Multiple pathways for high voltage-activated ca(2+) influx in anterior pituitary lactotrophs and somatotrophs.

The present study demonstrates that a significant proportion of high voltage-activated (HVA) Ca(2+) influx in native rat anterior pituitary cells is carried through non-L-type Ca(2+) channels. Using whole-cell patch-clamp recordings and specific Ca(2+) channel toxin blockers, we show that approximately 35% of the HVA Ca(2+) influx in somatotrophs and lactotrophs is carried through Ca(v) 2.1, Ca(v) 2.2 and Ca(v) 2.3 channels, and that somatotrophs and lactotrophs share similar proportions of these non-L-type Ca(2+) channels. Furthermore, experiments on mixed populations of native anterior pituitary cells revealed that the fraction of HVA Ca(2+) influx carried through these non-L-type Ca(2+) channels might even be higher (approximately 46%), suggesting that non-L-type channels exist in the majority of native anterior pituitary cells. Using western blotting, immunoblots for α(1C) , α(1D) , α(1A) , α(1B) and α(1E) Ca(2+) channel subunits were identified in native rat anterior pituitary cells. Additionally, using reverse transcriptase-polymerase chain reaction, cDNA transcripts for α(1C) , α(1D) , α(1A) and α(1B) Ca(2+) channel subunits were identified. Transcripts for α(1E) were nonspecific and transcripts for α(1S) were not detected at all (control). Taken together, these results clearly demonstrate the existence of multiple HVA Ca(2+) channels in the membrane of rat native anterior pituitary cells. Whether these channels are segregated among different membrane compartments was investigated further in flotation assays, demonstrating that Ca(v) 2.1, Ca(v) 1.2 and caveolin-1 were mostly localised in light fractions of Nycodenz gradients (i.e. in lipid raft domains). Ca(v) 1.3 channels were distributed among both light and heavy fractions of the gradients (i.e. among raft and nonraft domains), whereas Ca(v) 2.2 and Ca(v) 2.3 channels were distributed mostly among nonraft domains. In summary, in the present study, we demonstrate multiple pathways for HVA Ca(2+) influx through L-type and non-L-type Ca(2+) channels in the membrane of native anterior pituitary cells. The compartmentalisation of these channels among raft and nonraft membrane domains might be essential for their proper regulation by separate receptors and signalling pathways.

A conserved cis-acting element in the parathyroid hormone 3'-untranslated region is sufficient for regulation of RNA stability by calcium and phosphate.

Calcium and phosphate regulate parathyroid hormone (PTH) gene expression post-transcriptionally by changes in protein-PTH mRNA 3'-untranslated region (UTR) interactions, which determine PTH mRNA stability. We have identified the protein binding sequence in the PTH mRNA 3'-UTR and determined its functionality. The protein-binding element was identified by binding, competition, and antisense oligonucleotide interference. The sequence was preserved among species suggesting its importance. To study its functionality in the context of another RNA, a 63-base pair cDNA PTH sequence was fused to the growth hormone (GH) gene. There is no parathyroid (PT) cell line and therefore an in vitro degradation assay was used to determine the stability of transcripts for PTH, GH, and a chimeric GH-PTH 63 nucleotides with PT cytosolic proteins. The full-length PTH transcript was stabilized by PT proteins from rats fed a low calcium diet and destabilized by proteins from rats fed a low phosphate diet, correlating with PTH mRNA levels in vivo. These PT proteins did not affect the native GH transcript. However, the chimeric GH transcript was stabilized by low calcium PT proteins and destabilized by low phosphate PT proteins, similar to the PTH full-length transcript. Therefore, we have identified a PTH RNA-protein binding region and shown that it is sufficient to confer responsiveness to calcium and phosphate in a reporter gene. This defined element in the PTH mRNA 3'-UTR is necessary and sufficient for the regulation of PTH mRNA stability by calcium and phosphate.

Molecular mechanisms of secondary hyperparathyroidism.

Secondary hyperparathyroidism is a frequent complication of chronic renal failure (CRF) and a major factor in the pathogenesis of renal osteodystrophy. A high serum phosphate, decreased levels of serum 1,25(OH)2D3 and the subsequently low serum calcium are the major metabolic abnormalities in CRF, which lead to the secondary hyperparathyroidism. At the level of parathyroid hormone (PTH) secretion there is insensitivity to the ambient serum calcium. PTH mRNA levels are increased by a post-transcriptional mechanism that involves the binding of PT cytosolic proteins to the PTH mRNA 3'-untranslated region (UTR). In a dietary model of secondary hyperparathyroidism due to hypocalcemia there is increased binding of parathyroid proteins to the 3'-UTR and decreased degradation as determined by an in vitro degradation assay. Changes in serum phosphate also dramatically regulate PTH mRNA stability. There is also regulation at the level of PT cell proliferation. PT cell proliferation is increased by experimental hypocalcemia or hyperphosphatemia and decreased by hypophosphatemia and administered 1,25(OH)2D3. The understanding of the molecular mechanisms involved in the genesis of secondary hyperparathyroidism will allow the design of new effective strategies in the management of this troubling condition.

Identification of AUF1 as a parathyroid hormone mRNA 3'-untranslated region-binding protein that determines parathyroid hormone mRNA stability.

Parathyroid hormone (PTH) mRNA levels are post-transcriptionally increased by hypocalcemia and decreased by hypophosphatemia, and this is mediated by cytosolic proteins binding to the PTH mRNA 3'-untranslated region (UTR). The same proteins are also present in other tissues, such as brain, but only in the parathyroid is their binding regulated by calcium and phosphate. The function of the PTH mRNA 3'-UTR-binding proteins was studied using an in vitro degradation assay. Competition for the parathyroid-binding proteins by excess unlabeled 3'-UTR destabilized the full-length PTH transcript in this assay, indicating that these proteins protect the RNA from RNase activity. The PTH RNA 3'-UTR-binding proteins were purified by RNA affinity chromatography of rat brain S-100 extracts. The eluate from the column was enriched in PTH RNA 3'-UTR binding activity. Addition of eluate to the in vitro degradation assay with parathyroid protein extracts stabilized the PTH transcript. A major band from the eluate at 50 kDa was sequenced and was identical to AU-rich binding protein (AUF1). Recombinant AUF1 bound the full-length PTH mRNA and the 3'-UTR. Added recombinant AUF1 also stabilized the PTH transcript in the in vitro degradation assay. Our results show that AUF1 is a protein that binds to the PTH mRNA 3'-UTR and stabilizes the PTH transcript.

Dynein light chain binding to a 3'-untranslated sequence mediates parathyroid hormone mRNA association with microtubules.

The 3'-untranslated region (UTR) of mRNAs binds proteins that determine mRNA stability and localization. The 3'-UTR of parathyroid hormone (PTH) mRNA specifically binds cytoplasmic proteins. We screened an expression library for proteins that bind the PTH mRNA 3'-UTR, and the sequence of 1 clone was identical to that of the dynein light chain LC8, a component of the dynein complexes that translocate cytoplasmic components along microtubules. Recombinant LC8 binds PTH mRNA 3'-UTR, as shown by RNA electrophoretic mobility shift assay. We showed that PTH mRNA colocalizes with microtubules in the parathyroid gland, as well as with a purified microtubule preparation from calf brain, and that this association was mediated by LC8. To our knowledge, this is the first report of a dynein complex protein binding an mRNA. The dynein complex may be the motor that is responsible for transporting mRNAs to specific locations in the cytoplasm and for the consequent is asymmetric distribution of translated proteins in the cell.

Mechanism of increased parathyroid hormone mRNA in experimental uremia: roles of protein RNA binding and RNA degradation.

Patients with chronic renal failure develop secondary hyperparathyroidism with increased synthesis and secretion of parathyroid hormone (PTH) resulting in severe skeletal complications. In rats with secondary hyperparathyroidism due to 5/6 nephrectomy, there are increased PTH mRNA levels, and this mechanism was studied. Parathyroid glands were microdissected from control and 5/6 nephrectomy rats and analyzed for PTH mRNA and control genes, and the nuclei were used for nuclear run-on experiments. The cytosolic proteins of the parathyroids were used to study PTH mRNA protein binding by ultraviolet cross-linking and the degradation of the PTH transcript in vitro. Nuclear run-ons showed that the increase in PTH mRNA levels was posttranscriptional. Protein binding to the PTH mRNA 3'-UTR determines PTH mRNA stability and levels. Parathyroid proteins from uremic rats bound PTH mRNA similar to control rats by ultraviolet cross-linking. To determine the effect of uremia on PTH mRNA stability, an in vitro RNA degradation assay was performed with parathyroid proteins from uremic rats. When parathyroid proteins from control rats were incubated with PTH mRNA, there was transcript degradation already at 30 min, reaching 50% at 60 min and 90% at 180 min. With uremic parathyroid proteins, the PTH mRNA was not degraded at all at 120 min and was moderately decreased at 180 min. This decrease in degradation by uremic parathyroid proteins suggests a decrease in parathyroid cytosolic endonuclease activity in uremia resulting in a more stable PTH transcript. The increased PTH mRNA levels would translate into increased PTH synthesis and serum PTH levels, which would lead to metabolic bone disease in many patients with chronic renal failure.

Post-transcriptional regulation of the parathyroid hormone gene by calcium and phosphate.

Parathyroid hormone messenger RNA levels are regulated by calcium, phosphate and 1,25-dihydroxyvitamin D3. Dietary induced hypocalcaemia increases and hypophosphataemia decreases parathyroid hormone mRNA levels post-transcriptionally. This regulation is mediated by binding of parathyroid cytosolic proteins to the parathyroid hormone mRNA 3'-untranslated region, and in particular the terminal 60 nucleotides of the 3'-untranslated region, thereby determining RNA stability.

Protein-RNA interactions determine the stability of the renal NaPi-2 cotransporter mRNA and its translation in hypophosphatemic rats.

Hypophosphatemia leads to an increase in type II Na(+)-dependent inorganic phosphate cotransporter (NaPi-2) mRNA and protein levels in the kidney and increases renal phosphate reabsorption. Nuclear transcript run-on experiments showed that the effect of a low phosphate diet was post-transcriptional. In an in vitro degradation assay, renal proteins from hypophosphatemic rats stabilized the NaPi-2 transcript 6-fold compared with control rats and this was dependent upon an intact NaPi-2 3'-untranslated region (UTR). To determine an effect of hypophosphatemia upon NaPi-2 protein synthesis, the incorporation of injected [(35)S]methionine into renal proteins was studied in vivo. Hypophosphatemia led to increased [(35)S]methionine incorporation only into NaPi-2 protein. The effect of hypophosphatemia on translation was studied in an in vitro translation assay, where hypophosphatemic renal proteins led to increased translation of NaPi-2 and other transcripts. NaPi-2 RNA interaction with cytosolic proteins was studied by UV cross-linking and Northwestern gels. Hypophosphatemic proteins led to increased binding of renal cytosolic proteins to the 5'-UTR of NaPi-2 mRNA. Therefore, hypophosphatemia increases NaPi-2 gene expression post-transcriptionally, which correlates with a more stable transcript mediated by the 3'-UTR, and an increase in NaPi-2 translation involving protein binding to the 5'-UTR. These findings show that phosphate regulates gene expression by affecting protein-RNA interactions in vivo.

Regulation of the parathyroid hormone gene by vitamin D, calcium and phosphate.

Secondary hyperparathyroidism is a frequent complication of chronic renal failure resulting in severe bone disease. Secondary hyperparathyroidism is composed of increased in parathyroid hormone (PTH) synthesis and secretion due to an increase in PTH gene expression and parathyroid cell proliferation. PTH gene expression is regulated by calcium, phosphate and 1,25-dihydroxy vitamin D (1,25(OH)2D). 1,25(OH)2D3 injected to rats leads to a dramatic decrease in PTH gene transcription without any increase in serum calcium. Hypocalcemia leads to a large increase in PTH mRNA levels which is post-transcriptional. Hypophosphatemia leads to a marked decrease in PTH gene expression that is also post-transcriptional. The mechanisms of the post-transcriptional effects of calcium and phosphate on the PTH gene have shown to be due to changes in protein-RNA interactions at the PTH mRNA 3'-UTR. Hypocalcemia leads to increased binding of parathyroid cytosolic proteins to the PTH mRNA 3'-UTR and hypophosphatemia to decreased binding of these proteins to the PTH mRNA 3'-UTR. The binding of the parathyroid proteins stabilizes the PTH RNA in an in vitro degradation assay. In rats with experimental uremia due to 5/6 nephrectomy, there is an increase in PTH mRNA levels due to a decrease in degradation of the PTH RNA as determined by this assay. The characterization of the parathyroid cytosolic proteins that interact with the PTH mRNA 3'-UTR may lead to a clearer understanding of how changes in serum calcium and phosphate result in secondary hyperparathyroidism.

Transcriptional and post-transcriptional regulation of PTH gene expression by vitamin D, calcium and phosphate.

1,25(OH)(2)D(3) the biologically active metabolite of vitamin D is synthesized in the renal proximal tubules from the hepatic metabolite 25 (OH)D. Lack of 1,25(OH)(2)D(3) is relevant to the pathogenesis of secondary hyperparathyroidism, and 1,25(OH)(2)D(3) itself is used effectively in the management of renal failure patients to prevent secondary hyperparathyroidism. The scientific basis of this therapy is the finding that 1,25(OH)(2)D(3) potently decreases PTH gene transcription both in vitro and in vivo.

Calreticulin inhibits vitamin D's action on the PTH gene in vitro and may prevent vitamin D's effect in vivo in hypocalcemic rats.

1,25-dihydroxyvitaminD3 [1,25-(OH)2D3] and PTH both act to increase serum calcium. In addition, 1,25-(OH)2D3 decreases PTH gene transcription, which is relevant both to the physiology of calcium homeostasis and to the management of the secondary hyperparathyroidism of patients with chronic renal failure. In chronic hypocalcemia there is secondary hyperparathyroidism with increased levels of PTH mRNA and serum PTH despite markedly increased levels of 1,25-(OH)2D3. We have studied the role of calreticulin in this resistance to 1,25-(OH)2D3. Weanling rats fed a low-calcium diet were hypocalcemic and had increased PTH mRNA levels despite high serum 1,25-(OH)2D3 levels. 1,25-(OH)2D3 given by continuous minipump infusion to normal rats led to the expected decrease in PTH mRNA. The hypocalcemic rats had an increased concentration of calreticulin in the nuclear fraction of their parathyroids, but not in other tissues. Gel shift assays showed that a purified vitamin D receptor and retinoid X receptor-beta bound to the PTH promoter's chicken and rat vitamin D response element (VDRE), and this binding was inhibited by added pure calreticulin. Transfection studies with a PTH VDRE-chloramphenicol acetyltransferase (CAT) construct showed that 1,25-(OH)2D3 decreased CAT transcription. Cotransfection of PTH VDRE-CAT with a calreticulin expression vector in the sense orientation prevented the transcriptional effect of 1,25-(OH)2D3, but a calreticulin vector in the antisense orientation had no effect. These results show that calreticulin prevents the binding of vitamin D receptor-retinoid X receptor-beta to the PTH VDRE in gel retardation assays and prevents the transcriptional effect of 1,25-(OH)2D3 on the PTH gene. This is the first report of calreticulin inhibiting a down-regulatory function of a sterol hormone and may help explain the refractoriness of the secondary hyperparathyroidism of many chronic renal failure patients to 1,25-(OH)2D3.

RNA-Protein binding and post-transcriptional regulation of parathyroid hormone gene expression by calcium and phosphate.

Parathyroid hormone (PTH) regulates serum calcium and phosphate levels, which, in turn, regulate PTH secretion and mRNA levels. PTH mRNA levels are markedly increased in rats fed low calcium diets and decreased after low phosphate diets, and this effect is post-transcriptional. Protein-PTH mRNA binding studies, with parathyroid cytosolic proteins, showed three protein-RNA bands. This binding was to the 3'-untranslated region (UTR) of the PTH mRNA and was dependent upon the terminal 60 nucleotides. Parathyroid proteins from hypocalcemic rats showed increased binding, and proteins from hypophosphatemic rats decreased binding, correlating with PTH mRNA levels. There is no parathyroid cell line; however, a functional role was provided by an in vitro degradation assay. Parathyroid proteins from control rats incubated with a PTH mRNA probe led to an intact transcript for 40 min; the transcript was intact with hypocalcemic proteins for 180 min and with hypophosphatemic proteins only for 5 min. A PTH mRNA probe without the 3'-UTR, or just the terminal 60 nucleotides, incubated with hypophosphatemic proteins, showed no degradation at all, indicating that the sequences in the 3'-UTR determine PTH mRNA degradation. Hypocalcemia and hypophosphatemia regulate PTH gene expression post-transcriptionally. This correlates with binding of proteins to the PTH mRNA 3'-UTR, which determines its stability.

Where is the "inverting factor" in hormone secretion from parathyroid cells?

Secretion of hormones and transmitters in the body fall into two general categories. In the majority of the secreting cells, including the presynaptic terminals in the nervous system, an increase in the extracellular calcium causes an increase in secretion. There are two notable exceptions to this general rule: the parathyroid cells and the renal juxtaglomerular cells, where an increase in extracellular calcium leads to a decrease in secretion. Because these two cell types have a cardinal role in a wide variety of physiological and pathophysiological functions, it is of great importance to understand the regulation of their hormone secretion process. A key element to such an understanding is the identification of the location of the "inverting step," which makes the parathyroid cells behave in a fashion contrary to most other secretory cells. Whole cell imaging studies strongly suggested that the inversion factor is between the changes in intracellular calcium concentration ([Ca2+]i) and the secretion of the hormone. Surprisingly, confocal calcium imaging of the parathyroid cells did not support this dogma. It revealed that the interior of the parathyroid cell is a nonhomogeneous medium and that an increase in the extra-cellular calcium concentration produces changes in [Ca2+]i, in both the same and opposite directions, in different parts of the parathyroid cell.

Parathyroid hormone gene expression in Hyp mice fed a low-phosphate diet.

BACKGROUND: The murine analogue of X-linked hypophosphataemia is the Hyp mouse; it has chronic phosphate depletion from an inherited defect of renal tubular reabsorption. Phosphate directly regulates the parathyroid (PT) in normal rats and it is of interest whether this regulation is intact in Hyp mice. METHODS: Hyp mice were fed either a low-phosphate diet or control diet and PTH mRNA levels were measured. In addition changes in NMR-visible kidney and muscle intracellular phosphate potentials in normal and Hyp mice were determined. Mice were maintained on a low-phosphate (0.02%) or normal-phosphate (0.6%) diet for 24 and 72 h. RESULTS: On the normal diet, Hyp mice had hypophosphataemia, normocalcaemia, and normal PTH mRNA levels. Phosphate deprivation for 72 h led to a profound fall in plasma phosphate, a slight but significant rise in plasma calcium, and a dramatic decrease in PTH mRNA, similar to that of normal mice fed this diet. Changes in kidney and muscle intracellular phosphate measured by NMR spectroscopy were not affected by diet or genotype. CONCLUSION: Dietary phosphate deprivation decreased Hyp mice PTH mRNA levels and caused no change in intracellular phosphate potentials. Therefore Hyp mice parathyroids' adapt appropriately to phosphate deprivation albeit at a lower threshold compared to normal mice.

Regulation of parathyroid cell proliferation.

The parathyroid normally has very few cells in mitosis but it retains the potential to proliferate. In-vivo studies in rats have demonstrated that hypocalcaemia and high serum phosphate both lead to an increase in the number of proliferating cells, which is relevant to the increased parathyroid cell proliferation in chronic renal failure. Hypophosphataemia and 1,25(OH)2D3 decrease parathyroid cell proliferation. Genetic factors have been defined for multiple endocrine neoplasia which includes parathyroid cell hyperplasia, and in some parathyroid adenomas there are genetic rearrangements.

Calcium, phosphate, vitamin D, and the parathyroid.

The main factors which regulate parathyroid hormone (PTH) production are calcium, phosphate, vitamin D, and estrogens. Hypocalcemia leads to increased PTH secretion in seconds and minutes, gene expression in hours, and parathyroid (PT) cell number in weeks and months. Hypercalcemia leads to a decrease in PTH secretion by its action on the PT cell calcium receptor and no decrease in PTH mRNA levels. There is now convincing evidence that phosphate regulates the PT, independent of its effect on serum calcium and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. In vivo in rats hypophosphatemia markedly decreases PTH mRNA and serum intact PTH levels, independent of its effect on serum calcium and 1,25(OH)2D3. Clinical studies also indicate that phosphate regulates the PT independent of its effect on calcium and 1,25(OH)2D3; 1,25(OH)2D3 itself has a marked effect on the PT, where it decreases PTH gene transcription by a direct action on the PT. The application of basic science findings of how calcium, phosphate, and 1,25(OH)2D3 regulate the PT has led to an efficient and safe prescription for the management of the secondary hyperparathyroidism of chronic renal failure, which is the maintenance of a normal serum calcium and phosphate and the careful use of 1,25(OH)2D3.