Infliximab is an effective option in patients with ulcerative colitis previously exposed to full subcutaneous anti-TNF agent: Results from a real-world multicenter study.

BACKGROUND: Data on infliximab efficacy in bio-exposed patients with ulcerative colitis (UC) are limited. AIMS: To evaluate infliximab effectiveness and its predictors in UC patients with prior exposure to subcutaneous (SC) anti-TNF agent. METHODS: In this multicenter retrospective study (8 centers), we included all consecutive UC patients with prior exposure to subcutaneous anti-TNF, starting infliximab for symptomatic UC, excluding acute severe colitis. Corticosteroid-free clinical remission (CFREM) was assessed at week 14 (W14) and W52 while endoscopic improvement (CFREM + endoscopic Mayo score≤1) was evaluated at W14. RESULTS: Overall, 104 patients were included (pancolitis=54.8%, primary failure to subcutaneous anti-TNF=57.4%, concomitant immunosuppressant=53.8%, median partial Mayo score at baseline=7[5-8]). The rate of CFREM was 33.6% (35/104) at W14 and 40.4% (42/104) at W52. At W14, endoscopic improvement was achieved in 29.8%(31/104). In multivariable analysis, concomitant immunosuppressant was associated with higher rate of CFREM at W14(OR=2.83[1.06-7.54], p = 0.037) and W52(OR=2.68[1.16-6.22];p = 0.021), while primary failure to a previous subcutaneous anti-TNF agent led to lower rate of CFREM at W14 (OR=0.37[0.14-0.98], p = 0.046). After a median follow-up of 20.9 months[11.7-33.7]), 50.0%(52/104) patients had discontinued infliximab. CONCLUSION: Infliximab is an effective option in UC patients previously exposed to prior subcutaneous anti-TNF agent and should be used with concomitant immunosuppressant.

White matter integrity assessment in spinocerebellar ataxia type 2 (SCA2) patients.

AIM: To assess the burden of white matter (WM) damage in the cerebrum and cerebellum of spinocerebellar ataxia type 2 (SCA2) patients in an attempt to identify key regions affected by the neurodegenerative processes using diffusion tensor imaging (DTI). MATERIALS AND METHODS: Nine SCA2 patients and 16 age-matched healthy controls were examined twice (SCA2 patients 3.6 ± 0.7 years and controls 3.3 ± 1.0 years apart) on the same 1.5 T scanner by acquiring T1-weighted and diffusion-weighted (b-value = 1,000 s/mm2) images. Using tract-based spatial statistics, DTI analysis on fractional anisotropy (FA), mean diffusivity (MD), axial (AD)/radial (RD) diffusivity was performed. RESULTS: At baseline magnetic resonance imaging (MRI), FA was significantly decreased in SCA2 patients in the corticospinal tracts, inferior and superior cerebellar peduncles, middle cerebellar peduncles, cerebral peduncles, right superior and posterior corona radiata. RD was only significantly increased in SCA2 patients in the middle cerebellar peduncles. No significant AD and MD changes were observed. Tract-based spatial statistics (TBSS) analysis between SCA2 patients at baseline and at follow-up showed no significant changes in any of the DTI metrics. CONCLUSIONS: DTI is a sensitive tool for following the progression of WM neurodegeneration and severity assessment in patients with SCA2. These findings add to a better understanding of the neurological underpinnings of the symptoms experienced by SCA2 patients.

Home management of acute medical complications in cancer patients: a prospective pilot study.

BACKGROUND: The development of reliable alternatives to conventional hospitalization in patients with cancer would have great clinical and economical value. The aim of the present study was to assess the feasibility of a home-based nursing intervention model as a safe alternative for the management of acute medical complications in cancer patients who would otherwise require conventional hospitalization. PATIENTS AND METHODS: From October 2013 to October 2014, we prospectively evaluated the outcomes of consecutive acute medical episodes treated at home under the home-based intervention program named the Bridge Project (BP). Episodes were classified as "avoided hospitalization in outpatients" (AHO) vs. "reduced hospitalization in inpatients" (RHI). The primary end-point was to assess the rate and causes of BP intervention failure (unplanned hospital readmission or death). RESULTS: Two hundred and forty-six consecutive episodes (52 % AHO and 48 % RHI) involving 203 patients (55 % male; mean age 63 years) were enrolled. The main conditions managed at home were non-neutropenic infections (40 %), febrile neutropenia (20 %), and cancer-related complications (28 %). The median duration of the BP intervention was 5 days (range 1-16 days). No deaths were reported at home. Unplanned hospital readmissions occurred in 9 % of episodes (14 % in AHO vs. 4 % in RHI; p = 0.001). Five of the 22 readmitted patients (22.7 % of the BP failures; 2.5 % of the whole series) died during hospitalization. The BP intervention burden was 1353 days, representing a potential saving of 14 % of days of hospitalization during the study period. CONCLUSIONS: The BP is a safe intervention which can potentially avoid or reduce the length of hospitalization in selected cancer patients with acute medical complications. Our findings support further development of innovative home-based clinical approaches to promote potentially avoidable hospitalization in this setting.

Molecular diagnosis of Pneumocystis jiroveci pneumonia in immunocompromised patients.

Pneumocystis jiroveci is the major cause of pneumonia in immunocompromised patients. To evaluate the performance of single and nested-polymerase chain reaction (PCR) methods compared with immunofluorescent assay (IFA) and cytological staining for diagnosis of P. jiroveci infection, the bronchoalveolar lavage (BAL) and sputum samples from 60 immunocompromised patients were studied. Between January 2005 and March 2008, 75 respiratory specimens (41 BAL and 34 sputum samples) were examined for P. jiroveci identification. We used the clinical classification as our diagnostic standard and we considered true positive the definite or probable Pneumocystis pneumonia. Fourteen patients (23.3%) developed Pneumocystis pneumonia. Eleven patients had a positive IFA but only nine were positive by cytological staining. Sixteen patients had a positive detection of P. jiroveci by PCR and nested-PCR. Thirteen of these patients were considered as having a definite Pneumocystis pneumonia and one patient with a probable Pneumocystis pneumonia. Five other patients had a positive detection only by nested-PCR. These patients were classified as no Pneumocystis pneumonia. PCR detection of P. jiroveci is a very sensitive test and will offer a powerful technique in clinical laboratories for the routine diagnosis of Pneumocystis pneumonia. Using the nested-PCR, additional clinical cases can be diagnosed, but there is then an obvious risk of detecting subclinical colonisation by P. jiroveci.

Usefulness of enzyme linked immunosorbent assays species specific in the detection of Chlamydia trachomatis and Chlamydophila pneumoniae IgG antibodies in patients with genital infections or respiratory tract infections.

Chlamydia trachomatis (Ct) and Chlamydophila pneumoniae (Cpn) are obligate intracellular bacteria causing genital tract infections (GTI) and respiratory tract infections (RTI), respectively. Antigenic cross-reactivity between the two species may complicate serologic diagnosis. In this study, we compared the performance of two ELISA tests in relation to microimmunofluorescence (MIF) for the detection of Ct and Cpn IgG antibodies. We also explored the degree of cross-reactivity by ELISA and MIF. Among 278 positive sera for Cpn and/or Ct IgG antibodies in the MIF, 153 were from patients with GTI and 125 were from patients with RTI. These sera were tested by our in house MIF test and by two commercial ELISA: SeroCP and SeroCT for the detection of anti-Cpn IgG antibodies and anti-Ct IgG antibodies, respectively. In sera from patients with RTI, correlation between MIF and SeroCP was 92%. The specificity of this test was 38.5%. In fact, among the 140 sera from patients with GTI and that cross-reacted in MIF, only six were confirmed by the two ELISA tests as having IgG antibodies to Ct. The correlation between MIF and SeroCT was 80%. The specificity of this test was 100%. Indeed, among the 65 sera from patients with RTI with cross-reactions in MIF, 30 sera showed a negative SeroCT test. SeroCT was highly specific and could diminish considerably the extent of cross-reactions. Whilst, SeroCP test was not specific enough to distinguish between the presence of IgG antibodies and Cpn or Ct.

[Candida glabrata perinephric abscess. A case report]. / Abcès périrénal à Candida glabrata.

We report a case of Candida glabrata perinephric abscess in a patient with diabetes mellitus who recently underwent ureteropelvic surgery for lithiasic urinary tract obstruction. Surgical drainage and amphotericin B treatment led to resolution of the infection. C. glabrata urinary infection has become more prevalent over the last decade in immunocompromised patients. Drainage is indicated for development of a fungal abscess in the perinephric area. Most authors recommend administration of an antifungal adjuvant treatment.

Beta-amyloid neurotoxicity in vitro: evidence of oxidative stress but not protection by antioxidants.

Recent data from several groups suggest that the primary mechanism of beta-amyloid neurotoxicity may be mediated by reactive oxygen species. To evaluate this hypothesis, we first compared the efficacy of antioxidant agents in preventing toxicity caused by oxidative insults (iron, hydrogen peroxide, and tert-butyl hydroperoxide) and beta-amyloid peptides in cultured rat hippocampal neurons. Tested antioxidants (propyl gallate, Trolox, probucol, and promethazine) generally provided significant protection against oxidative insults but not beta-amyloid peptides. Next, we examined whether beta-amyloid causes oxidative stress, by comparing levels of lipid peroxidation after exposure to either iron or beta-amyloid. In a cell-free system, iron but not beta-amyloid generated lipid peroxidation. In culture, both insults caused rapid increases in lipid peroxidation, with iron inducing higher levels at later time points. Pretreatment with the antioxidant probucol significantly reduced lipid peroxidation caused by both insults but only attenuated iron toxicity, suggesting that lipid peroxidation does not contribute directly to cell death induced by beta-amyloid. Finally, we observed that increasing basal levels of oxidative stress by pretreating cultures with subtoxic doses of iron significantly increased neuronal vulnerability to beta-amyloid. The ability of beta-amyloid to induce oxidative stress and the demonstration that oxidative stress potentiates beta-amyloid toxicity support the clinical use of antioxidants for AD. However, these data do not support the theory that the primary mechanism of beta-amyloid toxicity involves oxidative pathways, indicating a continued need to identify additional cellular responses to beta-amyloid that underlie its neurodegenerative actions.

beta-Amyloid increases enzyme activity and protein levels of glutamine synthetase in cultured astrocytes.

Previous studies have reported that beta-amyloid peptides induce properties of reactivity in cultured astrocytes. We report here that aggregated A beta peptides increase expression of the enzyme glutamine synthetase in cultured astrocytes, as assessed by enzyme assay, Western blot analysis, and immunocytochemistry. The enhanced enzyme levels occur gradually over a period of 4 days after A beta exposure and maintain peak values for at least several days thereafter. These data suggest that A beta-related reactive astrocytosis in Alzheimer's disease brain may benefit local neurons by enhancing glial capacity to regulate levels of the excitotoxin glutamate.

Characterization of the defect in the Na(+)-phosphate transporter in vitamin D-resistant hypophosphatemic mice.

Hypophosphatemic vitamin D-resistant rickets is the most common form of vitamin D-resistant rickets in man. The hypophosphatemic mouse model (Hyp) is phenotypically and biochemically similar to the human disease. Biochemically, hypophosphatemia is the hallmark of this disorder. The cause of the hypophosphatemia is thought to be secondary to a defect in the renal and/or intestinal Na(+)-phosphate transporter. The current studies were designed to investigate and characterize the localization of the defect in the Na(+)-phosphate transporter in this disorder. Phosphate uptake by renal brush border membrane vesicles (BBMV) showed a significant decrease in the slope of the initial rate of phosphate uptake in (Hyp) compared with control mice (0.009 versus 0.013, respectively). The slopes representing initial rates of phosphate uptake by jejunal BBMV were similar in (Hyp) and control mice (0.004 and 0.004, respectively). Kinetics of jejunal Na(+)-dependent phosphate uptake showed a Vmax of 0.63 +/- 0.12 and 0.64 +/- 0.12 nmol/mg protein/15 s in (Hyp) and control mice, respectively, whereas Km values were 0.12 +/- 0.08 and 0.2 +/- 0.11 mM, respectively. Similar kinetic analysis in the kidney showed a Vmax of 0.32 +/- 0.06 and 1.6 +/- 0.1 (p less than 0.01) and Km of 0.07 +/- 0.06 and 0.39 +/- 0.05 (p less than 0.02) in (Hyp) and control mice, respectively. Na(+)-dependent D-glucose uptake by BBMVs of intestine and kidney showed typical overshoot phenomena in (Hyp) and control mice. In order to explore these findings further, Na(+)-phosphate transporter expression from intestine and kidney was accomplished by microinjection of 50 ng of poly(A)+ RNA into Xenopus laevis oocytes. Na(+)-dependent phosphate uptake was expressed 6 days after the microinjection of intestinal and kidney poly(A)+ RNA from control mice. However, expression of the transporter from (Hyp) mice occurred only from the intestine, and not from the kidney. The decrease in the expression of the Na(+)-dependent phosphate transporter was not secondary to accelerated efflux of phosphate or decreased metabolism in oocytes injected with poly(A)+ RNA from (Hyp) mice.(ABSTRACT TRUNCATED AT 400 WORDS)

Ontogeny of mitochondrial calcium transport in spontaneously hypertensive (SHR) and WKY rats.

The current studies were designed to investigate calcium uptake by intestinal jejunal sacs as well as in intestinal mitochondria of spontaneously hypertensive rats and their genetically matched WKY control rats. Kinetics of jejunal calcium uptake by jejunal sacs of adult SHR and WKY rats showed a significant decrease in Vmax of calcium uptake in SHR (227 +/- 24 versus 423 +/- 22 nmol.g tissue-1.3 min-1) compared to WKY rats P less than 0.001. To explore the intracellular handling of calcium by the intestinal mitochondria, calcium uptake was characterized by intestinal mitochondria before (suckling and weanling periods) and after (adult period) development of hypertension. Calcium uptake by intestinal mitochondria was driven by ATP in the presence of succinate as a respiratory substrate. Calcium uptake was stimulated several fold by the presence of ATP compared to no ATP conditions. Maximal calcium uptake occurred between 15-30 min and was significantly greater in adult SHR and WKY rats compared to corresponding values in weanling and suckling rats. Maximal ATP dependent calcium uptake in adult, weanling and suckling WKY rats was significantly greater compared to corresponding mean values in each age group in SHR (P less than 0.001). Oligomycin (10 micrograms/mg protein) inhibited calcium uptake partially. Ruthenium red (0.25 microM), 1 mM sodium azide and 0.5 mM dinitrophenol inhibited calcium uptake by more than 80% in both SHR and WKY rats. Kinetic parameters for ATP stimulated calcium uptake at 10 s revealed a Vmax of 0.56 +/- 0.6, 3.46 +/- 0.23 and 3.95 +/- 0.52 nmol/mg protein/10 s in suckling, weanling and adult WKY rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Intestinal phosphate transport in spontaneously hypertensive rats and genetically matched controls.

Hypophosphatemia has been documented in patients with hypertension and in spontaneously hypertensive rats compared with genetically matched control Wistar-Kyoto rats. However, renal tubular reabsorption is increased in spontaneously hypertensive rats. Therefore, it was hypothesized that decreased serum phosphate levels in spontaneously hypertensive rats may be related to a decrease in the intestinal transport of phosphate. To test this hypothesis, sodium-dependent phosphate uptake by jejunal brush-border membrane vesicles of spontaneously hypertensive rats and genetically matched Wistar-Kyoto rats was determined. Phosphate uptake consisted of two components: sodium-independent passive diffusion across the brush border and sodium-dependent, carrier-mediated uptake. The initial rate of uptake in spontaneously hypertensive and Wistar-Kyoto rats was linear up to 20 seconds. The initial rate and time course of jejunal sodium-dependent phosphate uptake was decreased in adult spontaneously hypertensive rats compared with corresponding mean values in Wistar-Kyoto rats. This decrease was secondary to a decrease in Vmax rather than Km, suggesting tha the number and/or the activity of the sodium-phosphate transporters is decreased. Sodium-dependent phosphate uptake was pH dependent, with greater uptake at pH 6.0 than at pH 7.4. However, uptake values were lower in spontaneously hypertensive rats than in Wistar-Kyoto rats at all pH levels tested. In contrast, sodium-dependent phosphate uptake in weanling rats (prehypertensive state) was not significantly different between spontaneously hypertensive and Wistar-Kyoto rats. Vitamin D deficiency in both spontaneously hypertensive and Wistar-Kyoto rats decreased Vmax and Km of sodium-dependent phosphate uptake, whereas 1,25(OH)2 vitamin D3 administration increased Vmax and Km in both spontaneously hypertensive and Wistar-Kyoto rats. These results suggest that the hypophosphatemia seen in adult spontaneously hypertensive rats is secondary to a decrease in sodium-dependent phosphate uptake compared with controls. The sodium phosphate transporter in spontaneously hypertensive rats is responsive to vitamin D administration.

Glutamine transport by human intestinal basolateral membrane vesicle.

This study characterizes for the first time, by use of a well-validated technique, glutamine transport across human basolateral membrane vesicles. Glutamine transport represented uptake into an osmotically active intravesicular space without significant metabolism. Glutamine uptake was temperature- and pH-dependent with maximal uptake at pH 7.5, and it was mediated by sodium-dependent and -independent processes. The initial rate of uptake was linear up to 20 s, as depicted by the formula gamma (nmol/mg protein) = 0.0009 X (s) - 0.0011 (r = 0.99). Kinetic analysis of glutamine uptake at concentrations between 0.01 and 0.3 mmol/L at 5 s under sodium and potassium gradients showed a maximal transport capacity (Vmax) of 0.39 +/- 0.04 and 0.21 +/- 0.02 nmol.mg protein-1.5 s-1 for sodium-dependent and -independent processes, respectively (p less than 0.01). Km values were 0.17 +/- 0.04 and 0.06 +/- 0.2 mmol/L, respectively (p less than 0.05). Glutamine uptake under the sodium-gradient condition was electrogenic whereas under the potassium-gradient it was electroneutral. Neutral amino acids inhibited both sodium-dependent and -independent processes. This study confirms and characterizes the presence of carrier-mediated glutamine uptake at the basolateral membranes of human enterocytes.

Vitamin D-regulated, ATP-dependent calcium transport by intestinal Golgi vesicles during maturation in the rat.

The developmental aspects of calcium uptake by intestinal Golgi vesicles was determined using highly purified Golgi vesicles from enterocytes of suckling (2 wk old), weanling (3 wk old), and adolescent (6 wk old) rats. Calcium uptake by Golgi vesicles at all age groups represented transport into the intravesicular space as evident by temperature dependency and by calcium ionophore A23187-induced calcium efflux studies. Calcium uptake was driven by ATP at all age groups, however, maximal uptake at 15 min was significantly greater in Golgi vesicles of adolescent rats compared to mean values in Golgi vesicles of suckling rats (p less than 0.01). Calcium uptake in the absence of ATP was minimal. The requirement for the adenine base and the hydrolysis of the beta-gamma-phosphodiester was tested by replacement of ATP in the incubation media by CTP and the nonhydrolyzable ATP analogue, adenylyl-(beta-gamma-methylendiphosphonate). Both agents had no stimulatory effect on calcium uptake. Calcium uptake was linear up to 40 s. Kinetic parameters of calcium uptake at free calcium concentrations of 0.04 to 1.0 microM showed a maximal transport capacity of 0.99 +/- 0.05, 0.55 +/- 0.04, and 0.29 +/- 0.03 nmol/mg protein/15 s for adolescent, weanling, and suckling rats, respectively. Km values were 0.16 +/- 0.02, 0.12 +/- 0.03, and 0.07 +/- 0.02 microM for adolescent, weanling and suckling rats, respectively. Km and Vmax values were significantly different between adolescent and suckling rats (p less than 0.01). The calcium regulatory protein calmodulin has no effect on calcium uptake by Golgi vesicles. Vitamin D deficiency in all age groups decreased ATP-dependent calcium uptake. Administration of 1,25-(OH)2 vitamin D3 8 h before death enhanced ATP-dependent calcium uptake in all age groups studied. This enhancement was the result of increase in maximal transport capacity of ATP-dependent calcium uptake. This study demonstrates a vitamin D-regulated ATP-driven calcium uptake by intestinal Golgi vesicles at all age groups including the suckling period. This transport system shows developmental patterns in regard to its kinetic parameters.

Intestinal maturation: characterization of mitochondrial phosphate transport in the rat and its regulation by 1,25-(OH)2 vitamin D3.

The mitochondria play a major role in the regulation of oxidative phosphorylation within the cell. Despite the fact that the enterocytes receive the majority of absorbed phosphate and their high metabolic turnover rate, the role of the intestinal mitochondria in phosphate transport system during maturation is not known. Therefore, the current studies were designed to characterize phosphate transport by jejunal mitochondria of rats during maturation (suckling, weanling, and adolescent rats). The functional integrity of the intestinal mitochondria of suckling and adolescent rats was determined by oxygen consumption studies demonstrating respiratory control ratios of more than 3 when succinate was used as a test substrate. Phosphate uptake was significantly stimulated by the presence of 3 mM ATP at all age groups studied. Maximal phosphate uptake in the presence of 3 mM ATP and 2 mM succinate was 16.5 +/- 1.0, 20.5 +/- 1 and 28.7 +/- 0.4 nmol/mg protein (mean +/- SE) in suckling, weanling, and adolescent rats respectively. ATP-dependent phosphate uptake was inhibited by 80% with 100 microM p-MB. Kinetic parameters for ATP stimulated phosphate uptake at 10 s revealed a Km of 4 +/- 0.9, 2.8 +/- 0.4, and 0.9 +/- 0.1 mM and Vmax of 5 +/- 0.7, 9.5 +/- 1, and 11 +/- 0.7 nmol/mg protein per 10 s in suckling, weanling, and adolescent rats, respectively. Phosphate uptake was also stimulated by an inwardly directed pH gradient (pH out less than pH inside) compared to no pH gradient condition suggesting the presence of PO4-/OH- exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of calcium uptake by brush border membrane vesicles of human small intestine.

Calcium uptake was characterized in human duodenal, jejunal, and ileal brush border membrane vesicles. Calcium uptake into human intestinal brush border membrane vesicles represented uptake into intravesicular space as evidenced by studies of osmolality, temperature dependence, calcium ionophore A23187-induced efflux and influx, and lanthanum displacement. Calcium uptake into membrane vesicles was sodium-independent. Negative membrane potential induced by valinomycin and anion substitution studies indicated an electroneutral process. Initial rate of uptake of calcium was linear up to 30 s (Y = 0.11 + 0.02x, r = 0.99). Kinetic parameters were determined from uptake measurements at 7 s, well within the linear phase of uptake. Calcium uptake represented mediated and nonmediated components. These components showed changes along the intestinal tract. Km values of the mediated component increased aborally, being lowest in the duodenum and highest in the terminal ileum. Vmax was highest in the duodenum, followed by, in descending order, the ileum, terminal ileum, and jejunum. The nonmediated component was greatest in the duodenum and decreased aborally. The duodenum appears to have a high-affinity, high-capacity system for the transport of calcium in humans. These studies are the first to characterize calcium transport by brush border membranes of the human small intestine.

Intestinal maturation: characterization of mitochondrial calcium transport in the rat.

The mitochondria play a major role in the regulation of intracellular calcium. Despite the fact that the enterocytes receive the majority of absorbed calcium, the role of the intestinal mitochondria in calcium transport during maturation is not known. Therefore, the current studies were designed to characterize calcium pump activity of jejunal mitochondria of rats during maturation (suckling, weanling, and adolescent rats). The functional integrity of the intestinal mitochondria of suckling and adolescent rats was determined by oxygen consumption studies demonstrating respiratory control ratios of more than 3 when succinate was used as a test substrate. Ca++ uptake was significantly stimulated by the presence of 3 mM ATP at all age groups studied. Maximal Ca++ uptake in the presence of 3 mM ATP and 2 mM succinate was 31.1 +/- 0.4, 50.2 +/- 4.2, and 94.3 +/- 1.5 nmol/mg protein (mean +/- SE) in suckling, weanling, and adolescent rats, respectively. Rates of ATP hydrolysis were 15.5 +/- 1.5 and 2.9 +/- 0.3 nmol/ATP hydrolyzed/mg protein in adolescent and suckling rats, respectively (p less than 0.001). Ca++ uptake was completely inhibited by 0.25 microM ruthenium red, oligomycin (10 micrograms/mg protein), 0.5 mM dinitrophenol and 1 mM sodium azide at all age groups. Ca++ efflux in the presence of ruthenium red occurred by a Na+-dependent pathway, indicating a Ca++/Na+ exchange mechanism. Kinetic parameters for ATP stimulated Ca++ uptake at 10 s revealed a Km of 0.84 +/- 0.11, 0.65 +/- 0.17, and 0.57 +/- 0.03 microM and Vmax of 1.83 +/- 0.07, 3.62 +/- 0.26 and 14.15 +/- 0.21 nmol/mg protein/10 s in suckling, weanling, and adolescent rats, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphate transport by intestinal endoplasmic reticulum during maturation.

The transport of phosphate into jejunal endoplasmic reticulum vesicles isolated from suckling and adolescent rats was investigated using a rapid filtration technique. Intestinal endoplasmic reticulum from both ages were enriched with NADPH cytochrome-C-reductase whereas other markers for brush border, basolateral, mitochondrial, and Golgi apparatus were impoverished. Phosphate uptake represented an energy-dependent process as evident by more than 80% decrease in uptake values at 0 degrees C compared to 25 degrees C. Phosphate uptake was ATP dependent in both age groups, however, mean uptake values were significantly greater in suckling rats compared to adolescent rats. pH optimum for uptake was 7.2 p-Chloromercuribenzoate at 100 microM concentration inhibited phosphate uptake by more than 90%. Initial rate of phosphate uptake was linear up to 45 s. Kinetics of phosphate uptake at 30 s showed a Km of 0.7 +/- 0.1 and 0.15 +/- 0.1 suckling and adolescent rats, respectively. Vmax was 1.5 +/- 0.5 and 0.14 +/- 0.01 nmol/mg protein/30 s for both suckling and adolescent rats, respectively. Herein we provide evidence for the first time for the presence of a phosphate carrier in intestinal endoplasmic reticulum of rats. Endoplasmic reticulum of phosphate uptake was significantly greater in suckling rats compared to adolescent rats. This increase in uptake is due to a greater number and activity of phosphate carriers in suckling rats.

Active calcium transport by intestinal endoplasmic reticulum during maturation.

Calcium uptake by intestinal endoplasmic reticulum was determined during maturation in the rat. Calcium uptake was enhanced severalfold by the presence of ATP in suckling, weanling, and adolescent rats. Uptake values were higher during early life and decreased gradually with age. Calcium uptake represented transport into the intravesicular space of the microsomes as evident by marked decrease in the uptake of 0 degrees C compared with values at 25 degrees C and by rapid release of intravesicular calcium by the ionophore A23187. Calcium uptake was dependent on magnesium and media pH and was inhibited by vanadate. Sodium oxalate enhanced calcium uptake. Oligomycin and sodium azide did not inhibit calcium uptake by microsomes, suggesting that calcium uptake represents a property of the microsomes rather than mitochondrial uptake. Initial rate uptake was linear up to 30 s. Maximal uptake occurred at pH 7.2. Kinetic studies revealed a high-affinity, high-capacity system in microsomes from suckling rats (Vmax 2.26 +/- 0.2 nmol.mg protein-1.15 s-1 and Km 0.56 +/- 0.01 microM) compared with a low-capacity system in microsomes from adolescent rats (Vmax 0.72 +/- 0.1 nmol.mg protein-1.15 s-1 and Km 0.69 +/- 0.02 microM). These findings suggest that the endoplasmic reticulum of the enterocyte may play a major role in regulating intestinal cytosolic calcium homeostasis during early development.

Developmental maturation of calcium transport by rat brush border membrane vesicles.

The developmental aspects of calcium transport across the intestinal brush membrane vesicles was studied utilizing a millipore filtration technique. Calcium transport represented uptake into the intravesicular space as evidenced by osmolality studies, calcium release by the calcium ionophore A23187, and temperature dependency. Calcium transport in both suckling and adolescent rats appears to occur by a saturable mechanism. Calcium uptake was similar in the presence of sodium and potassium gradients, but decreased in the presence of choline gradient. The imposition of negative membrane potential did not enhance calcium uptake compared to voltage clamp conditions indicating an electroneutral process. The initial rate of calcium uptake was linear up to 15 s. Kinetic analysis of calcium uptake at 7 s showed lower Vmax and lower Km in suckling rats compared to adolescent rats. These studies are the first to demonstrate the maturational aspects of calcium entry at the brush border level and are consistent with our previous kinetic studies utilizing whole tissue.