Effect of high intensity exercise on peak oxygen uptake and endothelial function in long-term heart transplant recipients.

Coronary allograft vasculopathy is a well-known long-term complication after cardiac transplantation. Endothelial dysfunction is involved and may be prevented by aerobic exercise. The purpose of this study was to examine whether high intensity aerobic exercise improves peak oxygen uptake (VO(2 peak) ) and endothelial function in heart transplant (HT) recipients. Twenty-seven long-term HT recipients were randomized to either 8-weeks high intensity aerobic exercise or no training. Flow mediated dilation of the brachial artery (FMD) was measured by ultrasound and VO(2 peak) by the analysis of expired air. Blood pressure and biomarkers were measured before and after 8 weeks. VO(2 peak) increased significantly in the exercise group (VO(2 peak) 23.9 ± 1.79 to 28.3 ± 1.63 mL/kg/min compared to controls (VO(2 peak) 24.6 ± 1.38 to 23.4 ± 1.58, p < 0.001 exercise vs. control).FMD increased in the exercise group compared to controls (8.3 ± 1.1% to 11.4 ± 1.2% vs. 5.6 ± 1.0% to 5.3 ± 1.7%, p = 0.024). No increase in nitroglycerin-induced vasodilation was observed. Systolic blood pressure fell in the exercise group (142 ±4.2 mmHg to127 ± 3.4 mmHg, p = 0.01) and was unchanged in controls (141 ± 4.2 mmHg to 142 ±6.4 mmHg, NS). High intensity aerobic exercise reduces systolic blood pressure and improves endothelial function in HT recipients.

Identification and evaluation of Peruvian plants used to treat malaria and leishmaniasis.

Households in eleven geographically and ethnically distinct areas in Loreto, Peru, were interviewed about their knowledge and use of plants, for the treatment of malaria and leishmaniasis. The survey resulted in 988 use records representing 118 plant-taxa for malaria and 289 use-records representing 85 plant-taxa for leishmaniasis. In both cases the 10 most frequently reported taxa accounted for about half of all the use-records. Plant material was collected and extracts were screened for in vitro inhibition of Plasmodium and Leishmania parasites. In the case of Plasmodium, extracts of 11 of the 13 most frequently reported plants showed significant growth inhibitory activity, while only a few plant extracts inhibited the growth of Leishmania parasites.

The inflammatory and tumor-promoting sesquiterpene lactone, thapsigargin, activates platelets by selective mobilization of calcium as shown by protein phosphorylations.

We have studied the activation of human blood platelets by the inflammatory and tumor-promoting sesquiterpene lactone, thapsigargin. The effect of thapsigargin was compared with other common agonists (calcium ionophore A23187, phorbol ester TPA and thrombin). Platelet aggregation, serotonin release, raised cytoplasmic free calcium level and phosphorylation of platelet proteins was examined in platelet-rich plasma and washed platelet suspension. In contrast to A23187 and thrombin, the platelet activation induced by thapsigargin developed slowly, with maximal response obtained after 2-3 min. Both the thapsigargin- and the A23187-induced serotonin releases were synergistically increased by TPA. Studies of the phosphorylation of platelet proteins revealed that thapsigargin and A23187 equally well induced a selective phosphorylation of two proteins with apparent molecular masses of 20 kDa and 47 kDa. These proteins, which are substrates of myosin light-chain kinase and protein kinase C respectively, are known to be involved in platelet activation. The thapsigargin-induced platelet aggregation and serotonin release was completely inhibited by class I (nimodipine), class II (verapamil) and class III (diltiazem) calcium-channel blockers. The inhibitory activity of nimodipine was abolished by the corresponding 1,4-dihydropyridine calcium-channel agonist, BAY K 8644. These results shows that the thapsigargin-induced platelet activation is mediated by an increase in the cytoplasmic free calcium level, presumably obtained by stimulation of the passive calcium transport through specific channels. These thapsigargin-sensitive channels should predominantly be located in the membranes of intracellular calcium stores rather than in the plasma membrane, because removal of extracellular calcium by EGTA had only an insignificant effect on the thapsigargin-induced rise in cytoplasmic free calcium level.

Effect of thapsigargin on cytoplasmic Ca2+ and proliferation of human lymphocytes in relation to AIDS.

The tumor-promoting sesquiterpene lactone, thapsigargin, induced a dose-dependent increase of the cytoplasmic Ca2+ concentration ([ Ca2+]i) in human lymphocytes from a resting level between 100 and 150 nM up to about 1 microM. Half-maximum response was found at about 1 nM of thapsigargin, full response at 100 nM. The effect of thapsigargin on [Ca2+]i exceeded that of phytohaemagglutinin (PHA) which raised [Ca2+]i to maximum 300 nM. In combination with phorbol 12-myristate 13-acetate (PMA), thapsigargin stimulated the proliferation of normal lymphocytes to the same extent as did PHA, whereas the thapsigargin/PMA treatment could not restore the defective proliferation of AIDS lymphocytes in spite of the increased [Ca2+]i. Thapsigargin or PMA added separately had no stimulatory effects on cell proliferation. The thapsigargin/PMA treatment caused an increase in the interleukin-2 (IL-2) production of the lymphocytes, which was much higher than that caused by the PHA treatment, even in AIDS lymphocytes. Moreover, the thapsigargin/PMA treatment stimulated the expression of the IL-2 receptors on both normal and AIDS lymphocytes, similar to the effect of PHA. It is concluded that thapsigargin exerts its effects on lymphocyte proliferation by increasing [Ca2+]i, and that the general defect of AIDS lymphocytes, rather than being ascribed to the initiating signal systems, is associated with later events related to DNA synthesis and proliferation.

Synergistic stimulation of histamine release from rat peritoneal mast cells by 12-O-tetradecanoylphorbol 13-acetate (TPA)-type and non-TPA-type tumor promoters.

Thapsigargin, a non-TPA (12-O-tetradecanoylphorbol 13-acetate)-type tumor promoter, provoked histamine release from rat peritoneal mast cells at concentrations above 30 ng/ml, but not at 10 ng/ml. TPA-type tumor promoters such as TPA, teleocidin and aplysiatoxin released very little, if any, histamine even at 100 ng/ml. When mast cells were incubated in medium containing thapsigargin at 10 ng/ml and varying concentrations of TPA-type tumor promoters, histamine release was increased synergistically. Maximum synergistic effects were observed at 10 ng/ml of each TPA-type tumor promoter. Palytoxin, another non-TPA-type tumor promoter, having no effect on histamine release at up to 10 pg/ml, also induced histamine release in the presence of 10 ng/ml of each TPA-type tumor promoter. However, no synergistic effect on histamine release was observed when mast cells were incubated in medium containing two different non-TPA-type tumor promoters, e.g., 10 ng/ml thapsigargin and 10 pg/ml palytoxin, or in medium containing two different TPA-type tumor promoters, e.g., TPA and teleocidin, TPA and aplysiatoxin, or teleocidin and aplysiatoxin (all at 10 ng/ml). These results suggest that the release of histamine from mast cells is stimulated synergistically under the mutual influence of TPA-type tumor promoters and non-TPA-type tumor promoters.

A tool coming of age: thapsigargin as an inhibitor of sarco-endoplasmic reticulum Ca(2+)-ATPases.

Thapsigargin is the most widely used inhibitor of the ubiquitous sarco-endoplasmic reticulum Ca(2+)-ATPases in mammalian cells. Over the past ten years, this guaianolide compound of plant origin has become a popular tool in a host of studies directed at elucidating the mechanisms of intracellular Ca2+ signalling. Its remarkable potency and selectivity have been instrumental in widening our view of the function of intracellular Ca2+ stores to include such key aspects as store-operated Ca2+ entry or the involvement of the stores in protein synthesis or cell growth. In this article Marek Treiman, Casper Caspersen and Søren Brøgger Christensen review the key pharmacological features of thapsigargin action; they also discuss some of the ways in which its unique properties have shown to be important for obtaining new insights into the biology of Ca2+ stores, and how these properties might encompass a therapeutic potential. In parallel, attention is drawn to some of the limitations and pitfalls encountered when working with thapsigargin.

Calcium entry in Xenopus oocytes: effects of inositol trisphosphate, thapsigargin and DMSO.

Agonist- and inositol 1,4,5-trisphosphate (InsP3)-evoked responses in Xenopus oocytes utilize calcium mobilized from cellular stores as well as from the medium. We studied the effect of the status of Ca stores on InsP3-induced Ca entry. Thapsigargin (TG) caused a net increase of 45Ca2+ efflux from oocytes in a time and dose dependent manner (31 and 54% of total label, at 30 and 60 min, respectively). Incubation with TG (60 min) resulted in a complete loss of the response to InsP3 implying that InsP3-sensitive Ca stores were depleted. Challenge with 1.8 mM Ca2+ resulted in a large depolarizing chloride current (1231 +/- 101 nA) which was not further potentiated by InsP3. This suggested that extensive depletion of cellular Ca stores is sufficient to induce maximal entry of extracellular Ca (Cao). Following the injection of InsP3, a much more limited loss of cellular Ca was sufficient to produce large Ca entry. Dimethyl sulfoxide (DMSO) alone, the vehicle used to dissolve TG, did not cause increase in either efflux of 45Ca2+, nor in the Cao-evoked Cl- current. It did, however, markedly potentiate this current following the injection of InsP3. DMSO moderately inhibited InsP3-induced 45Ca2+ efflux from oocytes. Hence, apparent potentiation of Ca entry can be observed without additional depletion of cellular Ca. We conclude that Ca entry may be induced via either stimulation with InsP3 and limited Ca depletion or depletion of a specific and, possibly small, cellular Ca store alone. The mechanism of DMSO potentiation is unknown, but may be important in view of the universal use of this solvent as vehicle.

Thapsigargin-induced increase in cytoplasmic Ca2+ concentration and aldosterone production in rat adrenal glomerulosa cells: interaction with potassium and angiotensin-II.

Thapsigargin (Tg), a microsomal Ca2+ pump inhibitor, dose-dependently increases the cytoplasmic Ca2+ concentration and aldosterone production without having any striking effect on the formation of inositol phosphates in isolated rat adrenal glomerulosa cells. The interaction of Tg with the major Ca2(+)-mediated stimuli of glomerulosa cells on aldosterone production was also examined. The effects of Tg and the Ca2(+)-mobilizing angiotensin-II (AII) were additive. The aldosterone production stimulatory effect of potassium, which induces Ca2+ influx via voltage-operated Ca2+ channels, was potentiated by Tg. The positive interaction between Tg and potassium on aldosterone production raises the possibility that stimuli generating Ca2+ signal by depleting intracellular Ca2+ stores, such as Tg or AII, enhance the response of the cell to depolarization. Such an interaction between AII and potassium may have an important role in the physiological control of aldosterone production.

Influence of cigarette smoking on goiter formation, thyroglobulin, and thyroid hormone levels in women.

The possible influence of cigarette smoking on goiter formation, thyroglobulin (Tg) secretion, and thyroid hormone production was assessed by estimations of the presence of palpable goiter and by RIAs of Tg, T3, rT3, T4, and TSH in sera from 441 women (48-53 yr old), representing a normal population included in a study on the prevalence of thyroid disease. Smoking habits were evaluated by a questionnaire, and the women were then classified as never smokers (n = 192), smokers (n = 169), and exsmokers (n = 80). Smokers were subdivided as moderate (1-19 cigarettes/day) and heavy (greater than or equal to 20 cigarettes/day). Palpable goiter was found in 15% of the smokers, in contrast to only 4% of the exsmokers and 9% of the never smokers. Among smokers, 37% had serum Tg values over 30 micrograms/liter (third quartile), while such values were found in only 16% of the exsmokers and 18% of the never smokers. In addition, smokers were found to have higher serum T3 and lower rT3 concentrations than never smokers; this difference was most pronounced in heavy smokers. Serum T4 was not different, while TSH was insignificantly lower in smokers than in nonsmokers. Exsmokers did not differ significantly from never smokers in any of these parameters. It seems possible that cigarette smoking may have two, calorigenically opposed, effects on thyroid hormone production; it may be goitrogenic (possibly due to inhaled thiocyanate), but it may also enhance the formation of T3 at the expense of rT3 formation.

Derivatives of thapsigargin as probes of its binding site on endoplasmic reticulum Ca2+ ATPase. Stereoselectivity and important functional groups.

The naturally occurring sesquiterpene lactone thapsigargin is a potent and selective inhibitor of SERCA ATPases, a family of Ca(2+)-pumping ATPases present in the endoplasmic reticulum of all mammalian cells. We have studied some of the molecular features of thapsigargin responsible for its inhibitory action towards these Ca2+ ATPases. A series of thapsigargin analogues were synthesised and their inhibitory potencies determined using the uptake of 45Ca2+ in bovine cerebellar microsomes as a sensitive marker of Ca2+ ATPase activity. An attenuation of the inhibitory potency relative to the parent compound was found ranging from slight to over 3 orders of magnitude. The inhibitory activity showed a very strong configuration dependence, a major contribution from the ester groups at C3 and C10, and an apparently minor contribution from the lactone ring substituents. The data are consistent with thapsigargin fitting to a sterically discriminating cleft involving the hydrophobic transmembrane region of the ATPase, and is compatible with available kinetic evidence of thapsigargin-mediated inhibition.

ACTA, a fluorescent analogue of thapsigargin, is a potent inhibitor and a conformational probe of skeletal muscle Ca2+-ATPase.

Thapsigargin is a highly potent and selective inhibitor of sarco-endoplasmic reticulum (SERCA) family of Ca2+-ATPases and a useful tool in research concerning the function of intracellular Ca2+ stores. We describe here a novel fluorescent derivative (8-O-(4-aminocinnamoyl)-8-O-debutanoylthapsigargin, termed ACTA) of this compound, acting as a Ca2+-ATPase inhibitor with a potency approaching that of thapsigargin. Binding of ACTA to the skeletal muscle sarcoplasmic reticulum vesicles results in a strong fluorescence enhancement, approximately 66% of which depends on ACTA association with Ca2+-ATPase. This specific component of ACTA fluorescence is sensitive to the E1-E2 conformational equilibrium of the pump. The combined properties of high potency and binding-dependent fluorescence suggest ACTA to be a useful probe for a range of studies involving the SERCA class of ATPases.

Structure-activity relationships of analogues of thapsigargin modified at O-11 and O-12.

A number of analogues of thapsigargin have been synthesized by alkylating or acylating O-11 and O-12 in the lactol obtained by reducing thapsigargicin. Introduction of alpha-disposed substituents decreased the Ca(2+)-ATPase inhibitory potency of the analogue, whereas the enzyme was more tolerant toward beta-disposed substituents, indicating that the alpha-face of the lactone ring is in close contact with the binding site when the inhibitor is bound to the enzyme.

Antileishmanial chalcones: statistical design, synthesis, and three-dimensional quantitative structure-activity relationship analysis.

A large number of substituted chalcones have been synthesized and tested for antileishmanial and lymphocyte-suppressing activities. A subset of the chalcones was designed by using statistical methods. 3D-QSAR analyses using 67 (antileishmanial activity) and 63 (lymphocyte-suppressing activity) of the compounds for the training sets and 9 compounds as an external validation set were performed by using the GRID/GOLPE methodology. The Smart Region Definition procedure with subsequent region selection as implemented in GOLPE reduced the number of variables to approximately 1300 yielding 3D-QSAR models of high quality (lymphocyte-suppressing model, R2 = 0. 90, Q2 = 0.80; antileishmanial model, R2 = 0.73, Q2 = 0.63). The coefficient plots indicate that steric interactions between the chalcones and the target are of major importance for the potencies of the compounds. A comparison of the coefficient plots for the antileishmanial effect and the lymphocyte-suppressing activity discloses significant differences which should make it possible to design chalcones having a high antileishmanial activity without suppressing the proliferation of lymphocytes.

The crystal structure, absolute configuration, and phosphodiesterase inhibitory activity of (+)-1-(4-bromobenzyl)-4-(3-(cyclopentyloxy)- 4-methoxyphenyl)-pyrrolidin-2-one.

Chiral HPLC resolution of the phosphodiesterase IV (PDE IV) inhibitor rolipram (1) provided (-)-1, and this enantiomer was converted into its 1-(4-bromobenzyl) derivative, (+)-2. X-ray structural analysis of (+)-2 established the absolute configuration as R, which provides the first direct evidence for a previously assumed assignment of configuration. The crystal structure of (+)-2 and the PDE inhibitory activity of both enantiomers of 2 are discussed in the context of a previously proposed topological model.

Design, synthesis, and pharmacological evaluation of thapsigargin analogues for targeting apoptosis to prostatic cancer cells.

A series of thapsigargin (TG) analogues, containing an amino acid applicable for conjugation to a peptide specifically cleaved by prostate-specific antigen (PSA), has been prepared to develop the drug-moiety of prodrugs for treatment of prostatic cancer. The analogues were synthesized by converting TG into O-8-debutanoylthapsigargin (DBTG) and esterifying O-8 of DBTG with various amino acid linkers. The compounds were evaluated for their ability to elevate the cytosolic Ca(2+) concentration ([Ca(2+)](i)) in TSU-Pr1 cells, their ability to inhibit the rabbit skeletal muscle SERCA pump, and their ability to induce apoptosis in TSU-Pr1 human prostatic cancer cells. The activity of analogues, in which DBTG were esterified with omega-amino acids [HOOC(CH(2))(n)()NH(2), n = 5-7, 10, 11], increased with the linker length. Analogues with 3-[4-(L-leucinoylamino)phenyl]propanoyl, 6-(L-leucinoylamino)hexanoyl, and 12-(L-serinoylamino)dodecanoyl were considerably less active than TG, and analogues with 12-(L-alaninoylamino)dodecanoyl and 12-(L-phenylalaninoylamino)dodecanoyl were almost as active as TG. The 12-(L-leucinoylamino)dodecanoyl gave an analogue equipotent with TG, making this compound promising as the drug-moiety of a PSA sensitive prodrug of TG.

1,4-Cyclohexanecarboxylates: potent and selective inhibitors of phosophodiesterase 4 for the treatment of asthma.

Evaluation of a variety of PDE4 inhibitors in a series of cellular and in vivo assays suggested a strategy to improve the therapeutic index of PDE4 inhibitors by increasing their selectivity for the ability to inhibit PDE4 catalytic activity versus the ability to compete for high affinity [3H]rolipram-binding sites in the central nervous system. Use of this strategy led ultimately to the identification of cis-4-cyano-4-[3-(cyclopentyloxy)-4-methoxyphenyl]cyclohexane-1-carboxyl ic acid (1, SB 207499, Ariflo), a potent second-generation inhibitor of PDE4 with a decreased potential for side effects versus the archetypic first generation inhibitor, (R)-rolipram.

Localization of Tc-99m MDP in epiphyseal growth plates of rats.

The distribution and localization of Tc-99m methylene diphosphonate (Tc-MDP) in the epiphyseal growth plates of the rat were elucidated by contact and microautoradiography. The uptake of the tracer was found to be especially high in the calcified cartilage bars at the end of the vascular loops. In addition to areas of mineralization, increased uptake was found in the Howship's lacunae on the resorbing surfaces. This labeling corresponded with the fluorescence of tetracycline, which labeled both forming and resorbing surfaces, when given with short labeling interval. Distribution of Tc-MDP did not coincide with new production of collagen, as judged by H-3 proline labeling; nor was the uptake localized within cells with high alkaline phosphatase activity. The affinity of the tracer for the mineral phase was confirmed by decalcification of in vivo labeled sections with EDTA, which showed loss of radioactivity in contrast to sections incubated in water. By chromatography the activity in the decalcification medium could not be distinguished from that of Tc-MDP.

The ability of thapsigargin and thapsigargicin to activate cells involved in the inflammatory response.

The ability of thapsigargin and thapsigargicin to activate mast cells and leukocytes has been investigated. The thapsigargin-induced histamine release from rat peritoneal mast cells was found to be dependent on the concentration of thapsigargin, the purity of the mast cell preparations, and the number of mast cells in suspension. Thapsigargin induced histamine release from human basophil leukocytes. Thapsigargin induced beta-glucuronidase and lysozyme release from human neutrophil leukocytes. Thapsigargin caused a release of histamine from mesentery, lung, and heart mast cells of the rat, but only to a minor extent from the corresponding guinea-pig cells. Thapsigargicin induced histamine release from mesentery, lung, and heart mast cells of the rat at concentrations from 0.1 microM but provoked only a release from the corresponding guinea-pig cells in the concentration-range 0.16 to 1.6 microM. Thapsigargin increased the cytoplasmic free calcium level in intact human blood platelets at concentrations from 3.0 nM.

Analysis of the stimulative effect of thapsigargin, a non-TPA-type tumour promoter, on arachidonic acid metabolism in rat peritoneal macrophages.

1. At concentrations above 10 ng ml-1, the tumour promoter thapsigargin stimulates the release of radioactivity from [3H]-arachidonic acid-labelled macrophages harvested from rat peritoneal cavity. 2. The release of radioactivity from prelabelled macrophages was augmented more than additively when the cells were incubated in the medium containing both thapsigargin (10 ng ml-1) and other tumour promoters (10 ng ml-1), such as 12-O-tetradecanoylphorbol-13-acetate (TPA), teleocidin and aplysiatoxin. 3. Thapsigargin required extracellular Ca2+ for the stimulation of arachidonic acid release, while TPA did not. 4. Cytoplasmic free calcium level was increased by thapsigargin treatment but not by TPA treatment. 5. An inhibitor of protein kinases, H-7 inhibited the effect of TPA dose-dependently, whereas H-7 did not inhibit that of thapsigargin. 6. These results suggest that thapsigargin stimulates arachidonic acid release by a mechanism different from that of TPA, viz by acting as a selective Ca2+ mobilizer, but not by activating protein kinase C as TPA does.

Suppression of human inflammatory cell function by subtype-selective PDE4 inhibitors correlates with inhibition of PDE4A and PDE4B.

1. Of the four major phosphodiesterase 4 (PDE4) subtypes, PDE4A, PDE4B and PDE4D are widely expressed in human inflammatory cells, including monocytes and T lymphocytes. We explored the functional role of these subtypes using ten subtype-selective PDE4 inhibitors, each belonging to one of two classes: (i) dual PDE4A/PDE4B inhibitors or (ii) PDE4D inhibitors. 2. These compounds were evaluated for their ability to inhibit antigen-stimulated T-cell proliferation and bacterial lipopolysaccharide (LPS)-stimulated tumour necrosis factor alpha (TNFalpha) release from peripheral blood monocytes. 3. All compounds inhibited T-cell proliferation in a concentration-dependent manner; with IC50 values distributed over an approximately 50 fold range. These compounds also inhibited TNFalpha release concentration-dependently, with a wider ( approximately 1000 fold) range of IC50 values. 4. In both sets of experiments, mean IC50 values were significantly correlated with compound potency against the catalytic activity of recombinant human PDE4A or PDE4B when analysed by either linear regression of log IC50 values or by Spearman's rank-order correlation. The correlation between inhibition of inflammatory cell function and inhibition of recombinant PDE4D catalytic activity was not significant in either analysis. 5. These results suggest that PDE4A and/or PDE4B may play the major role in regulating these two inflammatory cell functions but do not rule out PDE4D as an important mediator of other activities in mononuclear leukocytes and other immune and inflammatory cells. Much more work is needed to establish the functional roles of the PDE4 subtypes across a broader range of cellular functions and cell types.