Expression of transforming growth factor alpha (TGF alpha) in differentiated rat mammary tumors: estrogen induction of TGF alpha production

Primary well-differentiated dimethylbenzene alpha-anthracene (DMBA)-or nitrosomethylurea (NMU)-induced rat mammary adenocarcinomas that are estrogen dependent possess biologically active and immunoreactive transforming growth factor alpha (TGF alpha), which can be detected in a sort agar growth-promoting assay and by a specific liquid-phase competitive RIA, respectively. In contrast, tissue extracts prepared from transplantable undifferentiated DMBA-I and NMU-II rat mammary carcinomas that are estrogen independent and metastatic exhibit low or undetectable levels of TGF alpha. In addition, the primary DMBA- and NMU-induced rat mammary adenocarcinomas express a specific 4.8-kilobase TGF alpha mRNA species, whereas little or no TGF alpha mRNA can be detected in the transplantable DMBA-I and NMU-II tumors. Primary tumors synthesize type IV basement membrane collagen, whereas the transplantable tumors elaborate very little type IV collagen. Either TGF alpha or estrogens can differentially enhance the synthesis of type IV collagen by 0.5- to 4-fold over total protein synthesis in primary cultures of normal mouse mammary epithelial cells or in primary NMU-induced tumor cells, respectively. Therefore, TGF alpha could function as an estrogen-inducible autocrine growth factor for well differentiated rat mammary tumor cells by its ability to selectively regulate type IV collagen synthesis. Estrogens can modulate TGF alpha production in vivo in primary DMBA-induced rat mammary tumors, because ovariectomy results in a rapid decline (within 6 h) of TGF alpha mRNA levels. This response to estrogens can also be observed in vitro. Primary DMBA- or NMU-induced rat mammary tumor cells cultured in the presence of 17 beta-estradiol (10(-8) M) for 4 days show an increase in the level of TGF alpha mRNA over cells not treated with estrogen. This increase in TGF alpha mRNA is paralleled by a 2- to 3-fold increase in the levels of immunoreactive TGF alpha that can be detected and in the conditioned medium from estrogen-treated cells. These results suggest that TGF alpha may be an adjunct marker for those mammary tumors that are well differentiated adenocarcinomas and estrogen dependent and that estrogen-independent tumors do not constitutively produce TGF alpha or express TGF alpha mRNA.     

Nuclear topoisomerase I and DNase activities in rat diethylnitrosamine-induced hepatoma, in regenerating and fetal liver.

DNase activity in the presence of Ca2+ + Mg2+, Mg2+ alone, Mn2+ alone, or EDTA, and topoisomerase I activity were measured in nuclear extracts of diethylnitrosamine (DEN)-induced hepatomas, regenerating, fetal, and normal rat livers. In hepatoma tissue, the Ca/Mg-dependent DNase activity was lower than in normal tissue and nearly the same as in fetal liver. In the poorly differentiated hepatomas, Mn-dependent DNase activity was higher than in both moderately and well differentiated ones and than in normal liver tissue. The activity of topoisomerase I in hepatomas and in regenerating liver was lower than in normal liver tissue.     

Cell nuclear DNases: multiplicity and heterogeneity

In order to determine the ratio of activities of major endonucleases of rat liver chromatin, a stepwise fractionation of cell nuclear extracts by chromatography on phosphocellulose and gel filtration through Toyopearl HW60 was carried out. This procedure resulted in partially purified preparations of Ca2+,Mg2+-dependent endonuclease (55 +/- 10 kD), Ca2+,Mg2+-dependent endonuclease (30 +/- 10 kD), Mn2+-dependent endonuclease (30 +/- 5 kD) and acid cation-independent endonuclease. The Ca2+,Mg2+-dependent endonuclease with Mr of 55 +/- 10 kD made up to 57% of the nuclear extract activity in the presence of Ca2+ + Mg2+ and revealed a high calcium-magnesium synergism. Under the same experimental conditions, the 30 +/- 10 kD enzyme made up to 33% of the nuclear extract activity and revealed a low synergism. The activity of Mn2+-dependent endonuclease made up to 26% of the total nuclear extract activity in the presence of Mn2+, that of acid endonuclease--11% of the extract activity in 1 mM EDTA at pH 5.0. It was assumed that the low molecular weight Ca2+,Mg2+-dependent endonuclease represents a product of limited proteolysis of high molecular weight Ca2+,Mg2+-dependent endonuclease.     

Ca2+-stimulated ribonuclease. A new marker enzyme of differentiated rat mammary tissues.

A unique ribonuclease, active only in the presence of Ca2+, was present in lactating mammary gland and milk of the rat. This enzyme was absent from virgin-rat mammary gland and non-mammary tissues of lactating rats. The presence of moderate activity in differentiated mammary tumours, together with an increase in activity in normal tissue parelleling development of mammary function, identify this enzyme as a marker of mammary differentiation in the rat.     

Evidence for the existence of actomyosin ATPase in the rat pancreas. Isolation and biochemical characterization

In a crude extract of rat pancreas, myosin was associated with a protein having the same electrophoretic mobility as actin. This myosin was purified after dissociation of the actomyosin complex with KI-ATP. On sodium dodecylsulfate/acrylamide gel electrophoresis, the isolated pancreatic myosin showed a major component of approximately 200 kDa, and two smaller components with apparent molecular weight of 22 and 15 kDa, respectively. This purified myosin exhibited high ATPase activity in the presence of K+ + EDTA or Ca2+ and very little activity in the presence of Mg2+. (K+ + EDTA)-ATPase activity showed one pH optimum at 8.0, while Ca2+-ATPase activity showed two pH optima at 6.0 and 9.0, respectively. (K+ + EDTA)-stimulated enzyme activity was specific for ATP whereas Ca2+-stimulated activity showed low specificity for nucleoside triphosphates.     

Antitumor activity and mechanism of action of different antiprogestins in experimental breast cancer models

Onapristone and other antiprogestins proved to possess a potent antitumor activity in several hormone-dependent experimental breast cancer models. This activity is as strong or even better than that of tamoxifen or ovariectomy in the MXT-mammary tumor of the mouse and the DMBA- and MNU-induced mammary tumor of the rat. The antitumor activity is evident in these models in spite of elevated serum levels of ovarian and pituitary hormones. The detailed analysis of all our data including the morphological (ultrastructure) studies of the mammary tumors of treated animals and the effects on growth and cell cycle kinetics using DNA flow cytometry indicates that the antitumor action of antiprogestins is mediated via the progesterone receptor and related to the induction of terminal cell differentiation leading to increased cell death. The strong antitumor activity of antiprogestins in our experimental breast cancer models does not primarily depend on a classical anti-hormonal mechanism. The antiprogestin-related reduction of the number of mammary tumor cells in the S-phase in our experimental tumor models (G₀G₁ arrest) emphasizes the unique innovative mechanism of action of these new agents in the treatment of human breast cancer.     

Purification and characterization of actinogelin, a calcium-sensitive actin-accessory protein, from rat liver

Although cell-free extracts prepared from several types of free-living cells, including Ehrlich tumor cells, macrophages and sea-urchin eggs, readily form gels under low Ca2+ conditions, no such ability to induce actin-related gel has been detected in tissue-cell extracts. Ca2+ -insensitive gelation activity was discovered, however, in several tissue-cell extracts, including liver and brain, provided that the extracts were supplemented with skeletal muscle actin. Based on sodium dodecylsulfate/polyacrylamide gel electrophoretic analysis of the gel, these extracts seem to contain both a Ca2+ -insensitive gelation factor and Ca2+ -sensitive one, actinogelin. A procedure for purification of actinogelin from rat liver was developed, and the properties of actinogelin thus purified were compared with those of Ehrlich tumor cell actinogelin. No appreciable difference was found in these two proteins, and Ca2+ sensitivity (50% inhibition of gelation at 1 microM) was very similar. Some of the molecular characteristics are described, and the importance of the presence of actinogelin in tissue cells is discussed.     

Purification and characterization of myosin from the clonal rat glial cell strain C-6.

A myosin was isolated from the clonal rat glial cell strain C-6 and compared with rat skeletal muscle myosin. After cell extracts were subjected to gel filtration chromatography in the presence of KI and magnesium pyrophosphate the C-6 myosin was rapidly purified by a procedure similar to that used for skeletal muscle myosin. The C-6 myosin resembles muscle myosin both physically and enzymatically. It contains heavy chains of 200,000 daltons and two classes of light chains of 17,000 and 19,000 daltons in approximately equal molar ratios. This myosin forms bipolar thick filaments in 0.1 M KCl and binds reversibly to skeletal muscle F-actin, the binding being inhibited by MgATP. Skeletal muscle F-actin stimulates the C-6 myosin adenosine triphosphatase 2- to 3-fold in the presence of KCl and Mg2+. The action activation of muscle myosin ATPase at low ionic strength is 10-fold greater than that of C-6 myosin. Ca2+ and EDTA stimulated the ATPase activities of both enzymes. When assayed in the presence of 0.6 M KCl and 1 mM EDTA the skeletal muscle myocin ATPase demonstrates substrate saturation while the C-6 myosin enzyme activity is stimulated by ATP concentrations above 2.5 mM.     

The presence of nitric oxide synthase in mucosal epithelial cells of the frog urinary bladder

In experiments on frog Rana temporaria L. urinary bladder, we investigated localization of NO-synthase (NOS) in urinary bladder slices and measured NOS activity in the suspension of mucosal epithelial cells. Intensive NADPH-diaphorase staining which is widely used as an indicator of NOS activity was found in mucosal epithelium. Almost all mucosal epithelial cells isolated in Ca2+ -free conditions demonstrated positive NADPH-diaphorase reactivity. Direct measurement of NOS activity in suspension of mucosal cells determined by the rate of conversion of L-arginine to L-citrullin showed that the enzyme activity was reduced in absence of external Ca2+ and was inhibited by L-NAME: non-specific NOS inhibitor, and 1400 W: a highly selective iNOS inhibitor (control: 754 +/- 184; L-NAME, 1 mM 329 +/- 87; 1400 W, 20 mM: 547 +/- 25; Ca2+ -free/EDTA: 490 +/- 184 cpm [3H]-citrullin/10(6) cells per 45 min, p < 0.05, n = 7-8). The data obtained demonstrate that frog urinary bladder mucosa epithelial cells provided antidiuretic hormone-induced increase of osmotic water permeability contain nitric oxide synthase. The presence of inducible (iNOS) as well as constitutive isoform(s) revealed in these cells allows to suggest involvement of NOS in intracellular signaling pathways regulated water transport across the epithelium.     

Phosphatidylinositol-4,5-bisphosphate phosphodiesterase and phosphomonoesterase activities of rat brain. Some properties and possible control mechanisms.

The phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] [and to a lesser extent, the phosphatidylinositol-4-phosphate (PtdIns4P)] phosphodiesterase and monoesterase activities of a rat brain supernatant have been studied by using 32P-labelled substrates prepared from human red blood cells. PtdIns(4,5)P2 monoesterase is maximally stimulated by Mg2+, though some activity is detectable in Ca2+/EDTA (Mg2+-free) buffers. The phosphodiesterase, however, is Ca2+-dependent, and in Ca2+/EDTA buffers with the pure lipid as substrate, shows maximal activity at 100 nM-Ca2+. If PtdIns(4,5)P2 is presented as a component of a lipid mixture of similar composition to that of the inner half of the lipid bilayer of a rat liver plasma membrane, the phosphodiesterase shows considerable activity at 1 microM-Ca2+, and is maximal at 100 microM-Ca2+. However, if it is assayed against the same substrate in Ca2+/EGTA buffers with 3mM-Mg2+ and 80 mM-KCl present (as an approximate parallel with the ionic environment in vivo), it shows no detectable activity below 100 microM-Ca2+, and is maximal at 1 mM-Ca2+. The monoesterase can hydrolyse PtdIns(4,5)P2 in such a lipid mixture at all Ca2+ concentrations with 1 or 3 mM-Mg2+ present. PtdIns(4,5)P2 phosphodiesterase can be induced to attack its substrate under ionic conditions similar to those in vivo (0.1-1 microM-Ca2+; 1 mM-Mg2+; 80 mM-KCl) by the conversion of its substrate into a non-bilayer configuration. If given such a substrate [by mixing PtdIns(4,5)P2 with an excess of phosphatidylethanolamine (PtdEtn)] it shows a shallow Ca2+-dependency curve from 0.1 to 100 microM and then a steep rise to 1 mM-Ca2+. Together these observations lead us to the suggestion that a perturbation in a membrane in vivo equivalent to a non-bilayer configuration would be sufficient to induce phosphodiesterase-catalysed PtdIns(4,5)P2 breakdown. When given substrates mixed with excess PtdEtn at pH 7.25 (or 5.5), 1 microM-Ca2+, 1 mM-Mg2+ and 80 mM-KCl, the rat brain supernatant phosphodiesterase activity hydrolysed PtdIns(4,5)P 50-100-fold faster than it hydrolysed phosphatidylinositol (PtdIns). If the supernatant was presented with such a non-bilayer mixture containing a ten-fold excess of PtdIns over PtdIns(4,5)P2, the latter phospholipid was still hydrolysed by phosphodiesterasic cleavage at nearly ten times the rate of the former. Receptor-stimulated phosphodiesterase cleavage of polyphosphoinositides is an early event in cell activation by many agonists. The properties of PtdIns(4,5)P2 phosphodiesterase in vitro suggest that a change in the presentation of its substrate would be a sensitive and sufficient control on the enzyme's activity in vivo.     

The Role of Bivalent Cations on the Isolation of Allantoinase from the Soybean Seed Extracts

Allantoinases (ALNase) in the water extracts distilled from seeds and leaves of soybean (Glycine max L. cv. Keyu 10) were. remarkably heat stable. However the enzyme and non-enzyme protein in the seed extract, but not leaf extract, lost their activity and were denaturated at 75℃ for 5 min in the presence of Ca2+, Mg2+. or Mn2+ions respecitively. At room temperature over 40%～50% of the non-enzyme proteins in the seed extract could be removed by the bivalent cations without affecting the enzyme activity. This effect was weakend by the increase of concentration. Both extracts had different responses to all sorts of insoluble Ca2. salts. For the seed extract about 50 % of the non-enzyme proteins were removed by 5 % CaSO4 (W/V), without effecting the enzyme activity, while the leaf extract was sensible to Ca3 (PO4) 2. After treatment with 5 % Ca3 (PO4) 2 about 50 % of the enzyme activities and about 70% of proteins were lost. Mn2+ ions could enhance the enzyme activity in crude seed extract, but had no effect on partially purified enzyme from seeds and enzyme in crude extract from leaves. Further, EDTA had no effect on enzyme activity in both extracts.     

Interconversion of androgen receptor forms by divalent cations and 8 S androgen receptor-promoting factor. Effects of Zn2+, Cd2+, Ca2+, and Mg2+

The effects of divalent cations (Zn2+, Cd2+, Ca2+, Mg2+) on the cytosol androgen receptor were determined by sedimentation into sucrose gradients. At low ionic strength (25 mM KCl, 50 mM Tris, pH 7.4), Zn2+ (200 microM total, which calculates to 130 nM free Zn2+ in 10 mM mercaptoethanol) causes a shift in the sedimentation coefficient of the rat Dunning prostate tumor (R3327H) cytosol receptor and rat ventral prostate cytosol receptor from 7.5 +/- 0.3 S to 8.6 +/- 0.3 S. Zn2+ stabilizes the 8.6 S receptor form in salt concentrations up to 0.15 M KCl in 50 mM Tris, pH 7.2. In low ionic strength gradients containing Ca2+ (greater than or equal to 200 microM) or Mg2+ (greater than or equal to 1 mM), the receptor sediments as 4.7 +/- 0.3 S. The dissociating effects of Ca2+ and Mg2+ can be fully reversed by sedimentation into gradients containing Zn2+ (200 microM total) or Cd2+ (10 microM total). In the presence of Zn2+ (200 microM total), Ca2+ (10 microM to 3 mM) converts the receptor to an intermediate form with sedimentation coefficient 6.2 +/- 0.2 S, Stokes radius 73 A, and apparent Mr approximately 203,000. The potentiating effect of Zn2+ on formation of the 8.6 S receptor (in the absence of Ca2+) and the 6.2 S receptor (in the presence of Ca2+) requires both the 4.5 S receptor and the 8 S androgen receptor-promoting factor. Sodium molybdate stabilizes the untransformed cytosol receptor but, unlike Zn2+, does not promote reconstitution of the 8.6 S receptor from its partially purified components. These results indicate that divalent cations alter the molecular size of the androgen receptor in vitro and thus may have a role in altering the state of transformation of the receptor.     

Purification of (Ca2+-Mg2+)-ATPase from rat liver plasma membranes

The Ca2+-stimulated, Mg2+-dependent ATPase from rat liver plasma membranes was solubilized using the detergent polyoxyethylene 9 lauryl ether and purified by column chromatography using Polybuffer Exchanger 94, concanavalin A-Sepharose 4B, and Sephadex G-200. The molecular weight of the enzyme, estimated by gel filtration in the presence of the detergent on a Sephadex G-200 column, was 200,000 +/- 15,000. The enzyme was purified at least 300-fold from rat liver plasma membranes and had a specific activity of 19.7 mumol/mg/min. Polyacrylamide gel electrophoresis under nondenaturing conditions of the purified enzyme indicated that the enzymatic activity correlated with the major protein band. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, one major band in the molecular weight range of 70,000 +/- 5,000 was seen. The isoelectric point of the purified enzyme was 6.9 +/- 0.2 as determined by analytical isoelectric focusing. The enzyme was activated by Ca2+ with an apparent half-saturation constant of 87 +/- 2 nM for Ca2+. Calmodulin and trifluoperazine at the concentration of 1 microgram/ml and 100 microM, respectively, had no effect on the enzymatic activity.     

An endogenous protein inhibitor of DNA polymerase alpha in normal and neoplastic rat mammary tissues

Extracts of whole tissue or isolated nuclei from lactating rat mammary gland that has diminished cell replication capacity were more active than the corresponding extracts of pregnant rat mammary gland that contains actively replicating cells in causing a dose-dependent inhibition of DNA polymerase alpha in vitro. Purification of the inhibitor from both tissue and nuclear extracts using a sequence of Sephacryl S200, DEAE-cellulose and CM52 columns confirmed the above assay results. Using the same assay and purification procedures, both tissue and nuclear extracts from the rapidly growing transplanted R3220AC mammary tumors exhibited very little or no inhibitor activity. The partially purified mammary inhibitor (mol. wt of 155kD, high A280 nm/A260 nm ratio, heat labile) was equally inhibitory to the purified DNA polymerase alpha from either R3230AC tumor or calf thymus, and to the nuclear matrix bound DNA polymerase alpha of R3230AC tumor.     

The antitumor potency of progesterone antagonists is due to their differentiation potential.

A new therapy for the progesterone receptor positive mammary carcinoma may be the treatment with progesterone antagonists. This new class of antihormones causes a strong inhibition of tumor growth comparable to the potency of ovariectomy in a panel of experimental mammary carcinomas. The mechanisms of the strong tumor-inhibiting action of progesterone antagonists on experimental mammary carcinomas mainly depends on a progesterone receptor mediated process leading to induction of terminal differentiation and a blockade of the cell cycle. To further characterize the antitumor mechanism of progesterone antagonists we analyzed the effects of Onapristone and ZK 112.993 on DMBA- and MNU-mammary tumors of the rat and MXT-tumors of the mouse after different therapy intervals. These hormone-dependent mammary tumors normally display intraductal growth in papillary, cribiform or solid formation, whereas after treatment periods of 2-6 weeks with progesterone antagonists they displayed dysplastic ductal and acinous formations, usually filled with secretory material. Whereas tumor size, mitotic index, and the grade of tumor malignancy decreased distinctly, the volume fraction of glandular structures in the tumors as well as the appearance of apoptosis increased 3-fold compared to the controls. In addition, the mammary glands of progesterone antagonist treated animals showed the morphological features of differentiation with the appearance of secretory activity. Interestingly, the staining pattern of some of the lectins used, especially UEA 1 binding pattern, fits to the concept of differentiation since recent studies revealed a higher degree of fucosylation only in benign lesions of human breast cancers. Therefore, these data underline the concept of a differentiation potential of progesterone antagonists on progesterone receptor positive mammary carcinomas.     

Detergent Effects on the Phosphatidylinositol-Specific Phospholipase C in Rat Brain Synaptosomes

In the presence of Ca2+ (2.5 mM) and using [14C]arachidonoyl phosphatidylinositol (PI) membrane as substrate, phosphatidylinositol-specific phospholipase C (PI-PLC) (EC 3.1.4.10) in rat brain synaptosomes was activated by deoxycholate but not taurocholate. Calcium stimulated enzymic hydrolysis by both detergents, but the stimulatory effect of taurocholate was less than that of deoxycholate. Peak stimulation for deoxycholate was observed at 1 mg/ml, whereas that for taurocholate was 4 mg/ml. When 1 mM EDTA was added to the taurocholate (4 mg/ml) and Ca2+ (3.5 mM) system, synaptosomal PI-PLC activity was greatly stimulated, to almost the same level as the deoxycholate + Ca2+ system. This system required the presence of all three factors, and EGTA could not effectively replace EDTA in the stimulatory action. The detergent-induced hydrolysis of synaptosomal PI by the deoxycholate + Ca2+ and the taurocholate + Ca2+ + EDTA systems was strongly inhibited by divalent metal ions such as Zn2+, Cu2+, Pb2+, and Fe2+, whereas Mg2+ and Ca2+ were ineffective. Nevertheless, only the deoxycholate + Ca2+ system was responsive to enzyme inhibition by membrane-perturbing agents such as lysophospholipids and free fatty acids. The specific requirement for EDTA in the taurocholate system may be due to the release of a pool of inhibitory divalent metal ions from the membranes.     

Generation of the Ca2(+)-independent form of Ca2+/calmodulin-dependent protein kinase II in cerebellar granule cells

Conditions that regulate the generation of the Ca2(+)-independent form of Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) in cultured rat cerebellar granule cells have been investigated. Under basal conditions, 4-5% of total CaM-kinase II activity, assayed in the presence of Ca2+/CaM, was the Ca2(+)-independent form active in the presence of EGTA. Depolarization with 56 mM K+ produced a transient increase to 9% Ca2+ independence within 15 s followed by a decline to 5-6% at 10 min. The divalent cation ionophore ionomycin elicited 10% Ca2+ independence, which remained elevated. Removal of Ca2+ from the Krebs-Ringer medium reduced basal Ca2+ independence to 1-2% and eliminated the elevation in response to K+ depolarization. Inclusion of 5 microM okadaic acid, a protein phosphatase inhibitor, in the incubation medium potentiated the levels of Ca2(+)-independent activity of CaM-kinase II. Additional studies in granule cell extracts indicated that there were both okadiac acid-sensitive and -insensitive protein phosphatases involved in the reversal of the Ca2+ independence of CaM-kinase II. Phosphopeptide mapping of the CNBr-cleaved 32P-labeled 58-60-kDa subunit of CaM-kinase II revealed that under basal conditions, the kinase contained phosphate in many sites. Conditions that promoted formation of the Ca2(+)-independent form of the kinase increased the 32P incorporation into multiple sites of the kinase. However, there was a good temporal correlation between 32P incorporation into CNBr peptide 1, which contains Thr-287, and generation of the Ca2(+)-independent kinase activity. These results indicate that formation of the Ca2(+)-independent species of CaM-kinase II is dynamically regulated in cerebellar granule cells by Ca2(+)-mobilizing agents and by protein phosphatase activity and is correlated with autophosphorylation of Thr-287.     

Cyclic nucleotide phosphodiesterase of rat pancreatic islets. Effects of Ca2+, calmodulin and trifluoperazine.

Cyclic nucleotide phosphodiesterase activity towards cyclic AMP and cyclic GMP was studied in extracts of rat islets of Langerhans. Biphasic Eadie plots [Eadie (1942) J. Biol. Chem. 146, 85-93] were obtained with either substrate suggesting the presence of both 'high'- and 'low'-Km components. The apparent Km values were 6.2 +/- 0.5 (n = 8) microM and 103.4 +/- 13.5 (6) microM for cyclic AMP and 3.6 +/- 0.3 (12) microM and 61.4 +/- 7.5 (13) microM for cyclic GMP. With cyclic AMP as substrate, phosphodeisterase activity was increased by calmodulin and Ca2+ and decreased by trifluoperazine, a specific inhibitor of calmodulin. With cyclic GMP as substrate, phosphodiesterase activity was decreased by omission of Ca2+ or addition of trifluoperazine. Addition of exogenous calmodulin had no effect on activity. The data suggest that Ca2+ may influence the islet content of cyclic AMP and cyclic GMP via effects on calmodulin-dependent cyclic nucleotide phosphodiesterase(s).     

Autophosphorylation of Ca2+/calmodulin-dependent protein kinase II. Effects on total and Ca2+-independent activities and kinetic parameters

Incubation of purified rat brain Ca2+/calmodulin-dependent protein kinase II for 2 min in the presence of Ca2+, calmodulin (CaM), Mg2+, and ATP converted the kinase from a completely Ca2+-dependent kinase to a substantially Ca2+-independent form with little loss of total activity. Subsequent addition of EGTA to the autophosphorylation reaction enhanced further autophosphorylation of the kinase which was associated with a suppression of total kinase activity to the Ca2+-independent value. Protein phosphatase 1 rapidly increased the suppressed total activity back to the control value and slowly decreased the Ca2+-independent activity. Kinetic analysis showed that the kinase not previously autophosphorylated had a Km for the synthetic peptide syntide-2 of 7 microM and Vmax of 9.8 mumol/min/mg when assayed in the presence of Ca2+ and CaM. The partially Ca2+-independent species, assayed in the presence of EGTA, had a Km of 21 microM and Vmax of 6.0. In the presence of Ca2+ and CaM the Km decreased and the Vmax increased to approximately control nonphosphorylated values. The completely Ca2+-independent form generated by sequential autophosphorylation first in the presence of Ca2+ and then EGTA had similar kinetic parameters to the partially independent species when assayed in the presence of EGTA, but addition of Ca2+ and CaM (up to 1 mg/ml) had little effect. These results suggest that separate autophosphorylation sites in the Ca2+/CaM-dependent protein kinase II are associated with formation of Ca2+-independent activity and suppression of total activity.     

Calcium uptake in isolated brush-border vesicles from rat small intestine.

Ca2+ uptake in brush-border vesicles isolated from rat duodena was studied by a rapid-filtration technique. Ca2+ uptake showed saturation kinetics, was dependent on the pH and ionic strength of the medium and was independent of metabolic energy. Uptake activity was readily inhibited by Ruthenium Red, La3+, tetracaine, EGTA, choline chloride and Na+ or K+. The effect of variations in medium osmolarity on Ca2+ uptake and the ionophore A23187-induced efflux of the cation from preloaded vesicles indicated that the Ca2+-uptake process involved binding to membrane components, as well as transport into an osmotically active space. Scatchard-plot analyses of the binding data suggested at least two classes of Ca2+-binding sites. The high-affinity sites, Ka = (2.7 +/- 1.1) x 10(4) M-1 (mean +/- S.D.) bound 3.2 +/- 0.8 nmol of Ca2+/mg of protein, whereas the low-affinity sites (Ka = 60 +/- 6 M-1) bound 110 +/- 17 nmol of Ca2+/mg of protein. In the presence of 100 mM-NaCl, 1.7 and 53 nmol of Ca2+/mg of protein were bound to the high- and low-affinity sites respectively. Decreased Ca2+-uptake activity was observed in vesicles isolated from vitamin D-deficient as compared with vitamin D-replete animals and intraperitoneal administration of 1,25-dihydroxycholecalciferol to vitamin D-deficient rats 16 h before membrane isolation stimulated the initial rate of Ca2+ uptake significantly. The data indicated that Ca2+ entry and/or binding was passive and may involve a carrier-mediated Ca2+-uptake component that is associated with the brush-border membrane. Altering the electrochemical potential difference across the membrane by using anions of various permeability and selected ionophores appeared to increase primarily binding to the membrane rather than transport into the intravesicular space. Since there is considerable binding of Ca2+ to the vesicle interior, a comprehensive analysis of the transport properties of the brush-border membrane remains difficult at present.