Activation of caspases in human spermatozoa during cryopreservation--an immunoblot study.

Cellular stress to ejaculated spermatozoa such as cryopreservation is known to induce caspase-derived, apoptotic signaling. Therefore, the proenzymes and active forms of caspases 1, 3, 8 and 9 were examined by western blot technique in unfrozen and frozen human spermatozoa of infertility patients and of healthy donors. Twenty-two semen samples derived from healthy donors and 26 semen samples of unselected infertility patients were divided into 3 parts, two of them were cryostored at -196 degrees C with 7% or 14% (v/v, final concentration) of glycerol. The caspases were detected by immunoblots with polyclonal rabbit-anti-caspases-antibodies after 15% sodium dodecyl sulfate-polyacrylgel electrophoresis (SDS-PAGE) under reducing conditions. For evaluation of the differences between amounts of caspase protein the luminol/H(2)O(2) method was applied. A significant increase of activated caspase-1 in donors, of caspase-8 in patients and caspase-9 in patients and donors after cryopreservation were found, whereas, the application of 14% glycerol resulted in higher amounts of activated caspase than did 7% glycerol. Possibly, glycerol may also contribute to activation of caspases via direct toxic effects to mitochondria during cryopreservation of spermatozoa. This finding strongly supports an hypothesis of a higher mitochondria-derived apoptosis-sensitivity of spermatozoa in patients than in healthy donors during cryopreservation. Inactive caspase-3 was reduced subsequent to cryopreservation in patients (p<0.05) and non-significant in donors (p<0.05). Active caspase-3 was detectable in all samples but without significant differences between the three assays. It is concluded that mechanisms associated with apoptotic processes deserve attention in cryopreservation of spermatozoa in order to conserve vital sperm functions after thawing.

Mechanism of cell death of rat cardiac fibroblasts induced by serum depletion.

Serum starvation has recently been shown to cause cell death of cardiac fibroblasts and increased synthesis of extracellular matrix proteins in the surviving cells. In the present study, events occurring in the dying cells were investigated. Cultured adult rat cardiac fibroblasts were exposed to serum-free medium. Cell number was measured using a Coulter Counter Channelyzer. The activity of the extracellular signal-regulated or mitogen-activated protein kinases (ERK1/2, p42/p44(MAPK)), the p38 kinase (p38(MAPK)), the c-Jun N-terminal kinases (p46/p54(JNK)), and Akt kinase was assessed by Western blotting and phospho-specific antibodies. Caspase 7-cleavage was investigated by Western blotting and specific antibodies. Caspase 3 activity was measured by detection of its cleaved substrate. The appearance of necrosis was studied by inclusion of trypan blue. Apoptosis was assessed by DNA ladder formation. The mRNA expression of Bax and Bcl-2 was investigated by quantitative real-time PCR. Serum withdrawal led to the death of 26% of cultured isolated cardiac fibroblasts during the first 5 h. The activity of the p42/ p44(MAPK) as well as of Akt kinase was partially reduced. For p46/p54(JNK) and p38(MAPK), elevated phosphorylation was measured. Inhibition of p46/p54(JNK) and p38(MAPK) activity by SB202190 did not affect the decrease in cell number. Cleavage of caspase 7 was detected after 90 min. However, no activation of caspase 3 was measured. DNA fragmentation was not found after serum depletion. Trypan blue staining, however, was observed in 16% of the cells after 5 h. The mRNA levels of both Bax and Bcl-2 were increased after 30 min. These results indicate the appearance of necrosis during serum starvation in cardiac fibroblasts. However, some processes typical of apoptosis were also detected.

Regulation of norepinephrine-induced proliferation in cardiac fibroblasts by interleukin-6 and p42/p44 mitogen activated protein kinase.

Norepinephrine (NE) is involved in many cardiovascular diseases such as congestive heart failure. We have recently reported that NE had a comitogenic effect in isolated cardiac fibroblasts, and that it activated p42/p44 mitogen activated protein kinase (MAPK). This study was designed to characterize a possible mechanism involved in the proliferative effect of NE. Isolated rat cardiac fibroblasts were exposed to NE (10 microM) for up to 8 h, and interleukin-6 (IL-6) expression was measured by Ribonuclease Protection Assay and Western blotting. The activity of p42/p44MAPK was analyzed by Western blotting. Cell number was assessed by use of a Coulter Counter. IL-6/GAPDH mRNA was increased by NE in a time-dependent manner reaching 23 fold stimulation after 1 h compared to untreated samples. Immunoreactivity to IL-6 was not found in controls. After 16 h of exposure to NE, IL-6 protein was detected. It further increased up to 48 h. The effect of NE on IL-6 mRNA was abolished by the beta-adrenoceptor blockers propranolol, metoprolol (beta1) and ICI 118.551 (beta2), but not by the alpha-adrenoceptor blockers prazosin (alpha1) and yohimbine (alpha2). The MAPK-inhibitor PD98059 suppressed the NE-induced MAPK activation in a concentration-dependent fashion after 5 min, attenuated the NE-induced IL-6 expression after 2 h, and suppressed the proliferative effect of NE from 53 to 18% after 48 h. Recombinant IL-6 caused an increase in proliferation by 31% after 48 h. Simultaneous application of the IL-6 antibody reduced the NE-induced proliferation to 34%, and completely prevented the IL-6 induced effect. These results suggest that NE induces proliferation of rat cardiac fibroblasts in part by increasing the expression of IL-6 through regulation of MAPK.

Catecholamine-induced cardiac hypertrophy in rats is associated with the activation of p70 kinase and c-Jun NH(2)-terminal kinase 2.

OBJECTIVE: To assess the effect of intravenous infusion of adrenergic, hypertrophic agonists on the activity of the ribosomal S6 (p70(S6)) kinase and the c-Jun NH(2)-terminal kinase 2 (JNK2) in adult rat hearts. ANIMALS AND METHODS: Noradrenaline (NA), isoproterenol (ISO), phenylephrine (PE), NA in combination with propranolol (NA+Prop) and NA in combination with prazosin (NA+Praz) were continuously intravenously infused in rat hearts for up to 72 h. The expression and phosphorylation status of JNK2 and p70(S6) kinase were investigated by western blotting and use of specific and phosphospecific antibodies. The activity of p70(S6) kinase was measured by incorporation of (32)P-labelled phosphate into the specific substrate S6 peptide. RESULTS: The ratio of left ventricular weight to body weight was increased by NA, and by alpha- (PE, NA+Prop) and beta-adrenergic (ISO, NA+Praz) stimulation. Right ventricular weight to body weight ratio was higher only after beta-adrenergic stimulation (ISO, NA+Praz). p70(S6) kinase activity was stimulated predominantly through beta-adrenoceptors in parallel with left and right ventricular hypertrophy. Activation of JNK2 was mainly dependent on alpha-adrenoceptor activation, which led to hypertrophy of only the left ventricle. CONCLUSIONS: The activation of p70(S6) kinase and JNK2 may be implicated in the development of catecholamine-induced cardiac hypertrophy in vivo.