Parathyroid hormone fragments inhibit active hormone and hypocalcemia-induced 1,25(OH)2D synthesis.

Carboxyl (C)-terminal fragments of parathyroid hormone (PTH) oppose the calcemic, phosphaturic, and bone-resorbing effects of active hormone. To study the action of these fragments on 1,25(OH)(2)D (1,25-dihydroxyvitamin D) synthesis, we infused parathyroidectomized rats with human or rat active 1-34 or 1-84 PTH at doses selected to produce similar calcemic responses. Human active PTH influenced neither phosphate nor 1,25(OH)(2)D concentrations. However, active 1-34 rat PTH decreased phosphate to the same level as vehicle-treated rats and increased 1,25(OH)(2)D to very high levels, whereas active 1-84 PTH decreased phosphate but maintained 1,25(OH)(2)D. As the latter effect could have been due to C-terminal fragment generation during its metabolic breakdown, we infused a mixture of rat C-terminal fragments alone or with rat 1-34. The C-terminal fragments decreased 1,25(OH)(2)D and prevented hypocalcemic-induced 1,25(OH)(2)D synthesis. When infused with active rat 1-34, they lowered the 1,25(OH)(2)D level to that seen with intact rat 1-84. The C-terminal fragments did not influence either basal or rat 1-34- or 1-84-induced CYP27B1 mRNA levels, suggesting that their inhibitory effects on 1,25(OH)(2)D synthesis appears to be post-transcriptional.

Effects of acute exercise on the gluconeogenic capacity of periportal and perivenous hepatocytes.

The present study was conducted to examine the effect of a single bout of exercise (rodent treadmill, 60 min at 26 m/min, 0% grade) on the gluconeogenic activity of periportal hepatocytes (PP-H) and perivenous hepatocytes (PV-H) in fasted (18 h) rats. Isolated PP-H and PV-H, obtained by selective destruction following liver perfusion with digitonin and collagenase, were incubated with saturating concentrations of alanine (Ala; 20 mM) or a mixture of lactate and pyruvate (Lac+Pyr; 20:2 mM) to determine the glucose production flux (J(glucose)) in the incubation medium. Results show that, in the resting conditions, J(glucose) from all exogenous substrates was significantly higher (P < 0.01) in PP-H than in PV-H. Exercise, compared with rest, resulted in a higher J(glucose) (P < 0.01) from Lac+Pyr substrate in the PV-H but not in the PP-H, resulting in the disappearance of the difference in J(glucose) between PP-H and PV-H. Exercise, compared with rest, led to a higher J(glucose) (P < 0.01) from Ala substrate in both PP-H and PV-H. However, the exercise-induced increase in J(glucose) (gluconeogenic activity) from Ala substrate was higher in PV-H than in PP-H, resulting, as from Lac+Pyr substrate, in the disappearance (P > 0.05) of the difference of J(glucose) between PP-H and PV-H. It is concluded that exercise differentially stimulates the gluconeogenic activity of PV-H to a larger extent than PP-H, indicative of a heterogeneous metabolic response of hepatocytes to exercise.

1,25-Dihydroxyvitamin D(3) downregulates the rat intestinal vitamin D(3)-25-hydroxylase CYP27A.

The vitamin D(3)-25-hydroxylase CYP27A is located predominantly in liver, but its expression is also detected in extrahepatic tissues. Our aim was to evaluate the regulation of CYP27A by vitamin D(3) (D(3)) or its metabolites in rat duodena. Vitamin D-depleted rats were repleted with D(3), 25-hydroxyvitamin D (25OHD), or 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] or acutely injected 1,25(OH)(2)D(3) to investigate the mechanisms of action of the hormone. All D(3) compounds led to a progressive decrease in CYP27A mRNA, with levels after D(3) representing 20% of that observed in D depletion. 25OHD decreased CYP27A mRNA by 55%, whereas 1,25(OH)(2)D(3) led to a 40% decrease, which was accompanied by a 31% decrease in CYP27A protein levels and an 89% decrease in enzyme activity. Peak circulating 1,25(OH)(2)D(3) concentrations were, however, the highest in D(3)-repleted, followed by 25OHD- and 1,25(OH)(2)D(3)-repleted animals. 1,25(OH)(2)D(3) resulted in a decrease in both CYP27A mRNA half-life and transcription rate. Our data illustrate that the intestine expresses the D(3)-25-hydroxylase and that the gene is highly regulated in vivo through a direct action of 1,25(OH)(2)D(3) or through the local production of D(3) metabolites.

Expression of CYP27A, a gene encoding a vitamin D-25 hydroxylase in human liver and kidney.

Vitamin D3 (D3) is not active but must be hydroxylated at C-25 in liver before acquiring its hormonal potential in the kidney. The sterol-27 hydroxylase (gene symbol: CYP27A) catalyses the oxidation of sterol side chain in bile acid synthesis but the enzyme is also known as a D3-25 hydroxylase. The study examined the expression of the gene encoding CYP27A in adult and fetal human livers and kidneys. Thirty-nine adults (18 men and 21 women; mean age 58 years in men and 57 years in women) and three normal fetuses gestational age 17-19 weeks were studied. Tissue CYP27A mRNA and serum 25OHD concentrations were measured. Normal specimens: CYP27A transcript was found to be higher in adult than in fetal livers but its expression was similar in adult and fetal kidneys. In fetuses, no difference was observed between CYP27A levels in livers and kidneys. In adult livers CYP27A levels were higher in women than in men. Hepatic CYP27A mRNA and serum 25OHD concentrations were both found to be higher in summer than in winter. Multiple linear regression analyses indicate that the season of the year and the serum 25OHD concentrations (but not 1,25(OH)2D concentrations) are the best predictors of CYP27A mRNA abundance in normal adult livers. In situ hybridization illustrates a clear label in hepatocytes which increases in intensity in the perivenous region of the hepatic acinus. Pathological specimens: In one man with an hepatic carcinoma there was a very large increase in CYP27A (> 1000 fold) compared to the level found in the normal liver. In that patient, serum 25OHD concentrations were found to be high considering the level of CYP27A mRNA in the normal hepatic area suggesting that the neoplastic tissue contributed to the C-25 hydroxylation of vitamin D. Specimens obtained from two patients suffering from focal hepatic hyperplasia indicate that in one case the level of CYP27A mRNA was twice as high in the pathological than in the normal area while in the other its levels were similar in both areas. No difference in the CYP27A transcript was observed between specimens obtained from normal areas and those obtained form either an hepatic adenoma or from two intrahepatic colonic metastases. CYP27A is present not only in the human adult liver but also in the adult kidney, and in the fetal liver and kidney. Our findings illustrate that CYP27A can be significantly upregulated in certain pathological situations such as in hepatic carcinoma and that the neoplastic tissue could contribute to the circulating concentration of 25OHD.

Extracellular calcium-sensing receptor is expressed in rat hepatocytes. coupling to intracellular calcium mobilization and stimulation of bile flow.

Liver cells respond to changes in Ca(2+)(o). The hepatic functions affected include bile secretion, metabolic activity, liver regeneration, and the response to xenobiotics. In the present study, we demonstrate the presence, in the liver, of the extracellular calcium-sensing receptor (CASR), described previously in the parathyroid and thyroid glands and kidney. CASR mRNA was specifically expressed in hepatocytes and was absent in nonparenchymal liver cells (stellate, endothelial, and Kupffer cells). Western blot analysis using a specific CASR antibody showed staining in both whole liver and hepatocyte extracts. Immunohistochemistry and in situ hybridization of rat liver sections showed expression of CASR protein and mRNA by a subset of hepatocytes. The known agonists of the CASR, gadolinium (Gd(3+); 0.5-3.0 mm) and spermine (1.25-20 mm), in the absence of Ca(2+)(o), elicited dose-related increases in Ca(2+)(i) in isolated rat hepatocytes loaded with Fura-2/acetoxymethyl ester. There was a greatly attenuated response to a second challenge with either agonist. The response was also abrogated when inositol 1,4,5-trisphosphate (IP(3))-sensitive calcium pools had been depleted by pretreatment with either thapsigargin or phenylephrine, an alpha(1)-adrenergic receptor agonist known to mobilize Ca(2+)(i) from IP(3)-sensitive pools. Addition of the deschloro-phenylalkylamine compound, NPS R-467, but not the S enantiomer, NPS S-467, increased the sensitivity of the Ca(2+)(i) mobilization response to 1.25 mm spermine. Bile flow ceased after Ca(2+)(o) withdrawal, and its recovery was enhanced by spermine in isolated perfused liver preparations. The CASR agonists Ca(2+) and Gd(3+) increased bile flow, and the response to a submaximal Ca(2+) concentration was enhanced by NPS R-467 but not the S compound. Thus, the data indicate that rat hepatocytes harbor a CASR capable of mobilizing Ca(2+)(i) from IP(3)-sensitive stores and that activation of the CASR stimulates bile flow.

Influence of the in vivo calcium status on cellular calcium homeostasis and the level of the calcium-binding protein calreticulin in rat hepatocytes.

Little attention has been given to the consequences of the in vivo calcium status on intracellular calcium homeostasis despite several pathological states induced by perturbations of the in vivo calcium balance. The aim of these studies was to probe the influence of an in vivo calcium deficiency on the resting cytoplasmic Ca2+ concentration and the inositol-1,4,5-trisphosphate-sensitive Ca2+ pools. Studies were conducted in hepatocytes (a cell type well characterized for its cellular Ca2+ response) isolated from normal and calcium-deficient rats secondary to vitamin D depletion. Both resting cytoplasmic Ca2+ concentration and Ca2+ mobilization from inositol-1,4,5-trisphosphate-sensitive cellular pools were significantly lowered by calcium depletion. In addition, Ca deficiency was shown to significantly reduce calreticulin messenger RNA and protein levels but calcium entry through store-operated calcium channels remained unaffected, indicating that the Ca2+ entry mechanisms are still fully operational in calcium deficiency. The effects of calcium deficiency on cellular calcium homeostasis were reversible by repletion with oral calcium feeding alone or by the administration of the calcium-regulating hormone 1,25-dihydroxyvitamin D3, further strengthening the tight link between extra- and intracellular calcium. These data, therefore, challenge the currently prevailing hypothesis that extracellular Ca2+ has no significant impact on cellular Ca2+ by demonstrating that despite the large Ca2+ gradient between extra- and intracellular Ca2+ concentrations, calcium deficiency in vivo significantly alters the hormone-sensitive cellular calcium homeostasis.

Effect of cyclosporine A on cytochrome P-450-mediated drug metabolism in the partially hepatectomized rat.

Despite its hepatotoxic potential, cyclosporine A (CsA) has been reported to positively influence compensatory liver growth. To probe the physiological consequences of CsA on the recovery of liver function, studies were initiated in the 2/3 partially hepatectomized (PHx) rat, taking the recovery of cytochromes P-450-dependent drug metabolism as primary outcome. CsA was administered at a dose of 3. 33 mg/kg/day for 10 days. Drug metabolism was evaluated by the recovery of 14CO2 after administration of isotopically labeled model drugs and by studying the expression of the P-450 transcripts involved in their biotransformation before and 24 to 96 h after PHx. Before PHx, neither the steady-state mRNA nor the in vivo disposition of caffeine (CYP1A2), erythromycin (CYP3A2 and 3A1), or aminopyrine (CYP2B1 and 2C11) were influenced by CsA. Studies 24 h after PHx revealed a 29 to 39% reduction in the elimination of [14C]aminopyrine and [14C]erythromycin, which was unaffected by CsA. Their metabolism at 48 to 96 h after PHx also remained unaffected by CsA. By contrast, postPHx, [14C]caffeine elimination decreased to a level closely proportional to the loss in liver mass. In addition, CsA accelerated the recovery and/or prevented the decrease of caffeine elimination 24 h after PHx but not at later time points, indicating an early, but unsustained, beneficial effect of CsA on the recovery of CYP1A2-mediated activities. These data show that at the critical time of greatest loss in liver mass, CsA has only a selective influence on the biotransformation of cytochrome P-450 protein-dependent activities and that its effect on the regeneration process does not translate into an overall accelerated recovery of the hepatic drug-metabolizing function.

Metabolic and hormonal responses to exercise in partially hepatectomised rats.

To characterise how the liver affects metabolic and hormonal exercise responses, hepatectomised (70%; HX) rats were submitted to a 30- or 50-min treadmill exercise (26 m/min, 0% slope) 48 hr or 7 days after surgery (reduced or normal liver mass, respectively). To determine whether metabolic effects of liver mass reduction during exercise were caused by reduced capacity of the liver to produce glucose, metabolic and hormonal responses to the same exercise protocol were measured in 48-hr HX rats. Euglycemia, maintained by exogenous glucose infusion, produced attenuated lactate, insulin, and glucagon values in 48-hr HX rats but did not affect FFA, glycerol, and plasma catecholamine responses. Results indicate that metabolic and hormonal exercise responses are amplified in 48-hr HX rats. Maintaining euglycemia in 48-hr HX rats during exercise does not reduce all responses. Intrahepatic events, similar to those in a short-term (48-hr) HX liver, may influence metabolic and hormonal exercise responses.

Adaptation of parathyroid function to intravenous 1,25-dihydroxyvitamin D3 or partial parathyroidectomy in normal dogs.

Parathyroid function was studied in 14 normal dogs 1 month before and after daily i.v. administration of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) (eight dogs), or about 50% parathyroidectomy (six dogs), to test the hypothesis that degradation of newly synthesized intact parathyroid hormone (I-PTH) is involved in parathyroid gland adjustment to a modified demand for I-PTH. Parathyroid function was studied through i.v. infusions of Na2EDTA and CaCl2 and measurement of ionized calcium (Ca2+), I-PTH and carboxyl-terminal PTH (C-PTH) at various time points. The C-PTH/I-PTH ratio was used as an index for change in the relative proportion of circulating C-PTH vs I-PTH, 1 month prior to and following each intervention. This ratio was further validated by looking at the HPLC profile of I- and C-PTH in hypo- and hypercalcemia under experimental conditions. Basal Ca2+ was unaltered 1 month after surgery, and was maintained constant in the 1,25-(OH)2D3-treated group by gradually decreasing 1,25-(OH)2D3 doses over time from 0.25 to 0.13 microgram twice daily during the last week of the experimental protocol. In this same group, basal 1,25-(OH)2D3 was increased by 65% (P < 0.0001) and basal I-PTH was decreased by 40% (P < 0.05), while basal C-PTH and the C-PTH/I-PTH ratio remained unchanged. Stimulated and non-suppressible I- and C-PTH followed the same pattern with, this time, an increase of stimulated and non-suppressible C-PTH/I-PTH ratio of 60% (P < 0.05) and 85% (P < 0.05) respectively. There was no change in basal I-PTH, C-PTH, or C-PTH/I-PTH ratio after surgery. However, stimulated I- and C-PTH were decreased by 45% (P < 0.005) and 65% (P < 0.005) respectively, with a 30% (P < 0.005) decrease of stimulated C-PTH/I-PTH ratio. There was no change in non-suppressible I-PTH, while non-suppressible C-PTH decreased by 55% (P < 0.005), with a 55% (P < 0.05) decrease in non-suppressible C-PTH/I-PTH ratio. The HPLC profiles of I- and C-PTH obtained in hypo- and hypercalcemia disclosed a similar distribution of the immuno-reactivity into peaks before and after i.v. administration of 1,25-(OH)2D3 as well as partial parathyroidectomy. This indicated that C-PTH/I-PTH ratio changes were related to different circulating levels of I- and C-PTH rather than to a different composition of I- and C-PTH. These data indicate a shift in the circulating PTH profile toward more PTH carboxyl-terminal fragments after 1 month of i.v. 1,25-(OH)2D3, but toward more intact PTH 1 month after about 50% parathyroidectomy, possibly reflecting adjustments in PTH degradation induced by a modified demand for I-PTH. Although these changes are most likely modulated at the parathyroid gland level, we cannot formally eliminate participation of the hormone's peripheral metabolism.

Influence of the vitamin D status on the early hepatic response to carcinogen exposure in rats.

Although 1,25-dihydroxyvitamin D3 has been shown to promote the differentiation of cancer cells and cell lines in vitro, its protective effect against a chemical insult known to induce neoplastic growth in vivo has not been evaluated. The aim of this study was to investigate, in vivo, the influence of the vitamin D status on the early response to an insult known to induce morphological and functional changes leading to hepatocarcinogenesis. The influence of vitamin D status on the susceptibility of rat liver to carcinogenesis was studied after the administration of diethylnitrosamine and 2-acetylaminofluorene, in association with a partial hepatectomy (Solt-Farber protocol), to normal or vitamin D-depleted rats. Preneoplastic foci (gamma-glutamyltranspeptidase-positive and glucose-6-phosphatase-negative) appeared in both groups of animals as early as 1 week after 2-acetylaminofluorene withdrawal and continued to increase during the subsequent weeks. Livers from vitamin D-depleted rats exhibited a significant increase in the number of foci over that observed in normal rats at weeks 1 and 5 after 2-acetylaminofluorene withdrawal. However, the main effect of vitamin D depletion was on focus size, which was found to be significantly greater in vitamin D-depleted rat livers at weeks 2 to 6; focus area (volume fraction) was also found to be consistently larger in livers of vitamin D-depleted rats than in those of normal rats. Labeling of oval cells, a cell compartment possibly associated with the repopulation of the liver parenchyma, was significantly reduced by vitamin D depletion. Control rat livers of both groups showed normal liver histology, and no foci, nodules or oval cells were detected in either group. The present data suggest that vitamin D depletion leads to increased in vivo susceptibility to chemicals known to induce hepatocarcinogenesis. Long-term studies must be conducted to evaluate the effect of vitamin D status on the evolution of preneoplastic foci into frank hepatocellular carcinoma.

Hepatic expression of regeneration marker genes following partial hepatectomy in the rat. Influence of 1,25-dihydroxyvitamin D3 in hypocalcemia.

BACKGROUND/AIMS: Vitamin D (D) depletion is a common feature of chronic liver diseases. In past years, disturbances in calcium metabolism involving inadequate D and parathyroid hormone status have been reported to significantly impair the hepatic regeneration process following partial hepatectomy in the rat. The purpose of this study was to investigate how hypocalcemia and D deficiency affect specific cell markers of hepatic compensatory growth. METHODS: Steady-state mRNA levels of gene markers of the regeneration process were investigated following 2/3 partial hepatectomy. The response of hypocalcemic D-depleted rats was compared to that of animals whose calcium status had been normalized by repletion with the active D hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). RESULTS: The transcript for the major hepatic mitogen HGF increased in both groups after partial liver resection but the increase was significantly lower as well as delayed in livers obtained from calcium deficient rats in the prereplicative phase of the regeneration process. TGF alpha mRNA levels were also found to be significantly lower in calcium deficient rats at all time-points following partial hepatectomy, while the relative behavior of the tandem TGF alpha-EGFR indicated an early dominant effect in normocalcemic 1,25(OH)2D3-repleted animals. HGF-c-met mRNA levels also indicated that the 1,25(OH)2D3-repleted animals reacted more promptly to the regeneration stimuli. Indeed, while relative (1,25(OH)2D3/D-Ca- ratio) maximum mRNA levels were observed 12 h following liver resection in 1,25(OH)2D3-treated animals, relative peak levels were only apparent 24 h post-surgery in hypocalcemic rats. Maximum cyclin D1 (a marker of the G1 phase of the cell cycle) mRNA occurred between 8-18 h after partial hepatectomy in 1,25(OH)2D3-repleted animals to return to base-line value thereafter, but in hypocalcemic rats the transcript levels remained significantly below 1,25(OH)2D3-repleted animals during the prereplicative period with increases above initial values between 12-24 h post-surgery. Both cyclin A (an S phase marker) transcripts (1.8 and 2.9 kb) were influenced by the regeneration process. The transcripts significantly and sharply increased in hypocalcemia between 30-36 h following partial hepatectomy to decrease thereafter, while the increase was observed between 24-30 h, and at 48 h (1.8 kb) in 1,25(OH)2D3-repleted animals. Liver weight recovery was also found to be decreased in D-depleted rats over the 48 h period of observation. CONCLUSIONS: Our data further confirm the presence of an impaired regeneration process in hypocalcemia of D deficiency which seems to be associated with gene markers indicating an inefficient transit across the G1 phase of the cell cycle.

Comparative in vivo expression of the calcitriol-24-hydroxylase gene in kidney and intestine.

Although the kidney and intestine are among the major organs involved in both the biotransformation and action of vitamin D3, they exhibit very distinct roles in calcium and D3 homeostasis. The aim of the present studies was to investigate the relative in vivo responsiveness of renal and intestinal 1,25(OH)2D3-24-hydroxylase (24-hydroxylase) mRNA levels to calcitriol (1,25(OH)2D3) following acute or chronic 1,25(OH)2D3 exposure using hypocalcemic vitamin D-depleted rats as an experimental model. Intestinal 24-hydroxylase mRNA levels were very responsive to a single i.v. injection of 2.4, 12 or 120 nmol 1,25(OH)2D3/kg but in kidney the mRNA levels only increased following exposure to the highest 1,25(OH)2D3 concentration, and exhibited a maximum response only 30% of that in the intestine despite similar tissue uptake of the hormone. To evaluate whether the kidney might preferentially respond to endogenously produced 1,25(OH)2D3, animals received increasing doses of 25(OH)D3. Although the intestinal 24-hydroxylase transcript was highly induced, the renal transcript was unresponsive to 25(OH)D3 treatment despite circulating 1,25(OH)2D3 concentrations of 24 nmol/l. By contrast, intestinal 24-hydroxylase mRNA levels were largely unresponsive to longterm calcitriol administration while the renal transcript, although insensitive to a physiological dose, responded to pharmacological 1,25(OH)2D3 doses. However, when challenged acutely with 1,25(OH)2D3 following chronic exposure, the kidney 24-hydroxylase mRNA levels remained largely unresponsive in contrast to the intestinal transcript which was markedly induced. These data indicate that significant differences exist in the in vivo tissue responsiveness of the 24-hydroxylase mRNA. Indeed, the gene exhibited high intestinal responsiveness to acutely, but not chronically, administered 1,25(OH)2D3, while in the kidney it only responded to high exogenous 1,25(OH)2D3 delivered either acutely or chronically. In addition, these site-specific regulatory mechanisms governing the expression of the 24-hydroxylase gene are independent of the endocrine calcium status and render the kidney relatively resistant to endogenously produced 1,25(OH)2D3.

Oral calcium transiently increases calbindin9k gene expression in adult rat duodena.

In rat intestine, the 9 kilodalton calbindin (CaBP9K) is significantly increased in vivo by 1,25-dihydroxyvitamin D3 (1, 25(OH)2D3) through a vitamin D (D) response element located in the 5'-flanking region of the gene. However, in vitro calcium has also been reported to increase CaBP9K gene expression in fetal duodenal culture preparations. The aim of the studies was to investigate whether calcium feeding alone can influence CaBP9K gene expression in vivo in adult rat duodena by evaluating the pattern of expression of its mRNA following short- or long-term exposure to oral calcium, comparing the data to exposure to the known inducer of the gene, 1, 25(OH)2D3. Hypocalcemic D-depleted rats were acutely or chronically supplemented with calcium per os, or with 1,25(OH)2D3 in the presence or absence of oral calcium. Short-term calcium feeding was shown to significantly increase the expression of the CaBP9K gene to a level similar to that observed in 1,25(OH)2D3-treated rats but no additive effect between oral calcium and 1,25(OH)2D3 on the level of its mRNA was observed. Moreover, the calcium effect on CaBP9K gene expression was shown to be independent of the circulating ionized calcium concentration and, contrary to the effect of 1,25(OH)2D3, not sustained following long-term exposure. Our data clearly indicate that oral calcium alone has a significant but only transient effect of the expression of the adult rat intestinal CaBP9K gene in vivo and that maintenance of its expression requires normalization of the D endocrine system.

Intravenous 1,25(OH)2D therapy increases the intact parathyroid hormone secretion set point in hemodialyzed patients.

We have studied the effect of intravenous calcitriol [1,25(OH)2D] therapy (1 microgram at the end of each dialysis session) on parathyroid secretory curves of hemodialyzed patients with near-normal basal intact (< 10 pmol/l, n = 7; NNBI) or elevated basal intact (> 10 pmol/l, n = 6; EBI) parathyroid hormone (PTH; iPTH) levels. These results were compared with those obtained in matched normal individuals (N). Our main objective was to define the influence of intravenous 1,25(OH)2D therapy on the set point of iPTH stimulation in relation to the severity of secondary hyperparathyroidism. A complete parathyroid function was obtained by CaCl2 and Na2EDTA infusions in 14 N and by modification of the dialysate calcium content in 13 hemodialyzed patients. Ionized calcium (Ca2+) and iPTH were measured regularly during hypo- and hypercalcemia. Parathyroid secretory curves were derived from these data. Both groups of patients had lower basal Ca2+ (NNBI 1.16 +/- 0.05; EBI 1.10 +/- 0.03; N 1.25 +/- 0.04 mmol/l; p < 0.001) and higher basal iPTH (NNBI 6.3 +/- 2.5; EBI 49.2 +/- 39.5; N 2.5 +/- 0.8 pmol/l; p < 0.01) levels than N with more extreme values in EBI than in NNBI patients (p < 0.001). NNBI patients had stimulated iPTH levels similar to N (18.4 +/- 7.1 vs. 17.3 +/- 7.2 pmol/l), while these levels were markedly increased in EBI patients (80.7 +/- 46.0 pmol/l; p < 0.001). After 1,25(OH)2D therapy, Ca2+ increased to 1.16 +/- 0.03 mmol/l in EBI and normalized in NNBI patients (1.25 +/- 0.07 mmol/l). Stimulated iPTH decreased by 30% in NNBI (p < 0.05) and by 21% in EBI patients (NS). These two factors contributed to a decrease in basal iPTH by 52% in NNBI (p < 0.05) and by 40% in EBI (p < 0.01). The set point of iPTH stimulation was lower than in N (1.18 +/- 0.04 mmol/l) and increased with intravenous 1,25(OH)2D therapy from 1.09 +/- 0.03 to 1.16 +/- 0.05 mmol/l in NNBI (p < 0.05) and from 1.08 +/- 0.04 to 1.12 +/- 0.04 mmol/l in EBI patients (p < 0.05). The set points and changes in set point were correlated with basal Ca2+ (r = 0.56; p = 0.003) and changes in basal Ca2+ (r = 0.64; p = 0.04) observed before and during therapy. The starting position of each patient on his secretory curve before and after 1,25(OH)2D therapy was inversely related to his starting Ca2+ concentration (n = 26; r = -0.66; p = 0.0003). Taking this into account improved the relationship between Ca2+ concentration and the set point of iPTH stimulation by Ca2+ in a stepwise regression (R2 = 0.62; p = 0.0003). However, no correlation was found between set points and stimulated iPTH values. We concluded that 1,25(OH)2D therapy induced an increase in the set point of PTH stimulation in hypocalcemic hemodialyzed patients related to a similar increase in basal Ca2+ concentration. This is in part related to the starting position of each patient on his secretory curve which will affect his set point in relation to the hysteresis phenomenon in iPTH secretion. But the set point of PTH stimulation is also related to the basal ionized calcium concentration by mechanisms yet to be elucidated.

Hypocalcemia modifies the intracellular calcium response to the alpha 1-adrenergic agent phenylephrine in rat hepatocytes.

In vivo, extracellular calcium ([Ca2+]e) homeostasis is maintained within a very narrow range by the calcium regulating hormones. At the cellular level, the response to many agents is transduced by changes in cytosolic Ca2+ ([Ca2+]i) which involves both mobilization of cellular pools and entry of [Ca2+]e through plasma membrane channels. To investigate the cellular effects of chronic hypocalcemia (Ca-) on [Ca2+]i homeostasis, hepatocytes, a cell type well characterized for its [Ca2+]i response, were used. Data indicate that Ca- leads to a significant shift to the left in the basal resting cytosolic Ca2+ concentration distribution curve with half-maximum cumulative frequency of 119 versus 149 nM in Ca- and normal rats (N) respectively (P < 0.0001). The response to the alpha 1-adrenergic agonist phenylephrine (Phe) was also influenced by Ca- with a dampening of the dose-response curve, a significant decrease in the frequency of sustained responses (P < 0.001), and significant changes in the oscillation pattern. Indeed, hepatocytes obtained from Ca- exhibited a higher frequency of large amplitude, low frequency oscillations than N most particularly at the 2 and 5 microM Phe dose while N predominantly exhibited low amplitude, high frequency oscillations on sustained plateaus (P < 0.001). IP3 receptor (IP3R) binding studies and Ca2+ mobilization from IP3-sensitive pools showed that IP3R was highly sensitive to the prevailing Ca2+ with, in the range of resting [Ca2+]i, R affinity significantly lower in Ca- than in N. Upon exposure of permeabilized cells to 25 microM IP3, Ca2+ mobilization from the IP3-sensitive intracellular pool was significantly reduced by Ca- (P < 0.05) suggesting a decrease in the IP3-mobilizable Ca2+ pool in Ca-. Our results indicate that hypocalcemia significantly alters [Ca2+]i signalling by perturbing the initial response to agonist and the [Ca2+]i response pattern. In addition, the decrease in Ca2+ mobilization from IP3-sensitive pools suggests that hypocalcemia may also lead to a decrease in the Ca2+ content of intracellular pools.

Accumulation of a non-(1-84) molecular form of parathyroid hormone (PTH) detected by intact PTH assay in renal failure: importance in the interpretation of PTH values.

A molecular form of PTH different from PTH-(1-84) and present in normal serum is recognized by two-site intact (I-) PTH assays; it responds to Ca2+ changes in the same way that PTH carboxyl-terminal fragments do. To evaluate the impact of this finding, we have compared basal, stimulated, and nonsuppressible I-PTH values in 14 normal subjects and 15 renal failure patients, subdivided into 8 patients with low (< 12 pmol/L; LBI) and 7 with high (> 12 pmol/L; HBI) basal I-PTH. Samples obtained under various calcemic conditions in these 3 groups were further fractionated by high performance liquid chromatography (HPLC) and assayed for I-PTH, and the various peaks observed were quantitated by planimetry. Differences among the 3 groups were reinterpreted knowing the exact composition of I-PTH. Basal I-PTH was greatly increased in HBI (mean +/- SD, 44.1 +/- 38.6 pmol/L) compared to that in normal subjects (2.5 +/- 0.8 pmol/L; P < 0.001) or LBI (6.1 +/- 2.4 pmol/L; P < 0.001); the difference was less in these last 2 groups (P < 0.01). Similar differences were observed for stimulated and nonsuppressible I-PTH, except for stimulated I-PTH, which was similar in normal and LBI subjects. Two I-PTH HPLC molecular forms accounted for I-PTH immunoreactivity in the 3 groups. In normal subjects, PTH-(1-84) accounted for 74.9 +/- 4.3%, 79.0 +/- 3.0%, and 87.2 +/- 1.0% of I-PTH in hyper-, normo-, and hypocalcemia, respectively, but only for 44.6 +/- 2.5%, 50.5 +/- 0.7%, and 63.6 +/- 0.1% in renal failure patients, with similar results in HBI and LBI. The accumulation of a non-(1-84) PTH peak accounted for the difference between normal subjects and renal failure patients. When basal, stimulated, and nonsuppressible I-PTH values were separated into their 2 components, prior differences between HBI and LBI or normal subjects remained unchanged because of very high I-PTH values in HBI, but differences between normal and LBI subjects were entirely explained by the accumulation of the non-(1-84) PTH peak [basal, 3.0 +/- 1.2 vs. 0.5 +/- 0.2 pmol/L (P < 0.001); stimulated, 6.8 +/- 2.3 vs. 2.3 +/- 1.0 pmol/L (P < 0.001); nonsuppressible, 1.3 +/- 0.7 vs. 0.2 +/- 0.08 pmol/L (P < 0.001)]; PTH-(1-84) values were similar (basal, 3.1 +/- 1.2 vs. 2.0 +/- 0.6 pmol/L; stimulated, 12.0 +/- 3.9 vs. 15.5 +/- 6.6 pmol/L; nonsuppressible, 1.1 +/- 0.6 vs. 0.52 +/- 0.22 pmol/L). Thus, a non-(1-84) PTH molecular form detected by two-site I-PTH assays accumulates in renal failure and accounts for a larger proportion of I-PTH than that in normal subjects. Levels of I-PTH 1.57 times higher than those in normocalcemic subjects are thus required in renal failure to achieve similar PTH-(1-84) concentrations. The composition of I-PTH is also identical in all hemodialyzed patients.

Effect of phalloidin on cholestasis, hemodynamics, and microcirculation in isolated perfused rat liver.

In this study, the possible role of the hepatic microcirculation in phalloidin-induced cholestasis and hepatotoxicity was examined in isolated perfused rat livers (IPRL). Administration of a phalloidin bolus (1 mg/kg body weight) through the portal vein induced an immediate reduction of bile flow. In 16.9 minutes, bile flow was 50% lower than basal values. Portal pressure was only increased in 60 minutes after phalloidin injection and increased sharply from this time up to the end of perfusion (90 minutes). Under these conditions, phalloidin did not induce liver cell cytolysis, as assessed by aspartate transaminase (AST) and lactate dehydrogenase (LDH) release in the perfusate effluent. Under electron microscopy, hepatocytic vacuolization was mild 15 minutes after phalloidin administration but increased with time. At the end of perfusion, the hepatic architecture was markedly altered; erythrocyte accumulation was observed in both sinusoids and hepatocyte vacuoles. Evaluation by multiple indicator dilution curves showed that extravascular volume (EVV) was significantly affected by phalloidin. It was augmented in 30 minutes after phalloidin administration with values increasing gradually over time. Neither vascular nor cellular volume was altered. The hepatic swelling may be attributed to enlargement of the extravascular space of the liver. These results indicate that changes in the liver microcirculation are not the primary cause of phalloidin-induced cholestasis in the IPRL.

Effects of vitamin D3 and cyclosporin A on HgCl2-induced autoimmunity in brown Norway rats.

BACKGROUND: Mercuric chloride (HgCl2) induces a lymphoproliferative disorder and autoimmune glomerulonephritis in Brown Norway (BN) rats. This syndrome is the consequence of T cell-dependent polyclonal B cell activation and autoantibody production. We have previously shown that HgCl2-induced autoimmune perturbations can be prevented in BN rats by the administration of cyclosporin A (CsA). The most potent vitamin D3 metabolite 1,25(OH)2 D3 (Vit D3) shares certain immunomodulatory properties with CsA. We therefore chose to compare the effects of Vit D3 to those of CsA in BN rats treated with HgCl2 in order to establish whether Vit D3 either alone or in combination with CsA can attenuate an autoimmune syndrome in vivo. METHODS: BN rats were treated with HgCl2 according to a standard protocol. Subgroups of rats were also given CsA alone, Vit D3 or synthetic analogues of Vit D3 alone, or combinations of both agents. Different doses and routes of administration were compared. The following markers of disease activity were evaluated: mortality, peak proteinuria, serum IgE concentrations, and renal immunoglobulin deposition. RESULTS: Disease activity was markedly attenuated in all rats treated with CsA alone. Vit D3 and certain of its synthetic analogues administered alone also tempered the autoimmune process, but to a lesser extent than did CsA. The effect of CsA alone was so potent, that no additive or synergistic effects could be demonstrated when CsA was administered in combination with Vit D3. CONCLUSIONS: Despite similar described immunomodulatory effects in vitro, CsA is clearly more effective than Vit D3 in preventing HgCl2 autoimmune disease in BN rats. This suggests that there is a difference in the cellular targets of these two agents in vivo, and/or a difference in the potency with which HgCl2-triggered immune activation is suppressed.

Expression of the 1,25-dihydroxyvitamin D3-24-hydroxylase gene in rat intestine: response to calcium, vitamin D3 and calcitriol administration in vivo.

The 25(OH)D3/1,25(OH)2D3 24-hydroxylase (24-hydroxylase) displays an induction profile responsive to vitamin D (D) abundance and is hence only observed in normal extracellular Ca2+ concentrations. However, the participation of Ca2+ in the expression of the 24-hydroxylase gene in vivo is not known. The present studies investigate the role played by the circulating Ca2+ and the D3 and/or 1,25(OH)2D3 status on the 1,25(OH)2D3-mediated inducibility of the 24-hydroxylase gene in rat duodenum. Hypocalcemic D-depleted rats were supplemented with calcium alone to normalize serum Ca2+ without normalizing the D3 status or were acutely or chronically supplemented with D3 or 1,25(OH)2D3. Messenger RNA for the 24-hydroxylase was undetectable in the intestine of hypocalcemic D-depleted rats, and short- or long-term calcium supplementation was completely unsuccessful in inducing its expression. By contrast, acute 1,25(OH)2D3 administration led to significant increases in the levels of expression of the gene which was independent of the calcium intake, the prevailing circulating Ca2+, and the D3 or 1,25(OH)2D3 status. Moreover, 24-hydroxylase gene expression was only found to respond to acutely administered 1,25(OH)2D3, the mRNA levels being unaltered following continuous exposure to physiological or pharmacological doses of the hormone for 7 days. Time-course studies revealed, however, that induction of the gene was clearly apparent early in the 1,25(OH)2D3 supplementation course but gradually faded by 3 days to return to basal uninduced levels by 7 days, suggesting the presence of intestinal adaptation mechanism(s) which down-regulated the responsiveness in the continuous presence of 1,25(OH)2D3. Our data show the lack of effect of calcium alone or in combination with 1,25(OH)2D3 on the in vivo induction of the 24-hydroxylase gene expression in rat intestine. By rapidly reacting to surges in 1,25(OH)2D3 concentrations, the 24-hydroxylase efficiently controls the amount of 1,25(OH)2D3 in intestine as the first step in the biotransformation pathway aimed at the irreversible clearance of the secosteroid hormone.

Hypocalcemia decreases the early and late responses to epidermal growth factor in rat hepatocytes.

Extreme variations in extracellular Ca2+ concentrations ([Ca2+]e) modify the signaling generated by many hormones and growth factors. However, the influence of physiological changes in [Ca2+]e on the response to hepatic mitogens remains largely unknown. To study the influence of [Ca2+]e on the response to epidermal growth factor (EGF), hepatocytes from normal rat livers were equilibrated in vitro at [Ca2+]e similar to those observed in normocalcemia or hypocalcemia. To further investigate the effect of hypocalcemia in vivo, hepatocytes were obtained from chronically hypocalcemic rats and kept in vitro at the [Ca2+]e prevailing in vivo. Intracellular Ca2+ concentrations ([Ca2+]i) and DNA synthesis were evaluated after increasing doses of EGF. [Ca2+]e strongly influenced the [Ca2+]i response to EGF with significantly smaller [Ca2+]i increases in hepatocytes of normal rats kept in low [Ca2+]e compared with those kept in normal [Ca2+]e. In hypocalcemic rat hepatocytes, the response was further decreased and found to be significantly lower than that obtained in control cells kept in vitro at either 1.25 mmol/L or 0.8 mmol/L [Ca2+]e. In normal [Ca2+]e, the EGF-induced increases in [Ca2+]i were abolished by inhibiting EGF receptor autophosphorylation and by blocking calcium channels. Low in vitro [Ca2+]e significantly dampened the EGF-mediated DNA synthesis in normal rat hepatocytes but hypocalcemia in vivo further reduced the proliferative response compared with that obtained in control rat hepatocytes maintained in normal, or low [Ca2+]e. Furthermore, the blunted responses in [Ca2+]i mobilization and DNA synthesis associated with hypocalcemia could not be overcome by increasing concentrations of EGF nor by normalization of [Ca2+]e in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)