Daily activity rhythms and the stress-related response in the wedge sole (Dicologoglossa cuneata Moreau, 1881).

To gain further insights on the wedge sole (Dicologoglossa cuneata Moreau, 1881) physiology and behavior, we evaluated its daily feeding and locomotor activity rhythms and compared three different feeding strategies: self-feeding (SF), diurnal feeding schedule (DS) and nocturnal feeding schedule (NS). 450 fish divided into three groups (three replicates each), were kept during 65days. SF had free access to self-feeders whereas DS and NS were fed four times a day. Physiological stress parameters as plasma cortisol, glucose, lactate, proteins and triglycerides were determined. Under the SF setting, the 91% of feeding demands occurred during the dark phase. Furthermore, locomotor activity was also higher during the scotophase (64% of the total activity). Significantly higher values for specific growth rate (SGR) and feed efficiency rate (FER) were observed in NS (0.49 and 0.48%day-1, respectively); whereas SF consumed much less food than the rest and presented a high mortality rate (46%). Plasma cortisol levels were dramatically increased in SF and DS compared to NS (21.8±6.1, 65.8±30.3 and 0.3±0.1ngmL-1, respectively). In summary, the wedge sole appears as a species with nocturnal locomotor and feeding behaviors and NS as the most appropriate feeding strategy. These new findings appear as key information for both the preservation of natural stocks of this species and its rearing.

Differential ontogenetic patterns of in vitro desensitization to GHRH in fetal and neonatal anterior pituitary.

The aim of this study was to assess the ontogenetic changes in vitro in both the responsiveness of anterior pituitary tissue to growth hormone-releasing hormone (GHRH) and the critical role of GHRH in the long-term regulation of pulsatile GH secretion during perinatal porcine life. A superfusion system was used to apply three consecutive 10-min pulses of GHRH (the first of 1 nM and the other two of 10 nM) for 3 consecutive days in pituitary glands isolated from fetal (95- and 110-day) and neonatal (12-day) male pigs. In fetuses, total GHRH-induced GH release decreased progressively over the 3 days. However, in neonates, GH did not decrease until day 3, but remained higher than in fetuses. When each GH pulse was assessed individually, fetuses showed a similar pattern. GH secretion induced by the first GHRH pulse on days 1 and 2 was lower than that induced by the second and third pulses. By day 3, GH release lowered dramatically after all pulses. In contrast, in neonates no differences were observed among the GH levels induced by the three GHRH pulses at any day, although day 3 showed lower GH rates. In conclusion, during perinatal development, a desensitizing effect to long-term repetitive GHRH pulses was observed in both fetuses and neonates, but this effect was delayed in neonates. Thus, the capacity of somatotrope cells to maintain GH response to GHRH seems to be developmentally regulated during perinatal stages. Furthermore, the frequency of GHRH pulses, rather than the concentrations, might be a key factor to elicit desensitization.

Dinámica in vitro de la secreción de hormonaparatiroidea regulada por calcio y efecto sobre el ciclo celular: adenoma frente a hiperplasiaparatiroidea / In vitro dynamics of parathyroid hormone secretion regulated by calcium and effects on the cell cycle: parathyroid hyperplasia versus adenoma

Objetivo: Comparar in vitro la dinámica de la secreción de hormona paratiroidea (PTH) regulada por calcio y su relación con el ciclo celular en adenomas frente a hiperplasia de paratiroides. Material y métodos: Ocho adenomas de paratiroides y 23 glándulas hiperplásicas procedentes de 8 pacientes con hiperparatiroidismo primario y 7 pacientes con hiperparatiroidismo secundario, respectivamente. Para el estudio de la dinámica de secreción, pequeños fragmentos de tejido paratiroideo se transfirieron secuencialmente a intervalos de una hora a pocillos con concentraciones variables de calcio: 0,4, 0,6, 0,8, 1,0, 1,25, y 1,35 o 1,5 mM. Se determinaron las concentraciones de iPTH en el medio. Células paratiroides se aislaron sin el uso de enzimas y el ciclo celular paratiroideo se analizó por el método de Vindelov. El núcleo se adquirió por citometría de flujo y se analizó usando un software CELLFIT. Resultados: En los tejidos paratiroideos de las glándulas hiperplásicas, el aumento del calcio extracelular produjo una disminución de la secreción de PTH manifestada con valores de calcio de0,8 mM y una inhibición máxima de secreción de PTH con un calcio de 1,25 mM. Por el contrario, en los tejidos de los adenomas de paratiroides se requirieron concentraciones de Ca de 1,2 mM para provocar una mínima disminución de la secreción de PTH. En los adenomas no hubo correlación entre las fases del ciclo celular y la calcemia o el set point (r = 0,914; p <0,005) y el calcio sérico basal (r = 0,862; p <0,02). Conclusiones: La regulación de la secreción de PTH por el calcio extracelular in vitro es menos sensible en adenomas que en glándulas hiperplásicas paratiroideas. En hiperplasia paratiroidea la proliferación celular parece es- tar regulada por la concentración de calcio extracelular (a mayor calcemia menor proliferación) (AU)

Aim: To compare the dynamics of calcium-regulated PTH secretion in vitro from adenomatous versus hyperplastic glands and to investigate the relationship between the parathyroid cell cycle and the calcium-regulated PTH secretion in these glands. Materials and methods: A total of31 parathyroid glands (8 adenomatous and 23 hyperplastic) from 8 patients with primary hyperparathyroidism and7 with secondary hyperparathyroidism respectively were studied. For the evaluation of calcium-regulated PTH secretion, small parathyroid pieces of 1 mm were sequentially transferred to wells with varying Ca concentrations:0.4, 0.6, 0.8, 1, 1.25 and 1.35 or 1.5 mM. PTH concentrations were determined in the medium. For the parathyroid cell cycle studies, parathyroid cells were isolated without the use of enzymes and cell cycle was analyzed using the method described by Vindelov. The nuclei were acquired by flow cytometer and analyzed using the CELL- FIT software. Results: In parathyroid tissues from hyper- plastic glands, the increase in extracellular calcium pro- duced a decrease in PTH secretion which was apparent with a calcium level as low as 0.8 mM and the maximal in- hibition of PTH secretion was obtained with a calcium of 1.25 mM, by the contrary, adenomatous glands required a calcium of 1.2 mM to produce a minimal decrease in PTH secretion. In hyperplastic parathyroid glands but not in parathyroid adenomas there was a significant correlation between the percentage of cells in G0/G1 phase with the set point (r = 0.914; P <0.005) and the basal serum Ca (r = 0.862; P <0.02). Conclusions: The control of the extracellu- lar calcium-PTH release in vitro is less sensitive in parathy- roid adenomas than hyperplasic parathyroid glands. In parathyroid hyperplasia the cell proliferation may be reg- ulated by the extracellular calcium concentration (higher calcemia less proliferation) (AU)

[In vitro dynamics of parathyroid hormone secretion regulated by calcium and effects on the cell cycle: parathyroid hyperplasia versus adenoma]. / Dinámica in vitro de la secreción de hormona paratiroidea regulada por calcio y efecto sobre el ciclo celular: adenoma frente a hiperplasia paratiroidea.

AIM: To compare the dynamics of calcium-regulated PTH secretion in vitro from adenomatous versus hyperplastic glands and to investigate the relationship between the parathyroid cell cycle and the calcium-regulated PTH secretion in these glands. MATERIALS AND METHODS: A total of 31 parathyroid glands (8 adenomatous and 23 hyperplastic) from 8 patients with primary hyperparathyroidism and 7 with secondary hyperparathyroidism respectively were studied. For the evaluation of calcium-regulated PTH secretion, small parathyroid pieces of 1 mm were sequentially transferred to wells with varying Ca concentrations: 0.4, 0.6, 0.8, 1, 1.25 and 1.35 or 1.5 mM. PTH concentrations were determined in the medium. For the parathyroid cell cycle studies, parathyroid cells were isolated without the use of enzymes and cell cycle was analyzed using the method described by Vindelov. The nuclei were acquired by flow cytometer and analyzed using the CELLFIT software. RESULTS: In parathyroid tissues from hyperplastic glands, the increase in extracellular calcium produced a decrease in PTH secretion which was apparent with a calcium level as low as 0.8 mM and the maximal inhibition of PTH secretion was obtained with a calcium of 1.25 mM, by the contrary, adenomatous glands required a calcium of 1.2 mM to produce a minimal decrease in PTH secretion. In hyperplastic parathyroid glands but not in parathyroid adenomas there was a significant correlation between the percentage of cells in G0/G1 phase with the set point (r = 0.914; P < 0.005) and the basal serum Ca (r = 0.862; P < 0.02). CONCLUSIONS: The control of the extracellular calcium-PTH release in vitro is less sensitive in parathyroid adenomas than hyperplasic parathyroid glands. In parathyroid hyperplasia the cell proliferation may be regulated by the extracellular calcium concentration (higher calcemia less proliferation).

Dinámica de la secreción de PTH regulada por calcio en el hiperparatiroidismo secundario: comparación de la respuesta in vivo frente a in vitro / Dynamics of calcium-regulated PTH secretion in secondary hyperparathyroidism: comparison between «in vivo» vs. «in vitro» responses

Objetivo: Comparar la dinámica de la secreción de calcio-PTH in vivo e in vitro de glándulas paratiroideas hiperplásicas. Materiales y métodos: Se estudiaron 7 pacientes con hiperparatiroidismo secundario y las 23 glándulas hiperplásicas obtenidas tras paratiroidectomía de estos mismos pacientes. Estudios in vivo de la curva de secreción de PTH se obtuvieron con inducción de hipocalcemia e hipercalcemia con infusiones intravenosas continuas de EDTA sódico y gluconato de calcio, respectivamente. Para los estudios in vitro se emplearon pequeñas piezas de paratiroides de 1 mm que se transfirieron secuencialmente a concentraciones de calcio variables: 0,4, 0,6, 0,8, 1, 1,25 y 1,50 mM, determinándose la concentración de PTHi en el medio. Resultados: Las curvas de secreción de PTH in vivo e in vitro fueron sigmoidales y similares, aunque el set point in vivo era más alto que el in vitro (1,57 ± 0,05 frente a 1,27 ± 0,07 mM; p <0,001). El grado de inhibición máxima de PTH fue similar en ambas circunstancias (30,5 ± 8,1 frente a 33,6 ± 5,4%; p = NS), con una correlación directa significativa (r = 0,901; p <0,01). El set point in vivo no se correlacionaba con las concentraciones de PTH basales, aunque se correlacionó significativamente con las concentraciones basales de calcio sérico (r = 0,62; p <0,02). Conclusiones: El set point in vivo del calcio está más relacionado con la concentración sérica de calcio que con la concentración basal de PTHi. Aunque hay diferencias entre el set point de calcio in vivo e in vitro, el grado máximo de inhibición de PTH y la curva sigmoidal fueron similares en las dos circunstancias (AU)

Aim: To compare the dynamics in vivo and in vitro calcium-PTH release of uremic patients with secondary hyperparathyroidism and their hyperplasic parathyroid glands after parathyroidectomy. Materials and methods: Seven patients with secondary HPT and their 23 hyperplasic glands obtained after surgical parathyroidectomy were evaluated. In vivo studies of the PTH secretion curve were obtained by induction of hypocalcemia and hypercalcemia with a continuous endovenous infusion of sodium EDTA and Ca gluconate, respectively. For the in vitro studies, small parathyroid pieces of 1 mm were sequentially transferred to wells with varying Ca concentrations: 0.4, 0.6, 0.8, 1, 1.25 and 1.5 mM. iPTH concentrations were determined in the medium. Results: The in vivo set point did not correlate with the basal, maximal or minimal PTH concentrations, although it correlated significantly with the basal serum Ca concentration (r = 0.62, p <0.02). Both in vivo and in vitro PTH secretion curves were sigmoidal, although the in vivo set point was higher than the in vitro (1.57 ± 0.05 vs. 1.27 ± 0.07 mM, p <0.001). The degree of maximal PTH inhibition were similar in both circumstances (30.5 ± 8.1 vs. 33.6 ± 5.4 %; p = NS) with a significant direct correlation (r = 0.901; p <0.01). Conclusions: The in vivo set point of calcium is more closely related to serum calcium concentration than to basal iPTH concentration. Although there are differences between the in vivo and in vitro calcium set point the maximal degree of PTH inhibition was similar in both circumstances (AU)

[Dynamics of calcium-regulated PTH secretion in secondary hyperparathyroidism: comparison between "in vivo" vs. "in vitro" responses]. / Dinámica de la secreción de PTH regulada por calcio en el hiperparatiroidismo secundario: comparación de la respuesta in vivo frente a in vitro.

AIM: To compare the dynamics in vivo and in vitro calcium-PTH release of uremic patients with secondary hyperparathyroidism and their hyperplasic parathyroid glands after parathyroidectomy. MATERIALS AND METHODS: Seven patients with secondary HPT and their 23 hyperplasic glands obtained after surgical parathyroidectomy were evaluated. In vivo studies of the PTH secretion curve were obtained by induction of hypocalcemia and hypercalcemia with a continuous endovenous infusion of sodium EDTA and Ca gluconate, respectively. For the in vitro studies, small parathyroid pieces of 1 mm were sequentially transferred to wells with varying Ca concentrations: 0.4, 0.6, 0.8, 1, 1.25 and 1.5 mM. iPTH concentrations were determined in the medium. RESULTS: The in vivo set point did not correlate with the basal, maximal or minimal PTH concentrations, although it correlated significantly with the basal serum Ca concentration (r = 0.62, p < 0.02). Both in vivo and in vitro PTH secretion curves were sigmoidal, although the in vivo set point was higher than the in vitro (1.57 +/- 0.05 vs. 1.27 +/- 0.07 mM, p < 0.001). The degree of maximal PTH inhibition were similar in both circumstances (30.5 +/- 8.1 vs. 33.6 +/- 5.4 %; p = NS) with a significant direct correlation (r = 0.901; p < 0.01). CONCLUSIONS: The in vivo set point of calcium is more closely related to serum calcium concentration than to basal iPTH concentration. Although there are differences between the in vivo and in vitro calcium set point the maximal degree of PTH inhibition was similar in both circumstances.

The calcimimetic R-568 increases vitamin D receptor expression in rat parathyroid glands.

We previously demonstrated that extracellular calcium regulates vitamin D receptor (VDR) expression by parathyroid cells. Since the calcimimetic R-568 potentiates the effects of calcium on the calcium-sensing receptor, it was hypothesized that administration of R-568 may result in increased VDR expression in parathyroid tissue. In vitro studies of the effect of R-568 on VDR mRNA and protein were conducted in cultures of whole rat parathyroid glands and human hyperplastic parathyroid glands. In vivo studies in Wistar rats examined the effect of R-568 and calcitriol alone and in combination. Incubation of rat parathyroid glands in vitro with R-568 (0.001-1 microM) resulted in a dose-dependent decrease in parathyroid hormone (PTH) secretion and an increase in VDR expression (mean +/- SE). Incubation in 1 mM calcium + 0.001 microM R-568 elicited an increase in VDR mRNA (306 +/- 46%) similar to the maximum increase detected with 1.5 mM calcium (330 +/- 42%). In vivo, VDR mRNA was increased after administration of R-568 (168 +/- 9%, P < 0.001 vs. control) or calcitriol (198 +/- 16%, P < 0.001 vs. control). Treatment with R-568 also increased VDR protein in normal rat parathyroid glands and in human parathyroid glands with diffuse, but not nodular, hyperplasia. In conclusion, the present study shows that the calcimimetic R-568 exerts a stimulatory effect on VDR expression in the parathyroid glands of study models and provides additional evidence for the use of calcimimetics in the treatment of secondary hyperparathyroidism.

(99m)Tc-sestamibi scintigraphy and cell cycle in parathyroid glands of secondary hyperparathyroidism.

Double-phase parathyroid MIBI ((99m)Tc-sestamibi) was performed in 27 patients with secondary hyperparathyroidism (SPT). Focal areas of increased uptake were scored for intensity on a three-point scale. All patients underwent subtotal parathyroidectomy (SPTx), and a total of 78 glands were removed at operation. Blood was obtained from the jugular vein before and after SPTx to measure the parathyroid hormone (PTH) levels. The volume and weight of the glands were calculated. The tissue was divided, with one aliquot being used for cell cycle analysis. The nuclei were acquired by flow cytometry and analyzed using CELLEIT software. Cell viability was assessed by flow cytometry and analyzed with LYSIS II software. Positive MIBI uptake was observed in 88.8% of patients. Focal MIBI uptake of one, two, or three glands was observed in 6, 11, and 8 patients, respectively. All patients experienced an 86% decrease in PTH blood level after SPTx compared to that before excision. A correlation was found between the volume of glands and the blood levels of intact PTH (iPTH) (r = 0.5, p < 0.05). A positive correlation was observed between MIBI uptake and the iPTH levels before SPTx (p < 0.01) and between the uptake of MIBI in the parathyroid glands and the cell cycle phases; low-grade uptake correlated with the G(0) phase and higher uptake with G(2)+S phase (r = 7, p < 0.01). No correlation was observed between MIBI uptake and the weight of the glands. MIBI scintigraphy accurately reflects the functional status of the hyperplastic parathyroid glands: Higher uptake grades correlated with the active growth phase. MIBI uptake does not reveal parathyroid enlargement; rather, it identifies the presence of hyperfunctioning autonomous glands. SPTx and total parathyroidectomy with autografting (TPTx+A) are the most widely accepted surgical approaches for patients with SPT. Reoperation for recurrence is necessary in 6% to 15% of cases. MIBI is now considered to be the radionuclide of reference for parathyroid gland scanning, although it is widely accepted that it produces poor results when trying to detect hyperplastic glands.

High phosphate level directly stimulates parathyroid hormone secretion and synthesis by human parathyroid tissue in vitro.

Phosphate retention plays an important role in the pathogenesis of secondary hyperparathyroidism in patients with renal failure. In in vitro studies, high extracellular phosphate levels directly stimulate PTH secretion in rat and bovine parathyroid tissue. The present study evaluates the effect of high phosphate levels on the secretion of PTH and the production of prepro PTH mRNA in human hyperplastic parathyroid glands. The study includes parathyroid glands obtained from patients with primary adenomas and from hemodialysis and kidney-transplant patients with diffuse and nodular secondary hyperplasia. The experiments were performed in vitro using small pieces of parathyroid tissue. The ability of high calcium levels to decrease PTH secretion was less in adenomas than in secondary hyperplasia; among the secondary hyperplasia, nodular was less responsive to an increase in calcium than diffuse hyperplasia. In diffuse hyperplasia, PTH secretion was increased in response to 3 and 4 mM phosphate compared with 2 mM phosphate, despite a high calcium concentration in the medium; prepro PTH mRNA levels increased after incubation in 4 mM phosphate. Similar results were obtained with nodular hyperplasia, except that the elevation of PTH secretion in response to 3 mM phosphate did not attain statistical significance. In adenomas, high calcium concentrations (1.5 mM) did not result in inhibition of PTH secretion, independent of the phosphate concentration, and the prepro PTH mRNA was not significantly increased by high phosphate levels. In conclusion, first, the PTH secretory response to an increase in calcium concentration is less in nodular than diffuse hyperplasia; second, high phosphate levels directly affect PTH secretion and gene expression in patients with advanced secondary hyperparathyroidism.

The effect of a high phosphorus diet on the parathyroid cell cycle.

High extracellular phosphorus directly increases parathyroid hormone (PTH) secretion and gene transcription. The present study evaluates the effect of high phosphorus diet on the parathyroid cell cycle in rats with normal renal function. Rats were divided into two groups, receiving either a high phosphorus diet (HPD, P=1.2%) or a normal phosphorus diet (NPD, P=0.6%). The dietary calcium content was 0.6% in both diets. Rats were pair fed and sacrificed on days 0, 1, 5, 10 and 15 after initiation of the diet. The parathyroid glands were removed and parathyroid cells dispersed for evaluation of cell cycle and apoptosis by flow cytometry. Serum calcium, phosphorus, PTH and calcitriol were measured. As compared with NPD, the ingestion of a HPD resulted in an increased number of cells in the S phase of the cell cycle from day 1 to 10 (1.2+/-0.09% vs 0.6+/-0.04% for day 1, 1.2+/-0.11% vs 0.6+/-0.06% for day 5 and 1.0+/-0.09% vs 0.5+/-0.04% for day 10, P<0.01). By day 15, the percentage of cells in the S phase in NPD and HPD were not different. In the rats fed the HPD, serum PTH increased significantly from day 5 through 15 (P<0.01). Parathyroid cell apoptosis was minimal and unaffected by the diet. At day 15, the parathyroid gland size in HPD was increased by 27% as compared with NPD (P<0.05). This increase should be attributed to cell proliferation since parathyroid cell size remained unchanged. Serum calcitriol and calcium were not significantly different in the two groups. In HPD, an increase in serum phosphorus was observed only on day 1. The results show that an HPD results in the stimulation of the parathyroid cell cycle independently of changes in calcium and calcitriol.

Direct effect of phosphorus on PTH secretion from whole rat parathyroid glands in vitro.

Phosphorus retention is an important factor in the development of hyperparathyroidism secondary to renal failure. In vivo manipulation of phosphorus is associated with changes in serum calcium and calcitriol levels which in turn can modify parathyroid hormone synthesis and secretion. The present in vitro study evaluates whether high extracellular phosphorus has a direct effect on parathyroid hormone secretion. Fresh rat parathyroid glands were incubated in a media with phosphorus concentrations of 1, 2, 3, and 4 mM and subsequently exposed to calcium levels ranging from 0.4 to 1.35 mM. In 1.25 mM calcium, the parathyroid hormone secretion rate was similar in 1 and 2 mM phosphorus; however, a phosphorus concentration of 3 and 4 mM produced a 3- and 4-fold increase in the parathyroid hormone secretion, respectively, as compared with 1 mM phosphorus. While in 1 or 2 mM phosphorus an increase in calcium from 0.6 to 1.35 mM reduced parathyroid hormone secretion to 37%, in 4 mM phosphorus the same increase in calcium only inhibited parathyroid hormone secretion to 75%. Furthermore, the addition of arachidonic acid 20 microM, a substrate for inhibitory intracellular signal pathway, to the 4 mM phosphorus-1.35 mM calcium incubation media reduced the parathyroid hormone secretion to 34.5% (p < 0.05). In conclusion, our results indicate that in vitro, high phosphorus directly increases parathyroid hormone secretion.

NADH-ascorbate free radical and -ferricyanide reductase activities represent different levels of plasma membrane electron transport.

Plasma membranes isolated from rat liver by two-phase partition exhibited dehydrogenase activities for ascorbate free radical (AFR) and ferricyanide reduction in a ratio of specific activities of 1:40. NADH-AFR reductase could not be solubilized by detergents from plasma membrane fractions. NADH-AFR reductase was inhibited in both clathrin-depleted membrane and membranes incubated with anti-clathrin antiserum. This activity was reconstituted in plasma membranes in proportion to the amount of clathrin-enriched supernatant added. NADH ferricyanide reductase was unaffected by both clathrin-depletion and antibody incubation and was fully solubilized by detergents. Also, wheat germ agglutinin only inhibited NADH-AFR reductase. The findings suggest that NADH-AFR reductase and NADH-ferricyanide reductase activities of plasma membrane represent different levels of the electron transport chain. The inability of the NADH-AFR reductase to survive detergent solubilization might indicate the involvement of more than one protein in the electron transport from NADH to the AFR but not to ferricyanide.

Thiol groups are involved in NADH-ascorbate free radical reductase activity of rat liver plasma membrane.

Plasma membranes purified by two-phase partition from rat liver showed an NADH-ascorbate free radical reductase activity of about 14 nmoles NADH oxidized/min/mg protein. This activity was inhibited by N-ethyl maleimide, iodoacetate and iodoacetamide, reagents that covalently block thiol groups. NADH-ascorbate free radical reductase was also inhibited by reduced glutathione and the inhibitions observed with blocking reagents and reduced compounds were additive. These results support the involvement of sulphydryl groups in NADH-AFR reductase and point out the idea that a balance between reduced sulfhydryls and oxidized disulfides is required for the optimal function of this activity, considered as part of the transplasma membrane electron transport system.

Litiasis vesicular en el niño. Estudio de 20 casos / Vesicular lithiasis in child. A 20 case study

De los 20 pacientes estudiados, 7 presentaron antecedentes personales de sicklemia; en el cuadro clónico predominaron el dolor y los vómitos. El dolor predominó también en el exámen físico, mientras que en la valoración nutricional prevaleció la obesidad. De gran ayuda diagnóstica fueron los exámenes simples y contrastados de las vías biliares. Se constata que la intervención quirúrgica electiva se realizó en mayor número de pacientes que la urgente, la de elección fue la colecistectomía, y la evolución posoperatoria fue muy satisfactoria (AU)