Calmodulin in interferon preparations: effect of interferon on calmodulin bioactivity

Heat-stable calmodulin immunoreactivity and bioactivity were detected in crude preparations of various types of human, murine and chicken interferons (IFNs). Calmodulin containing HuIFN-alpha was retained on a trifluorophenothiazine-Sepharose column. The two activities were separated by serial elutions with 50 microM Ca2+ (HuIFN-alpha) followed by 2 mM EGTA (calmodulin). While maintaining its full antiviral activity, calmodulin free HuIFN-alpha inhibited enhancement of Ca2+-ATPase activity in vitro by authentic purified eukaryote calmodulin. These results indicate that IFNs are calmodulin-binding proteins and that the secretion of both IFNs and calmodulin occurs from IFN-induced cells.     

Insulin release and protein phosphorylation: possible role of calmodulin

Both Ca2+ and cyclic AMP (cAMP) are implicated in the regulation of insulin release in the pancreatic beta cell. In hamster insulinoma cells used in our laboratory to study the mechanism of insulin release, Ca2+ and cAMP trigger secretion independently. Concomitant with stimulation of the secretory apparatus both cAMP and Ca2+ promote phosphorylation of distinct insulinoma cell proteins. Calmodulin may be involved in the stimulation of insulin release and protein phosphorylation induced by Ca2+ influx. The Ca2+-dependent protein kinase of the insulinoma cell is activated by exogenous calmodulin and blocked by trifluoperazine, and inhibitor of calmodulin action. This drug also inhibits glucose-induced insulin release in pancreatic islets. In insulinoma cells trifluoperazine blocks Ca2+ influx-mediated insulin release and protein phosphorylation with no effect on basal or cAMP-mediated insulin release and protein phosphorylation with no effect on basal or cAMP-mediated secretion. Inhibition of Ca2+ influx-mediated insulin release and protein phosphorylation occurs with nearly identical dose dependence. Inasmuch as trifluoperazine affects voltage-dependent Ca2+ uptake in insulinoma cells, an involvement of calmodulin cannot be directly inferred. The evidence suggests that protein phosphorylation may be involved in the activation of the secretory apparatus by both cAMP and Ca2+. It is proposed that stimulation of insulin release by cAMP and Ca2+ is mediated by cAMP-dependent protein kinase and calmodulin-dependent protein kinase, respectively.     

Calmodulin activation and inhibition of skeletal muscle Ca2+ release channel (ryanodine receptor).

The calmodulin-binding properties of the rabbit skeletal muscle Ca2+ release channel (ryanodine receptor) and the channel's regulation by calmodulin were determined at < or = 0.1 microM and micromolar to millimolar Ca2+ concentrations. [125I]Calmodulin and [3H]ryanodine binding to sarcoplasmic reticulum (SR) vesicles and purified Ca2+ release channel preparations indicated that the large (2200 kDa) Ca2+ release channel complex binds with high affinity (KD = 5-25 nM) 16 calmodulins at < or = 0.1 microM Ca2+ and 4 calmodulins at 100 microM Ca2+. Calmodulin-binding affinity to the channel showed a broad maximum at pH 6.8 and was highest at 0.15 M KCl at both < or = 0.1 MicroM and 100 microM Ca2+. Under condition closely related to those during muscle contraction and relaxation, the half-times of calmodulin dissociation and binding were 50 +/- 20 s and 30 +/- 10 min, respectively. SR vesicle-45Ca2+ flux, single-channel, and [3H]ryanodine bind measurements showed that, at < or = 0.2 microM Ca2+, calmodulin activated the Ca2+ release channel severalfold. Ar micromolar to millimolar Ca2+ concentrations, calmodulin inhibited the Ca(2+)-activated channel severalfold. Hill coefficients of approximately 1.3 suggested no or only weak cooperative activation and inhibition of Ca2+ release channel activity by calmodulin. These results suggest a role for calmodulin in modulating SR Ca2+ release in skeletal muscle at both resting and elevated Ca2+ concentrations.     

Biochemical characterization of the inhibitory effect of CsA on cytolytic T lymphocyte effector functions

We have examined the effects of cyclosporine A (CsA) on a number of CTL effector functions. CsA partially inhibited the CTL-mediated lysis of Ag-bearing target cells. Both target cell- and anti-TCR mAb-induced granule exocytosis were markedly inhibited by CsA. In addition, marked inhibition of PMA and calcium ionophore (A23187) induced granule exocytosis was produced by CsA suggesting that the inhibitory effects of CsA on granule exocytosis involve biochemical events after protein kinase C activation and increases in intracellular free Ca2+. CsA had no inhibitory effects on TCR-mediated phosphatidylinositol metabolism. The inhibitory effects of CsA were not mediated by the cAMP-dependent protein kinase inhibitory pathway and no effect of CsA on the Ca2+-induced binding of calmodulin to calmodulin-binding proteins could be demonstrated. CsA was also a potent inhibitor of IgE receptor-mediated exocytosis in rat basophil leukemia cells. CsA had no effect on receptor-mediated phosphatidylinositol hydrolysis; 400 ng/ml CsA resulted in a 90% inhibition of serotonin release but had no effect on phosphatidylinositol hydrolysis. These results indicate that CsA may inhibit some common event in Ca2+-dependent secretory cells. Taken together, these results suggest that CsA does not inhibit signal transduction but rather interferes with the biochemical events in the later stages of Ca2+-dependent reactions that follow the binding of calmodulin to cytoskeletal or cytoplasmic calmodulin binding proteins.     

Identification and characterization of calmodulin-binding proteins in islet secretion granules

The binding of 125I-calmodulin to intact secretion granules and protein gel blots of secretion granules from pancreatic islet tissue was examined. Binding of 125I-calmodulin to intact secretion granules was Ca2+-dependent and inhibited by the calmodulin inhibitors trifluoperazine and calmidazolium. Binding was inhibited by excess (200 nM) unlabeled calmodulin, but not by parvalbumin, a Ca2+-binding protein which has little sequence homology to calmodulin. In order to study the binding of calmodulin to specific secretion granule proteins, secretion granules were solubilized, and the solubilized proteins were resolved on sodium dodecyl sulfate-polyacrylamide gels, electrophoretically transferred to nitrocellulose, and incubated with 125I-calmodulin. Autoradiograms of the protein gel blots revealed the presence of three major calmodulin-binding proteins with approximate molecular weights of 73,000, 64,000, and 58,000. These proteins reversibly bound calmodulin in a calcium-dependent manner. Unlabeled calmodulin in the range of 0.1-1.0 nM competed with 125I-calmodulin for binding to these proteins, whereas troponin and parvalbumin were 100 and 1000-fold less effective, respectively. Trifluoperazine blocked binding to the granule proteins in a range of 10(-4) to 10(-5) M, and calmidazolium was effective between 10(-5) and 10(-6) M. Trypsin, at a concentration which did not lyse granules, markedly inhibited calmodulin binding to intact secretion granules. Protein blots from trypsin-treated granules showed that the three major calmodulin-binding proteins were absent. These results indicate that Ca2+-dependent calmodulin-binding proteins are present on the cytoplasmic surface of islet secretion granules and are consistent with the hypothesis that these proteins may play a role in secretion granule exocytosis.     

Calmodulin-binding sites on adenylyl cyclase type VIII

Ca2+ stimulation of adenylyl cyclase type VIII (ACVIII) occurs through loosely bound calmodulin. However, where calmodulin binds in ACVIII and how the binding activates this cyclase have not yet been investigated. We have located two putative calmodulin-binding sites in ACVIII. One site is located at the N terminus as revealed by overlay assays; the other is located at the C terminus, as indicated by mutagenesis studies. Both of these calmodulin-binding sites were confirmed by synthetic peptide studies. The N-terminal site has the typical motif of a Ca2+-dependent calmodulin-binding domain, which is defined by a characteristic pattern of hydrophobic amino acids, basic and aromatic amino acids, and a tendency to form amphipathic alpha-helix structures. Functional, mutagenesis studies suggest that this binding makes a minor contribution to the Ca2+ stimulation of ACVIII activity, although it might be involved in calmodulin trapping by ACVIII. The primary structure of the C-terminal site resembles another calmodulin-binding motif, the so-called IQ motif, which is commonly Ca2+-independent. Mutagenesis and functional assays indicate that this latter site is a calcium-dependent calmodulin-binding site, which is largely responsible for the Ca2+ stimulation of ACVIII. Removal of this latter calmodulin-binding region from ACVIII results in a hyperactivated enzyme state and a loss of Ca2+ sensitivity. Thus, Ca2+/calmodulin regulation of ACVIII may be through a disinhibitory mechanism, as is the case for a number of other targets of Ca2+/calmodulin.     

Multiple transduction mechanisms are likely involved in calcium-mediated exocrine secretory events in rat parotid cells

The parotid gland of the aged rat provides an example of an altered alpha 1-adrenergic physiologic response (K+ efflux) resulting from a postreceptor perturbation in signal transduction mechanisms (Ito, H., Baum, B. J., Uchida, T., Hoopes, M. T., Bodner, L. & Roth, G. S. (1982) J. Biol. Chem. 257, 9532-9538). This alteration in gland function can be completely circumvented by eliciting K+ efflux via the Ca2+-ionophore, A23187, at several Ca2+ concentrations (ibid.). Since Ca2+ is purported to mediate other secretory events in the rat parotid, we have probed neurotransmitter regulated Ca2+ mobilization and secretory mechanisms in this tissue by employing an aging paradigm. The responses studied were alpha-adrenergic- and muscarinic-cholinergic-mediated K+ efflux, 45Ca2+ release, and amylase secretion. No differences were detected between young (3 months) and old (24 months) cell preparations for any muscarinic-cholinergic agonist-induced response studied. Following alpha-adrenergic stimulation, K+ efflux and 45Ca2+ release from old cell preparations were reduced markedly, while no changes were found for the amylase secretion response. These results suggest that 1) alpha-adrenergic and cholinergic signal transduction mechanisms for K+ efflux and 45Ca2+ release are dissociated in cells of the rat parotid gland, and 2) following alpha 1-adrenergic stimulation, signal transduction likely proceeds by at least two pathways, one which is apparently involved in protein excytosis (intact in cells from old rats) and the other which is apparently involved in K+ efflux and 45Ca2+ release (perturbed in old cells).     

Characterization of Ca2+-stimulated secretion in permeable GH3 pituitary cells

In this report, the secretory response to Ca2+ in GH3 rat pituitary cells permeabilized by electric field discharge has been compared in both magnitude and Ca2+ sensitivity to prolactin (PRL) release from intact GH3 cells. The half-maximally effective [Ca2+] for stimulating PRL release in permeable cells was approximately 0.5 microM, and maximal stimulation was obtained at 3-10 microM Ca2+. The magnitude of Ca2+ stimulation in permeable cells was in the same range as that obtained from an equal number of intact cells stimulated by depolarizing K+. Moreover, the Ca2+ sensitivity of PRL release in intact GH3 cells (measured by Quin 2 fluorescence) closely resembled the Ca2+ sensitivity determined in permeable cells. Release of a sulfated proteoglycan whose release is stimulated by secretagogues in intact cells was stimulated by Ca2+ in permeable cells with the same Ca2+ sensitivity as for PRL release. Maximal Ca2+ stimulation of PRL release in permeable cells required the addition of MgATP. Other energy sources (ADP, GTP, and inorganic phosphate) also supported Ca2+-stimulated secretion but were less effective. The above results indicated that PRL release from permeable cells resembles the physiological process in intact cells. The permeable cell system should prove useful in investigating the mechanism mediating the effect of Ca2+ on secretion, although our studies with pharmacological agents have so far proved inconclusive. Among calmodulin antagonists tested, only trifluoroperazine inhibited Ca2+-stimulated secretion, whereas pimozide and calmidazolium did not.     

Protein phosphorylation in permeabilized pancreatic islet cells.

A system of digitonin-permeabilized islet cells was developed to characterize Ca2+- and calmodulin-dependent protein phosphorylation further and to determine whether activation of this membrane-bound process was sufficient for initiation of Ca2+-stimulated insulin secretion. The efficacy of digitonin in permeabilizing the plasma membrane was assessed by Trypan Blue exclusion, by extracellular leakage of lactate dehydrogenase, and by permeability to [gamma-32P]ATP. This treatment did not detectably alter the ultrastructure of the permeabilized cells. Digitonin was equally effective when presented to islet cells that had been previously dispersed or directly to intact isolated islets. The Ca2+- and calmodulin-dependent phosphorylation of endogenous membrane-bound substrates could be demonstrated in the permeabilized cells incubated with [gamma-32P]ATP. This activity displayed characteristics that were similar to those described for the protein kinase measured in subcellular fractions and was dependent on addition of exogenous calmodulin, indicating that calmodulin had been removed from the kinase by permeabilization of the cells. Ca2+-dependent insulin release by the digitonin-permeabilized islet was demonstrated, with half-maximal release occurring at 0.1 microM-free Ca2+ and maximal secretion at 0.2 microM-free Ca2+. Under these conditions, calmodulin did not further enhance insulin release, although a stimulatory effect of calmodulin was observed in the absence of free Ca2+. These studies indicate that the permeabilized-islet model will be useful in dissecting out the factors involved in Ca2+-activated insulin secretion.     

Calmodulin is involved in catecholamine secretion from digitonin-permeabilized bovine adrenal medullary chromaffin cells.

The role of calmodulin in exocytotic secretion was studied using digitonin-permeabilized bovine adrenal medullary chromaffin cells. Addition of calmodulin to the permeabilized cells increased Ca(2+)-dependent norepinephrine release in a dose-dependent manner. Unlike calmodulin, addition of caldesmon, actin or bovine serum albumin did not increase the release. Calmodulin increased the release at Ca2+ concentrations of more than 10(-6) M and its effect increased with increase in Mg2+ concentration. Th release of norepinephrine enhanced by calmodulin was inhibited by tetanus toxin, which specifically inhibits exocytotic secretion. These results indicate directly that calmodulin plays an important role in exocytotic secretion from chromaffin cells.     

Caldesmon: A calmodulin — Binding actin — Regulatory protein

The protein caldesmon, originally isolated from smooth muscle tissue where it is the most abundant calmodulin-binding protein, has since been shown to have a wide distribution in actin- and myosin- containing cells where it is localized in sub-cellular structures concerned with motility, shape changes and exo- or endo-cytosis. Caldesmon is believed to be an actin- regulatory protein, and binds with high affinity to actin or actin-tropomyosin. Caldesmon inhibits the activation by actin-tropomyosin of myosin MgATPase activity, and the inhibition can be reversed by Ca2+.calmodulin. The binding of caldesmon to smooth muscle proteins has been studied in detail, enabling a model to be constructed which could account for the observed Ca2+ regulation of smooth muscle thin filaments. The abundance of caldesmon, and the Ca2+-regulation of its activity via calmodulin, mean that it is potentially an important intracellular regulator of processes such as smooth muscle contraction, cell motility and secretion.     

The calmodulin-binding domain mediates the self-association of the plasma membrane Ca2+ pump.

Dimerization (oligomerization) of the plasma membrane Ca2+ pump increases its activity (Kosk-Kosicka, D., Bzdega, T., and Wawrzynow, A. (1989) J. Biol. Chem. 264, 19495-19499). Fluorescence titration on preparations of the purified eosin-labeled human erythrocyte ATPase has been used to monitor the oligomerization process. Calmodulin inhibits oligomerization, although it can bind to the oligomerized enzyme. Synthetic peptides corresponding to the calmodulin-binding domain of the pump stimulate its ATPase activity, indicating the formation of heterooligomers of the peptides with the pump. The oligomerization is prevented by the preincubation of the ATPase with calmodulin. Polyclonal antibodies against the synthetic calmodulin-binding domain inhibit its basal and its calmodulin-stimulated ATPase activity and prevent the formation of the oligomers. ATPase preparations truncated at the COOH terminus with calpain to a fragment of 124 kDa which does not contain the calmodulin-binding domain fail to oligomerize with the intact ATPase. The results show that the calmodulin-binding domain mediates the oligomerization of the Ca2+ pump.     

A new method for cell permeabilization reveals a cytosolic protein requirement for Ca2+ -activated secretion in GH3 pituitary cells

Ca2+ is a major regulator of exocytosis in secretory cells, however, the biochemical mechanisms underlying regulation remain to be identified. To render the secretory apparatus accessible for biochemical studies, we have developed a cell permeabilization method (cell cracking) which utilizes mechanical shear. GH3 pituitary cells subjected to cracking were permeable to macromolecules but retained a normal cytoplasmic ultrastructure including secretory granules. Incubation of the permeable cells at 30-37 degrees C with 0.1-1.0 microM Ca2+ and millimolar MgATP resulted in the release of the secretory proteins, prolactin (PRL) and a proteoglycan, but not lysosomal enzymes. Extensively washed permeable cells were incapable of releasing PRL in response to Ca2+ and MgATP addition. However, addition of cytosol was found to restore Ca2+-activated, MgATP-dependent PRL release. The cytosolic factor responsible for activity was thermolabile and protease sensitive. The protein was partially purified, and its molecular mass was estimated to be equivalent to that of a globular protein of 200-350 kDa by molecular sieve chromatography. Inhibitors of calmodulin or protein kinase C (trifluroperazine, calmidazolium, H-7) failed to inhibit Ca2+-activated PRL release, and the required cytosolic protein could not be replaced by purified calmodulin, calmodulin-dependent protein kinase II, protein kinase C, or calpactin I. Further purification and characterization of the cytosolic protein should reveal the nature of biochemical events involved in regulated secretory exocytosis.     

Calmodulin- and protein phosphorylation-independent release of catecholamines from PC-12 cells

Catecholamine secretion from PC-12 cells can be triggered by agents that increase intracellular Ca2+ and is enhanced by phorbol esters and agents that elevate intracellular cAMP concentrations. In mutant PC-12 cells lacking cAMP-dependent protein kinase (PK-A) in which protein kinase C (PK-C) was down-regulated, Ca2+-dependent secretion occurred normally but was no longer enhanced by cAMP or phorbol esters. In digitonin-permeabilized PC-12 cells that lacked PK-C and PK-A, a range of calmodulin (CaM) inhibitors failed to block Ca2+-triggered catecholamine release. Moreover, Mn2+, a CaM activator, failed to trigger catecholamine release whereas Ba2+, which does not activate CaM, supported secretion. These results indicate that the basic mechanism of stimulus/secretion coupling in PC-12 cells does not absolutely require a regulated protein phosphorylation- or calmodulin-dependent step.     

Cardiac sarcoplasmic-reticulum calmodulin-binding proteins. Modulation of calmodulin binding to phospholamban by phosphorylation.

The gel-overlay technique with 125I-labelled calmodulin allowed the detection of several calmodulin-binding proteins of Mr 280 000, 150 000, 97 000, 56 000, 35 000 and 24 000 in canine cardiac sarcoplasmic reticulum. Only two calmodulin-binding proteins could be identified unambiguously. Among them, the 97 000-Mr protein that undergoes phosphorylation in the presence of Ca2+ and calmodulin, is likely to be glycogen phosphorylase. In contrast, the (Ca2+ + Mg2+)-activated ATPase did not appear to bind calmodulin under our experimental conditions. The second known calmodulin target is dephosphophospholamban, which migrates with an apparent Mr of 24 000. The dimeric as well as the monomeric form of phospholamban was found to bind calmodulin. Phospholamban shifts the apparent Kd of erythrocyte (Ca2+ + Mg2+)-activated ATPase for calmodulin, suggesting thus a tight binding of calmodulin to the proteolipid. Interestingly enough, phospholamban phosphorylation by either the catalytic subunit of cyclic AMP-dependent protein kinase or the Ca2+/calmodulin-dependent phospholamban kinase was found to inhibit calmodulin binding.     

钙调蛋白及其结合蛋白对神经递质释放的调控作用

钙离子（Ca2+）是调节突触前神经递质的胞吐释放的关键离子信号.作为胞内最普遍存在的钙离子感受器的钙调蛋白（CaM）被发现能通过与多种蛋白的相互作用,调控着突触小泡的生发、运输及再填充,从而传递胞内Ca2+浓度变化的信号,对神经递质的释放及突触电生理活动起到至关重要的调控作用.本文综述了CaM及其结合蛋白是如何参与对突触小泡的胞吐释放和胞吞恢复的调控,并探讨了其中可能的分子机制.     

Calmodulin-independent inhibition of platelet phospholipase A2 by calmodulin antagonists

We tested the effects of calmodulin, two types of calmodulin antagonists, and various phospholipids on the phospholipase A2 activities of intact platelets, platelet membranes, and partially purified enzyme preparations. Trifluoperazine, chlorpromazine (phenothiazines) and N-(6-amino-hexyl)-5-chloro-1-naphthalenesulfonamide (W-7), at concentrations which antagonize the effects of calmodulin, significantly inhibited thrombin- and Ca2+ ionophore-induced production of arachidonic acid metabolites by suspensions of rabbit platelets and Ca2+-induced arachidonic acid release from phospholipids of membrane fractions, but not phospholipase A2 activity in purified enzyme preparations. The addition of acidic phospholipids, but not calmodulin, stimulated phospholipase A2 activity in purified enzyme preparations while decreasing its Km for Ca2+. The dose-response and kinetics of inhibition by calmodulin antagonists of acidic phospholipid-activated phospholipase A2 activity in purified preparations were similar to those of Ca2+-induced arachidonic acid release from membrane fractions. Calmodulin antagonists were also found to inhibit Ca2+ binding to acidic phospholipids in a similar dose-dependent manner. Our results suggest that the platelet phospholipase A2 is the key enzyme involved in arachidonic acid mobilization in platelets and is regulated by acidic phospholipids in a Ca2+-dependent manner and that calmodulin antagonists inhibit phospholipase A2 activity via an action on acidic phospholipids.     

Transmitter release increases intracellular calcium in perisynaptic Schwann cells in situ.

Glial cells isolated from the nervous system are sensitive to neurotransmitters and may therefore be involved in synaptic transmission. The sensitivity of individual perisynaptic Schwann cells to activity of a single synapse was investigated, in situ, at the frog neuromuscular junction by monitoring changes in intracellular Ca2+ in the Schwann cells. Motor nerve stimulation induced an increase in intracellular Ca2+ in these Schwann cells; this increase was greatly reduced when transmitter release was blocked. Furthermore, local application of the cotransmitters acetylcholine and ATP evoked Ca2+ responses even in the absence of extracellular Ca2+. Successive trains of nerve stimuli or applications of transmitters resulted in progressively smaller Ca2+ responses. We conclude that transmitter released during synaptic activity can evoke release of intracellular Ca2+ in perisynaptic Schwann cells. This Ca2+ signal may play a role in the maintenance or modulation of a synapse. These data show that synaptic transmission involves three cellular components with both postsynaptic and glial components responding to transmitter secretion.     

Effect of Ca2+-antagonists on virally-induced cell-permeability changes

Sendai virus-mediated permeability changes in cells are affected by extracellular Ca2+ or Mn2+ as follows: the lag period to onset of permeability changes is lengthened and the subsequent extent of leakage is reduced. Drugs that block Ca2+ action in excitable cells, such as verapamil and prenylamine, and drugs that inhibit the action of calmodulin, such as trifluoperazine and R24571, have an effect opposite to that of Ca2+: lag is shortened and extent of leakage is increased. The concentration at which either type of drug shows 50% of maximal effect is similar to the concentration at which 50% of binding by drug to calmodulin is achieved. It is concluded that calmodulin may be involved in protecting cells against virally-mediated membrane damage; alternatively the action of calmodulin-binding drugs may not be as specific as currently thought.     

Divergent pharmacological responses to two calmodulin inhibitors, chlorpromazine and W-7, in rat pancreatic acinar cells

The possible role of calmodulin in exocytotic secretion is supported by the presence of this calcium-binding protein in secretory cells, and by the antisecretory effects in intact secretory cells of substances which can antagonize calmodulin-stimulated enzymes in broken cell preparations. In this study, two in vitro calmodulin antagonists, W-7 and chlorpromazine, were found to produce both similar and different pharmacological effects on the secretory process in rat exocrine pancreas. Both substances blocked amylase secretion in response to carbachol or cholecystokinin octapeptide, but only chlorpromazine inhibited the ability of carbachol to stimulate 45Ca efflux from isotope-preloaded cells. Only W-7 could inhibit the secretory response to vasoactive intestinal peptide (VIP); but both W-7 and chlorpromazine were equipotent partial antagonists of VIP-stimulated cyclic AMP synthesis. Chlorpromazine increased the secretory response to melittin but W-7 did not. The divergence in biological responsiveness to W-7 and chlorpromazine makes it difficult to extrapolate the in vitro effects of these agents to similar actions in intact cell systems.