Cecilia S. Koenig

Pex3p-dependent peroxisomal biogenesis initiates in the endoplasmic reticulum of human fibroblasts.

The mechanisms of peroxisomal biogenesis remain incompletely understood, specially regarding the role of the endoplasmic reticulum (ER) in human cells, where genetic disorders of peroxisome biogenesis lead to Zellweger syndrome (ZS). The Pex3p peroxisomal membrane protein (PMP) required for early steps of peroxisome biogenesis has been detected in the ER in yeast but not in mammalian cells. Here, we show that Pex3p‐GFP expressed in a new ZS cell line (MR), which lacks peroxisomes due to a mutation in the PEX3 gene, localizes first in the ER and subsequently in newly formed peroxisomes. Pex3p bearing an artificial N‐glycosylation site shows an electrophoretic shift indicative of ER targeting while en route to preformed peroxisomes in normal fibroblast. A signal peptide that forces its entry into the ER does not eliminate its capability to drive peroxisome biogenesis in ZS cells. Thus, Pex3p is able to drive peroxisome biogenesis from the ER and its ER pathway is not privative of ZS cells. Cross‐expression experiments of Pex3p in GM623 cells lacking Pex16p or Pex16p in MR cells lacking Pex3p, showed evidence that Pex3p requires Pex16p for ER location but is dispensable for the ER location of Pex16p. These results indicate that Pex3p follows the ER‐to‐peroxisomal route in mammalian cells and provides new clues to understand its function. J. Cell. Biochem. 107: 1083–1096, 2009.

Modulation of hepatic content and biliary excretion of P-glycoproteins in hepatocellular and obstructive cholestasis in the rat

BACKGROUND/AIMS Release into bile of canalicular membrane enzymes, such as alkaline phosphatase and gamma-glutamyl transpeptidase, is significantly increased in rats subjected to experimental models of hepatocellular or obstructive cholestasis. This effect appears to be related to a greater susceptibility of these membrane intrinsic proteins to the solubilizing effects of secreted bile acids. It is not known whether canalicular membrane transport proteins, such as P-glycoprotein isoforms, involved in ATP-dependent xenobiotic biliary excretion and phospholipid secretion, are excreted into bile and whether this process is modified in cholestasis. The aims of this work have been to investigate in the rat: a) whether P-glycoproteins are normally excreted into bile, b) whether their excretion is modified in two experimental models of cholestasis, i.e., hepatocellular cholestasis induced by ethynylestradiol and obstructive cholestasis, and c) whether observed changes correlate with bile acid and phospholipid secretion and enzyme release into bile and with relative P-glycoprotein content in hepatic tissue and isolated and purified canalicular membranes. METHODS P-glycoproteins in bile and hepatic tissue were identified and quantitated by Western-blotting and immunohistochemistry using the C219 MAb. Changes in total mdr mRNA were analyzed by Northern-blotting. RESULTS Like canalicular membrane enzymes, P-glycoproteins are normally excreted into bile. Ethynylestradiol-induced cholestasis was associated with a 4.9-fold increase in P-glycoprotein excretion compared with controls while, in contrast, the excretion of the carrier decreased markedly in obstructive cholestasis to 2% of control values. P-glycoprotein excretion per nmol of secreted bile acids increased 4.4-fold in ethynylestradiol-induced cholestasis but decreased to 2% of control values in obstructive cholestasis. Total mdr mRNA levels in hepatic tissue were markedly increased (3.4-fold) in rats subjected to obstructive cholestasis and moderately increased (1.6-fold) in the ethynylestradiol group, compared with controls. P-glycoprotein content in isolated canalicular membranes was slightly decreased by 15% in ethynylestradiol-induced cholestasis, while it increased 4.7-fold in obstructive cholestasis. Immunohistochemistry of rat livers showed that P-glycoprotein reaction at the canalicular domain of hepatocytes at acinar zone 1 was decreased in ethynylestradiol-treated rats and markedly increased in obstructive cholestasis. CONCLUSIONS Ethynylestradiol-induced cholestasis is associated with increased P-glycoprotein biliary excretion and decreased hepatic content. In contrast, obstructive cholestasis results in decreased P-glycoprotein biliary excretion and increased hepatic content. These results suggest that biliary P-glycoprotein excretion might be a modulating factor in canalicular membrane P-glycoprotein content. Increased P-glycoprotein release into bile in ethynylestradiol-treated rats is apparently not a consequence of cholestasis, but it might be a primary event and play a pathogenetic role in ethynylestradiol-induced cholestasis.

Effect of Dimboa, a Hydroxamic Acid from Cereals, on Peroxisomal and Mitochondrial Enzymes from Aphids: Evidence for the Presence of Peroxisomes in Aphids

Abstract2,4-Dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), a hydroxamic acid involved in the resistance of cereals to aphids, was administered to adult individuals of the aphid Sitobion avenae in artificial diets. Effects on the cellular metabolism were inferred from the evaluation of several organelle marker enzymes. Catalase from peroxisomes and cytochrome c oxidase from mitochondria increased their activities about twofold when aphids were fed with 2 mM DIMBOA. The role of these enzymes in the metabolizing of xenobiotics by aphids is discussed. Biochemical and cytochemical evidences for the presence of peroxisomes in aphids are reported here for the first time.

Enhanced biliary excretion of canalicular membrane enzymes in ethynylestradiol-induced cholestasis. Effects of ursodeoxycholic acid administration

Cholestasis is associated with a marked increase in the release of canalicular membrane enzymes into bile. This phenomenon has been related to an increased lability of these canalicular membrane integral proteins to the solubilizing effects of secreted bile salts. To further characterize the effects of oral ursodeoxycholic acid (UDCA) administration on ethynylestradiol (EE)-induced cholestasis, the influence of this bile acid on changes in biliary excretion of membrane-bound enzymes was investigated. Bile flow, basal bile salt and biliary lipid secretory rates, the maximum secretory rate of taurocholate (TC SRm), and the biliary excretion of the canalicular membrane-bound ectoenzymes alkaline phosphatase (ALP) and gamma-glutamyl transpeptidase (GGT) were measured in rats after EE and/or UDCA administration. The activities of ALP, GGT and Na+,K(+)-ATPase in purified isolated canalicular and sinusoidal membrane fractions and the ultrastructure of hepatic acinus, including histochemical studies of ALP distribution, were also examined. EE significantly reduced bile flow, bile salt and biliary lipid secretory rates, and TC SRm, and caused dilatation and loss of microvilli at the canalicular pole of hepatocytes. Biliary excretion of ALP increased 2-fold, whereas biliary excretion of GGT was unchanged. The relationship between biliary excretion of ALP or GGT and bile salt secretion (units of enzyme activity secreted per nanomole of bile salt) was greater in EE-treated rats compared with controls (2.1- and 1.5-fold greater for ALP and GGT, respectively), indicating that in EE-induced cholestasis more enzyme was released into bile per nanomole of bile salt. Na+,K(+)-ATPase activity in sinusoidal membrane fraction was reduced significantly, whereas ALP activity increased in both membrane fractions in EE-treated rats. The histochemical distribution of ALP in the acinus showed a strong reaction in acinar zone 3 and at both the canalicular and sinusoidal membranes. Oral administration of UDCA prevented EE-induced bile secretory failure by normalizing bile flow, bile salt and biliary phospholipid secretory rates, and TC SRm. UDCA also prevented the EE-induced changes in the biliary excretion of enzymes. On the contrary, UDCA did not modify either the enzyme activity in isolated membrane fractions or the morphological or ALP histochemical changes associated with EE administration. These data indicate that in EE-induced cholestasis changes occur at the canalicular membrane, enabling this portion of the plasma membrane to be more susceptible to the solubilizing effect of bile salt, and that oral administration of UDCA prevents bile secretory failure and changes in the biliary excretion of ALP and GGT in EE-treated rats.

α2-Adrenoceptors control the release of noradrenaline but not neuropeptide Y from perivascular nerve terminals

Neuropeptide Y (NPY) and noradrenaline (NA) are co-transmitters at many sympathetic synapses, but it is not yet clear if their release is independently regulated. To address this question, we quantified the electrically evoked release of these co-transmitters from perivascular nerve terminals to the mesenteric circulation in control and drug-treated rats. 6-Hydroxydopamine reduced the tissue content and the electrically evoked release of ir-NPY and NA as well as the rise in perfusion pressure. A 0.001 mg/kg reserpine reduced the content of ir-NPY and NA, but did not modify their release nor altered the rise in perfusion pressure elicited by the electrical stimuli. However, 0.1mg/kg reserpine reduced both the content and release of NA but decreased only the content but not the release of ir-NPY; the rise in perfusion pressure was halved. Clonidine did not affect the release of ir-NPY while it lowered the outflow of NA, not altering the rise in perfusion pressure elicited by the electrical stimuli. Yohimbine, did not modify the release of ir-NPY but increased the NA outflow, it antagonized the clonidine effect. Therefore, presynaptic alpha2-adrenoceptors modulate the release of NA but not NPY, implying separate regulatory mechanisms.

A critical study of the histochemical lead method for localization of Mg-ATPase at cell boundaries

SynopsisThe staining obtained at cell boundaries by the Wachstein-Meisel technique for ATPases in the gastric gland of the toad (Bufo spinolosus) was studied. Homogenates and sections of the tissue fixed in glutaraldehyde exhibit considerable ATP hydrolytic activity in media with and without lead ions. Attempts to establish a correlation between intensity of staining and the rate of non-enzymatic hydrolysis of ATP by lead were not successful. Good staining could be obtained in media with high ATP:Pb2+ ratios where no non-enzymatic hydrolysis could be detected.

Modulatory effects of histamine on cat carotid body chemoreception

Histamine has been proposed to be an excitatory transmitter between the carotid body (CB) chemoreceptor (glomus) cells and petrosal ganglion (PG) neurons. The histamine biosynthetic pathway, its storage and release, as well as the presence of histamine H1, H2 and H3 receptors have been found in the CB. However, there is only indirect evidence showing the presence of histamine in glomus cells, or weather its application produces chemosensory excitation. Thus, we studied the histamine immunocytochemical localization in the cat CB, and the effects of histamine, and H1, H2 and H3 receptor blockers on carotid sinus nerve (CSN) discharge, using CB and PG preparations in vitro. We found histamine immunoreactivity in dense-cored vesicles of glomus cells. Histamine induced dose-dependent increases in CSN discharge in the CB, but not in the PG. The H1-antagonist pyrilamine reduced the CB responses induced by histamine, the H2-antagonists cimetidine and ranitidine had no effect, while the H3-antagonist thioperamide enhanced histamine-induced responses. Present data suggests that histamine plays an excitatory modulatory role in the generation of cat CB chemosensory activity.

Induction of peroxisomal enzymes and a 64-kDa peptide in cultured mouse macrophages treated with clofibrate.

The presence of catalase-containing particles was demonstrated in mouse macrophages of the cell line J774.3. These cells were incubated with clofibrate, a hypolipidemic drug known to cause peroxisome proliferation in rodent hepatocytes. The specific activities of catalase and fatty acid oxidase were significantly increased after 48 h incubation with 2 mM clofibrate. This response proved to be time and dose dependent. Other organelle marker enzymes showed little change in activity. Unexpectedly, clofibrate treatment also produced an increase in a 64-kDa peptide in these cells.

Actin and myosin in oxyntic cell: Gelation and contraction of crude extracts in vitro☆

Abstract Extracts prepared from purified toad oxyntic cells undergo temperature-dependent, cytochalasin B (CB)-sensitive gelation. In the presence of adenosine triphosphatase (ATP), the interaction between gelled actin and myosin produces a contracted gel. Such association ceases spontaneously after shrinkage is completed. A stable myosin-actin interaction takes place in the absence of ATP, but no contraction of the gel is observed. A correlation between this actomyosin system present in oxyntic cells and the high mobility of the secretory pole, resulting in the changes in shape observed during the onset of HCl secretion, is proposed.

Cytochemical study of the distribution of adenosine triphosphatase in the pancreas of the dog.

Dog pancreatic tissue, incubated in a modified Wachstein-Meisel medium, showed two different adenosine triphosphatase activities. One of them is located at the apical border of the cells lining the intralobular ducts and of the centroacinar cells and is stimulated by HCO3-, depressed by SCN- and OCN- and completely abolished by CN-. The other is located at the intracellular clefts of the epithelium lining the interlobular ducts and is stimulated by Mg++. These findings correlate well with the results of incubation of homogenates of fresh and fixed tissues. Their significance with respect to the role of different segments of the duct system in the formation of the pancreatic juice is discussed.