Increased NHE2 expression in rat intestinal epithelium during ontogeny is transcriptionally mediated

1998 ◽  
Vol 275 (4) ◽  
pp. C1143-C1150 ◽  
Author(s):  
James F. Collins ◽  
Pawel R. Kiela ◽  
Hua Xu ◽  
Jiamin Zeng ◽  
Fayez K. Ghishan
Keyword(s):  
Northern Blot ◽  
Membrane Vesicle ◽  
Mrna Levels ◽  
Rat Intestine ◽  
Functional Protein ◽  
Adult Rats ◽  
Specific Staining ◽  
Western Blots ◽  
Intestinal Membrane ◽  
Old Rats

We have previously described changes in intestinal brush-border membrane vesicle (BBMV) Na+/H+exchange activity and characterized Na+/H+exchanger (NHE3) expression during rat ontogeny. The current studies were designed to investigate developmental changes in NHE2 expression in rat intestine. In previous studies, pH-dependent uptake of Na+ in jejunal BBMV utilizing HOE-694 inhibition demonstrated that NHE2 functional protein levels were lowest in 2-wk-old rats, higher in 3-wk-old and adult rats, and highest in 6-wk-old rats [Collins et al. Am. J. Physiol. 273 ( Cell Physiol. 42): C1937–C1946, 1997]. In the current investigation, Northern blot analyses showed that NHE2 mRNA levels in the jejunum were similar in 6-wk-old, adult, and 3-wk-old rats and three- to fivefold lower in 2-wk-old rats. In situ hybridization of 2- and 6-wk-old rat intestine with NHE2-specific probes confirmed Northern blot observations. Polyclonal antibodies were developed against an NHE2-specific peptide from amino acids 652–661. Western blots with NHE2 antiserum showed that the intensity of a specific 90-kDa band was lowest in 2-wk-old animals and four- to sixfold higher in 3- and 6-wk-old and adult animals. Immunohistochemical analysis showed specific staining of NHE2 antiserum to only the apical intestinal membrane. Furthermore, nuclear run-on analyses showed a 1.7-fold higher NHE2 transcription rate in 6-wk-old rats than in 2-wk-old rats. Overall, the current data suggest that increases in NHE2 expression upon weaning are mediated by increased gene transcription.

Functional and molecular characterization of NHE3 expression during ontogeny in rat jejunal epithelium

1997 ◽  
Vol 273 (6) ◽  
pp. C1937-C1946 ◽  
Author(s):  
James F. Collins ◽  
Hua Xu ◽  
Pawel R. Kiela ◽  
Jiamin Zeng ◽  
Fayez K. Ghishan
Keyword(s):  
Northern Blot ◽  
Polyclonal Antibodies ◽  
Membrane Vesicle ◽  
Age Groups ◽  
Fold Increase ◽  
Jejunal Mucosa ◽  
Mrna Levels ◽  
Adult Rats ◽  
Intestinal Brush Border Membrane ◽  
Protein Levels

Ontogenic changes occur in intestinal brush-border membrane vesicle (BBMV) Na+/H+exchange activity. The present studies were designed to investigate ontogenic changes in Na+/H+exchanger (NHE) isoform 3 in rat jejunum. pH-dependent Na+ uptake was assayed in four age groups of rats in the presence of 0, 50, or 800 μM HOE-694, a specific NHE inhibitor with differential sensitivities for NHE2 [inhibition constant ( K i) = 5 μM in PS120 fibroblasts] and NHE3 ( K i = 650 μM). Results showed that NHE2 and NHE3 contribute to basal BBMV uptake at all ages. Uptake levels were highest in 6-wk-old rats, lower in adult rats, and lowest in 2-wk-old (suckling) and 3-wk-old (weanling) rats. NHE3 contribution ranged from 92% at 6 wk of age to 59% at 2 and 3 wk of age. NHE3 inhibition by 800 μM HOE-694 was 38–45%. Statistical analysis showed that HOE-694 had a significant effect at both concentrations at all ages and that differences were present between all ages except 2- and 3-wk rats (at all HOE-694 concentrations). Northern blot analyses of jejunal mucosa showed lowest NHE3 mRNA levels in 2-wk animals and higher levels in all other age groups. Polyclonal antibodies were developed against an NHE3 COOH-terminal fusion protein, and antiserum was characterized with NHE3-transfected PS120 cells and by immunohistochemistry. Western blot analyses showed lowest protein levels in 2-wk animals and higher levels in the other ages. Suckling rats were subcutaneously injected with methylprednisone (MP) for 2 days and killed 1 day later. Northern blot analyses showed a twofold increase in NHE3 mRNA expression with MP treatment. Immunoblot analyses showed a 2.5-fold increase in NHE3 immunoreactive protein levels with MP injection. Overall, these data suggest that NHE3 is regulated during ontogeny and that ontogenic changes are most apparent around the time of weaning. Furthermore, the data suggest that NHE3 is regulated at transcriptional and posttranscriptional levels during mammalian intestinal development.

Expression of acetylcholine receptor mRNAs in atrophying and nonatrophying skeletal muscles of old rats

1998 ◽  
Vol 85 (5) ◽  
pp. 1903-1908 ◽  
Author(s):  
Ronald R. Gomes ◽  
Frank W. Booth
Keyword(s):  
Skeletal Muscle ◽  
Acetylcholine Receptor ◽  
Biceps Brachii ◽  
Male Rats ◽  
Brown Norway ◽  
Mrna Levels ◽  
Adult Rats ◽  
Γ Subunit ◽  
Age Related ◽  
Old Rats

We examined the age-related association in skeletal muscle between atrophy and expression of mRNAs encoding both the γ-subunit of the nicotinic acetylcholine receptor (AChR), and myogenin, a transcription factor that upregulates expression of the γ-subunit promoter. Gastrocnemius and biceps brachii muscles were collected from young (2-mo-old), adult (18-mo-old), and old (31-mo-old) Fischer 344/Brown Norway F1 generation cross male rats. In the gastrocnemius muscles of old vs. young and adult rats, lower muscle mass was accompanied by significantly elevated AChR γ-subunit and myogenin mRNA levels. In contrast, the biceps brachii muscle exhibited neither atrophy nor as drastic a change in AChR γ-subunit and myogenin mRNA levels with age. Expression of the AChR ε-subunit mRNA did not change with age in either gastrocnemius or biceps brachii muscles. Thus changes in skeletal muscle AChR γ-subunit and myogenin mRNA levels may be more related to atrophy than to chronological age in old rats.

scholarly journals Myostatin and insulin-like growth factor-I and -II expression in the muscle of rats exposed to the microgravity environment of the NeuroLab space shuttle flight

2000 ◽  
Vol 167 (3) ◽  
pp. 417-428 ◽  
Author(s):  
R Lalani ◽  
S Bhasin ◽  
F Byhower ◽  
R Tarnuzzer ◽  
M Grant ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Mass ◽  
Skeletal Muscle Mass ◽  
Recovery Period ◽  
Microgravity Environment ◽  
Mrna Levels ◽  
Adult Rats ◽  
Male Adult ◽  
Western Blots ◽  
Igf I

The mechanism of the loss of skeletal muscle mass that occurs during spaceflight is not well understood. Myostatin has been proposed as a negative modulator of muscle mass, and IGF-I and IGF-II are known positive regulators of muscle differentiation and growth. We investigated whether muscle loss associated with spaceflight is accompanied by increased levels of myostatin and a reduction in IGF-I and -II levels in the muscle, and whether these changes correlate with an increase in muscle proteolysis and apoptosis. Twelve male adult rats sent on the 17-day NASA STS-90 NeuroLab space flight were divided upon return to earth into two groups, and killed either 1 day later (R1) or after 13 days of acclimatization (R13). Ground-based control rats were maintained for the same periods in either vivarium (R3 and R15, respectively), or flight-simulated cages (R5 and R17, respectively). RNA and protein were isolated from the tibialis anterior, biceps femoris, quadriceps, and gastrocnemius muscles. Myostatin, IGF-I, IGF-II and proteasome 2c mRNA concentrations were determined by reverse transcription/PCR; myostatin and ubiquitin mRNA were also measured by Northern blot analysis; myostatin protein was estimated by immunohistochemistry; the apoptotic index and the release of 3-methylhistidine were determined respectively by the TUNEL assay and by HPLC. Muscle weights were 19-24% lower in the R1 rats compared with the control R3 and R5 rats, but were not significantly different after the recovery period. The myostatin/beta-actin mRNA ratios (means+/-s.e.m. ) were higher in the muscles of the R1 rats compared with the control R5 rats: 5.0-fold in tibialis (5.35 +/- 1.85 vs 1.07 +/- 0.26), 3.0-fold in biceps (2.46+/-0.70 vs 0.81 +/- 0.04), 1.9-fold in quadriceps (7.84 +/- 1.73 vs 4.08 +/- 0.52), and 2.2-fold in gastrocnemius (0.99 +/- 0.35 vs 0.44 +/- 0.17). These values also normalized upon acclimatization. Our antibody against a myostatin peptide was validated by detection of the recombinant human myostatin protein on Western blots, which also showed that myostatin immunostaining was increased in muscle sections from R1 rats, compared with control R3 rats, and normalized upon acclimatization. In contrast, IGF-II mRNA concentrations in the muscles from R1 rats were 64-89% lower than those in R3 animals. With the exception of the gastrocnemius, IGF-II was also decreased in R5 animals maintained in flight-simulated cages, and normalized upon acclimatization. The intramuscular IGF-I mRNA levels were not significantly different between the spaceflight rats and the controls. No increase was found in the proteolysis markers 3-methyl histidine, ubiquitin mRNA, and proteasome 2C mRNA. In conclusion, the loss of skeletal muscle mass that occurs during spaceflight is associated with increased myostatin mRNA and protein levels in the skeletal muscle, and a decrease in IGF-II mRNA levels. These alterations are normalized upon restoration of normal gravity and caging conditions. These data suggest that reciprocal changes in the expression of myostatin and IGF-II may contribute to the multifactorial pathophysiology of muscle atrophy that occurs during spaceflight.

scholarly journals Three mRNA transcripts of the proopiomelanocortin gene in human placenta at term

2000 ◽  
pp. 533-536 ◽  
Author(s):  
SI Grigorakis ◽  
E Anastasiou ◽  
K Dai ◽  
A Souvatzoglou ◽  
M Alevizaki
Keyword(s):  
Pregnant Women ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Mode Of Delivery ◽  
Mrna Levels ◽  
Functional Protein ◽  
Mrna Transcripts ◽  
Gene Transcripts ◽  
Pomc Gene

The proopiomelanocortin (POMC) gene whose normal pituitary specific mRNA product is 1200 bases (b) is also expressed in placenta and its peptide derivatives such as ACTH and beta-endorphin may play an important role in the initiation of labor. So far, two mRNA transcripts, one small (800b) and one large (1380b) have been reported in placenta by Northern blot analysis, similar to other endocrine tissues and various extrapituitary tumors; however, it is questionable whether both of these transcripts are effectively translated to a functional protein. We examined by Northern blot analysis the size and the differential expression of placental POMC gene transcripts in pregnant women with different modes of delivery. Placental tissues were collected from two groups of pregnant women, six with vaginal delivery (VD) and five with cesarean section (CS). In both groups of placentae three POMC gene transcripts were detected of 800, 1200 and 1380 bases; the 1200b pituitary specific species often predominated and was always present. The 800b transcript was also always present, while the large transcript (1380b) was expressed in 3/6 VD and 2/5 CS placental tissues. No differences in the relative levels of any of these mRNA species showing effect of the mode of delivery were observed. We conclude that POMC gene transcription in placental tissue at term gives rise to three mRNA transcripts, thus resembling extrapituitary tumors. The reported changes in the levels of the derivative peptides according to the mode of delivery do not reflect changes in POMC mRNA levels and could be attributed to a post-translational effect.

scholarly journals Effect of IGF-I and PDGF administered in vivo on the expression of osteoblast-related genes in old rats

2002 ◽  
Vol 174 (1) ◽  
pp. 63-70 ◽  
Author(s):  
H Tanaka ◽  
A Wakisaka ◽  
H Ogasa ◽  
S Kawai ◽  
CT Liang
Keyword(s):  
Gene Expression ◽  
Growth Factors ◽  
Mrna Expression ◽  
Combined Treatment ◽  
Mrna Levels ◽  
Adult Rats ◽  
Igf I ◽  
Old Rats ◽  
Osteoblast Markers ◽  
Marrow Ablation

In order to establish the cellular basis for using growth factors as possible therapeutic agents for the age-dependent deficit in bone formation activity, we examined the individual and combined effects of IGF-I and/or platelet-derived growth factor (PDGF) on the gene expression of osteoblast-related markers in male rats. The expression of osteoblast markers was examined in the femurs of adult and old rats following marrow ablation, which amplifies gene expression activity. The mRNA levels of collagen(alpha1) (I) (COLI), alkaline phosphatase (AP), osteopontin (OP) and osteocalcin (OC) were significantly lower in the old as compared with the adult rats. To determine whether growth factors can abolish the age-related deficits in mRNA expression in old bone, PDGF and/or IGF-I were infused directly into the right femur for 5 days following marrow ablation. The contralateral femur was infused with vehicle only and used as a control. PDGF stimulated the expression of OP mRNA in both adult and old rats, whereas COLI, AP and OC mRNAs were not affected. IGF-I infusion did not have a significant effect on mRNA expression in adult rats. In contrast, treatment with IGF-I significantly enhanced the mRNA levels of COLI, AP and OP in old rats. To examine whether the combination of both factors could affect the expression of osteoblast markers synergistically, PDGF and IGF-I were infused together. In adult bones, the combined treatment with PDGF and IGF-I caused a slight increase in the level of OP gene expression but no change in AP, OC or COLI genes. Although neither IGF-I nor PDGF alone was effective in stimulating the expression of OC, the combined treatment in old bones enhanced OC expression significantly. The expression of COLI, AP and OP was also stimulated, but the stimulation was no different from that of IGF-I alone. In PDGF plus IGF-I treatment with a high dose, no dose-response effects were observed. Within the limits of the present study, it is suggested that IGF-I and, to a much lesser extent, PDGF may partially restore the deficit in the expression of osteoblast markers in old bones, and that the combination of both factors is slightly better than IGF-I alone in stimulating OC expression.

scholarly journals Interrelationship of glutathione–cystine transhydrogenase and glutathione reductase in developing rat intestine

1973 ◽  
Vol 132 (3) ◽  
pp. 623-631 ◽  
Author(s):  
Beatrice States ◽  
Stanton Segal
Keyword(s):  
Glutathione Reductase ◽  
Reductase Activity ◽  
Rat Intestine ◽  
Adult Rats ◽  
Adult Rat ◽  
Mucosal Layer ◽  
Old Rats ◽  
Developing Rat ◽  
Gssg Reductase

1. Glutathione reductase and glutathione–cystine transhydrogenase activity in supernatant fractions of whole homogenates and homogenates of mucosal and muscular layers were determined in developing rat intestine after determination of the optimum conditions for assay of the two enzymes. In jejunum from adult rat, the Km values for GSSG reductase and GSH–cystine transhydrogenase activities were 0.25mm-GSSG and 0.23mm-cystine respectively. 2. The two activities could be differentiated by stability studies since GSSG reductase was stable at 60°C for 10min and could be stored at 4°C for 24h without loss of activity. GSH–cystine transhydrogenase, on the other hand, was denatured at 60°C and completely inactive after 24h storage at 4°C. 3. Based on calculations of total activities, both enzymes increased from the eighteenth day until the animals were young adults. 4. Total GSSG reductase activity increased at a greater rate with age than total GSH–cystine transhydrogenase activity as evidenced by activity ratios for GSH–cystine transhydrogenase/GSSG reductase of 0.44 and 0.12 in ileum from suckling and adult rats respectively, and 0.31 and 0.24 in jejunum from suckling and adult rats respectively. 5. In mucosa from adult rats GSSG reductase was more active in the ileum than in the jejunum, whereas GSH–cystine transhydrogenase activity was higher in the jejunum. 6. GSH–cystine transhydrogenase was active only in the muscle cells of the ileum of 7-day-old rats but became localized primarily in the mucosal layer in the adult rat. However, GSSG reductase activity was distributed evenly between the two layers throughout the intestine.

Myogenic regulatory factors during regeneration of skeletal muscle in young, adult, and old rats

1997 ◽  
Vol 83 (4) ◽  
pp. 1270-1275 ◽  
Author(s):  
Daniel R. Marsh ◽  
David S. Criswell ◽  
James A. Carson ◽  
Frank W. Booth
Keyword(s):  
Skeletal Muscle ◽  
Young Adult ◽  
Muscle Regeneration ◽  
Tibialis Anterior Muscle ◽  
Brown Norway ◽  
Mrna Levels ◽  
Myogenic Regulatory Factors ◽  
Adult Rats ◽  
Regulatory Factors ◽  
Old Rats

Marsh, Daniel R., David S. Criswell, James A. Carson, and Frank W. Booth. Myogenic regulatory factors during regeneration of skeletal muscle in young, adult, and old rats. J. Appl. Physiol. 83(4): 1270–1275, 1997.—Myogenic factor mRNA expression was examined during muscle regeneration after bupivacaine injection in Fischer 344/Brown Norway F1 rats aged 3, 18, and 31 mo of age (young, adult, and old, respectively). Mass of the tibialis anterior muscle in the young rats had recovered to control values by 21 days postbupivacaine injection but in adult and old rats remained 40% less than that of contralateral controls at 21 and 28 days of recovery. During muscle regeneration, myogenin mRNA was significantly increased in muscles of young, adult, and old rats 5 days after bupivacaine injection. Subsequently, myogenin mRNA levels in young rat muscle decreased to postinjection control values by day 21 but did not return to control values in 28-day regenerating muscles of adult and old rats. The expression of MyoD mRNA was also increased in muscles at day 5 of regeneration in young, adult, and old rats, decreased to control levels by day 14 in young and adult rats, and remained elevated in the old rats for 28 days. In summary, either a diminished ability to downregulate myogenin and MyoD mRNAs in regenerating muscle occurs in old rat muscles, or the continuing myogenic effort includes elevated expression of these mRNAs.

Circadian regulation of uroguanylin and guanylin in the rat intestine

1999 ◽  
Vol 277 (6) ◽  
pp. C1177-C1183 ◽  
Author(s):  
Lawrence A. Scheving ◽  
Wen-Hui Jin
Keyword(s):  
Dark Period ◽  
Polyclonal Antibodies ◽  
Circadian Variation ◽  
Proximal Colon ◽  
Mrna Levels ◽  
Rat Intestine ◽  
Adult Rats ◽  
Circadian Expression ◽  
Circadian Time ◽  
Proximal Small Intestine

Uroguanylin (UGN) and guanylin (GN) are the endogenous intestinal ligands for guanylyl cyclase C (GC-C). We examined the circadian expression of UGN, GN, and GC-C in the jejunum, ileum, and proximal colon of young adult rats by Northern blot analyses. These assays revealed that UGN is more abundant in the proximal small intestine, whereas GN and GC-C are more abundant in the proximal colon. mRNA levels showed significant circadian variation for UGN (3- to 18-fold peak/trough difference), GN (2.1- to 2.8-fold peak/trough difference), and GC-C (3- to 5-fold peak/trough difference). The maximal abundance occurred in the dark period for all three mRNAs, although peak UGN and GN expression occurred later in the dark period in the jejunum relative to the ileum and colon. Immunoblot analyses using monospecific polyclonal antibodies against UGN and GN prohormones confirmed the regional and circadian variation detected by Northern assays. Thus the expression of these genes is regulated not only by histological position but also by circadian time.

Homeostatic control of manganese excretion in the neonatal rat

1987 ◽  
Vol 252 (5) ◽  
pp. R842-R847 ◽  
Author(s):  
N. Ballatori ◽  
E. Miles ◽  
T. W. Clarkson
Keyword(s):  
Trace Metal ◽  
Biliary Excretion ◽  
Intraperitoneal Injection ◽  
Neonatal Rat ◽  
Abrupt Increase ◽  
Adult Rats ◽  
Tracer Dose ◽  
Diminished Capacity ◽  
Carrier Free ◽  
Old Rats

Previous studies in neonatal and suckling animals showed that immature animals have a greatly diminished capacity to excrete manganese and therefore were considered to be unable to regulate tissue manganese concentrations. In contrast, the present studies indicate that suckling rats have the capacity to excrete excess manganese at rates nearly comparable to those of adults. Eight- to 10-day-old rats given a tracer dose of 54MnCl2 (essentially carrier free), either via gavage or by intraperitoneal injection showed little elimination of the 54Mn until the 18-19th day of life, when there was an abrupt increase in the rate of the metal's excretion. However, when manganese was given in doses of 1 and 10 mg/kg, the young animals excreted from 30-70% of the dose in only 4 days, at which time a new rate of excretion was achieved. This enhanced rate of excretion remained constant until the 18-19th day of life, when it was again accelerated. Biliary excretion of manganese, the primary route for the elimination of the metal, was only 30-60% lower in 14-day-old rats compared with adults at doses ranging from tracer to 10 mg 54Mn/kg. For both the 14-day-old and adult rats, an apparent biliary transport maximum was reached at a dose of 10 mg Mn/kg. These studies indicate that the excretory pathways for manganese are well developed in the neonatal rat. The avid retention of tracer quantities of manganese by the neonate may be a consequence of the scarcity of this essential trace metal in its diet.

scholarly journals The association of the sulphogalactosylglycerolipid of rat brain with myelination

1977 ◽  
Vol 166 (3) ◽  
pp. 421-428 ◽  
Author(s):  
Joanne Pieringer ◽  
G. Subba Rao ◽  
Paul Mandel ◽  
Ronald A. Pieringer
Keyword(s):  
Rat Brain ◽  
Adult Life ◽  
Developmental Pattern ◽  
Adult Rats ◽  
Jimpy Mouse ◽  
Myelin Fraction ◽  
Old Rats

The sulphogalactosylglycerolipid of rat brain is closely associated with the process of myelination, as demonstrated by the following observations. 1. The lipid is barely detectable in rat brain before 10 days of age, accumulates rapidly between age 10 and 25 days, and remains relatively constant in amount (between 0.3 and 0.4μmol per brain) thereafter into adult life. 2. The activity of adenosine 3′-phosphate 5′-sulphatophosphate–galactosyldiacylglycerol sulphotransferase is almost absent before 10 days of age, attains a maximum at age 20 days, and slowly decreases thereafter with increasing age. This developmental pattern correlates well with that of other myelin-specific metabolites. 3. Both the concentration of the sulphogalactosylglycerolipid and the activity of sulphotransferase are greatly decreased in the non-myelinating jimpy mouse. 4. The myelin fraction of rat brain contains most of the sulphogalactosylglycerolipid. The lipid occurs in a diacyl and an alkylacyl form. Determinations of the relative amount of each type in brain showed about a 1:1 mixture in both 21-day-old and adult rats. Rats injected with H235SO4 at 20 days of age lost35S from the diacyl form at a higher rate than from the alkylacyl compound over a 21-day period. These data suggest that the diacyl form has a higher turnover than the alkylacyl derivative. The percentage of the total sulpholipid content of brain contributed by the sulphogalactosylglycerolipid is 16% in 21-day-old rats and 8.4% in adult rats.

Sign in / Sign up

Export Citation Format

Share Document