Postnatal development of the serotonin signaling system in the mucosa of the guinea pig ileum.

BACKGROUND: Serotonin is an important neurohumoral molecule in the gut but its signaling system is not fully developed in the neonatal gastrointestinal (GI) tract. This study aimed to evaluate the postnatal maturation of serotonin signaling in the small intestine. METHODS: In vitro amperometry for real-time measurement of serotonin at the mucosal surface, immunoblot, immunohistochemistry and high-performance liquid chromatography (HPLC) were used to examine serotonin handling in ileal segments from guinea pigs of different ages. KEY RESULTS: Extracellular serotonin levels significantly declined over the first three postnatal weeks, after which the levels increased and reached their maximum at 9 weeks postnatally. Serotonin levels were insensitive to the inhibition of the serotonin transporter (SERT) until the animals reached 3 weeks old. Measurement of serotonin and its metabolite 5-hydroxyindole acetic acid (5-HIAA) in the mucosa revealed that the serotonin turnover was significantly lower in neonates. Immunoblot and immunohistochemistry showed that SERT expression was extremely low in the neonatal period. Serotonin staining in cross-section showed that enterochromaffin (EC) cells were preferentially localized in the crypt region in neonates and the number of EC cells was significantly higher in 9-week-old animals. CONCLUSIONS & INFERENCES: SERT expression is low in the neonatal intestine and serotonin signaling matures postnatally. Extracellular serotonin levels decrease during the first three neonatal weeks as SERT expression increases. Extracellular serotonin levels increase after 3 weeks (weaning) possibly due to an increase in EC cell numbers. Postnatal maturation of serotonin signaling coincides with dietary changes in the developing guinea pig.

Galectin-3 expression and subcellular localization in senescent human fibroblasts.

Galectin-3 is a galactose/lactose-binding protein (M(r) approximately 30,000), identified as a required factor in the splicing of pre-mRNA. In the LG1 strain of human diploid fibroblasts, galectin-3 could be found in both the nucleus and the cytoplasm of young, proliferating cells. In contrast, the protein was predominantly cytoplasmic in senescent LG1 cells that have lost replicative competence through in vitro culture. Incubation of young cells with leptomycin B, a drug that disrupts the interaction between the leucine-rich nuclear export signal and its receptor, resulted in the accumulation of galectin-3 inside the nucleus. In senescent cells, galectin-3 staining remained cytoplasmic even in the presence of the drug, thus suggesting that the observed localization in the cytoplasm was due to a lack of nuclear import. In heterodikaryons derived from fusion of young and senescent LG1 cells, the predominant phenotype was galectin-3 in both nuclei. These results suggest that senescent LG1 cells might lack a factor(s) specifically required for galectin-3 nuclear import.

The human LGALS3 (galectin-3) gene: determination of the gene structure and functional characterization of the promoter.

Galectin-3 is a beta-galactoside-specific lectin that is a pre-mRNA splicing factor. Here we report the genomic organization of the human LGALS3 (galectin-3) gene and functional characterization of the promoter. Southern blot analysis of genomic DNA revealed that galectin-3 is coded by a single gene in the human genome. The gene is composed of six exons and five introns, spanning a total of approximately 17 kilobases (kb). Based on primer extension and ribonuclease protection analyses, there are two transcription initiation sites located 52 and 50 nucleotides (nt) upstream of the exon I-intron 1 border, and defined here as +1a and +1b, respectively. The translation start site is in exon II. The ribonucleoprotein-like N-terminal domain, containing the proline-glycine-alanine-tyrosine (PGAY) repeat motif, is found entirely within exon III. The carbohydrate recognition sequence is found entirely within exon V. Genomic fragments encompassing -836 to +141 nt (relative to +1a) have significant promoter activity when linked to the luciferase reporter gene and transiently transfected into HeLa cells or human diploid fibroblasts. Quiescent fibroblasts have low promoter activity but the activity increases 100-fold following serum addition. Serum responsive activation regions in the promoter are located between -513 and -339 nt and between -339 and -229 nt; an additional activation region may be located between -105 and -15 nt. Because galectin-3 is an immediate-early gene whose expression is dependent on the proliferative state of the cell, this study provides the basis for determining the molecular mechanisms of transcriptional regulation in neoplasia or cellular senescence.

Platelet activity and phosphoinositide turnover increase with advancing age.

PURPOSE: Blood platelet activity increases with advancing age. This study was designed to determine if changes in a key signal-transducing mechanism in the platelet, phosphoinositide turnover, are associated with the enhanced platelet activity seen in aging. PATIENTS AND METHODS: Platelets were harvested from a total of 40 healthy, non-obese, 22- to 62-year-old individuals, free of any clinical evidence of atherosclerotic vascular disease, and having normal serum laboratory lipid levels. Studies of platelet activity included measurement of in vitro platelet aggregation and plasma beta-thromboglobulin (beta-TBG), a marker of in vivo platelet secretion. Basal and thrombin-stimulated phosphoinositide turnover was measured following [32P]-orthophosphate incorporation into the various phospholipids, isolation of the phosphoinositides and phosphatidic acid by thin-layer chromatography and autoradiography, and quantification by liquid scintillation spectroscopy of these radiolabeled phospholipids. RESULTS: There was a positive correlation with age for both adenosine diphosphate (ADP)-induced aggregation (1.25 microM, r = 0.464, p less than 0.001; 2.5 microM, r = 0.386, p less than 0.05) and plasma beta-TBG (r = 0.381, p less than 0.055). There was a time-dependent increase of [32P]orthophosphate (32Pi) incorporation into platelet phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4-phosphate (PIP), and isotopic equilibrium was reached by 120 minutes at 37 degrees C. A positive correlation was found between age and basal 32P-PIP2 (r = 0.640, p less than 0.001) and 32P-PIP (r = 0.676, p less than 0.0005). Basal 32Pi incorporation into PIP2 correlated positively with in vitro aggregation (1.25 microM ADP, r = 0.795, p less than 0.0001; 2.5 microM ADP, r = 0.755, p less than 0.0005) as did 32Pi incorporation into PIP (1.25 microM ADP, r = 0.815, p less than 0.0001; 2.5 microM ADP, r = 0.795, p less than 0.0001). There was also a positive correlation between plasma beta-TBG levels and basal 32P-PIP2 (r = 0.768, p less than 0.005) and 32P-PIP (r = 0.505, p less than 0.066). Finally, increasing age correlated with thrombin (4 U/mL)-stimulated 32P-PIP2 hydrolysis (r = 0.694, p less than 0.01) and phosphatidic acid formation (r = 0.556, p less than 0.05).

Corrected Q-T interval prolongation as diagnostic tool for assessment of cardiac autonomic neuropathy in diabetes mellitus.

A simple method for evaluating alterations in cardiac sympathetic innervation may be measurement of the Q-T interval. Seventy-three diabetic patients (46 insulin dependent and 27 non-insulin dependent) were separated into four groups based on the presence and degree of cardiac autonomic neuropathy (CAN) with noninvasive cardiovascular reflexes and blood pressure responses. None of the patients had evidence of ischemic heart disease, kidney disease, or the idiopathic long Q-T-interval syndrome. The corrected Q-T interval (Q-Tc) was determined at rest with Bazett's formula. As a group, diabetic patients with greater than or equal to 2 abnormalities of cardiac autonomic function had a longer Q-Tc interval than those with no evidence of CAN. Diabetic patients with greater than or equal to 1 abnormality had a prolonged Q-Tc interval compared with a control group of 96 healthy nondiabetic subjects (mean +/- SD 397 +/- 18). The frequency of prolonged (greater than 433 ms, normal mean + 2SD) resting Q-Tc intervals increased with the increasing number of abnormalities (0, 1, 2, greater than or equal to 3): 11, 25, 41, and 75%, respectively. Twenty-three of 25 (92%) patients with a Q-Tc greater than 433 ms had evidence of CAN. However, 57% (31 of 54) of the patients with CAN had a normal Q-Tc interval. These data provide further evidence of a relationship between the presence and severity of CAN and degree of Q-Tc interval prolongation.(ABSTRACT TRUNCATED AT 250 WORDS)

Decreased platelet phosphoinositide turnover and enhanced platelet activation in IDDM.

Individuals with diabetes mellitus may have increased in vivo platelet activity. Abnormal platelet function could contribute to the increased incidence of vascular disease in diabetes mellitus. The biochemical mechanism(s) for platelet hyperactivation is unknown. We examined the hypothesis that platelet phosphoinositide turnover, a key signal-transducing mechanism involved in platelet activation, was abnormal in diabetic subjects. Platelets were harvested from 16 subjects with insulin-dependent diabetes mellitus (IDDM) and 19 healthy, nondiabetic control subjects of comparable age. Plasma beta-thromboglobulin (beta-TBG), a specific marker of platelet activity in vivo, was increased in IDDM (67.1 +/- 7.3 ng/ml) compared with control (41.0 +/- 6.0 ng/ml) subjects (P less than .005). [32P]orthophosphate (32Pi) incorporation into the individual phosphoinositides and phosphatidic acid (PA) reached isotopic equilibrium by 120 min for IDDM and control subjects. Specific activity (dpm 32P/micrograms phosphorus) of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) was not different between IDDM and control subjects. Under these conditions, basal 32Pi incorporation into PIP2 and PIP but not phosphatidylinositol (PI) or PA was significantly lower in IDDM subjects. There was significantly decreased [32P]PIP2 and [32P]PIP hydrolysis and decreased [32P]PA formation in IDDM after platelet stimulation with 4 U/ml human thrombin. There were no differences in [32P]PI hydrolysis between the two groups. The mass of PIP2 was reduced (P less than .005) in the platelets from IDDM (0.71 +/- 0.23 nmol/10(9) platelets) compared with control (1.65 +/- 0.53 nmol/10(9) platelets) subjects. Similarly, PIP was lower (P less than .001) in IDDM (0.66 +/- 0.09 nmol/10(9) platelets) than in control (2.92 +/- 0.43 nmol/10(9) platelets) subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-sympathetic ganglia antibodies and postural blood pressure in IDDM subjects of varying duration and patients at high risk of developing IDDM.

We examined the sera of 94 subjects with insulin-dependent diabetes mellitus (IDDM) for the presence of complement-fixing sympathetic ganglia (CF-SG) antibodies. In a cross-sectional analysis (duration 0-43 yr), 22% had detectable CF-SG antibodies. Subjects at high risk for IDDM were also studied. Four groups were studied: group 1 (aged 4-64 yr) islet cell antibody-positive (ICA+) prediabetic subjects, 10 of 19 (53%) were CF-SG+; group 2 (aged 6-14 yr) ICA- prediabetic subjects (first-degree relatives of IDDM subjects with either transient hyperglycemia, impaired oral glucose tolerance, and/or first-phase insulin release after intravenous glucose tolerance testing), 4 of 9 (44%) were CF-SG+ (2 of the 4 ICA- CF-SG+ subjects have progressed to IDDM); group 3 (aged 1.5-43 yr) ICA+ IDDM subjects (less than or equal to 1 yr duration) 6 of 10 (60%) were CF-SG+; and group 4 (aged 8-59 yr) ICA- IDDM subjects (less than or equal to 1 yr duration), 2 of 11 (18%) were CF-SG+. All groups had increased CF-SG compared with controls. Postural blood pressure and simultaneous CF-SG antibody measurements were performed in 28 IDDM subjects. The drop in systolic blood pressure was greater in the CF-SG+ subjects (P less than .05), and the frequency of CF-SG was greater in the mean to -2SD group (P less than .03) when data were analyzed within mean +/- 2SD of the normal blood pressure response.