To test the involvement of the water channel aquaporin (AQP)-4 in gastric acid physiology, the human gastric cell line (HGT)-1 was stably transfected with rat AQP4. AQP4 was immunolocalized to the basolateral membrane of transfected HGT-1 cells, like in native parietal cells. Expression of AQP4 in transfected cells increased the osmotic water permeability coefficient (Pf) from 2.02 +/- 0.3 x 10-4 to 16.37 +/- 0.5 x 10-4 cm/s at 20 degrees C. Freeze-fracture EM showed distinct orthogonal arrays of particles (OAPs), the morphological signature of AQP4, on the plasma membrane of AQP4-expressing cells. Quantitative morphometry showed that the density of OAPs was 2.5 +/- 0.3% under basal condition and decreased by 50% to 1.2 +/- 0.3% after 20 min of histamine stimulation, mainly due to a significant decrease of the OAPs number. Concomitantly, Pf decreased by approximately 35% in 20-min histamine-stimulated cells. Both Pf and OAPs density were not modified after 10 min of histamine exposure, time at which the maximal hormonal response is observed. Cell surface biotinylation experiments confirmed that AQP4 is internalized after 20 min of histamine exposure, which may account for the downregulation of water transport. This is the first evidence for short term rearrangement of OAPs in an established AQP4-expressing cell line.

Animals↗

PubMed2001

An inhibitory role of Rho in the vasopressin-mediated translocation of aquaporin-2 into cell membranes of renal principal cells.

Vasopressin regulates water reabsorption in renal collecting duct principal cells by a cAMP-dependent translocation of the water channel aquaporin-2 (AQP2) from intracellular vesicles into the cell membrane. In the present work primary cultured inner medullary collecting duct cells were used to study the role of the proteins of the Rho family in the translocation of AQP2. Clostridium difficile toxin B, which inhibits all members of the Rho family, Clostridium limosum C3 toxin, which inactivates only Rho, and the Rho kinase inhibitor, Y-27632, induced both depolymerization of actin stress fibers and AQP2 translocation in the absence of vasopressin. The data suggest an inhibitory role of Rho in this process, whereby constitutive membrane localization is prevented in resting cells. Expression of constitutively active RhoA induced formation of actin stress fibers and abolished AQP2 translocation in response to elevation of intracellular cAMP, confirming the inhibitory role of Rho. Cytochalasin D induced both depolymerization of the F-actin cytoskeleton and AQP2 translocation, indicating that depolymerization of F-actin is sufficient to induce AQP2 translocation. Thus Rho is likely to control the intracellular localization of AQP2 via regulation of the F-actin cytoskeleton.

Amides↗

PubMed2001

Expression and localization of the aquaporin-8 water channel in rat testis.

Spermatogenesis and sperm maturation and storage are accompanied by significant movements of water, and multiple aquaporin transmembrane water channels (AQPs) have been recognized in the male reproductive tract. Nevertheless, the involvement of aquaporins in male reproductive physiology is mostly unknown. Here the expression and localization of AQP8 in rat spermatogenesis is defined and compared to that of AQP7, another aquaporin expressed in male germ cells. AQP8 mRNA was found in testis but not in epididymis, whereas the AQP7 transcript was present in both locations. By immunoblotting, the AQP8 protein was detected as a 25-kDa band and a 32- to 40-kDa diffuse component corresponding to the core and glycosylated protein, respectively. Membrane fractionation revealed AQP8 both in microsomal and plasma membrane-enriched fractions of rat testis while no apparent bands were detected in epididymis. AQP7 appeared as a 23- to 24-kDa band and was found both in testis and epididymis. By immunofluorescence, AQP8 labeling was found intracellularly as well as over the plasma membrane of germ cells throughout spermatogenesis. AQP7 was present in spermatids and spermatozoa and was predominant over the plasma membrane. AQP8 may be involved in the cytoplasmic condensation occurring during differentiation of spermatids into spermatozoa and in the generation of seminiferous tubule fluid.

Animals↗

PubMed2001

Rho inhibits cAMP-induced translocation of aquaporin-2 into the apical membrane of renal cells.

First published August 8, 2001; 10.1152/ajprenal.00091.2001.-We have recently demonstrated that actin depolymerization is a prerequisite for cAMP-dependent translocation of the water channel aquaporin-2 (AQP2) into the apical membrane in AQP2-transfected renal CD8 cells (29). The Rho family of small GTPases, including Cdc42, Rac, and Rho, regulates the actin cytoskeleton. In AQP2-transfected CD8 cells, inhibition of Rho GTPases with Clostridium difficile toxin B or with C. limosum C3 fusion toxin, as well as incubation with the Rho kinase inhibitor, Y-27632, caused actin depolymerization and translocation of AQP2 in the absence of the cAMP-elevating agent forskolin. Both forskolin and C3 fusion toxin-induced AQP2 translocation were associated with a similar increase in the osmotic water permeability coefficient. Expression of constitutively active RhoA induced formation of stress fibers and abolished AQP2 translocation in response to forskolin. Cytochalasin D induced both depolymerization of F-actin and AQP2 translocation, suggesting that depolymerization of F-actin is sufficient to induce AQP2 translocation. Together, these data indicate that Rho inhibits cAMP-dependent translocation of AQP2 into the apical membrane of renal principal cells by controlling the organization of the actin cytoskeleton.

Actins↗

PubMed2000

The effects of transdermal estradiol on the response to mental stress in postmenopausal women: a randomized trial.

PURPOSE: Estrogens inhibit adrenomedullary catecholamine release and catecholamine-mediated responses to stress. We examined whether estrogen supplementation reduces the sympathoadrenal response to mental stress in postmenopausal women. MATERIALS AND METHODS: We compared the effects of 3-week treatment with transdermal 17-beta-estradiol and placebo in 10 postmenopausal women using a randomized, blinded, crossover design. We measured plasma catecholamine levels and the cardiovascular and metabolic responses to a 15-minute stress with mental arithmetic. Treatments were compared using repeated measures analysis of variance. RESULTS: During placebo treatment, mean (+/- SD) epinephrine levels reached a peak of 431 +/- 135 pmol/liter after 15 minutes of stress; the epinephrine response was blunted during estradiol treatment, with a peak of 357 +/- 77 pmol/liter (P <0.05). Estradiol also blunted the diastolic blood pressure response to stress (baseline levels of 78 +/- 15 mm Hg vs peak of 90 +/- 6 mm Hg during placebo; baseline of 80 +/- 8 mm Hg vs peak of 84 +/- 6 mm Hg during estradiol; P <0.05). Estradiol treatment also blunted the decrease in the standard deviation of the mean of the electrocardiographic RR intervals and the increase in the ratio between the low-frequency and high-frequency bandwidths. CONCLUSION: We observed a moderate, although significant, reduction in markers of the stress response to mental arithmetic in postmenopausal women treated with transdermal 17-beta-estradiol.

Administration, Cutaneous↗

PubMed2000

The cultured human gastric cells HGT-1 express the principal transporters involved in acid secretion.

HGT-1 is a human cell line sharing a number of physiological features with gastric parietal cells. HGT-1 cell monolayers were able to secrete H+ when stimulated with histamine (calculated external pH variation, deltapH(e) 0.46+/-0.05) as assessed using the impermeant, pH-sensitive fluorescence dye 8-hydroxypyrene-1,3,6-trisulphonic acid, trisodium salt (HPTS). Treatment with 100 microM omeprazole inhibited the histamine-induced apical acidification by about 60%. Intracellular pH (pH(i)) measurements using the fluorescent pH-sensitive dye 2',7'-bis-carboxyethyl-5(6)-carboxyfluorescein (BCECF) demonstrated the expression of a functional, omeprazole-sensitive H+/K+-pump. A monoclonal antibody directed against the alpha subunit of the H+/K+-ATPase immunoprecipitated a 95-kDa protein from HGT-1 cells and human stomach which corresponds to the expected molecular size of the native protein. HGT-1 cells were also positive for the anion exchanger AE2 that is expressed in gastric parietal cells. In addition, we identified a histamine- and pH(i)-sensitive Na+/H+ exchanger in HGT-1 cells, which might correspond to the functional expression of the NHE4 isoform that has been detected in gastric epithelial cells as well as in primary cultured parietal cells. HGT-1 cells therefore display the principal features of parietal cells and might represent an interesting cell culture model for studying the regulatory mechanisms involved in acid secretion.

1-Methyl-3-isobutylxanthine↗

PubMed2000

Changes in HDL-cholesterol and lipoprotein Lp(a) after 6-month treatment with finasteride in males affected by benign prostatic hyperplasia (BPH).

Androgen effects on lipoproteins, mainly high density lipoprotein (HDL), could be exerted by a direct interaction of testosterone (T) or dihydrotestosterone (DHT) with liver androgen receptors. To assess if T needs to be converted into DHT to affect lipid metabolism, 13 patients were studied, affected with benign prostatic hyperplasia (BPH) and treated with an inhibitor of 5 alpha-reductase (finasteride). They were compared with 15 untreated controls. At baseline and after 3 and 6 months of therapy, each patient was evaluated as for lipoprotein and hormone concentrations, as well as for nutritional status. Body composition was assessed by anthropometry and bio-impedance analysis (BIA). Treatment was associated with a significant increase of HDL-cholesterol (HDL-C), mainly HDL3 subclass, and lipoprotein(a) (Lp(a)), as well as a decline of DHT, whereas no significant changes were apparent for T, estradiol (E2), sex hormone binding hormone (SHBG) and body composition indexes. However, no significant associations between DHT and lipid relative changes were apparent at bivariate correlation analysis. This finding was confirmed by comparing patient subsets identified by cluster analysis, according to HDL subclass individual responses. Rather, a slight association with E2 for HDL2 (positive) and HDL3 (negative) was found. In conclusion, finasteride can modify HDL and Lp(a) concentrations. However, by the data, these effects cannot be definitively attributed to the changes in DHT synthesis induced by finasteride, since a direct and non-specific interference of the drug on liver metabolism cannot be excluded.

Aged↗

PubMed2000

Evaluation of the circadian profiles of serum dehydroepiandrosterone (DHEA), cortisol, and cortisol/DHEA molar ratio after a single oral administration of DHEA in elderly subjects.

Aging is associated with a selective decline in circulating levels of dehydroepiandrosterone (DHEA) and its sulfate, with no major changes in cortisol secretion. In young subjects, serum levels of both DHEA and cortisol are regulated according to a circadian rhythm, and an age-related attenuation of DHEA, but not cortisol, circadian rhythmicity has been reported. Several trials have evaluated the effects of DHEA supplementation in elderly subjects, although the results are still controversial. However, no data are available on the 24-hour profile of DHEA circulating levels in elderly subjects with DHEA administration. In the present study, we evaluated the circadian rhythms of DHEA, cortisol, and the cortisol/DHEA molar ratio in old subjects treated with either placebo (old-PL) or a single 50-mg dose of DHEA (old-D), both administered orally at 0700 hours. For each variable, the circadian profiles were compared with those obtained in young control subjects. The group of young subjects displayed a circadian rhythm for both DHEA and cortisol serum concentrations but no rhythm for the cortisol/DHEA molar ratio. In the old-PL group, the circadian rhythm of DHEA was completely abolished, whereas significant rhythms for both cortisol and the cortisol/DHEA molar ratio were observed. Particularly, at each time point, the cortisol/DHEA molar ratio was significantly higher in these subjects versus the young group. In the old-D group, the circadian rhythm of DHEA was completely restored and was comparable to that observed in the young group. Analogous to the observations in young subjects, the profile of the cortisol/DHEA molar ratio in old-D subjects did not display any circadian rhythmicity, the values being almost completely comparable to those observed in young controls. Our data demonstrate that the circadian rhythm of DHEA is totally abolished in elderly subjects. A single 50-mg dose of DHEA administered orally at 0700 hours restores the circadian rhythmicity of serum DHEA and almost completely normalizes the 24-hour profile of the cortisol/DHEA molar ratio in old subjects without affecting the cortisol circadian rhythm.

Administration, Oral↗

PubMed2000

Physiological cyclic variation of the myocardial integrated backscatter signal in athlete's heart.

BACKGROUND: Left ventricular hypertrophy which realizes in athlete's heart could create some problems of acoustic reflection related to the increase of myocytic and not-myocytic elements of the heart. The aim of the present study was to analyze the ultrasonic backscatter myocardial indexes both as peak end-diastolic signal intensity and as its cardiac-cyclic variation in athlete's heart, compared to healthy sedentary controls. METHODS: Two groups of ten subjects each, all males of mean age (31.6+/-3.5), and of comparable weight and height were analyzed: group (A) comprised ten cyclists of good professional level and group (C) included ten healthy subjects acting as controls. A 2D-color Doppler echocardiography with a digital echograph Agilent Technologies (AT) Sonos 5500 was carried out on all subjects in the study for the conventional analysis of the left ventricular mass and function. The ultrasonic myocardial integrated backscatter signal (IBS) was analyzed with an "Acoustic Densitometry" module implemented on an AT echograph. The signal was also sampled with a R.O.I. placed at interventricular septum and at posterior left ventricular wall level. The systo-diastolic variation of the backscatter was also considered as Cyclic Variation Index (CVIibs). RESULTS: The left ventricular mass was significantly higher in athletes in comparison with controls (LVMbs: A: 154.5+/-18.7; C: 101.4+/-12.4; p<0.001). The end diastolic IBS signal did not show significant statistical differences between the two groups. The CVIibs both at septum (A: 30.5+/-5.3; C: 27.2+/-7.3; p<0.002) and posterior wall level (A: 43.7+/-9.1; C: 40.7+/-9.1; p<0.001) though was comparable in both groups. CONCLUSION: The conclusions reached in the present study confirmed the physiology of the left ventricular hypertrophy of the athlete's heart evaluated with an ultrasonic integrated backscatter tissue characterization, in particular through the cyclic variation of integrated backscatter myocardial signal. This finding is probably the expression of a preserved intramural myocardial function in the athlete's heart despite the increase of left ventricular mass induced by physical training.

Adult↗

PubMed2000

Ultrasonic myocardial textural analysis in subclinical hypothyroidism.

BACKGROUND: In subclinical hypothyroidism (sHT), a condition in which impaired hormone synthesis is compensated by thyroid-stimulating hormone (TSH) hypersecretion, previous studies have suggested the presence of disturbances in left ventricular (LV) function. OBJECTIVES: Our goal was to investigate LV structure and function through the combined use of conventional Doppler echocardiography and ultrasonic videodensitometry. METHODS: We studied 16 patients with sHT (aged 32+/-12 [mean +/- SD] years) who had raised TSH levels (> 3.6 mIU/L) but normal levels of free thyroid hormones (free thyroxine [FT(4)] and free triiodothyro-nine [FT(3)]), and 16 carefully age- and sex-matched euthyroid subjects. Transmitral flow Doppler analysis and quantitative analysis of the echocardiographic digitized images were performed in all study subjects. Textural parameters of the septum and posterior wall were obtained as mean gray levels, which were then used to calculate the cyclic variation index (CVI), that is, the percent change in mean gray levels between diastole and systole. RESULTS: Patients with sHT had a significantly higher LV mass index (92 +/- 16 versus 76 +/- 16 g.m(2), P<.01) and isovolumic relaxation time corrected for heart rate (IVRTc) (2.9 +/- 0.6 versus 2.5 +/- 0.6, P<.04) than did controls. On videodensitometry, patients had lower CVIs both for the septum (-5% +/- 22% versus 33% +/- 9%, P<.0001) and the posterior wall (10% +/- 26% versus 49% +/- 18%, P<.0001). IVRTc discriminated only 25% of the patients from the controls, whereas CVI analysis correctly identified 85% of the patients with sHT (P<.002). Furthermore, CVI values were found to be significantly related to serum FT(4) and FT(3) concentrations in a direct fashion, and to serum TSH levels in an inverse fashion. CONCLUSIONS: Subclinical hypothyroidism is associated with changes in videodensitometric myocardial structure. These changes, which are not accurately detected by conventional or Doppler echocardiography, are quantitatively related to loss of thyroid function and could represent an early sign of myocardial damage in hypothyroidism.

Adult↗

PubMed2000

Aging-related decline of gonadal function in healthy men: correlation with body composition and lipoproteins.

OBJECTIVE: To assess if androgen decline in physiological aging contributes to the concomitant changes in body composition and lipoprotein levels. DESIGN: Cross-sectional, observational study. SETTING: A university-based outpatient center. SUBJECTS: The study comprised 206 healthy volunteers (aged 18-95 years). MEASUREMENTS: Blood samples were drawn after an overnight fast for the assay of hormones (free testosterone (FT), estradiol (E2), and sex hormone-binding globulin (SHBG)) and lipids (total cholesterol, triglycerides, high-density lipoprotein cholesterol, and lipoprotein Lp(a)). At the same time, body composition was assessed by both anthropometry (fat mass percentage (FM%) estimated from four measures of skinfold thickness using the Durnin and Womersley equation and the Siri equation) and by bioimpedance analysis (FM% estimated using the Segal or Deurenberg equations, respectively, for subjects younger or older than 62 years). RESULTS: A significant age-related decline was found for FT and E2 concentrations, whereas SHBG levels were related positively with age. No significant association was apparent between hormonal changes and the concomitant modifications of body composition and lipoproteins. Only SHBG showed a significant inverse association between FM% and the waist-to-hip ratio, independent of age. The comparison between older hypogonadal (with FT levels below the lower limit of the normality range assessed in younger subjects) and eugonadal men did not show any significant differences in body composition or lipid profile. CONCLUSIONS: This study suggests that, in men, androgen decline caused by normal aging does not significantly affect some targets of testosterone action, such as body composition and lipid metabolism. Therefore, androgen supplementation in hypogonadal older men cannot be expected to influence nutritional status and body composition to the same extent that it does other main targets of testosterone action, such as sexual activity and muscle strength. However, we cannot exclude that selected subsets of older patients with low testosterone levels, especially if affected by catabolic disease, could benefit from the effects of androgen administration on nutritional status.

Adolescent↗

PubMed2000

The phosphatase inhibitor okadaic acid induces AQP2 translocation independently from AQP2 phosphorylation in renal collecting duct cells.

Phosphorylation by kinases and dephosphorylation by phosphatase markedly affect the biological activity of proteins involved in intracellular signaling. In this study we investigated the effect of the serine/threonine phosphatase inhibitor okadaic acid on water permeability properties and on aquaporin2 (AQP2) translocation in AQP2-transfected renal CD8 cells. In CD8 cells both forskolin alone and okadaic acid alone increased the osmotic water permeability coefficient P(f) by about 4- to 5-fold. In intact cells, in vivo phosphorylation studies revealed that forskolin stimulation resulted in a threefold increase in AQP2 phosphorylation. In contrast, okadaic acid treatment promoted only a 60% increase in AQP2 phosphorylation which was abolished when this treatment was performed in the presence of 1 microM H89, a specific protein kinase A (PKA) inhibitor. Nevertheless, in this latter condition, confocal microscopy analysis revealed that AQP2 translocated and fused to the apical membrane. Okadaic acid-induced AQP2 translocation was dose dependent having its maximal effect at a concentration of 1 microM. In conclusion, our results clearly indicate that okadaic acid exerts a full forskolin-like effect independent from AQP2 phosphorylation. Thus AQP2 phosphorylation is not essential for water channel translocation in renal cells, indicating that different pathways might exist leading to AQP2 apical insertion and increase in P(f).

Actins↗