Reperfusion but Not Acute Ischemia in Pig Small Intestine Induces Transcriptionally Mediated Heat Shock Response in situ

Abstract

Background: Although there is data on the cytoprotective role of heat shock proteins in intestinal ischemia-reperfusion, the effects of ischemia and reperfusion per se on the small intestinal heat shock response have been poorly characterized. Methods: Four female pigs were subjected to 60-min ischemia by superior mesenteric artery occlusion followed by 360-min reperfusion. Systemic and local hemodynamics were monitored. Samples from the jejunal mucosa and muscularis were obtained for histology and for time series molecular biologic analyses of heat shock transcription factor 1 (HSF1), hsp70 mRNA and Hsp70 protein. Results: A 30-min reperfusion of jejunum after a preceding 1-hour ischemia results in a significantly increased DNA-binding activity of HSF1, in a 10-fold increase of hsp70 mRNA in the mucosal and in a 7-fold increase in the muscular layers. Translational activation and accumulation of Hsp70 protein occurs after 60 min of reperfusion in the intestine. Nevertheless, a 60-min ischemia inducing mucosal detachment does not induce the heat shock response at any level analyzed. Conclusions: Ischemia alone is insufficient to induce the heat shock response, whereas subsequent reperfusion induces the response via transcriptionally mediated induction of Hsp70 synthesis both in the mucosal and muscular layers.

Exercise-induced oxidative stress and muscle stress protein responses in trotters

“…and quantified using image analysis software (NIH-Image, USA). A gel mobility shift assay was performed as previously described Oksala et al 2002). The protein extracts were prepared similarly to those in the Western blot and mixed with isotope-labelled probes corresponding to the two overlapping heat shock elements.…”

Heat Shock Preconditioning and Eicosanoid Pathways Modulate Caspase 3-Like Activity in Superficially Injured Isolated Guinea Pig Gastric Mucosa

“…The analysis was performed as described previously by our group [9,10,26,27]. Briefly, the tissue samples were homogenized and sonicated and protein extracts (30 Ìg protein per lane) were electrophoresed on SDS-PAGE and transferred to a nitrocellulose membrane (Millipore, Bedford, Mass., USA) which was stained with Ponceau S solution (Sigma, St. Louis, Mo., USA), and checked for an even loading and transfer.…”

“…The tissue samples were homogenized and sonicated similarly as in Western blot [9,10,26,27]. The samples were centrifuged and supernatants collected.…”

Heat Shock Preconditioning Induces Protein Carbonylation and Alters Antioxidant Protection in Superficially Injured Guinea Pig Gastric Mucosa In Vitro

“…The tissue samples were rinsed with + 4 • C Ringer's solution, frozen in liquid nitrogen, homogenized and sonicated 3 times for 5 s each in a solution containing 25% glycerol, 0.42 mol/L NaCl, 1.5 mmol/L MgCl 2 , 0.2 mmol/L ethylenediaminetetraacetic acid, 20 mmol/L N-2-hydroxyethylpiperazine-N -2-ethanesulfonic acid, 5 µmol/L dithiotreitol, and 5 µmol/L phenylmethylsulphoxide at + 4 • C as described by our group [7,8,10,[24][25][26]. Protein extracts (30 µg protein per lane) together with molecular weight markers were electrophoresed on SDS/PAGE (sodium dodecyl sulfate/8 or 10% polyacrylamide gel electrophoresis) and transferred to a nitrocellulose membrane (Millipore, Bedford, MA).…”