Ethinyl estradiol decreases acidification of rat liver endocytic vesicles

Treatment with ethinyl estradiol is known to impair bile formation, bile acid transport and Na,K-ATPase activity, to alter receptor-mediated endocytosis and transcytosis of IgA and asialoorosomucoid and to affect membrane lipid composition and fluidity. Because appropriate sorting and trafficking of asialoorosomucoid requires adequate acidification of endocytic vesicles by a lipid-sensitive electrogenic proton pump, we examined the effects of 5 days of treatment with ethinyl estradiol (5 mg/kg body wt, subcutaneously) on acidification of early endosomes prepared from male rat livers. Littermate control animals received equal volumes of the solvent propylene glycol. Pretreatment with ethinyl estradiol reduced ATP-dependent initial rates of endosome acidification by 11% to 25% when measured in potassium medium containing 0 to 140 mmol/L chloride; these differences were significant at four of six chloride concentrations tested. The proton pumps of ethinyl estradiol and propylene glycol endosomes exhibited similar Michaelis-Menten constants for MgATP (Michaelis-Menten constant of 63 and 66 Μmol/L in the absence of chloride and 101 and 126 Μol/L in the presence of chloride, respectively). Acidification of ethinyl estradiol and propylene glycol endosomes changed in the same manner when various cations or anions were substituted for potassium gluconate, although the effects of ethinyl estradiol were less marked in the absence of K + . Kinetics of inhibition for ethinyl estradiol and propylene glycol endosomes were similar for the proton pump inhibitors N-ethylmaleimide (50% inhibitory concentrations of 13.5 and 18.1 Μmol/L), dicyclohexylcarbodiimide (50% inhibitory concentrations of 206 and 216 Μmol/L) and bafilomycin A (50% inhibitory concentrations of 11 and 6 nmol/L). Although initial rates of acidification were slower in ethinyl estradiol endosomes, ATP-dependent steady-state vesicle interior pH was the same as that of propylene glycol endosomes over a range of chloride concentrations; this appeared to be due mainly to a trend toward decreased proton leak rates in ethinyl estradiol endosomes. Overall, ethinyl estradiol treatment modestly decreased initial rates of acidification and vesicle proton leakage, perhaps because of changes in endosome lipid composition; differences in the number, density or activation state of proton pumps; or differences in endosome geometry. Because the decrease in acidification rates was small, the effects of estrogen on the efficiency of uncoupling of endocytosed ligands such as asialoorosomucoid from their receptors in early endosomes; thus the rates of sorting and distribution of ligands remain unclear. (HEPATOLOGY 1993;18:604–613.)Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38399/1/1840180320_ftp.pd

Role of Na,K-ATPase in regulating acidification of early rat liver endocytic vesicles

Endocytic vesicles are acidified by an electrogenic proton pump and a parallel chloride conductance; however, acidification might be decreased if electrogenic transporters, such as Na,K-ATPase, that increase vesicle interior-positive membrane potential were also present. We examined this issue in early rat liver endosomes using ion substitution and inhibitors to alter Na,K-ATPase activity. These early endosomes, labeled for 2 min with the fluorescent fluid-phase marker fluorescein isothiocyanate-dextran, consistently acidified faster than endosomes similarily labeled for a 10-min period. In chloride-free media initial rates of acidification of early endosomes were faster in K + media than in Na + medium, although addition of K + to Na + or Na + to K + media to allow Na,K-ATPase to function did not decrease the rate of acidification. In chloride-containing media, rates were the same regardless of cation composition. The Na,K-ATPase inhibitor vanadate was prepared from orthovanadate by several methods, all of which inhibited liver ATPase activity. Two hundred Μmol/L vanadate, prepared Cl − -free, tended to decrease rates of acidification in all media tested and these effects achieved statistical significance in Cl − -free media containing 150 mmol/L K + or mixtures of Na + and K + and in 145 mmol/L KCl/5 mmol/L NaCl medium. Vanadate stocks pH-adjusted with hydrogen chloride increased rates of acidification in sodium gluconate buffers, probably as a result of the effects of the included Cl − . Five mmol/L ouabain (loaded into vesicles by endocytosis) and the membrane-permeable analog strophanthidin (2 mmol/L) both markedly inhibited endosome acidification, regardless of buffer ion composition. Collectively, these results suggest that Na,K-ATPase does not regulate acidification of rat liver early endocytic vesicles, that vanadate may modestly inhibit endosome acidification and that ouabain at high concentrations may inhibit acidification from the vesicle interior face. (H EPATOLOGY 1994;19:1034–1043.)Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38406/1/1840190432_ftp.pd

BHPR research: qualitative1. Complex reasoning determines patients' perception of outcome following foot surgery in rheumatoid arhtritis

Background: Foot surgery is common in patients with RA but research into surgical outcomes is limited and conceptually flawed as current outcome measures lack face validity: to date no one has asked patients what is important to them. This study aimed to determine which factors are important to patients when evaluating the success of foot surgery in RA Methods: Semi structured interviews of RA patients who had undergone foot surgery were conducted and transcribed verbatim. Thematic analysis of interviews was conducted to explore issues that were important to patients. Results: 11 RA patients (9 ♂, mean age 59, dis dur = 22yrs, mean of 3 yrs post op) with mixed experiences of foot surgery were interviewed. Patients interpreted outcome in respect to a multitude of factors, frequently positive change in one aspect contrasted with negative opinions about another. Overall, four major themes emerged. Function: Functional ability & participation in valued activities were very important to patients. Walking ability was a key concern but patients interpreted levels of activity in light of other aspects of their disease, reflecting on change in functional ability more than overall level. Positive feelings of improved mobility were often moderated by negative self perception ("I mean, I still walk like a waddling duck”). Appearance: Appearance was important to almost all patients but perhaps the most complex theme of all. Physical appearance, foot shape, and footwear were closely interlinked, yet patients saw these as distinct separate concepts. Patients need to legitimize these feelings was clear and they frequently entered into a defensive repertoire ("it's not cosmetic surgery; it's something that's more important than that, you know?”). Clinician opinion: Surgeons' post operative evaluation of the procedure was very influential. The impact of this appraisal continued to affect patients' lasting impression irrespective of how the outcome compared to their initial goals ("when he'd done it ... he said that hasn't worked as good as he'd wanted to ... but the pain has gone”). Pain: Whilst pain was important to almost all patients, it appeared to be less important than the other themes. Pain was predominately raised when it influenced other themes, such as function; many still felt the need to legitimize their foot pain in order for health professionals to take it seriously ("in the end I went to my GP because it had happened a few times and I went to an orthopaedic surgeon who was quite dismissive of it, it was like what are you complaining about”). Conclusions: Patients interpret the outcome of foot surgery using a multitude of interrelated factors, particularly functional ability, appearance and surgeons' appraisal of the procedure. While pain was often noted, this appeared less important than other factors in the overall outcome of the surgery. Future research into foot surgery should incorporate the complexity of how patients determine their outcome Disclosure statement: All authors have declared no conflicts of interes