Administration Of Staurosporine Research Articles

Exploring the MEN1 dependent modulation of caspase 8 and caspase 3 in human pancreatic and murine embryo fibroblast cells

MEN1 mutation causes pancreatic neuroendocrine neoplasia and benign malignancies of the parathyroid, the adrenal cortex and pituitary gland. The transcriptional activity of its product menin promotes the expression of genes deputed to several cellular mechanism including cell death. Here, we focused on its implication in the activation of the initiator and executioner caspases after staurosporine mediated cell death in 2D and 3D human and murine cell models. The administration of staurosporine, a well-known inducer of apoptotic cell death, caused a significant reduction of BON1, QGP1 and HPSC2.2 cell viability. The transient knockdown of MEN1, performed by using a specific siRNA, caused a significant down-regulation of CDKN1A and TP53 transcripts. The treatment with 1 µM of staurosporine caused also a significant down-regulation of MEN1 and was able to restore the basal expression of TP53 only in QGP1 cells. Transient or permanent MEN1 inactivation caused a decrease of caspase 8 activity in BON1, HPSC2.2 cells and MEN1−/− MEFs treated with staurosporine. Caspase 3/7 activity was suppressed after administration of staurosporine in MEN1 knocked down HPSC2.2 and MEN1−/− MEFs as well. The cleaved caspase 8 and caspase 3 decreased in human cells after MEN1 knockdown and in MEN1−/− MEFs. The treatment with staurosporine caused a reduction of the size of MEN1+/+ MEFs spheroids. Instead, MEN1−/− MEFs spheroids did not show any significant reduction of their size. In conclusion, MEN1 controls the activity of the initiator caspase 8 and the executioner caspase 3 in human and murine cells. Restoring of a functional MEN1 and interfering with the apoptotic mechanism could represent a future strategy for the treatment of MEN1-related malignancies.

Effects of High Glucose on AVP-Induced Hyperplasia, Hypertrophy, and Type IV Collagen Synthesis in Cultured Rat Mesangial Cells

Introduction. Hyperglycemia is a principal characteristic of diabetes and influences many cellular functions. Diabetic nephropathy is characterized by glomerular mesangial expansion which could result from increased mesangial cell extracellular matrix synthesis induced by hyperglycemia. Methods. To investigate whether the physiological functions of mesangial cells are altered in a diabetic environment, we evaluated the effect of high extracellular glucose concentration on thymidine/leucine incorporation, hyperplasia/hypertrophy, and type IV collagen synthesis, induced by vasopressin (AVP), in cultured rat mesangial cells. Results. The exposure of mesangial cells to a high glucose concentration (30 mM) significantly reduced AVP-induced thymidine incorporation and hyperplasia compared with normal glucose (10 mM). By contrast, treatment of mesangial cells with AVP in the presence of high extracellular glucose significantly increased leucine incorporation, hypertrophy, and type IV collagen synthesis compared with those at normal glucose levels. The administration of staurosporine, a protein kinase C inhibitor, reversed these effects of high-glucose conditions. Furthermore, the nonpeptide AVP V1A receptor-selective antagonists potently inhibited these AVP-induced physiological responses in mesangial cells cultured in high-glucose conditions. Conclusions. These results demonstrate that high glucose suppresses mesangial cell proliferation but enhances hypertrophy and type IV collagen synthesis induced by AVP. This increased mesangial cell hypertrophy and extracellular matrix synthesis may play a crucial role in the glomerular mesangial expansion common to diabetic nephropathy.

Role of protein kinase C-ε (PKCε) in isoflurane-induced cardioprotection

Volatile anaesthetics precondition the heart against infarction, an effect partly mediated by activation of the epsilon isoform of protein kinase C (PKCepsilon). We investigated whether cardioprotection by activation of PKCepsilon depends on the isoflurane concentration. Anaesthetized rats underwent 25 min of coronary artery occlusion followed by 120 min of reperfusion and were randomly assigned to the following groups (n=10 in each group): isoflurane preconditioning induced by 15 min administration of 0.4 minimal alveolar concentration (MAC) (0.4MAC), 1 MAC (1MAC) or 1.75 MAC (1.75MAC) followed by 10 min washout before ischaemia. Each protocol was repeated in the presence of the PKC inhibitor staurosporine (10 microg kg(-1)): 0.4MAC+S, 1MAC+S and 1.75MAC+S. Controls were untreated (CON) and additional hearts received staurosporine without isoflurane (S). In a second set of experiments (n=6 in each group) hearts were excised before the infarct inducing ischaemia, and phosphorylation and translocation of PKCepsilon were determined by western blot analysis. Isoflurane reduced infarct size from a mean of 61(SEM 2)% of the area at risk in controls to 20(1)% (0.4MAC), 26(3)% (1MAC) and 30(1)% (1.75MAC) (all P<0.01 vs CON or S). This protection was partially reversed by administration of staurosporine in the 0.4MAC+S group (30[2]%; P<0.05 vs 0.4MAC) group, but not after administration of 1 MAC or 1.75 MAC isoflurane (26[2]% and 31[2]%, respectively). Thus 0.4MAC increased PKCepsilon phosphorylation, and this effect was blocked by staurosporine. Higher concentrations of isoflurane did not change PKCepsilon phosphorylation. PKCepsilon was translocated to the membrane fraction after administration of 0.4 MAC isoflurane, but not after 1.0 or 1.75 MAC. Although isoflurane preconditioning resulted in a reduction in infarct size at all concentrations used, the protection was mediated by phosphorylation and translocation of PKCepsilon only at 0.4 MAC.

Effect of the protein kinase C inhibitor, staurosporine, on the high dose of methamphetamine-induced behavioral sensitization to dizocilpine (MK-801)

In our preliminary study, methamphetamine (METH) at 2.5 mg/kg, but not at 1.0 mg/kg, induced a delayed increase in glutamate levels in the nucleus accumbens (NAc). We hypothesize that repeated increases in glutamate levels produces behavioral sensitization to a selective uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, dizocilpine (MK-801), and that an activation of protein kinase C (PKC) plays an important role for this sensitization. This study was conducted to confirm delayed increases in glutamate levels induced by a higher dose of METH (2.5 mg/kg), and to examine the effect of straurosporine, a PKC inhibitor, on the higher dose of METH-induced sensitization to dizocilpine. The effects of METH on extracellular glutamate levels in the NAc were studied using in vivo microdialysis. Locomotor activity was measured by using an infrared sensor. METH at 2.5 mg/kg, but not at 1.0 mg/kg, induced delayed increases in glutamate levels. The acute administration of staurosporine did not affect the locomotor activity by a single injection of METH (2.5 mg/kg). Repeated METH administrations (2.5 mg/kg, once in every other day, for five times) developed behavioral sensitization to the locomotion-inducing effect of dizocilpine (0.2 mg/kg), a selective uncompetitive NMDA receptor antagonist. Staurosporine (0.1 mg/kg), given 120 min later for every METH treatment, inhibited the development of behavioral sensitization to dizocilpine. These results suggest the involvement of increased glutamate levels and an activation of PKC in delayed-induced synaptic and cellular plasticity underlying the higher dose of METH-induced behavioral sensitization to dizocilpine.

Mechanical overload-induced apoptosis: a study in cultured neonatal ventricular myocytes and fibroblasts.

Apoptosis of cardiac myocytes has been implicated in cardiac dysfunction due to chronic hemodynamic overload. Reports on the role of apoptosis in the transition from hypertrophy to decompensated heart failure are not unequivocal. In this study we analysed the direct relationship between mechanical overload and induction of apoptosis in an in vitro model of cultured heart cells. Cyclic mechanical stretch was applied to cultured neonatal rat ventricular myocytes and fibroblasts. Several indicators of apoptosis were examined, such as morphological features, caspase-3 activity and DNA fragmentation. Mechanical strain did not induce any significant change in these parameters as compared to non-stretched myocytes or fibroblasts. However, administration of staurosporine, a known inducer of apoptosis, induced massive apoptosis in myocytes as well as fibroblasts. We conclude that this in vitro cell model system lacks a direct link between mechanical stretch and apoptosis. The three-dimensional structure-function relationship of myocardial tissue in the intact heart may elicit stretch-induced molecular signaling cascades in a much more complex way than in monolayer cultures of cardiac cells.

μ-Opioid and GABA B receptors modulate different types of Ca 2+ currents in rat nodose ganglion neurons

Whole-cell patch-clamp recordings were obtained from nodose ganglion neurons acutely dissociated from 10–30-day-old rats to characterize the Ca 2+ channel types that are modulated by GABA B and μ-opioid receptors. Five components of high-threshold current were distinguished on the basis of their sensitivity to blockade by ω-conotoxin GVIA, nifedipine, ω-agatoxin IVA and ω-conotoxin MVIIC. Administration of the μ-opioid agonist H-Tyr-D-Ala-Gly-Phe(N-Me)-Gly-ol (0.3–1 mM) or the GABA B agonist baclofen in saturating concentrations suppressed high-threshold Ca 2+ currents by 49.9±2.4% n=69) and 18.7±2.1% n=35), respectively. The inhibition by H-Tyr-D-Ala-Gly-Phe(N-Me)-Gly-ol exceeded that by baclofen in virtually all neurons that responded to both agonists (67%), and occlusion experiments revealed that responses to μ-opioid and GABA B receptor activation were not linearly additive. In addition, administration of staurosporine, a non-selective inhibitor of protein kinase A and C, did not affect the inhibitory responses to either agonist or prevent the occlusion of baclofen-induced current inhibition by H-Tyr-D-Ala-Gly-Phe(N-Me)-Gly-ol. Blockade of N-type channels by ω-conotoxin GVIA eliminated current suppression by baclofen in all cells tested n=11). μ-Opioid-induced inhibition in current was abolished by ω-conotoxin GVIA in 12 of 30 neurons tested, but was only partially reduced in the remaining 18 neurons. In the latter cells administration of ω-agatoxin IVA reduced, but did not eliminate the μ-opioid sensitive current component that persisted after blockade of N-type channels. This residual component of μ-opioid-sensitive current was blocked completely by ω-conotoxin MVIIC in nine neurons, whereas responses to H-Tyr-D-Ala-Gly-Phe(N-Me)-Gly-ol were still recorded in the remaining cells after administration of these Ca 2+ channel toxins and nifedipine. Dihydropyridine-sensitive (L-type) current was not affected by activation of μ-opioid or GABA B receptors in any of the neurons. These data indicate that in nodose ganglion neurons μ-opioid receptors are negatively coupled to N-, P- and Q-type channels as well as to a fourth, unidentified toxin-resistant Ca 2+ channel. In contrast, GABA B receptors are coupled only to N-type channels. Furthermore, the results do not support a role for either protein kinase C or A in the modulatory pathway(s) coupling μ-opioid and GABA B receptors to Ca 2+ channels, but rather lend credence to the notion that the signalling mechanisms utilized by these two receptors might simply compete for inhibitory control of a common pool of N-type channels.

Local Intraosseous Injection of Staurosporine Increased Bone Formation in Rats

The effects of local intraosseous injection of staurosporine on bone formation were investigated in the weaning Sprague-Dawley rats. The left tibia received administration of staurosporine (0.5 pmole/day for 2 weeks) into the metaphysis. The contralateral right tibia received injection with DMSO vehicle and saline was used for the control. Another group of rats which received no injection provided another normal control. The results showed that the body weight gain was decreased by staurosporine, but the tibial length was not affected. Histological examination showed that local injection of staurosporine significantly increased the metaphyseal bone trabeculae by 36%, as compared to the controls. In addition, periosteal bone formation was demonstrated in the staurosporine-treated limbs. The number of osteoblasts in primary spongiosa of the staurosporine-injected limbs and those of contralateral limbs was not different, so was the osteoclasts. However, the number of osteoblasts and that of osteoclasts in secondary spongiosa in the staurosporine-injected limbs increased, as compared to the controls. This finding confirmed a stimulatory role of staurosporine in bone formation.

A role of protein kinase C in the alteration of renal glucose-6-phosphatase activity caused by fluoride.

In this study, protein kinase C was demonstrated to operate as a down-regulator of glucose-6-phosphatase in the kidney, at least. Renal glucose-6-phosphatase activity reached a maximum level in 3 h after the administration of fluoride to rats. The incremental increase of renal glucose-6-phosphatase activity caused by fluoride administration was markedly amplified by the administration of staurosporine (66 micrograms/kg, i.p.), which has inhibitory activity against protein kinase C, or by the administration of H-7 (5 mumol/kg, i.p.), a specific and strong inhibitor of protein kinase C. Interestingly, the finding indicated that protein kinase C operates as a down regulator of renal glucose-6-phosphatase activity. The finding was reconfirmed by the result that fluoride-stimulated glucose-6-phosphatase activity was further enhanced by treatment with calphostin C (200 nmol/kg, i.p.), a specific and strong inhibitor of protein kinase C, but the change was small. Moreover, calmodulin was indicated as being possibly concerned with the down regulation of renal glucose-6-phosphatase activity by using W-7 (5 mumol/kg, i.p.), a calmodulin specific inhibitor.

Effects of Staurosporine on the Pressor Responses to Alpha-Adrenoceptor Agonists in Pithed Rats: A Comparison with Nifedipine

The effects of the calcium channel antagonist nifedipine and the protein kinase C (PKC) inhibitor, staurosporine were examined on the pressor actions of a full, cirazoline, and a partial, St587, alpha 1-adrenoceptor agonist as well as the alpha 2-adrenoceptor agonist B-HT 920 in the pithed rat preparation. Administration of nifedipine or staurosporine significantly reduced the diastolic blood pressure of pithed rats. Staurosporine displaced the dose-diastolic pressure response curves for B-HT 920 and St587 to the right in a dose-dependent manner. The ED50 value for the dose-response curves to B-HT 920 and St587 were found to be significantly increased after the administration of staurosporine. Staurosporine also caused a depression of the maximum response to B-HT 920 and St587. The presence of nifedipine resulted in an increase in the ED50 value of the dose-response curve to B-HT 920 and St587, and this was accompanied by significant reductions of the maximum response, whereas the administration of either staurosporine or nifedipine did not significantly affect the calculated ED50 value of the dose-response curve to cirazoline. In the presence of the calcium channel antagonist but not the PKC inhibitor the maximum response to cirazoline was significantly depressed. As judged from the slope function of the dose-response curves the nature of the inhibition produced by nifedipine compared to staurosporine also appeared to differ. Thus, with nifedipine as the antagonist, the slope function of the dose-response curve for alpha-agonists was significantly reduced. Moreover, in contrast to the actions of staurosporine, nifedipine reduced the maximum response to cirazoline in a dose-independent manner. This study supports the hypothesis that activation of alpha-adrenoceptors that have a substantial dependence on extracellular calcium for vasoconstriction are susceptible not only to the action of calcium channel antagonists but also to the actions of the PKC inhibitor staurosporine, thus suggesting that PKC may modulate directly/indirectly calcium channel activity in vascular smooth muscle.

Staurosporine, a protein kinase inhibitor, attenuates basal forebrain-lesion-induced amnesia and cholinergic neuronal deficit

We have investigated whether administration of staurosporine, which has been reported to induce differentiation in the human neuroblastoma cell in vitro, attenuates amnesia induced by basal forebrain lesion in rats. Multiple dosage of staurosporine at the doses of 0.05 and 0.1 mg/kg (i.p.) attenuated the impaired performance of the water maze task. Moreover, staurosporine (0.1 mg/kg) reversed the decrease of choline acetyltransferase activity in the fronto-parietal cortex. These results suggest that staurosporine attenuates amnesia through reversal of deficits in cholinergic neurons induced by basal forebrain lesion, and that neurotrophic factor-like substances may open the way for novel therapeutic approaches to Alzheimer's disease.