scholarly journals Ivermectin as an inhibitor of the plasma membrane and the sarcoplasmic/endoplasmic reticulum Ca2+-ATPases in rat vas deferens

2014 ◽  
Vol 23 (2) ◽  
pp. 75
Author(s):  
Humberto Muzi-Filho ◽  
Dominick Rodrigues Alves de Souza ◽  
Christianne Bretas Vieira Scaramello ◽  
Valeria Do Monti Nascimento Cunha
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Atpase Activity ◽  
Vas Deferens ◽  
Physiological Function ◽  
Binding Protein ◽  
Rat Vas Deferens ◽  
Release Channel ◽  
Fk506 Binding Protein ◽  
Fertility Status

ObjectiveThe present work investigated the effect of ivermectin on Ca2+ content and on the Ca2+-ATPase activity (represented by the plasma membrane Ca2+-ATPase and the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase present in rat vas deferens.MethodsThe assays were carried out using ultracentrifuged homogenate preparations from rat vas deferens in the presence or absence of the 12-kDa FK506-binding protein-Ca2+ release channel complex. Measures of Ca2+ content and Ca2+ ATPase activity were then carried out in function of different concentrations of ivermectin. ResultsThe data show that ivermectin (10 μM) reduces the sarcoplasmic reticulum Ca2+ content in FK506-binding protein (+) and FK506-binding protein (-) fractions of ultracentrifuged homogenate from rat vas deferens (inhibition of 50% and 40%, respectively, p<0.05) and inhibits both the activities of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase and plasma membrane Ca2+-ATPases pumps (33% and 16%, respectively, p<0.05).ConclusionThese data suggest that ivermectin effects Ca2+ handling in the rat vas deferens, indicating that this drug could alter the contractility of this smooth muscle. Therefore, ivermectin could be an interesting pharmacological tool to alter the physiological function of vas deferens and to manipulate the fertility status of male rats.Indexing terms: Calcium. Ivermectin. Rats.

Heterologous expression of Na+-K+-ATPase in insect cells: intracellular distribution of pump subunits

2001 ◽  
Vol 281 (3) ◽  
pp. C982-C992 ◽  
Author(s):  
Craig Gatto ◽  
Scott M. McLoud ◽  
Jack H. Kaplan
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Membrane Protein ◽  
Atpase Activity ◽  
Insect Cells ◽  
Expression System ◽  
Β Subunit ◽  
Α Subunit ◽  
Enzyme Preparations ◽  
High Five Cells

The Na+-K+-ATPase is a heterodimeric plasma membrane protein responsible for cellular ionic homeostasis in nearly all animal cells. It has been shown that some insect cells (e.g., High Five cells) have no (or extremely low) Na+-K+-ATPase activity. We expressed sheep kidney Na+-K+-ATPase α- and β-subunits individually and together in High Five cells via the baculovirus expression system. We used quantitative slot-blot analyses to determine that the expressed Na+-K+-ATPase comprises between 0.5% and 2% of the total membrane protein in these cells. Using a five-step sucrose gradient (0.8–2.0 M) to separate the endoplasmic reticulum, Golgi apparatus, and plasma membrane fractions, we observed functional Na+ pump molecules in each membrane pool and characterized their properties. Nearly all of the expressed protein functions normally, similar to that found in purified dog kidney enzyme preparations. Consequently, the measurements described here were not complicated by an abundance of nonfunctional heterologously expressed enzyme. Specifically, ouabain-sensitive ATPase activity, [3H]ouabain binding, and cation dependencies were measured for each fraction. The functional properties of the Na+-K+-ATPase were essentially unaltered after assembly in the endoplasmic reticulum. In addition, we measured ouabain-sensitive 86Rb+ uptake in whole cells as a means to specifically evaluate Na+-K+-ATPase molecules that were properly folded and delivered to the plasma membrane. We could not measure any ouabain-sensitive activities when either the α-subunit or β-subunit were expressed individually. Immunostaining of the separate membrane fractions indicates that the α-subunit, when expressed alone, is degraded early in the protein maturation pathway (i.e., the endoplasmic reticulum) but that the β-subunit is processed normally and delivered to the plasma membrane. Thus it appears that only the α-subunit has an oligomeric requirement for maturation and trafficking to the plasma membrane. Furthermore, assembly of the α-β heterodimer within the endoplasmic reticulum apparently does not require a Na+pump-specific chaperone.

scholarly journals The Rough Endoplasmic Reticulum-resident FK506-binding Protein FKBP65 Is a Molecular Chaperone That Interacts with Collagens

2008 ◽  
Vol 283 (46) ◽  
pp. 31584-31590 ◽  
Author(s):  
Yoshihiro Ishikawa ◽  
Janice Vranka ◽  
Jackie Wirz ◽  
Kazuhiro Nagata ◽  
Hans Peter Bächinger
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Molecular Chaperone ◽  
Binding Protein ◽  
Fk506 Binding Protein

scholarly journals Localization of the FK506-binding protein, FKBP 13, to the lumen of the endoplasmic reticulum

1993 ◽  
Vol 294 (2) ◽  
pp. 511-515 ◽  
Author(s):  
S K Nigam ◽  
Y J Jin ◽  
M J Jin ◽  
K T Bush ◽  
B E Bierer ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Intracellular Calcium ◽  
Microsomal Fraction ◽  
Staining Pattern ◽  
Binding Protein ◽  
Ion Regulation ◽  
Fk506 Binding Protein ◽  
Calcium Storage ◽  
Intracellular Calcium Ion ◽  
Cloned Cdna

The function of the immunophilins, FKBP 12 and FKBP 13, which are binding proteins for the immunosuppressant drug FK506 and rapamycin, remains poorly defined, although it has been suggested that immunophilins and immunophilin-like proteins may play a role in protein sorting/folding and intracellular calcium ion regulation. As a first step towards understanding the function of FKBP 13, we studied its subcellular localization by immunoblotting of well-defined subcellular fractions from a canine pancreatic homogenate and immunocytochemical analysis of an overexpressed cloned cDNA for FKBP 13. Whereas FKBP 12 fractionated entirely into the cytosol, virtually all FKBP 13 was found in the rough microsomal fraction which consisted of highly purified rough endoplasmic reticulum (ER), along with several well-characterized ER markers [the immunoglobulin heavy-chain binding protein (BiP), grp 94 and ribophorin I]. Moreover, FKBP 13 co-banded with the ER markers on isopycnic sucrose gradients. By immunofluorescence, the overexpressed cDNA for FKBP 13 in Hela cells gave an ER-staining pattern highly similar to that of known ER proteins. Addition of the ligand FK506 did not appear to alter the distribution of FKBP 13. Separation of the ER luminal contents and membrane revealed FKBP 13 to be a luminal ER protein. Since the lumen of the ER is where the folding of membrane and secreted proteins occurs, as well as a major site of intracellular calcium storage, it seems possible that FKBP 13 may be involved in one of these functions.

scholarly journals The Golgi Apparatus Maintains Its Organization Independent of the Endoplasmic Reticulum

2006 ◽  
Vol 17 (12) ◽  
pp. 5372-5380 ◽  
Author(s):  
Matthew Y. Pecot ◽  
Vivek Malhotra
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Binding Protein ◽  
Physiological Conditions ◽  
Fk506 Binding Protein ◽  
Golgi Membranes ◽  
Intermediate Compartment ◽  
Tight Association

Under artificial conditions Golgi enzymes have the capacity to rapidly accumulate in the endoplasmic reticulum (ER). These observations prompted the idea that Golgi enzymes constitutively recycle through the ER. We have tested this hypothesis under physiological conditions through use of a procedure that captures Golgi enzymes in the ER. In the presence of rapamycin, which induces a tight association between FKBP (FK506-binding protein) and FRAP (FKBP-rapamycin–associated protein), an FKBP-tagged Golgi enzyme can be trapped when it visits the ER by an ER-retained protein fused to FRAP. We find that although FKBP-ERGIC-53 of the ER-Golgi intermediate compartment (ERGIC) rapidly cycles through the ER (30 min), FKBP-Golgi enzyme chimeras remain stably associated with Golgi membranes. We also demonstrate that Golgi dispersion upon nocodazole treatment mainly occurs through a mechanism that does not involve the recycling of Golgi membranes through the ER. Our findings suggest that the Golgi apparatus, as defined by its collection of resident enzymes, exists independent of the ER.

scholarly journals Induction of the FK506-binding protein, FKBP13, under conditions which misfold proteins in the endoplasmic reticulum

1994 ◽  
Vol 303 (3) ◽  
pp. 705-708 ◽  
Author(s):  
K T Bush ◽  
B A Hendrickson ◽  
S K Nigam
Keyword(s):  
Endoplasmic Reticulum ◽  
Mdck Cells ◽  
Binding Protein ◽  
Significant Similarity ◽  
Fk506 Binding Protein ◽  
Unfolded Protein ◽  
Immunoglobulin Binding Protein ◽  
The Unfolded Protein Response ◽  
Protein Response ◽  
Immunoglobulin Binding

In order to determine whether the endoplasmic reticulum (ER) luminal FK506-binding protein, FKBP13, shares properties of ER molecular chaperones, MDCK cells were treated with either tunicamycin or Ca2+ ionophores. By Northern-blot analysis, tunicamycin resulted in a 2-fold rise in FKBP13 mRNA, whereas ionophores (A23187 and ionomycin) caused a more impressive rise in FKBP13 mRNA (up to 5-fold with ionomycin). Actinomycin D chase experiments in ionomycin-treated cells revealed no change in the half-life of FKBP13 mRNA, indicating that the increase in FKBP13 mRNA observed was not due to greater message stability. Moreover, sequencing of the 5′ flanking region of the gene for murine FKBP13 revealed significant similarity to similar regions in human BiP (immunoglobulin-binding protein) and the human glucose-regulated protein grp94, including a 37 bp sequence in FKBP13 with approximately 50% identity with the unfolded protein response element of the BiP gene. Together, these data suggest a role for FKBP13 in ER protein folding.

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