thapsigargin and Hypertrophy

The negative functional effects of cyclic GMP are controlled by the sarcoplasmic reticulum calcium-ATPase (SERCA). The effects of cyclic GMP are blunted in cardiac hypertrophy. We tested the hypothesis that the interaction between cyclic GMP and SERCA would be reduced in hypertrophic cardiac myocytes. Myocytes were isolated from 7 control and 7 renal-hypertensive hypertrophic rabbits. Control and hypertrophic myocytes received 8-bromo-cGMP (8-Br-cGMP; 10(-7), 10(-6), 10(-5) mol/l), the SERCA blocker thapsigargin (10(-8) mol/l) followed by 8-Br-cGMP, or the SERCA blocker, cyclopiazonic acid (CPA; 10(-7) mol/l) followed by 8-Br-cGMP. Percent shortening and maximal rate of shortening and relaxation were recorded using a video edge detector. Changes in cytosolic Ca2+ were assessed in fura 2-loaded myocytes. In controls, 8-Br-cGMP caused a significant 36% decrease in percent shortening from 5.8 +/- 0.4 to 3.7 +/- 0.3%. Thapsigargin and CPA did not affect basal control or hypertrophic myocyte function. When 8-Br-cGMP was given following thapsigargin or CPA, the negative effects of 8-Br-cGMP on control myocyte function were reduced. In hypertrophic myocytes, 8-Br-cGMP caused a smaller but significant 17% decrease in percent shortening from 4.7 +/- 0.2 to 3.9 +/- 0.1%. When 8-Br-cGMP was given following thapsigargin or CPA, no significant changes occurred in hypertrophic cell function. Intracellular Ca2+ transients responded in a similar manner to changes in cell function in control and hypertrophic myocytes. These results show that the effects of cyclic GMP were reduced in hypertrophic myocytes, but this was not related to SERCA. In presence of SERCA inhibitors, the responses to cyclic GMP were blunted in hypertrophic as well as control myocytes.

Topics: Animals; Calcium; Cyclic GMP; Heart Ventricles; Hypertension, Renal; Hypertrophy; In Vitro Techniques; Indoles; Myocytes, Cardiac; Rabbits; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin

We analyzed the hypomethylation of DNA and the sensitivity to apoptosis of tonsillar cells and peripheral blood lymphocytes (PBL) in twenty children with either recurrent tonsillitis (RT) or tonsillar hypertrophy (TH). We found no significant differences in DNA methylation of PBL obtained from RT and TH groups. Hypomethylation of DNA extracted from tonsillar tissue was higher in TH than in RT and was associated with lower spontaneous and thapsigargin-induced apoptosis. By contrast, RT showed a low level of DNA hypomethylation and was associated with high sensitivity to spontaneous and thapsigargin-induced apoptosis.

Topics: Apoptosis; Child; Child, Preschool; DNA Methylation; Humans; Hypertrophy; Infant; Lymphocytes; Palatine Tonsil; Recurrence; Thapsigargin; Tonsillitis

Transgenic mice with cardiac-specific overexpression of active Akt (TG) not only exhibit hypertrophy but also show enhanced left ventricular (LV) function. In 3-4-month-old TG, heart/body weight was increased by 60% and LV ejection fraction was elevated (84 +/- 2%, p < 0.01) compared with nontransgenic littermates (wild type (WT)) (73 +/- 1%). An increase in isolated ventricular myocyte contractile function (% contraction) in TG compared with WT (6.1 +/- 0.2 versus 3.5 +/- 0.2%, p < 0.01) was associated with increased Fura-2 Ca2+ transients (396 +/- 50 versus 250 +/- 24 nmol/liter, p < 0.05). The rate of relaxation (+dL/dt) was also enhanced in TG (214 +/- 15 versus 98 +/- 18 microm/s, p < 0.01). L-type Ca2+ current (ICa) density was increased in TG compared with WT (-9.0 +/- 0.3 versus 7.2 +/- 0.3 pA/pF, p < 0.01). Sarcoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) protein levels were increased (p < 0.05) by 6.6-fold in TG, which could be recapitulated in vitro by adenovirus-mediated overexpression of Akt in cultured adult ventricular myocytes. Conversely, inhibiting SERCA with either ryanodine or thapsigargin affected myocyte contraction and relaxation and Ca2+ channel kinetics more in TG than in WT. Thus, myocytes from mice with overexpressed Akt demonstrated enhanced contractility and relaxation, Fura-2 Ca2+ transients, and Ca2+ channel currents. Furthermore, increased protein expression of SERCA2a plays an important role in mediating enhanced LV function by Akt. Up-regulation of SERCA2a expression and enhanced LV myocyte contraction and relaxation in Akt-induced hypertrophy is opposite to the down-regulation of SERCA2a and reduced contractile function observed in many other forms of LV hypertrophy.

Topics: Adenoviridae; Alkaline Phosphatase; Animals; Blotting, Western; Body Weight; Calcium; Calcium-Transporting ATPases; Calsequestrin; Dose-Response Relationship, Drug; Down-Regulation; Echocardiography; Electrophysiology; Enzyme Inhibitors; Fura-2; Heart Ventricles; Hypertrophy; Inhibitory Concentration 50; Kinetics; Lysophospholipase; Mice; Mice, Transgenic; Muscle Cells; Organ Size; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Reverse Transcriptase Polymerase Chain Reaction; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin; Time Factors; Transfection; Transgenes; Up-Regulation

The pathogenic mechanism of tonsillar hypertrophy is unknown and lacks a proper infectious or immunological explanation. Epidemiological studies point to polluted environments as the main cause of tonsillar hypertrophy in the adaptation of the juvenile organism. Tonsils and adenoids of 67 children aged 2-16 years (mean 5.9 years) were divided into three groups: recurrent tonsillitis (n = 21), recurrent tonsillitis with tonsillar hypertrophy (n = 21) and tonsillar hypertrophy without history of tonsillitis (n = 25). The following biological markers were studied: anti-streptolysin O antibody and anti-deoxy ribonuclease B antibody serology, microbiology and cell count of granulocytes in tonsils and adenoids as well as lymphocyte subsets and "ex vivo" endonuclease activity in tonsils. Anti-streptolysin O antibody and anti-deoxyribonuclease B antibody titres were significantly raised in recurrent tonsillitis. Positive bacterial cultures for Streptococcus pyogenes were rare in cases of tonsillar hypertrophy. T-lymphocytes counts were lower and the proportion of basophils was higher in hypertrophic tonsils than in recurrent tonsillitis. Two parameters of apoptosis were studied; the activation of endonuclease, inducing breakdown of DNA resulting in cell death, and the sensitivity to thapsigargin, known to trigger the cleavage of DNA by apoptotic endonuclease. In children with tonsillar hypertrophy both parameters were decreased contrasting with those with recurrent tonsillitis where apoptosis is increased. It may be speculated that the increase of basophils in children with tonsillar hypertrophy results in increased release of interleukin-4, which could prevent lymphoid apoptosis and lead to cell proliferation in tonsillar tissue.. Whereas recurrent tonsillitis is characterised by apoptotic death of lymphoid tissue, tonsillar hypertrophy is caused by environmental pollution agents that trigger the chronic inflammatory process without apoptotic cell death.

Topics: Apoptosis; Biomarkers; Child; Child, Preschool; DNA; Endonucleases; Female; Humans; Hypertrophy; Lymphocytes; Male; Palatine Tonsil; Recurrence; T-Lymphocyte Subsets; Thapsigargin; Tonsillitis