Fracture liaison services for osteoporosis in the Asia-Pacific region: current unmet needs and systematic literature review.

The analysis aimed to identify the treatment gaps in current fracture liaison services (FLS) and to provide recommendations for best practice establishment of future FLS across the Asia-Pacific region. The findings emphasize the unmet need for the implementation of new programs and provide recommendations for the refinement of existing ones. The study's objectives were to evaluate fracture liaison service (FLS) programs in the Asia-Pacific region and provide recommendations for establishment of future FLS programs. A systematic literature review (SLR) of Medline, PubMed, EMBASE, and Cochrane Library (2000-2017 inclusive) was performed using the following keywords: osteoporosis, fractures, liaison, and service. Inclusion criteria included the following: patients ≥ 50 years with osteoporosis-related fractures; randomized controlled trials or observational studies with control groups (prospective or retrospective), pre-post, cross-sectional and economic evaluation studies. Success of direct or indirect interventions was assessed based on patients' understanding of risk, bone mineral density assessment, calcium intake, osteoporosis treatment, re-fracture rates, adherence, and mortality, in addition to cost-effectiveness. Overall, 5663 unique citations were identified and the SLR identified 159 publications, reporting 37 studies in Asia-Pacific. These studies revealed the unmet need for public health education, adequate funding, and staff resourcing, along with greater cooperation between departments and physicians. These actions can help to overcome therapeutic inertia with sufficient follow-up to ensure adherence to recommendations and compliance with treatment. The findings also emphasize the importance of primary care physicians continuing to prescribe treatment and ensure service remains convenient. These findings highlight the limited evidence supporting FLS across the Asia-Pacific region, emphasizing the unmet need for new programs and/or refinement of existing ones to improve outcomes. With the continued increase in burden of fractures in Asia-Pacific, establishment of new FLS and assessment of existing services are warranted to determine the impact of FLS for healthcare professionals, patients, family/caregivers, and society.

Synthesis, characterization and platelet adhesion studies of novel ion-containing aliphatic polyurethanes.

Two novel ion-containing aliphatic polyurethanes based on 4,4'-methylene dicyclohexyl diisocyanate (H12MDI), polytetramethyl oxide (PTMO) were synthesized using either sulfonated or carboxylated chain extender. The nonionic polyurethane chain extended with 1,4-butanediol, which is denoted as H-M-BD, was synthesized. Pellethane, a biomedical-grade polyurethane, was also studied for comparison. The polymer's bulk, surface, and platelet-contacting properties were studied using Fourier transform infrared spectrophotometry, differential scanning calorimetry, water absorption analysis, electron spectroscopy for chemical analysis, static contact angle analysis, and in vitro platelet adhesion experiments. The effects of ion incorporation on the morphology, surface properties and blood compatibility are discussed. Unlike MDI-based Pellethane, all H12MDI-based polyurethanes are not composed of crystalline hard segment domain but are amorphous. The ionic polyurethanes exhibit a smaller fraction of hydrogen-bonded carbonyl groups, poorer phase separation, smaller fraction of PTMO residing at the surface, and smaller contact angle; however, significant higher water absorption value than H-M-BD and Pellethane. The in vitro platelet adhesion experiments indicated that ion incorporation, especially for carboxylate, significantly reduced the number and the degree of activation of the adherent platelets.

Synthesis, characterization, and platelet adhesion studies of novel aliphatic polyurethaneurea anionomers based on polydimethylsiloxane-polytetramethylene oxide soft segments.

Two novel aliphatic polyurethaneurea anionomers were synthesized based on polydimethylsiloxane (PDMS)-polytetramethylene oxide (PTMO) soft segments. The hard segments consisted of either 4,4'-methylene dicyclohexyl diisocyanate (H12MDI), sulfonic acid-containing diol and 1,4-butandiol (BD) or H12MDI, carboxylic acid-containing diol and BD. The nonionic counterpart chain extended with BD was prepared. In addition, the base nonionic polyurethaneurea containing a pure PDMS soft segment, which is denote H-D-BD, was also studied for comparison. The effects of soft segment type and ion incorporation on the physical properties, surface properties, and plateled adhesion are discussed. The ionic polyurethaneureas exhibited poor phase separation, a smaller fraction of PTMO present at the surface, and a smaller contact angle. On the other hand, it also showed a larger fraction of PDMS present at the surface and a higher water absorption value than its nonionic counterpart. H-D-BD had more phase-separated structure, a larger fraction of PDMS present at the surface, and larger contact angle but lower water absorption value than the PTMO-containing polyurethaneureas. The in vitro platelet adhesion experiments indicated that the ionic groups, especially for carboxylate, and surface enrichment PDMS soft segment could effectively inhibit platelet adhesion.

Differential regulation of cardiac actomyosin S-1 MgATPase by protein kinase C isozyme-specific phosphorylation of specific sites in cardiac troponin I and its phosphorylation site mutants.

The significance of site-specific phosphorylation by protein kinase C (PKC) isozymes alpha and delta and protein kinase A (PKA) of troponin I (TnI) and its phosphorylation site mutants in the regulation of Ca(2+)-stimulated MgATPase activity of reconstituted actomyosin S-1 was investigated. The genetically defined TnI mutants used were T144A, S43A/S45A, S43A/S45A/T144A (in which the PKC phosphorylation sites Thr-144 and Ser-43/Ser-45 were respectively substituted by Ala) and N32 (in which the first 32 amino acids in the NH2-terminal sequence containing Ser-23/Ser-24 were deleted). Although the PKC isozymes displayed different substrate phosphorylation kinetics, PKC-alpha phosphorylated equally well TnI wild type and all mutants, whereas N32 was a much poorer substrate for PKC-delta. Furthermore, the two PKC isozymes exhibited discrete specificities in phosphorylating distinct sites in TnI and its mutants, either as individual subunits or as components of the reconstituted troponin complex. Unlike PKC-alpha, PKC-delta favorably phosphorylated the PKA-preferred site Ser-23/Ser-24 and hence, like PKA, reduced the Ca2+ sensitivity of the reconstituted actomyosin S-1 MgATPase. In contrast, PKC-alpha preferred to phosphorylate Ser-43/Ser-45 (common sites for all isozymes) and thus reduced the maximal Ca(2+)-stimulated activity of the MgATPase. In this respect, PKC-delta, by cross-phosphorylating the PKA sites, functioned as a hybrid of PKC-alpha and PKA. The site specificities and hence functional differences between PKC-alpha and -delta were most evident at low phosphorylation (1 mol of phosphate/mol) of TnI wild type and were magnified when S43A/S45A and N32 were used as substrates. The present study has demonstrated, for the first time, that distinct functional consequences could arise from the site-selective preferences of PKC-alpha and -delta for phosphorylating a single substrate in the myocardium, i.e., TnI.

Phosphorylation specificities of protein kinase C isozymes for bovine cardiac troponin I and troponin T and sites within these proteins and regulation of myofilament properties.

Protein kinase C (PKC) isozymes alpha, delta, epsilon, and zeta, shown to be expressed in adult rat cardiomyocytes, displayed distinct substrate specificities in phosphorylating troponin I and troponin T subunits in the bovine cardiac troponin complex. Thus, because they have different substrate affinities, PKC-alpha, -delta, and -epsilon phosphorylated troponin I more than troponin T, but PKC-zeta conversely phosphorylated the latter more than the former. Furthermore, PKC isozymes exhibited discrete specificities in phosphorylating distinct sites in these proteins as free subunits or in the troponin complex. Unlike other isozymes, PKC-delta was uniquely able to phosphorylate Ser-23/Ser-24 in troponin I, the bona fide phosphorylation sites for protein kinase A (PKA); and consequently, like PKA, it reduced Ca2+ sensitivity of Ca2+-stimulated MgATPase of reconstituted actomyosin S-1. In addition, PKC-delta, like PKC-alpha, readily phosphorylated Ser-43/Ser-45 (sites common for all PKC isozymes) and reduced maximal activity of MgATPase. In this respect, PKC-delta functioned as a hybrid of PKC-alpha and PKA. In contrast to PKC-alpha, -delta, and -epsilon, PKC-zeta exclusively phosphorylated two previously unknown sites in troponin T. Phosphorylation of troponin T by PKC-alpha resulted in decreases in both Ca2+ sensitivity and maximal activity, whereas phosphorylation by PKC-zeta resulted in a slight increase of the Ca2+ sensitivity without affecting the maximal activity of MgATPase. Most of the in vitro phosphorylation sites in troponin I and troponin T were confirmed in situ in adult rat cardiomyocytes. The present study has demonstrated for the first time distinct specificities of PKC isozymes for phosphorylation of two physiological substrates in the myocardium, with functional consequences.

Bacterial expression of the linker region of human MDR1 P-glycoprotein and mutational analysis of phosphorylation sites.

Phosphorylation may play a role in modulating multidrug resistance by P-glycoprotein (P-gp). The linker region between the two homologous halves of human P-gp harbors several serine residues which are phosphorylated by protein kinase C (PKC) in vitro. We used the glutathione S-transferase gene fusion system to express and purify a series of fusion proteins containing the relevant portion (residues 644-689) of the linker region of the human MDR1 gene product. The fusion proteins were subjected to in vitro phosphorylation and phosphopeptide mapping analysis to identify specific phosphorylation sites. On the basis of a mutational strategy in which individual serine residues were systematically replaced with nonphosphorylatable alanine residues, Ser-661 and Ser-667 were identified as major PKC sites and Ser-683 was identified as a minor PKC site. Ser-661 and Ser-667 were also found to be the primary sites of phosphorylation for a novel membrane-associated P-gp specific kinase isolated from the multidrug-resistant KB-V1 cell line. Individual phosphorylation sites were recognized independently of each other. These data show that the linker region of P-gp represents a target for multisite phosphorylation not only for PKC but also for the P-gp specific V1 kinase. Specific serine phosphorylation sites are identified, and evidence is presented that the V1 kinase has a specificity which overlaps, but is more restricted than, that of PKC. In addition, these studies also suggest that the use of GST fusion peptides may be applicable for the analysis of multisite and ordered protein phosphorylation in other systems.

Cardiac troponin I mutants. Phosphorylation by protein kinases C and A and regulation of Ca(2+)-stimulated MgATPase of reconstituted actomyosin S-1.

The significance of site-specific phosphorylation of cardiac troponin I (TnI) by protein kinase C and protein kinase A in the regulation of Ca(2+)-stimulated MgATPase of reconstituted actomyosin S-1 was investigated. The TnI mutants used were T144A, S43A/S45A, and S43A/S45A/T144A (in which the identified protein kinase C phosphorylation sites, Thr-144 and Ser-43/ Ser-45, were, respectively, substituted by Ala) and S23A/S24A and N32 (in which the protein kinase A phosphorylation sites Ser-23/Ser-24 were either substituted by Ala or deleted). The mutations caused subtle changes in the kinetics of phosphorylation by protein kinase C, and all mutants were maximally phosphorylated to various extents (1.3-2.7 mol of phosphate/mol of protein). Protein kinase C could cross-phosphorylate protein kinase A sites but the reverse essentially could not occur. Compared to wild-type TnI and T144A, un-phosphorylated S43A/S45A, S43A/S45A/T144, S23A/ S24A, and N32 caused a decreased Ca2+ sensitivity of Ca(2+)-stimulated MgATPase of reconstituted actomyosin. S-1. Phosphorylation by protein kinase C of wild-type and all mutants except S43A/S45A and S43A/S45A/T144A caused marked reductions in both the maximal activity of Ca(2+)-stimulated MgATPase and apparent affinity of myosin S-1 for reconstitued (regulated) actin. It was further noted that protein kinase C acted in an additive manner with protein kinase A by phosphorylating Ser-23/Ser-24 to bring about a decreased Ca2+ sensitivity of the myofilament. It is suggested that Ser-43/Ser-45 and Ser-23/Ser-24 in cardiac TnI are important for normal Ca2+ sensitivity of the myofilament, and that phosphorylation of Ser-43/Ser-45 and Ser-23/Ser-24 is primarily involved in the protein kinase C regulation of the activity and Ca2+ sensitivity, respectively, of actomyosin S-1 MgATPase.

Lysophosphatidylcholine increases vascular superoxide anion production via protein kinase C activation.

We tested the hypothesis that lysophosphatidylcholine (lyso-PC) could activate protein kinase C in intact vascular segments and sought to examine some of the physiological consequences of this activation. In segments of rabbit aorta, the patterns of protein phosphorylation determined by two-dimensional electrophoresis stimulated by lyso-PC and 12-O-tetradecanoylphorbol 13-acetate (TPA) were similar. Activation of protein kinase C can stimulate superoxide anion (O2-) production in other tissues, and we found that lyso-PC-treated rabbit aortas produced twofold more O2- than control vessels. Calphostin C, a potent and specific inhibitor of protein kinase C, attenuated O2- production in lyso-PC-treated vessels but had no effect in control vessels. The effect of lyso-PC on O2- production was mimicked by TPA. In separate bioassay studies, release of the endothelium-derived vascular relaxing factor (EDRF) quantified by the response of detector vessels was markedly impaired after exposure of donor rabbit aortic segments to lyso-PC. After incubation with calphostin C, EDRF release in response to acetylcholine from lyso-PC-treated donor vessels was restored significantly. Thus, lyso-PC can activate protein kinase C in intact vessels, leading to an increase in O2- production. Activation of protein kinase C by lyso-PC may also play a role in altering the release of EDRF in response to acetylcholine. Increased O2- production in response to lyso-PC may have important consequences in the atherogenic process.

Phosphorylation by protein kinase C and cyclic AMP-dependent protein kinase of synthetic peptides derived from the linker region of human P-glycoprotein.

Specific sites in the linker region of human P-glycoprotein phosphorylated by protein kinase C (PKC) were identified by means of a synthetic peptide substrate, PG-2, corresponding to residues 656-689 from this region of the molecule. As PG-2 has several sequences of the type recognized by the cyclic AMP-dependent protein kinase (PKA), PG-2 was also tested as a substrate for PKA. PG-2 was phosphorylated by purified PKC in a Ca2+/phospholipid-dependent manner, with a Km of 1.3 microM, and to a maximum stoichiometry of 2.9 +/- 0.1 mol of phosphate/mol of peptide. Sequence analysis of tryptic fragments of PG-2 phosphorylated by PKC identified Ser-661, Ser-667 and Ser-671 as the three sites of phosphorylation. PG-2 was also found to be phosphorylated by purified PKA in a cyclic AMP-dependent manner, with a Km of 21 microM, and to a maximum stoichiometry of 2.6 +/- 0.2 mol of phosphate/mol of peptide. Ser-667, Ser-671 and Ser-683 were phosphorylated by PKA. Truncated peptides of PG-2 were utilized to confirm that Ser-661 was PKC-specific and Ser-683 was PKA-specific. Further studies showed that PG-2 acted as a competitive substrate for the P-glycoprotein kinase present in membranes from multidrug-resistant human KB cells. The membrane kinase phosphorylated PG-2 mainly on Ser-661, Ser-667 and Ser-671. These results show that human P-glycoprotein can be phosphorylated by at least two protein kinases, stimulated by different second-messenger systems, which exhibit both overlapping and unique specificities for phosphorylation of multiple sites in the linker region of the molecule.

Human leukemia K562 cell mutant (K562/OA200) selected for resistance to okadaic acid (protein phosphatase inhibitor) lacks protein kinase C-epsilon, exhibits multidrug resistance phenotype, and expresses drug pump P-glycoprotein.

A human leukemia K562 cell mutant (K562/OA200) selected for resistance to okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A (PP1/PP2A), has been established. In wild type cells, the cytotoxicity of OA was associated with mitotic arrest and concentration- and time-dependent DNA fragmentation, a hallmark of apoptosis. The mutant was 100-fold more resistant to OA in terms of effects on these parameters. Although the synthesis of several proteins was altered, enzyme assay and immunoblot analysis indicated that the levels of PP1 and PP2A were unchanged in the mutant. Protein kinase C (PKC) assays and immunoblot analysis of calcium-dependent (cPKC) and calcium-independent (nPKC) isoforms revealed that nPKC-epsilon was strikingly absent in the mutant, which otherwise expressed in comparable amounts all other isotypes (cPKC-alpha, cPKC-beta, and nPKC-zeta) also present in the wild type. Northern blot analysis confirmed an absence of PKC-epsilon mRNA in the mutant cells. The OA200 cells were cross-resistant not only to another PP1/PP2A inhibitor, calyculin A, but also to structurally unrelated anticancer drugs (such as vinblastine and taxol) and furthermore, overexpressed the verapamil-sensitive drug pump P-glycoprotein at both the protein and mRNA levels. The mutant, however, was not cross-resistant to several PKC inhibitors tested including cardiotoxin, mastoparan, staurosporine, and an alkylphospholipid. Cardiotoxin, at a subtoxic concentration, enhanced by 6-fold vinblastine cytotoxicity in OA200 cells. These findings indicate that the multidrug resistance phenotype can be induced by cytotoxic agents other than conventional anticancer drugs, show that the development of multidrug resistance is not necessarily associated with increased cPKC activity, and identify certain PKC inhibitors that have potential as resistance modulators.

Role of protein kinase C in the phosphorylation of cardiac myosin light chain 2.

The role of protein kinase C (PKC) in the phosphorylation of myosin light chain 2 (MLC2) in adult rat heart cells has been investigated. PKC-mediated phosphorylation of MLC2 in adult rat cardiac myofibrils in vitro occurs with a stoichiometry (0.7 mol of phosphate/mol of protein) similar to that mediated by myosin light chain kinase (MLCK). Two-dimensional tryptic phosphopeptide mapping of MLC2 following phosphorylation by PKC or MLCK in vitro yields the same major phosphopeptides for each protein kinase. These sites are also 32P-labelled in situ when isolated cardiomyocytes are incubated with [32P]P(i). 32P labelling of MLC2 in cardiomyocytes is increased by 5-fold in 10 min upon incubation with the phosphatase inhibitor calyculin A, demonstrating the existence of a rapidly turning over component of MLC2 phosphorylation in these cells. 32P label is completely removed from MLC2 when myocytes are exposed to 2,3-butanedione monoxime, an inhibitor of cardiac contraction known to desensitize the myofilaments to activation by Ca2+. 32P labelling of MLC2 is also decreased by 50-100% following exposure to the PKC-selective inhibitors calphostin C and chelerythrine, suggesting that PKC, and not MLCK, is primarily responsible for incorporation of rapidly turning over phosphate into MLC2 in situ. Taken together, these data implicate PKC in the phosphorylation of MLC2 in heart cells and support the hypothesis that phosphorylation of cardiac MLC2 has a role in determining myofibrillar Ca2+ sensitivity.

Structure-function relationships of alkyl-lysophospholipid analogs in selective antitumor activity.

This investigation was initiated in order to delineate the structure-function relationship of the anticancer alkyl-lysophospholipids and assess their degree of selective cytotoxicity toward neoplastic cells. A series of glycerol phosphocholine analogs with varying substitutions in the sn-1 and sn-2 position were tested for their inhibitory activity as measured by thymidine incorporation, clonogenic assays and effects on protein kinase C activity against a series of human leukemic cell lines and healthy bone marrow progenitor cells. The IC50 was determined for each of the compounds in each cell line and healthy bone marrow cells following a 4-h incubation. The data indicated that a 16-18 carbon chain at the sn-1 coupled with a short substitution at sn-2 had the broadest antitumor activity and was the least toxic to normal bone marrow cells. The results provide a number of useful leads toward the design and development of potentially more active phospholipid compounds.

Phosphorylation of cardiac myosin light chain 2 by protein kinase C and myosin light chain kinase increases Ca(2+)-stimulated actomyosin MgATPase activity.

Myosin light chain 2 (MLC2) phosphorylation in rat cardiac whole myosin by cardiac myosin light chain kinase (MLCK) or by protein kinase C (PKC) resulted in increased actin-stimulated myosin MgATPase activity. The phosphorylation also increased Ca(2+)-stimulated myofibrillar MgATPase activity upon substitution of the phosphorylated myosin into myofibrils. In addition, phosphorylation of MLC2 in myofibrils by MLCK increased both the Ca(2+)-sensitivity and maximum activity of the myofibrillar Ca(2+)-stimulated MgATPase activity. The latter effect was inhibited by PKC-phosphorylation of troponin I, troponin T and C-protein. A role for both PKC and MLCK in regulating cardiac myofibrillar activity, via phosphorylation of various contractile proteins, is indicated.

Cobra venom cardiotoxin (cytotoxin) isoforms and neurotoxin: comparative potency of protein kinase C inhibition and cancer cell cytotoxicity and modes of enzyme inhibition.

Effects of cobra cardiotoxin (cytotoxin) and its isoforms, and neurotoxin, on protein kinase C (PKC) and cancer cells were investigated. A positive correlation existed between hydrophobicities and activities of the toxins to inhibit PKC activity (assayed using phosphatidylserine vesicle, arachidonate monomer, and Triton/phosphatidylserine mixed micelle systems), phorbol ester binding to PKC, proliferation of several cancer cell lines, and phorbol ester-induced HL60 cell differentiation. Their relative hydrophobicities and activities, in a decreasing order, were cardiotoxin-1 approximately cardiotoxin-3 > cardiotoxin (a mixture of isoforms) > cardiotoxin-4 >> neurotoxin (inactive). Under the mixed micelle assay system (containing 0.3% Triton X-100, 8 mol % phosphatidylserine, 2 mol % diolein, and 200 microM CaCl2), cardiotoxin inhibited PKC competitively with respect to phosphatidylserine (apparent Ki of about 0.06 mol % or 2.5 microM), and in a mixed-type manner with respect to both diolein (apparent Ki of about 0.04 mol % or 1.7 microM) and Ca2+ (apparent Ki of about 2.9 microM). On the basis of findings that IC50 (approximately 0.3 microM) of cardiotoxin for inhibition of HL60 cell proliferation and differentiation was lower than its IC50 (9 microM) for PKC inhibition in vitro in the phosphatidylserine vesicle system and that PKC inhibition was the only known molecular mechanism of cardiotoxin, it was suggested that cardiotoxin might be highly membrane interacting and that the observed cellular effects of cardiotoxin might be mediated, in part, via PKC inhibition.

Identification of specific sites in human P-glycoprotein phosphorylated by protein kinase C.

Phosphorylation of P-glycoprotein (Pgp) by protein kinase C occurs on apparently the same sites in vitro and in intact cells (in situ) and is implicated in modulation of Pgp function. The region of the molecule which contains the in vitro phosphorylation sites and two specific sites within this region are now determined by peptide sequencing. Membrane vesicles from multidrug-resistant human KB-V1 cells were incubated with purified protein kinase C and [gamma-32P]ATP, and Pgp (containing 1 mol of phosphate/mol of protein) was purified to apparent homogeneity. Phosphorylation occurred exclusively on serine residues. Phosphopeptides were generated by digestion with Lys-C endoproteinase or trypsin, partially purified by high performance liquid chromatography, and further purified with strategies developed for individual phosphopeptides. Sequence analysis by Edman degradation and comparison with the deduced amino acid sequence of human (mdr 1) Pgp identified serines 661 and 671, and one or more of serines 667, 675, and 683, as sites of phosphorylation. These sites are clustered in the linker region located between the two homologous halves of Pgp. Our results identify a previously undefined, phosphorylatable domain of Pgp, smaller in size but analogous in location to the R-domain of the cystic fibrosis transmembrane conductance regulator. These data provide a basis for a better understanding of the role of phosphorylation in the mechanism of action and regulation of this important multidrug pump protein.

Protein kinase C-mediated phosphorylation of troponin I and C-protein in isolated myocardial cells is associated with inhibition of myofibrillar actomyosin MgATPase.

Phosphorylation of cardiac myofibrillar proteins by protein kinase C (PKC) in isolated adult rat cardiomyocytes has been compared with that mediated by the cAMP-dependent protein kinase (PKA). PKA activation by beta-adrenoreceptor (isoproterenol) stimulation results in stoichiometric phosphorylation of troponin I (TnI) and C-protein. PKC activation by either 12-O-tetradecanoylphorbol-13-acetate (TPA) or by alpha-adrenoreceptor (phenylephrine plus propranolol) stimulation results in phosphorylation of the same two proteins to similar extents. Two-dimensional phosphopeptide mapping shows that the same sites in TnI are modified by PKC in vitro and in TPA- or alpha-agonist-stimulated cells. These sites are distinct from those phosphorylated in isoproterenol-stimulated cells or by PKA in vitro. Phosphopeptide mapping analysis of C-protein shows that PKC and PKA phosphorylate identical residues in this protein in vitro and in situ. TPA-stimulated phosphorylation in myocytes is associated with a reduction in maximal activity of myofibrillar Ca(2+)-dependent actomyosin MgATPase. Isoproterenol-stimulated phosphorylation has no effect on maximal activity but reduces the Ca2+ sensitivity of the MgATPase. These data demonstrate that TnI and C-protein are phosphorylated in myocardial cells by both PKA and PKC, resulting in different functional consequences in each case.

Multidrug-resistant human KB carcinoma cells are highly resistant to the protein phosphatase inhibitors okadaic acid and calyculin A. Analysis of potential mechanisms involved in toxin resistance.

In this study we show that multidrug-resistant (MDR) human KB-V1 cells are highly resistant to the cytotoxicity of okadaic acid and calyculin A, 2 toxins from marine sponges that are potent inhibitors of type-1 and type-2A protein phosphatases (PP1 and PP2A). Cytotoxicity and colony-forming assays indicated that, relative to parental drug-sensitive KB-3 cells, KB-V1 cells are 35-fold more resistant to okadaic acid and 70-fold more resistant to calyculin A. Cytotoxicity of the toxins was associated with mitotic arrest characterized by chromosome scattering and over-condensation, with KB-3 cells being more sensitive than KB-V1 cells and calyculin A being more potent than okadaic acid. The resistance of KB-V1 cells to both okadaic acid and calyculin A was completely reversed by verapamil, suggesting that the toxins may be transported by P-glycoprotein (P-gp). To further assess the possibility of an interaction with P-gp, the toxins were employed as potential modulators of the photoaffinity labeling of P-gp by [3H]azidopine. Relative to vinblastine, which effectively competed with [3H]azidopine for P-gp photolabeling, calyculin A was 100-fold less potent and okadaic acid did not inhibit photolabeling at concentrations up to 50 microM. To determine whether the resistance mechanism involved differences in toxin-sensitive phosphatase activity, the activity was assayed in extracts from both cell lines and found to be slightly higher (1.6-fold) in KB-V1 than in KB-3 cells. Our results demonstrate a novel, marked resistance of MDR KB-V1 cells to these phosphatase inhibitors and suggest that a major mechanism of resistance may involve toxin transport by P-gp at sites apparently different from those which bind azidopine.

Protein kinase C phosphorylation of cardiac troponin I and troponin T inhibits Ca(2+)-stimulated MgATPase activity in reconstituted actomyosin and isolated myofibrils, and decreases actin-myosin interactions.

The inhibitory effects of the phosphorylation of bovine cardiac troponin I (TnI) and troponin T (TnT) by protein kinase C (PKC) on the activity of Ca(2+)-stimulated MgATPase of reconstituted actomyosin complex, as a function of the concentration of myosin or myosin subfragment 1 (S-1), were investigated. Phosphorylation of TnI and/or TnT invariably decreased the Ca(2+)-stimulated enzyme activity of reconstituted actomyosin or actomyosin S-1, regardless of the concentration of whole myosin or S-1. The inhibition due to phosphorylated TnI was partially overcome as the concentration of myosin or S-1 increased, suggesting simple competition of phosphorylated TnI with myosin or S-1 for actin binding sites. Inhibition due to phosphorylated TnT, however, remained constant at all concentrations of myosin or S-1, suggesting that phosphorylated TnT may inhibit full Ca(2+)-activation of the thin filament. Both phosphorylated TnI and TnT inhibited the Ca(2+)-stimulated binding of S-1.ADP to regulated actin, consistent with the notion that the effects of phosphorylation of TnI and TnT affected interactions of the thin filament with the thick filament. Effects of PKC phosphorylation of the contractile components in adult rat cardiac myofibrils were also investigated. PKC phosphorylation of TnI and TnT, as well as other proteins in the contractile complex, resulted in the inhibition of Ca(2+)-stimulated MgATPase activity with little change in the Ca(2+)-sensitivity. Thus, the negative inotropic effects attributable to activation of PKC by phorbol esters, as reported by others, could be explained in part through PKC mediated phosphorylation of components of the contractile apparatus.

Protein kinase C phosphorylation of cardiac troponin T decreases Ca(2+)-dependent actomyosin MgATPase activity and troponin T binding to tropomyosin-F-actin complex.

Effects of phosphorylation of bovine cardiac troponin T (TnT) by protein kinase C on the Ca(2+)-stimulated MgATPase activity of reconstituted actomyosin complex and the binding of TnT to tropomyosin(Tm)-F-actin were investigated. The Ca(2+)-stimulated MgATPase of actomyosin containing phosphorylated TnT (1.8 mol of P/mol), compared with that containing unphosphorylated TnT, was decreased by up to 48%. Phosphorylation of TnT also decreased (up to 48%) its maximum binding to Tm-F-actin, which was accompanied by a decrease (up to 3.5-fold) in its apparent binding affinity. The findings indicate that the effects of phosphorylated TnT in decreasing actomyosin MgATPase might be secondary to its decreased interactions with the other components of the thin filament, representing a new mechanism underlying the negative inotropic responses of various cardiac preparations to protein kinase C-activating phorbol esters.

Comparative effects of protein phosphatase inhibitors (okadaic acid and calyculin A) on human leukemia HL60, HL60/ADR and K562 cells.

Inhibitors of protein phosphatases 1/2A (okadaic acid and calyculin A) exhibited differential cytotoxicity toward three human leukemia cell lines, in an increasing order of resistance, HL60 less than HL60/ADR less than K562 cells. Cytotoxicity of the toxins was associated with marked mitotic arrest of the cells, characterized by chromatid scattering/overcondensation and abnormal mitotic spindles. In all cases, calyculin A was more potent than okadaic acid. Protein phosphorylation experiments in intact cells revealed that HL60/ADR, the adriamycin-resistant variant, showed a higher overall phosphorylation of nuclear proteins than the drug-sensitive parental HL60, and that phorbol ester (protein kinase C activator) and calyculin A appeared to more specifically stimulate phosphorylation of p66 and p60, respectively. It was suggested that the toxins might be useful in delineating mechanisms underlying certain properties of cancer cells (such as multidrug resistance, mitosis and differentiation) related to protein phosphorylation/dephosphorylation reactions.