pd-150606 and Hypoxia

The goal of this study was to evaluate the effects of anti-inflammatory cytokine, interleukin-10 (IL-10), and calpain inhibitor, PD150606, on the expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits in rat hippocampal slices exposed to repeated brief hypoxic episodes. We studied both individual and combinatory effects of PD150606 and IL-10 on the expression of AMPA receptor subunits under hypoxic conditions for GluA1 and GluA2 as well as their phosphorylated forms - pSer831-GluA1 and pSer880-GluA2. Additionally, we studied whether brief hypoxic episodes and IL-10 may affect mRNA expression of transcriptional factors such as hypoxia-inducible factor-1α and nuclear factor κB (NF-κB). Western blotting analysis of hippocampal slice homogenates revealed that IL-10 and PD150606, both individually and in combination, ameliorate hypoxia-induced decrease in the expression of GluA1 and pSer831-GluA1, with different level of efficiency measured at 10, 50, and 90 min after hypoxia induction. Interestingly, brief hypoxic episodes did not induce any changes in the expression of GluA2 and pSer880-GluA2 subunits, whereas PD150606 showed biphasic effect, decreasing the expression of GluA2 and pSer880-GluA2 at 10 min and potentiating it at 90 min after hypoxia induction. IL-10 alone did not show any effect but was able to reverse PD150606 action on the expression of pSer880-GluA2 at 10 min and further potentiated it for GluA2 at 90 min after hypoxia. Finally, PCR analysis revealed that modulation of GluA1 and GluA2 expressions by hypoxia, and IL-10 was not associated with changes in the expression of hypoxia-inducible factor-1α and nuclear factor-κB (NF-κB) transcriptional factors.

Topics: Acrylates; Animals; Central Nervous System Agents; Gene Expression; Hippocampus; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Interleukin-10; Male; NF-kappa B; Rats, Wistar; Receptors, AMPA; RNA, Messenger; Time Factors; Tissue Culture Techniques

Excessive growth of pulmonary arterial (PA) smooth muscle cells (SMCs) is a major component of PA hypertension (PAH). The calcium-activated neutral cysteine proteases calpains 1 and 2, expressed by PASMCs, contribute to PH but are tightly controlled by a single specific inhibitor, calpastatin. Our objective was to investigate calpastatin during pulmonary hypertension (PH) progression and its potential role as an intracellular and/or extracellular effector. We assessed calpains and calpastatin in patients with idiopathic PAH and mice with hypoxic or spontaneous (SM22-5HTT(+) strain) PH. To assess intracellular and extracellular roles for calpastatin, we studied effects of the calpain inhibitor PD150606 on hypoxic PH in mice with calpastatin overexpression driven by the cytomegalovirus promoter (CMV-Cast) or C-reactive protein (CRP) promoter (CRP-Cast), inducing increased calpastatin production ubiquitously and in the liver, respectively. Chronically hypoxic and SM22-5HTT(+) mice exhibited increased lung calpastatin and calpain 1 and 2 protein levels and activity, both intracellularly and extracellularly. Prominent calpastatin and calpain immunostaining was found in PASMCs of remodeled vessels in mice and patients with PAH, who also exhibited increased plasma calpastatin levels. CMV-Cast and CRP-Cast mice showed similarly decreased PH severity compared with wild-type mice, with no additional effect of PD150606 treatment. In cultured PASMCs from wild-type and CMV-Cast mice, exogenous calpastatin decreased cell proliferation and migration with similar potency as PD150606 and suppressed fibronectin-induced potentiation. These results indicate that calpastatin limits PH severity via extracellular mechanisms. They suggest a new approach to the development of treatments for PH.

Topics: Acrylates; Animals; Calcium-Binding Proteins; Calpain; Cell Movement; Cell Proliferation; Cytomegalovirus; Disease Progression; Extracellular Space; Heart Function Tests; Humans; Hypertension, Pulmonary; Hypoxia; Intracellular Space; Male; Mice, Inbred C57BL; Mice, Transgenic; Myocytes, Smooth Muscle; Promoter Regions, Genetic; Pulmonary Artery

The goal of the present study was to determine the role of calpain in changes in plasma membrane permeability and cytoskeleton-associated paxillin, vinculin, talin, and alpha-actinin levels during acute renal cell death. The mitochondrial inhibitor antimycin A or hypoxia produced graded plasma membrane permeability in renal proximal tubules (RPTs), first allowing propidium iodide (PI, molecular mass 668 Da) influx and then lactate dehydrogenase (LDH, molecular mass 130 kDa) release. Cytoskeleton-associated paxillin levels decreased concomitantly with PI influx and before LDH release, whereas cytoskeleton-associated talin and vinculin levels decreased concomitantly with LDH release. Cytoskeleton-associated alpha-actinin levels did not change during antimycin A exposure or hypoxia. Purified micro-calpain cleaved paxillin, talin, vinculin, but not alpha-actinin. The dissimilar calpain inhibitors 3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid (PD150606) or chloroacetic acid N'-[6,7-dichloro-4-phenyl)-3-oxo-3,4-dihydroquinoxalin-2-yl] hydrazide (SJA7029) preserved cytoskeleton-associated paxillin, talin, and vinculin levels and prevented PI influx and LDH release in antimycin A-exposed or hypoxic RPTs. These results suggest that calpain mediates increased plasma membrane permeability and hydrolysis of cytoskeleton-associated paxillin, vinculin, and talin during renal cell death.

Topics: Acrylates; Actinin; Animals; Anti-Bacterial Agents; Antimycin A; Calpain; Cell Death; Cell Membrane Permeability; Coloring Agents; Cysteine Proteinase Inhibitors; Cytoskeletal Proteins; Dogs; Female; Hypoxia; Immunoblotting; Kidney; Paxillin; Phosphoproteins; Propidium; Rabbits; Swine; Talin; Vinculin

The effect of the newly developed, nonpeptide, calpain inhibitor, PD 150606, on hypoxia and ionomycin-induced increases in calpain activity in rat proximal tubules (PT) was determined. PD150606 inhibited both hypoxia and ionomycin-induced calpain activity as determined by the fluorescent substrate N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methyl coumarin (N-succinyl-Leu-Leu-Val-Tyr-AMC). This decrease in calpain activity was accompanied by dose-dependent cytoprotection against hypoxia and ionomycin-induced cell membrane damage. PD150606 had no effect on cathepsin B and L activity in PT as measured by the fluorescent substrate, benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methyl coumarin (Z-Phe-Arg-AMC). The effects of low intracellular pH (pHi) or low free cytosolic calcium [Ca2+]i on this hypoxia-induced calpain activity were also determined. Both low pHi and low [Ca2+]i attenuated the hypoxia-induced increase in calpain activity. This attenuation of calpain activity was observed early before hypoxia-induced membrane damage and was associated with marked reduction in the typical pattern of hypoxia-induced cell membrane damage observed in this model. To identify the isoform of calpain activated in rat proximal tubules, normoxic, hypoxic and ionomycin treated tubules were fractionated by MONO-Q anion exchange chromatography and the fractions were assayed for calpain activity. A single peak of calpain activity characteristic of mu-calpain was found. The calcium dependency of the calpain activity was in the nanomolar range, further confirming that the activity was the low Ca(2+)-sensitive mu-calpain. The present study suggests that in rat proximal tubules: (1) PD 150606 is a specific inhibitor of calpain and not cathepsins B and L; (2) the cytoprotective effects of low pHi and low [Ca2+]i are mediated, at least in part, by inhibition of calpain activity; and (3) the predominant active form of calpain is the isoenzyme mu-calpain.

Topics: Acrylates; Analysis of Variance; Animals; Calcium; Calpain; Cathepsins; Dose-Response Relationship, Drug; Hydrogen-Ion Concentration; Hypoxia; In Vitro Techniques; Kidney Tubules, Proximal; L-Lactate Dehydrogenase; Male; Rats; Rats, Sprague-Dawley