2+ was reversed in the presence of either 5 or 25 microM calmodulin or troponin C. These data show that (i) Ca2+, at concentrations similar to those which activate smooth muscle contraction, inhibits the stimulation of adenylate cyclase by several activators; (ii) Ca2+ and Mg2+ compete for a common site on the smooth muscle adenylate cyclase complex; and (iii) the reversal of Ca2+-dependent inhibition by Ca2+-binding proteins may be produced by chelation of the metal by these proteins.     

Ca2+-stimulated, Mg2+-dependent ATPase in bovine thyroid plasma membranes

An isolated plasma membrane fraction from bovine thyroid glands contained a Ca2+-stimulated, Mg2+-dependent adenosine triphosphatase ((Ca2+ + Mg2+)-ATPase) activity which was purified in parallel to (Na+ + K+)-ATPase and adenylate cyclase. The (Ca2+ + Mg2+)-ATPase activity was maximally stimulated by approx. 200 microM added calcium in the presence of approx. 200 microM EGTA (69.7 +/- 5.2 nmol/mg protein per min). In EGTA-washed membranes, the enzyme was stimulated by calmodulin and inhibited by trifluoperazine.     

Inhibition of a Low Km GTPase Activity in Rat Striatum by Calmodulin

In rat striatum, the activation of adenylate cyclase by the endogenous Ca2+-binding protein, calmodulin, is additive with that of GTP but is not additive with that of the nonhydrolyzable GTP analog, guanosine-5'-(beta, gamma-imido)triphosphate (GppNHp). One possible mechanism for this difference could be an effect of calmodulin on GTPase activity which has been demonstrated to "turn-off" adenylate cyclase activity. We examined the effects of Ca2+ and calmodulin on GTPase activity in EGTA-washed rat striatal particulate fractions depleted of Ca2+ and calmodulin. Calmodulin inhibited GTP hydrolysis at concentrations of 10(-9)-10(-6) M but had no effect on the hydrolysis of 10(-5) and 10(-6) M GTP, suggesting that calmodulin inhibited a low Km GTPase activity. The inhibition of GTPase activity by calmodulin was Ca2+-dependent and was maximal at 0.12 microM free Ca2+. Maximal inhibition by calmodulin was 40% in the presence of 10(-7) M GTP. The IC50 for calmodulin was 100 nM. In five tissues tested, calmodulin inhibited GTP hydrolysis only in those tissues where it could also activate adenylate cyclase. Calmodulin could affect the activation of adenylate cyclase by GTP in the presence of 3,4-dihydroxyphenylethylamine (DA, dopamine). Calmodulin decreased by nearly 10-fold the concentration of GTP required to provide maximal stimulation of adenylate cyclase activity by DA in the striatal membranes. The characteristics of the effect of calmodulin on GTPase activity with respect to Ca2+ and calmodulin dependence and tissue specificity parallel those of the activation of adenylate cyclase by calmodulin, suggesting that the two activities are closely related.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microtubule-associated adenylate cyclase

Twice-cycled bovine brain or rat brain microtubule protein contains an adenylate cyclase activity that passes 0.2 micron filters, is activated 2-7-fold by 30 microM forskolin, shows modest stimulation by fluoride (especially in the presence of added AI3+), but is virtually insensitive to added guanine nucleotides. The activity is insensitive to various hormones or Ca2+/calmodulin. The adenylate cyclase is active with both Mg2+ and Mn2+ but activity is less in the presence of Mg2+ than with Mn2+. The cyclase is inhibited by agonists of the adenosine P site. It is proposed that the catalytic unit of adenylate cyclase and probably small quantities of the guanine nucleotide regulatory protein, Ns, are cycled along with microtubules.     

Calmodulin activation of adenylate cyclase in the mouse B16 melanoma.

Calmodulin antagonists inhibited hormone-stimulated cyclic AMP accumulation in both cultured cells and cell lysates of mouse B16 melanoma. Particulate preparations of B16 melanoma contained 34-45% of total cell calmodulin, which could not be dissociated by extensive washing irrespective of the presence of EGTA in the buffer. The adenylate cyclase activity in such preparations was unaffected by the addition of exogenous calmodulin. However, the rare-earth-metal ion La3+, which can mimic or replace Ca2+ in many systems, produced an immediate inhibition of agonist-stimulated adenylate cyclase activity and preincubation of particulate preparations was La3+ followed by washing with La3+-free buffer dissociated calmodulin (96% loss) from particulate preparations. The loss of calmodulin from particulate preparations was associated with a decrease in agonist responsiveness (74%) and a marked change in the Ca2+-sensitivity of the enzyme, low concentrations of calcium (approx. 10 nM) now failing to stimulate enzyme activity, high concentrations of calcium (greater than or equal to 100 nM) producing greater-than-normal inhibition of enzyme activity. Direct activation of adenylate cyclase by the addition of pure calmodulin was now demonstrable in such calmodulin-depleted particulate preparations. Half-maximal stimulation of agonist-responsive adenylate cyclase occurred at 80 nM-calmodulin in the presence of 10 microM free Ca2+. Maximal stimulation by calmodulin (at 300-600 nM) restored enzyme activity to 89 +/- 5% (mean +/- S.E.M., n = 7) of the activity in untreated, calmodulin-intact, preparations.     

Calcium-independent activation of adenylate cyclase by calmodulin

Adenylate cyclase of Bordetella pertussis is stimulated by calmodulin by two distinct interactions. At low activator concentrations (approximately equal to 1 nM) the process is Ca2+-dependent (i.e. inhibited by EGTA added before calmodulin). High activator concentrations (approximately equal to 0.1-10 microM) stimulate adenylate cyclase also in the presence of EGTA, an effect not accounted for by residual Ca2+ or low concentrations of Ca X calmodulin, which thus appears to be due to calcium-free calmodulin. Some calmodulin dose-response curves show both phases of stimulation, separated by a plateau of activity, and half-maximal activating concentrations differ by 100-300-fold. Both effects are on the V and not the Km for ATP and are not mimicked by 10(5)-fold greater concentrations of parvalbumin or by various polyanions. In addition, adenylate cyclase stimulation at high calmodulin concentrations is greater in the presence of EGTA than in its absence. This enhancement is also produced by 1,10-phenanthroline and 8-hydroxyquinoline but not by non-chelating isomers. These compounds are poor Ca2+ chelators, stimulate at any calmodulin concentration (unlike EGTA), and suggest regulation of this adenylate cyclase by a second metal ion.     

Activation and stabilization of the catalytic unit of adenylate cyclase.

The requirements for stability and activity of the catalytic unit (C) of adenylate cyclase were investigated. After solubilization of bovine brain membranes in the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulphonate (Chaps), the catalytic unit was separated from the stimulatory guanine-nucleotide-binding protein (Gs) by gel filtration on Ultrogel AcA-34. The partially purified C unit was rapidly inactivated at 30 degrees C; 0.25 mM-ATP stabilized activity. Although C-unit activity was dependent on Mg2+ or Mn2+, stabilization by ATP did not require bivalent cations. Activity of the Ultrogel-AcA-34-purified C unit was increased by Ca2+ plus calmodulin and by phosphatidylcholine plus lysophosphatidylcholine; activity in the presence of both activators was significantly greater than with each alone. Calmodulin plus Ca2+ and phospholipids also stabilized C unit. The column-purified C unit was activated by forskolin; the effect of forskolin was additive to those of calmodulin plus Ca2+ and phospholipids. p[NH]ppG-stimulated adenylate cyclase activity was reconstituted by mixing samples from the gel-filtration column containing Gs with C unit. Activation by Ca2+ plus calmodulin and Gs plus p[NH]ppG was additive; Ca2+ plus calmodulin did not alter the concentration of p[NH]ppG required for half-maximal activation. Results were similar with forskolin and Gs plus p[NH]ppG; the presence of one activator did not alter the effect of the other. These studies define conditions for separation of C unit and Gs from brain adenylate cyclase and demonstrate that ATP (in the absence of bivalent cations), phospholipids, calmodulin plus Ca2+, and forskolin all interact with C unit in a manner that is independent of functional Gs.     

Lipids associated with bovine kidney glomerular basement membranes.

1. The activities of the enzymes involved in the metabolism of cyclic nucleotides were studied in sarcolemma prepared front guinea-pig heart ventricle; the enzyme activities reported here were linear under the assay conditions. 2. Adenylate cyclase was maximally activated by 3mM-NaF; NaF increased the Km for ATP (from 0.042 to 0.19 mM) but decreased the Ka for Mg2+ (from 2.33 to 0.9 mM). In the presence of saturating Mg2+ (15 mM), Mn2+ enhanced adenylate cyclase, whereas Co2+ was inhibitory. beta-Adrenergic amines (10-50 muM) stimulated adenylate cyclase (38+/-2%). When added to the assay mixture, guanyl nucleotides (GTP and its analogue, guanylyl imidophosphate) stimulated basal enzyme activity and enhanced the stimulation by isoproterenol. By contrast, preincubation of sarcolemma with guanylyl imidodiphosphate stimulated the formation of an 'activated' form of the enzyme, which did not reveal increased hormonal sensitivity. 3. The guanylate cyclase present in the membranes as well as in the Triton X-100-solubilized extract of membranes exhibited a Ka for Mn 2+ of 0.3 mM; Mn2+ in excess of GTP was required for maximal activity. Solubilized guanylate cyclase was activated by Mg2+ only in the presence of low Mn2+ concentrations; Ca2+ was inhibitory both in the absence and presence of low Mn2+. Acetylcholine as well as carbamolycholine stimulated membrane-bound guanylate cyclase. 4. Cylic nucleotide phosphodiesterase activities of sarcolemma exhibited both high-and low-Km forms with cyclic AMP and with cyclic GMP as substrate. Ca2+ ions increased the Vmax. of the cyclic GMP-dependent enzyme.     

Potent and cooperative feedback inhibition of adenylate cyclase activity by calcium in pituitary-derived GH3 cells

Calcium (Ca2+) ion concentrations that are achieved intracellularly upon membrane depolarization or activation of phospholipase C stimulate adenylate cyclase via calmodulin (CaM) in brain tissue. In the present study, this range of Ca2+ concentrations produced unanticipated inhibitory effects on the plasma membrane adenylate cyclase activity of GH3 cells. Ca2+ concentrations ranging from 0.1 to 0.8 microM exerted an increasing inhibition on enzyme activity, which reached a plateau (35-45% inhibition) at around 1 microM. This inhibitory effect was highly cooperative for Ca2+ ions, but was neither enhanced nor dependent upon the addition of CaM (1 microM) to EGTA-washed membranes. The inhibition was greatly enhanced upon stimulation of the enzyme by vasoactive intestinal peptide (VIP) and/or GTP. Prior exposure of cultured cells to pertussis toxin did not affect the inhibition of plasma membrane adenylate cyclase activity by Ca2+, although in these membranes, hormonal (somatostatin) inhibition was significantly attenuated. Maximally effective concentrations of Ca2+ and somatostatin produced additive inhibitory effects on adenylate cyclase. The addition of phosphodiesterase inhibitors demonstrated that inhibitory effects of Ca2+ were not mediated by Ca2(+)-dependent stimulation of a phosphodiesterase activity. These observations provide a mechanism for the feedback inhibition by elevated intracellular Ca2+ levels on cAMP-facilitated Ca2+ entry into GH3 cells, as well as inhibitory crosstalk between Ca2(+)-mobilizing signals and adenylate cyclase activity.     

Calmodulin regulation of adenylate cyclase activity in human platelet membranes

The mechanism of calmodulin dependent regulation of adenylate cyclase has been studied in human platelet membranes. Calmodulin activated adenylate cyclase exhibited a biphasic response to both Mg2+ and Ca2+. A stimulatory effect of Mg2 on adenylate cyclase was observed at all Mg2+ concentrations employed, although the degree of activation by calmodulin was progressively decreased with increasing concentrations of Mg2+. These results demonstrate that the Vmax of calmodulin dependent platelet adenylate cyclase can be manipulated by varying the relative concentrations of Mg2+ and Ca2+. The activity of calmodulin stimulated adenylate cyclase was always increased 2-fold above respective levels of activity induced by GTP, Gpp(NH)p and/or PGE. The stimulatory influence of calmodulin was not additive but synergistic to the effects of PGE1, GTP and Gpp(NH)p. GDP beta S inhibited GTP-and Gpp(NH)p stimulation of adenylate cyclase but was without effect on calmodulin stimulation. Since the inhibitory effects of GDP beta S have been ascribed to apparent reduction of active N-protein-catalytic unit (C) complex formation, these results suggest that the magnitude of calmodulin dependent adenylate cyclase activity is proportional to the number of N-protein-C complexes, and that calmodulin interacts with preformed N-protein-C complex to increase its catalytic turnover. Our data do not support existence of two isoenzymes of adenylate cyclase (calmodulin sensitive and calmodulin insensitive) in human platelets.     

Calmodulin-dependent elevation of calcium transport associated with calmodulin-dependent phosphorylation in cardiac sarcoplasmic reticulum

The rate of calcium transport by sarcoplasmic reticulum vesicles from dog heart assayed at 25 degrees C, pH 7.0, in the presence of oxalate and a low free Ca2+ concentration (approx. 0.5 microM) was increased from 0.091 to 0.162 mumol . mg-1 . min-1 with 100 nM calmodulin, when the calcium-, calmodulin-dependent phosphorylation was carried out prior to the determination of calcium uptake in the presence of a higher concentration of free Ca2+ (preincubation with magnesium, ATP and 100 microM CaCl2; approx. 75 microM free Ca2+). Half-maximal activation of calcium uptake occurs under these conditions at 10-20 nM calmodulin. The rate of calcium-activated ATP hydrolysis by the Ca2+-, Mg2+-dependent transport ATPase of sarcoplasmic reticulum was increased by 100 nM calmodulin in parallel with the increase in calcium transport; calcium-independent ATP splitting was unaffected. The calcium-, calmodulin-dependent phosphorylation of sarcoplasmic reticulum, preincubated with approx. 75 microM Ca2+ and assayed at approx. 10 microM Ca2+ approaches maximally 3 nmol/mg protein, with a half-maximal activation at about 8 nM calmodulin; it is abolished by 0.5 mM trifluperazine. More than 90% of the incorporated [32P]phosphate is confined to a 9-11 kDa protein, which is also phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase and most probably represents a subunit of phospholamban. The stimulatory effect of 100 nM calmodulin on the rate of calcium uptake assayed at 0.5 microM Ca2+ was smaller following preincubation of sarcoplasmic reticulum vesicles with calmodulin in the presence of approx. 75 microM Ca2+, but in the absence of ATP, and was associated with a significant degree of calmodulin-dependent phosphorylation. However, the stimulatory effect on calcium uptake and that on calmodulin-dependent phosphorylation were both absent after preincubation with calmodulin, without calcium and ATP, suggestive of a causal relationship between these processes.     

Regulation of axonemal Mg2+-ATPase from Paramecium cilia: effects of Ca2+ and cyclic nucleotides

Ciliary activity is regulated by Ca2+ and cyclic nucleotides, but the molecular mechanisms of the regulation are unknown. We have tested the ability of Ca2+ and cyclic nucleotides to alter ciliary Mg2+-ATPase or to stimulate phosphorylation of axonemal dynein. Mg2+-ATPase activity in cilia and axonemes from Paramecium was stimulated 2-fold by micromolar Ca2+, but this Ca2+ sensitivity was lost upon solubilization of the dyneins from the axoneme. The Ca2+-sensitive component of ciliary Mg2+-ATPase activity was inhibited by the dynein inhibitors vanadate and Zn2+, but was insensitive to the calmodulin antagonists calmidazolium and melittin. Dynein activity in the high-salt extract from axonemes was also insensitive to calmidazolium. Calmodulin did not sediment with 22 S or 12 S dyneins on sucrose gradients containing Ca2+, but it did sediment in the region from 19 S to 14 S. Mg2+-ATPase activity in ciliary fractions was unaltered in the presence of cAMP or cGMP. However, polypeptides associated with the 22 S and 12 S dyneins, as well as proteins of 19 S, 15 S, and 8 S, were substrates for endogenous ciliary kinases. High molecular weight polypeptides that sedimented at 22 S and 19 S were phosphorylated in a cyclic nucleotide-stimulated manner.     

Calmodulin-dependent adenylate cyclase activity in rat cerebral cortex: effects of divalent cations, forskolin and isoprenaline

The role of calcium-calmodulin (Ca2+-CaM) in the modulation of beta-adrenergic adenylate cyclase activity in rat cerebral cortex has been studied. In addition, the effects of manganese (Mn2+) and forskolin on CaM-dependent enzyme activity were investigated. At 2 mM magnesium (Mg2+) low concentrations of Ca2+ stimulated the enzyme activity (Ka 0.25 +/- 0.08 microM), whereas higher Ca2+ levels (greater than 2 microM) inhibited the activity. No activating effect of Ca2+ was observed in CaM-depleted membranes, but the inhibitory effect persisted and the stimulatory action of Ca2+ could be restored by addition of exogenous CaM. The ability of Ca2+ to activate the enzyme was reduced by increasing concentrations of Mg2+. At 10 mM Mg2+ the apparent Ka of Ca2+ was 0.55 +/- 0.16 microM and half-maximal inhibition was observed at 80-120 microM Ca2+. A synergistic effect was observed between Ca2+ and isoprenaline on the adenylate cyclase activity. Calcium did not alter the apparent Ka of isoprenaline (0.9 +/- 0.27 microM) and isoprenaline did not change the apparent Ka of Ca2+. However, isoprenaline decreased the apparent Ka of CaM; 0.11 +/- 0.07 micrograms vs. 0.32 +/- 0.1 micrograms (0.5 ml assay mixture)-1, with and without isoprenaline, respectively. A synergistic effect was also observed between Ca2+ and forskolin, but no change in their apparent Ka values was found. Furthermore, Mn2+ was found to activate the enzyme through CaM. These data demonstrate that Ca2+ -CaM potentiates beta-adrenergic adenylate cyclase activity and thus is able to modulate neurotransmitter stimulation in cortex. Furthermore, both forskolin and Mn2+ affect CaM-dependent enzyme activity. Forskolin potentiates Ca2+-CaM stimulation, while Mn2+ increases the activity by activating the enzyme through CaM.     

Distinct interactions between Ca2+/calmodulin and neurotransmitter stimulation of adenylate cyclase in striatum and hippocampus

1. Ca2+ and cAMP both act as intracellular second messengers of receptor activation. In neuronal tissue, Ca2+ acting via calmodulin can elevate cAMP levels. This regulation by Ca2+ provides a means whereby the elevation of intracellular [Ca2+] might modulate cAMP generation. 2. In the present studies, the impact of the Ca2+/calmodulin regulation on receptor-mediated stimulation of activity is compared in striatum and hippocampus--regions of differing sensitivity to Ca2+/camodulin. Ca2+/calmodulin stimulated striatal and hippocampal adenylate cyclase activity by 1.4- and 2.7-fold respectively, while dopamine and vasoactive intestinal peptide (VIP) stimulated the enzyme activity of these respective regions by 1.3- and 2-fold. 3. In the presence of Ca2+/calmodulin, the dopamine dose-response curve in the striatum was shifted upward, without alteration of the slope of the curve or of the maximal stimulation of activity elicited by dopamine. In the hippocampus, the ability of VIP to stimulate adenylate cyclase activity was reduced by the presence of calmodulin. 4. The dose dependence of these actions of calmodulin was examined. In the striatum, the stimulation of adenylate cyclase activity by 0.1 to 0.3 microM calmodulin obscured dopamine stimulation, while 1 to 10 microM was additive with the dopamine stimulation. In the hippocampus, all concentrations of calmodulin (0.1 to 10 microM) reduced VIP-mediated stimulation of enzyme activity. 5. These data suggest that the ratio of calmodulin-sensitive to calmodulin-insensitive adenylate cyclase activity varies in different rat brain regions and that, in those regions in which this ratio is low (e.g., rat striatum and most peripheral systems), calmodulin- and receptor-mediated activation of adenylate cyclase activity will be additive, while in those systems in which this ratio is high (e.g., most of the central nervous system), calmodulin will reduce receptor-mediated stimulation of enzyme activity.     

Ca2+-ATPase activity in pancreatic acinar plasma membranes. Regulation by calmodulin and acidic phospholipids

A high degree of ATP hydrolytic activity present in purified rat pancreatic acinar cells was localized to plasma membranes. This activity was stimulated almost equally by Mg2+ or Ca2+. Kinetic analysis revealed that the enzyme had a higher affinity for Ca2+ (Kd = 1.73 microM) than Mg2+ (Kd = 2.98 microM) but a similar maximal rate of activity. A comparison of substrate requirements revealed very similar profiles for the Mg2+- and Ca2+-stimulated activities. Combinations of saturating concentrations of Mg2+ or Ca2+ produced the same degree of maximal activity. Investigation of the partial reactions of the ATPase activity revealed two phosphoprotein intermediates (Mr = 115,000 and 130,000) in the presence of Ca2+ and Mg2+. A significant stimulation of the Ca2+-ATPase activity by calmodulin was observed (Kd = 0.7 microM). Calmodulin increased the Ca2+-sensitivity of this enzyme system; Mg2+ appeared to be required for this effect. The Ca2+-ATPase activity was also stimulated by acidic phospholipids. Using an 125I-labeled calmodulin gel overlay technique, calmodulin was shown to bind in a Ca2+-dependent fashion to 133,000- and 230,000-dalton proteins present in the plasma membrane-enriched fraction. Under conditions that favor Ca2+-dependent kinase activity, calmodulin enhanced the phosphorylation of a 30,000- and 19,000-dalton protein. The major ATP hydrolytic activity in pancreatic acinar plasma membranes was present as an ectoenzyme.     

Synthetic peptides based on the calmodulin-binding domain of myosin light chain kinase inhibit activation of other calmodulin-dependent enzymes

Nanomolar concentrations of synthetic peptides corresponding to the calmodulin-binding domain of skeletal muscle myosin light chain kinase were found to inhibit calmodulin activation of seven well-characterized calmodulin-dependent enzymes: brain 61 kDa cyclic nucleotide phosphodiesterase, brain adenylate cyclase, Bordetella pertussis adenylate cyclase, red blood cell membrane Ca++-pump ATPase, brain calmodulin-dependent protein phosphatase (calcineurin), skeletal muscle phosphorylase b kinase, and brain multifunctional Ca++ (calmodulin)-dependent protein kinase. Inhibition could be entirely overcome by the addition of excess calmodulin. Thus, the myosin light chain kinase peptides used in this study may be useful antagonists for studying calmodulin-dependent enzymes and processes.     

Calmodulin plays a dominant role in determining neurotransmitter regulation of neuronal adenylate cyclase

Ca2+, through the mediation of calmodulin, stimulates the activity of brain adenylate cyclase. The growing awareness that fluctuating Ca2+ concentrations play a major role in intracellular signalling prompted the present study, which aimed to investigate the implications for neurotransmitter (receptor) regulation of enzymatic activity of this calmodulin regulation. The role of Ca2+/calmodulin in regulating neurotransmitter-mediated inhibition and stimulation was assessed in a number of rat brain areas. Ca2+/calmodulin stimulated adenylate cyclase activity in EGTA-washed plasma preparations from each region studied--from 1.3-fold (in striatum) to 3.4-fold (in cerebral cortex). The fold-stimulation produced by Ca2+/calmodulin was decreased in the presence of GTP, forskolin, or Mn2+. In EGTA-washed membranes, receptor-mediated inhibition of adenylate cyclase was strictly dependent upon Ca2+/calmodulin stimulation in all regions, except striatum. A requirement for Mg2+ in combination with Ca2+/calmodulin to observe neurotransmitter-mediated inhibition was also observed. In contrast, receptor-mediated stimulation of activity was much greater in the absence of Ca2+/calmodulin. The findings demonstrate that ambient Ca2+ concentrations, in concert with endogenous calmodulin, may play a central role in dictating whether inhibition or stimulation of adenylate cyclase by neurotransmitters may proceed.     

Calcium and calmodulin in the regulation of human thyroid adenylate cyclase activity.

TSH (thyrotropin)-stimulated human thyroid adenylate cyclase has a biphasic response to Ca2+, being activated by submicromolar Ca2+ (optimum 22nM), with inhibition at higher concentrations. Calmodulin antagonists caused an inhibition of TSH-stimulated adenylate cyclase in a dose-dependent manner. Inhibition of TSH-and TSIg-(thyroid-stimulating immunoglobulins)-stimulated activity was more marked than that of basal, NaF- or forskolin-stimulated activity. This inhibition was not due to a decreased binding of TSH to its receptor. Addition of pure calmodulin to particulate preparations of human non-toxic goitre which had not been calmodulin-depleted had no effect on adenylate cyclase activity. EGTA was ineffective in removing calmodulin from particulate preparations, but treatment with the tervalent metal ion La3+ resulted in a loss of up to 98% of calmodulin activity from these preparations. Addition of La3+ directly to the adenylate cyclase assay resulted in a partial inhibition of TSH- and NaF-stimulated activity, with 50% inhibition produced by 5.1 microM and 4.0 microM-La3+ respectively. Particulate preparations with La3+ showed a decrease of TSH- and NaF-stimulated adenylate cyclase activity (approx. 40-60%). In La3+-treated preparations there was a decrease in sensitivity of TSH-stimulated adenylate cyclase to Ca2+ over a wide range of Ca2+ concentrations, but most markedly in the region of the optimal stimulatory Ca2+ concentration. In particulate preparations from which endogenous calmodulin had been removed by La3+ treatment, the addition of pure calmodulin caused an increase (73 +/- 22%; mean +/- S.E.M., n = 8) in TSH-stimulated thyroid adenylate cyclase activity. This was seen in 8 out of 13 experiments.     

Characterization of a Ca2+-dependent guanylate cyclase in the excitable ciliary membrane from Paramecium

The membraneous guanylate cyclase of cilia from Paramecium tetraurelia used MgGTP and MnGTP as substrate with Michaelis constants for GTP of 71.5 microM and 36 microM, respectively. A linear Arrhenius plot indicated that a single enzyme entity exists not sensitive to possible phase transitions of membrane lipids. Guanylate cyclase is activated by low concentrations (less than 100 microM) and inhibited by high concentrations (greater than 100 microM) of calcium, half-maximal effects were obtained with 8 microM and 500 microM Ca2+, respectively. Only strontium ions displayed partial activating and inhibiting potency, all other divalent cations tested, Ba2+, Fe2+, Co2+, Mn2+, Sn2+ and Ni2+ had no effect on guanylate cyclase activity. Ca2+ activation increased V; Km remained identical. The Ca2+ stimulated activity was not inhibited by trifluoperazine, tentatively suggesting that the stimulation may not be mediated by calmodulin. Ca2 inhibition was due to a single binding site of Ca2+ at the guanylate cyclase as evidence by a Hill coefficient h = -1 and was noncompetitive. The lanthanides La3+, Ce3+ and Tb3+ were powerful inhibitors of guanylate cyclase, with La3+ the half-maximal effect was obtained with 0.6 microM, it was kinetically a mixed-type inhibition. La3+ and CA2+ competed for the same binding site on the guanylate cyclase as determined by detailed kinetic analysis. Addition of EDTA reversed the activation and inhibition by Ca2+ and the inhibition by La3+. It is discussed that guanylate cyclase may be the initial target enzyme in the cilia for the calcium transient of the calcium-potassium action potential of Paramecium.