Apoptosis in parathyroid hyperplasia of patients with primary or secondary uremic hyperparathyroidism.

BACKGROUND: Chronic oversecretion of parathyroid hormone (PTH) is associated with parathyroid hyperplasia, reflecting a disturbed balance between cell proliferation and apoptosis. This study addressed the unsolved issue of apoptosis in hyperparathyroidism. METHODS: Parathyroid glands from 19 patients with primary (1 degrees ) and 11 patients with secondary (2 degrees ) uremic hyperparathyroidism, as well as 13 normal parathyroid glands, were examined. Apoptosis was evaluated by terminal deoxynucleotidyl transferase (Tdt)-mediated dUTP nick end-labeling assay (TUNEL). Because the apoptotic process is regulated by several oncoproteins, the expression of Bcl-2 and Bax was analyzed by immunohistochemistry. RESULTS: The numbers of apoptotic cells in 1 degrees parathyroid adenoma (0.99 +/- 0.03 per 1000 cells, mean +/- SE, P < 0.009) and 2 degrees parathyroid hyperplasia (1.20 +/- 0.54 per 1000 cells, P < 0.005) were significantly higher than in normal parathyroid tissue (0.13 +/- 0. 06 per 1000 cells). Light microscopy examination of hyperplastic parathyroid tissue from a uremic patient showed the presence of nuclei with dense chromatin characteristic of apoptosis. Bcl-2 staining was strong in normal tissues but weak or negative in several sections of 1 degrees and 2 degrees hyperparathyroid tissues, mostly in nodular areas. Bax staining was homogeneous in normal tissue but patchy in several hyperplastic tissues. CONCLUSION: These results suggest that hyperparathyroidism is associated with a compensatory increase in apoptosis, possibly favored by a diminished Bcl-2/Bax ratio. This renders highly improbable the hypothesis that parathyroid hyperplasia is due to a decreased rate of apoptosis.

Apoptosis of rat kidney cells after 241-americium administration.

Tumors induction by americium is well known but there are no data on the biological effects of this radionucleide at subcellular level. In order to study the possible ultrastructural lesions induced by this element, a group of rats were injected with 241-Americium-citrate (9 kBq), once a week for five weeks and sacrificed 7 days after the last injection. We describe the alterations observed in the cortex kidney using cytochemical (TUNEL reaction) and histochemical (PAS staining) methods for light microscopy as well as electron microscopy techniques. Various types of lesions were detected: condensation of nuclear chromatine, fragmentation of the nuclei, swollen mitochondria, disappearance of mitochondrial crests and skrinking of the cytoplasm. This study clearly demonstrated the induction of apoptosis by americium in rat cortex kidney cells.

Persistence of Ca2+-sensing receptor expression in functionally active, long-term human parathyroid cell cultures.

An original human parathyroid cell culture model from uremic patients with IIo hyperparathyroidism has been developed, with its main feature being long-term functionally active viability up to 5 months, as assessed by persistent responsiveness to changes of extracellular Ca2+ concentrations ([Ca2+]e). In addition to the inhibitory effect of increasing [Ca2+]e, increasing extracellular phosphate exerted a biphasic effect on parathyroid hormone (PTH) secretion. The presence of the Ca2+-sensing receptor (CaR), on which depends the response to [Ca2+]e and its persistence, has been demonstrated in our culture system both by direct detection and by inhibition of its activity. CaR protein was detected by Western blot analysis with a specific anti-CaR antibody. CaR gene transcripts have been identified by reverse transcription-polymerase chain reaction analysis. mRNA (by in situ hybridization) and protein (by immunocytochemistry) expression were detected for both CaR and PTH. Adding a specific anti-CaR antibody to the medium induced a marked reduction of low [Ca2+]e-stimulated PTH release, which decreased to levels equivalent to those obtained in high [Ca2+]e medium. The described long-term functionality could be due to several factors, including the clustered cell type of culture yielded by our preparation procedure, the growth characteristics of hyperplastic uremic tissue, and the use of a phosphate-rich medium. The present model, because of its long-term functionality, is a unique tool for the exploration of PTH synthesis and secretion and for studies of parathyroid cell growth in vitro.

Depressed expression of calcium receptor in parathyroid gland tissue of patients with hyperparathyroidism.

The factors involved in abnormal parathyroid cell secretory function and growth in patients with primary (I degree) and secondary (II degree) hyperparathyroidism are still incompletely understood. We compared the expression of the calcium-sensing receptor (CaR) at the gene message and the protein level in parathyroid tissue obtained from patients with I degree non-uremic or II degree uremic hyperparathyroidism with that in normal parathyroid tissue, using in situ hybridization and immunohistochemistry techniques. The expression of the CaR mRNA and protein was reduced in most cases of I degree adenoma and II degree hyperplasia, compared with strong expression normal parathyroid tissue. In II degree hyperparathyroidism, expression of both receptor mRNA message and protein was often particularly depressed in nodular areas, compared with adjacent non-nodular hyperplasia. Decreased Ca-R expression in adenomatous and hyperplastic parathyroid glands would be compatible with a less efficient control of PTH synthesis and secretion by plasma calcium than in normal parathyroid tissue.

De novo expression of transforming growth factor-alpha in parathyroid gland tissue of patients with primary or secondary uraemic hyperparathyroidism.

BACKGROUND: The factors involved in abnormal parathyroid cell secretory function and growth in patients with primary and secondary hyperparathyroidism are still incompletely understood. PATIENTS AND METHODS: We studied the expression of transforming growth factor-alpha (TGF-alpha), epidermal growth factor (EGF) and EGF receptor (EGF-R) at the gene message and the protein level in parathyroid tissue obtained from six patients with primary hyperparathyroidism, 15 patients with secondary uraemic hyperparathyroidism and five subjects with normal parathyroid tissue, using in situ hybridization and/or immunostaining technique. RESULTS: We found a consistent expression of TGF-alpha mRNA and protein in parathyroid endocrine cells of all six cases of primary parathyroid adenoma and in nearly all cases of secondary hyperplasia, in contrast to the absence of expression in normal parathyroid tissue. A marked expression of EGF-R mRNA and protein was also found in four of five tissue samples of primary parathyroid adenoma, in 13 of 15 tissue samples of secondary parathyroid hyperplasia and in most samples of normal parathyroid gland tissue. EGF mRNA and protein expression was undetectable in the majority of parathyroid tissue samples examined. CONCLUSION: Since TGF-alpha is known to bind to the EGF-R, the finding of an increased expression of TGF-alpha at the gene message and the protein level, together with a strong expression of EGF-R, in hyperplastic and adenomatous parathyroid glands suggests that this growth factor interacts with its receptor to promote parathyroid cell proliferation, perhaps by an autocrine mechanism.

Abnormal regulation of intestinal calbindin (CaBP9k) and calmodulin in the spontaneously hypertensive rat.

This review focuses particularly on abnormalities of Ca-binding proteins in transporting epithelia, which have been observed in various models of experimental hypertension. The enterocyte content of integral membrane Ca(2+)-binding protein (IMCAL) and calbindin-9k and the renal tubular calbindin-28k content have been shown to be decreased in the spontaneously hypertensive rat (SHR), compared with normotensive control rats (WKY). Similarly, calmodulin content was decreased in several tissues including the intestine; however, calmodulin activity was increased. In recent studies, the authors examined the response of intestinal calbindin-9k, calbindin-9k mRNA, and calmodulin contents to low-Ca and high-Ca diets in these two rat strains. It was shown that the SHR, unlike the WKY, was unable to augment intestinal calbindin-9k and calbindin-9k mRNA levels in response to a low-Ca diet of short duration. On the other hand, a high-Ca diet led to a similar decrease in calbindin-9k content in both SHR and WKY rats. Enterocyte calmodulin content was also diminished in the WKY, but not the SHR, fed such a high-Ca diet. Therefore, abnormal Ca-binding proteins could play a role in the disturbed Ca metabolism of arterial hypertension.

Duodenal calcium binding protein and active calcium transport in rats: are they functionally related?

The effects of calcitriol and a novel calcitriol analogue, 22-oxacalcitriol (OCT) on duodenal Ca transport, calbindin-D9k mRNA, and calbindin-D9k content were studied in two animal models reflecting common human pathologies, namely arterial hypertension and chronic renal failure, as well as in normal rats. The hormone or its analogue were administered intraperitoneally to vitamin-D-replete rats. Active Ca transport was increased in both spontaneously hypertensive rats (SHR) and in normotensive control WKY rats 5 h after calcitriol dosing of either 60 and 600 ng per rat. In WKY, calbindin-D9k content was slightly increased after the injection of 60 ng calcitriol, but not of 600 ng calcitriol whereas calbindin-D9k mRNA stayed essentially unchanged. In contrast, active Ca transport was significantly stimulated after the higher dose of 600 ng calcitriol. In SHR, while both doses of calcitriol increased active Ca transport, they had no stimulatory effect on calbindin-D9k mRNA or protein. In chronically uraemic rats, active Ca transport, duodenal calbindin-D9k and calbindin-D9k mRNA were stimulated after the injection of two subsequent doses of 300 ng calcitriol per rat. OCT treatment at same dosage led to a similar stimulation of calbindin-D9k and calbindin-D9k mRNA, but failed to induce an increase in active Ca transport. These results show that the stimulation of intestinal active Ca transport and calbindin-D9k can be entirely dissociated at the protein synthesis and the mRNA expression level (1) after calcitriol administration to normal and hypertensive rats, and (2) after OCT administration to uraemic rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Impaired duodenal response to short-term dietary calcium restriction in adolescent spontaneously hypertensive rats.

The response pattern of plasma calcitriol level and related intestinal adaptation to short-term moderate calcium (Ca) restriction was examined in adolescent male, spontaneously hypertensive rats (SHR) and normotensive WKY control rats. Twelve-week-old SHR and WKY fed a low (0.1%) Ca diet for 3, 6, or 12 days were compared with rats of either strain fed a normal (1.0%) Ca diet. Plasma calcitriol response was measured and duodenal adaptation to Ca restriction was investigated by evaluating active Ca transport, calbindin-D9K (CaBP9K) protein, CaBP9K mRNA, and alkaline phosphatase activity (ALP). Under the normal Ca diet, no significant difference between strains was observed for all five parameters. In response to the low Ca diet, the SHR and WKY showed a similar increase (nearly 50%) in plasma calcitriol, starting at day 3 of this diet. However, only the WKY displayed the expected duodenal adaptation: active Ca transport increased at day 6 and CaBP9K as well as ALP increased at day 3 of the low Ca diet. The stimulation of the latter three parameters was maintained until day 12 of Ca restriction. Moreover, CaBP9K mRNA was increased in WKY after 3 days of Ca restriction. In contrast, the SHR had either no or only a minor increase of duodenal parameters in response to Ca restriction. Finally, a significant and positive correlation between Ca transport and plasma calcitriol and between Ca transport and CaBP9K was found in WKY but not in SHR.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormal intestinal regulation of calbindin-D9K and calmodulin by dietary calcium in genetic hypertension.

Using isolated duodenal cells from spontaneously hypertensive rats (SHR) and their normotensive controls, Wistar-Kyoto rats (WKY), we previously showed that cellular calcium flux was decreased in SHR and that increasing dietary calcium (from 1 to 2%) eliminated strain differences in Ca2+ fluxes. The present study was carried out to investigate the role of calbindin-D9K and calmodulin in the flux difference and dietary calcium effects. Calbindin-D9K and calmodulin were separated by sodium dodecyl sulfate (SDS) gel electrophoresis in duodenal protein extracts of SHR and WKY (12-14 and 24-26 wk old) fed either a 1 or 2% calcium diet and measured by a ligand blotting (45Ca) technique. Young SHR had a significantly lower calbindin-D9K (P less than 0.001) than did WKY on either diet. Calmodulin was significantly lower in young SHR than in WKY (P less than 0.002). There was no strain difference in calmodulin in older rats fed the normal calcium diet. Calbindin-D9K was significantly decreased by the high-calcium diet in both strains at both ages. There was a significant correlation between duodenal calbindin-D9K and plasma levels of calcitriol (r = +0.80, P less than 0.001) in WKY but not in SHR. Calmodulin was significantly decreased by dietary calcium in mature WKY (4.8 +/- 0.2 vs. 3.7 +/- 0.4 micrograms/mg cell protein, P less than 0.03), demonstrating a potential regulation by dietary calcium of this protein. Finally, there was a significant correlation between calbindin-D9K and calmodulin (r = 0.59, P less than 0.001) in WKY but not in SHR.(ABSTRACT TRUNCATED AT 250 WORDS)

A functional cell surface type receptor is required for the early action of 1,25-dihydroxyvitamin D3 on the phosphoinositide metabolism in rat enterocytes.

1,25-Dihydroxyvitamin D3 (1,25-(OH)2-D3) (0.1 pM to 2 nM) induces a concomitant very rapid (within 5 s) and transient release of inositol trisphosphate and diacylglycerol in enterocytes from 3-month-old rats. This high level is not maintained but declines to a lower level 60 s after stimulation. The stimulating effect is dose-dependent and biphasic with a maximum effect at 10 pM. 1,25-(OH)2-D3 induces a rapid (within 10 s) accumulation of inositol bisphosphate and its effect on inositol monophosphate is delayed (120 s). The primary action of 1,25-(OH)2-D3 is to initiate hydrolysis of phosphatidyl 4,5-bisphosphate to yield inositol trisphosphate and diacylglycerol. In contrast, 1,25-(OH)2-D3, for any of the concentrations and the incubation time periods tested, has no effect on inositol trisphosphate and diacylglycerol formation in enterocytes from 16-day-old rats at a time when specific binding sites for 1,25-(OH)2-D3 cannot be detected. In conclusion, the early (within 5-60 s) effects of 1,25-(OH)2-D3 on small intestinal epithelium may be mediated via the phosphoinositide transduction system and require the presence of functioning cell membrane-type receptors.

Rapid stimulation of calcium uptake by isolated rat enterocytes by 1,25(OH)2D3.

Evidence is accumulating that 1,25(OH)2D3 may stimulate calcium transport from the intestinal lumen extremely rapidly by a mechanism which appears independent of de novo protein synthesis. To investigate this rapid action of 1,25(OH)2D3, the rate of calcium uptake by isolated enterocytes from duodena of young rats was determined in vitro as the uptake of 45Ca from 1-15 min. Prior in vitro exposure of cells to 1,25(OH)2D3 (100 pM) for 20 min significantly increased the rate of calcium uptake (p less than 0.001), an effect unaltered by 50 microM cycloheximide. Incubation with 100 pM 1-alpha-hydroxyvitamin D3 produced the same effect (p less than 0.01). In contrast, exposure to 10 pM 1,25(OH)2D3, as well as to 100 pM or to 1,000 pM 25-hydroxyvitamin D3 induced no significant change. Because both 1,25(OH)2D3 and starvation may stimulate key enzymes in polyamine metabolism, we investigated the effects of (i) difluoromethyl-ornithine (CHF2-Orn), a specific irreversible inhibitor of ornithine decarboxylase and (ii) varying the timing of feeding prior to sacrifice. Both in vitro CHF2-Orn and feeding prior to sacrifice significantly decreased the baseline rate of calcium uptake (p less than 0.05) and reduced the effect of 1,25(OH)2D3. Increased duration of starvation significantly increased the baseline rate of calcium uptake (p less than 0.02) without changing the increment in rate of calcium uptake induced by 1,25(OH)2D3. The study suggests (i) that the early action of 1,25(OH)2D3 on the influx process of intestinal calcium transport may involve a different molecular specificity from that involved in the genomic action of 1,25(OH)2D3 and (ii) that changes in polyamine metabolism may play a part in this process.

Decreased duodenal enterocyte calcium flux rates in the spontaneously hypertensive rat.

We have previously reported serum 1,25(OH)2 vitamin D3 (calcitriol) and active transduodenal calcium absorption measured in the Ussing chamber to be reduced in 12- to 14-week-old male Okamoto-Aoki spontaneously hypertensive rats (SHR). In the present study, we compared rates of calcium influx and efflux in isolated duodenal enterocytes in SHR and corresponding controls, Wistar-Kyoto rats (WKY). The early (0 to 1 minute) and the late (1 to 15 minute) phase of calcium influx rates at 1.0 mmol Ca2+ in the incubation medium were lower in the SHR than in the WKY (mean +/- SEM): 1.93 +/- 0.22 v 2.85 +/- 0.41 nmol/mg protein/min, n = 8 and n = 7 experiments, respectively, P less than .05; and 0.334 +/- 0.025 v 0.488 +/- 0.059 nmol/mg protein/min, n = 14 pairs, P less than .01. The calcium efflux rate constant of the SHR was reduced: 34.3 +/- 1.4 v 51.9 +/- 1.4% per hour, n = 11 pairs, P less than .01. However, in the absence of sodium or the presence of ouabain (4.0 mmol) in the incubation medium, a decrease in this constant was observed in the WKY but not in the SHR. These data, at the cellular level, support our previous observation in intact tissue of reduced active transduodenal calcium transport in the 12- to 14-week-old SHR. Whether the primary defect in calcium transport involves the luminal or the basolateral membrane of the enterocyte, or whether both disturbances are due to a common primary perturbation cannot be deduced from the present experiments.