Renal Expression of the Brain and Muscle Isoforms of Glycogen Phosphorylase in Different Cell Types

Abstract

Kidney contains glycogen. Glycogen is degraded by glycogen phosphorylase (GP). This enzyme comes in three isoforms, one of which, the brain isozyme (GP BB), is known to occur in kidney. Its pattern of distribution in rat kidney was studied in comparison to that of the muscle isoform (GP MM) with the aim to see if for GP BB and GP MM there were functional similarities in brain and kidney. In immunoblotting and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) experiments, both isozymes and their respective mRNAs were found in kidney homogenates. GP BB was immunocytochemically detected in collecting ducts which were identified by the marker protein aquaporin-2. GP MM was localized exclusively in interstitial cells of cortex and outer medulla. These cells were identified as fibroblasts by their expression of 5'-ectonucleotidase (cortex) or by their morphology (outer medulla). The physiological role of both isozymes is discussed in respect to local demands of energy and of proteoglycan building blocks.

Expression of the Brain and Muscle Isoforms of Glycogen Phosphorylase in Rat Heart

“…Application on heart of the fixation method employed for nervous tissue [immersion into 3.5% (w/v) paraformaldehyde/PBS at 4°C for 24 h] [5] resulted in intense autofluorescence as already reported for kidney sections [17]. Therefore the tissue had to be treated otherwise, i.e., the fresh cryosections were fixed with 4% (w/v) N-(3-dimethylaminopropyl)-N 0 -ethylcarbodiimideÁHCl/PBS (EDC) (Sigma-Aldrich, Steinheim, Germany) at room temperature for 1 h [19] and then washed with PBS.…”

“…Supernatants from homogenates were prepared as described for rat kidney [17] and taken for Western blotting experiments. For immunocytochemical experiments ventricular and atrial slices of up to 5 mm thickness were shockfrozen in liquid nitrogen and stored at -80°C.…”

“…The culture supernatant containing the monoclonal antibody against 5 0 -ectonucleotidase [18] was a kind gift of Dr. Brigitte Kaissling, University of Zuerich. Also second antibodies were used as described elsewhere [17].…”

“…Therefore the tissue had to be treated otherwise, i.e., the fresh cryosections were fixed with 4% (w/v) N-(3-dimethylaminopropyl)-N 0 -ethylcarbodiimideÁHCl/PBS (EDC) (Sigma-Aldrich, Steinheim, Germany) at room temperature for 1 h [19] and then washed with PBS. This fixation yielded better morphological preservation than the fixation procedure used for kidney sections [17] and was suitable for the use of antisera against GP BB and GP MM [5]. The sections were incubated with antisera as described for nervous [5] and renal tissues [17].…”

“…Other organs express GP MM only in very low concentrations [16,17]. Even in heart muscle the level of GP MM is considerably lower than in skeletal muscle [16].…”