Direct regulation by calcium of cytoplasmic messenger ribonucleic acid coding for pre-proparathyroid hormone in isolated bovine parathyroid cells.

DNA complementary to bovine preproparathyroid hormone mRNA was cloned and labeled by nick translation in order to measure mRNA by molecular hybridization. Bovine parathyroid cells were maintained in primary tissue culture for periods up to 96 h at 0.5 mM, 1.25 mM, and 2.5 mM calcium, which was followed by extraction of cellular RNA. Levels of mRNA showed no differences at 0.5 or 1.25 mM calcium, but at high calcium levels, there was a reversible decrease that began at 16 h to a plateau at 30% of control after 72 h. These studies suggest that the glandular capacity to synthesize hormone may be at or near maximal at normal calcium, but at high calcium, there is a decrease over time in steady state levels of mRNA.

Direct release of parathyroid hormone fragments from functioning bovine parathyroid glands in vitro.

To determine the origin of circulating parathyroid hormone fragments, hormonal peptides released from bovine parathyroid tissue in a physiologically responsive in vitro "perifusion" system were analyzed by gel exclusion chromatography and region-specific radioimmunoassays. When exposed to low Ca++, the tissue released large quantities of intact hormone (parathyroid hormone 1--84) as well as amino- and carboxyl-terminal fragments. Fragments of the hormone were also released when the tissue was exposed to high Ca++, but the carboxyl fragments comprised a much greater proportion of the hormonal peptides released. Control experiments indicated that fragmentation of the hormone occurred within the gland and not after it was secreted. These experiments provide direct evidence, therefore, that release of fragments from the parathyroid gland may contribute to the immunologic heterogeneity of the hormone in the circulation.

Dimeric ("big") human placental lactogen. Immunological and biological activity.

Dimeric ("big") human placental lactogen has been isolated in near homogeneous form from placental tissue. It consists of a disulfide-linked (stable) form and a noncovalently associated (unstable) form of the native hormone. The two forms were separated by exposure to denaturing conditions and resolution by gel exclusion chromatography. Both forms retained immunological activity, ability to bind mammary membranes, and ability to induce mammary N-acetyllactosamine synthetase in vitro. On a molar basis, stable dimeric placental lactogen was more active than placental lactogen in the radioimmunoassay indicating that the immunological determinants on both monomeric units could bind to antibody. On a molar basis, stable dimeric placental lactogen was equally active with monomeric placental lactogen in competing for mammary gland membrane binding sites, indicating that only one active site in the molecule could interact with the membrane at a time. Stable dimeric placental lactogen was also active in an in vitro bioassay using the induction of N-acetyllactosamine synthetase. It is concluded that dimer formation does not alter the biologically active portion of the placental lactogen molecule. Since the carboxyl-terminal region (residues 182-191) is involved in the interchain disulfide bonds of dimeric placental lactogen, this portion of the molecule is probably not necessary for its biological activity.

Nucleotide sequence of the DNA complementary to avian (chicken) preproparathyroid hormone mRNA and the deduced sequence of the hormone precursor.

The nucleotide sequence of avian (chicken) prepro-PTH (prepro-PTH) mRNA was determined from a 2.3-kilobase fragment of complementary chicken parathyroid DNA cloned in E. coli MM 924. Northern blot analysis of chicken parathyroid mRNA, using both bovine and chicken cDNA probes, showed that the mRNA (2.3 kilobases) for chicken hormone precursor was approximately 3 times the size of mRNA for mammalian prepro-PTH. Cleavage of the cloned DNA with restriction endonuclease Pstl resulted in three fragments, each of which was subjected to sequence determination. The hormone sequence deduced from the DNA showed that chicken prepro-PTH mRNA encoded a 119-amino acid precursor which included a 25-amino acid signal sequence, a six-residue prohormone peptide, and an 88-amino acid hormone. The hormonal peptide was four residues longer than all known mammalian homologs and included gene deletions and insertions. There was significant homology of sequence in the biologically active 1-34 region with mammalian hormones, but much less in the middle and carboxyl-terminal regions. This is the first nonmammalian PTH sequence to be determined and should prove useful in studying evolution of the gene as well as structure-function relationships of the hormone.

1,25-Dihydroxyvitamin D3 has opposite effects on the expression of parathyroid secretory protein and parathyroid hormone genes.

Previous studies have shown that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] decreases levels of mRNA for prepro-PTH as well as PTH secretion after chronic exposure (24-48 h) of parathyroid cells in tissue culture. We have now extended these studies to determine the effects of the vitamin D3 metabolite on parathyroid secretory protein (PSP) gene expression. Primary cultures of bovine parathyroid cells were incubated with 10(-8) M 1,25-(OH)2D3 for periods of time ranging from 24-72 h. As observed in earlier experiments, prepro-PTH mRNA decreased to less than 50% of the control value after 72 h. In marked contrast, PSP mRNA showed a 2.5-fold increase by 24 h and greater than 7-fold stimulation by 72 h. In the same studies, PTH secretion was suppressed (to 60% of control), while PSP secretion was increased by 40% over control values. Exposure to high (2.5 mM) or low (0.5 mM) calcium had no effect on PSP mRNA, even though low calcium stimulated the secretion of PSP while high calcium suppressed secretion. These studies showed that 1,25-(OH)2D3 has opposite effects on the gene expression of PSP and PTH in bovine parathyroid cells in tissue culture.

Regulation by calcium and 1,25-(OH)2D3 of cell proliferation and function of bovine parathyroid cells in culture.

The effects of high calcium and 1,25-(OH)2D3 on parathyroid cell growth, PTH secretion, and steady-state levels of pre-proPTH mRNA in proliferating bovine parathyroid cells were examined. Cells were established in primary tissue culture and then tested in passages 2 and 5. Cell proliferation was suppressed by 10(-9)-10(-7) M 1,25-(OH)2D3 but not by high calcium (2.5 mM). Cells at passages 2 and 5 were grown to subconfluence and then exposed for 72 h to 2.5 mM calcium or 10(-7) M 1,25-(OH)2D3. Pre-proPTH mRNA was decreased to approximately 50% of control by 2.5 mM calcium compared with 0.3 and 1.0 mM calcium. PTH secretion, as tested by low calcium stimulation for 1 h at the end of 72 h incubation, was inhibited by 50% in cells that had been exposed to high calcium compared with control. Incubation with 10(-7) M 1,25-(OH)2D3 caused a decrease in the levels of pre-proPTH mRNA and PTH release to 50% of control at 72 h. These results suggest that cultured bovine parathyroid cells, at least in early passages, have responses to high calcium and 1,25-(OH)2D3 similar to those in primary nonproliferating cultures studied earlier and that 1,25-(OH)2D3 inhibits the proliferation of parathyroid cells in a dose-responsive fashion.

Suppression by 1,25(OH)2D3 of transcription of the pre-proparathyroid hormone gene.

When bovine parathyroid cells in culture are exposed to the active vitamin D metabolite 1,25(OH)2D3, a significant decrease in the steady-state levels of pre-proparathyroid hormone (pre-proPTH) mRNA occurs. The possibility that the fall in specific mRNA is due to a decrease in rate of transcription of the PTH gene was examined in this study. In the presence of 1,25(OH)2D3, there was a rapid and steady decline in PTH gene transcription rate which fell to a minimum of 10-15% of control at 24 h. The effect was observed at physiological levels (10(-11)M) and was also fully reversible.

Identification of "big" human placental lactogen in placenta and serum.

Because of increasing evidence for the heterogeneity of polypeptide hormones, studies of the molecular species of human placental lactogen (hPL) were initiated. When extracts of freshly delivered human placentas were passed over Sephadex G-100 in 0.05M ammonium carbonate, three immunoreactive peaks were detected. In addition to a peak corresponding to native hPL (Kav = 0.39) and one in the void volume, a consistent peak which eluted before hPL (Kav = 0.20) was present. The latter represented 2-25% of total hormonal activity and could be rerun without significant conversion to hPL. In 8M urea, the peak continued to behave as a large molecular weight form on both Sephadex chromatography and on polyacrylamide disc gel electrophoresis. Extraction procedures at both neutral and alkaline pH produced similar quantities of the larger material. [125I]iodo-hPL was not converted to the larger form by the conditions of extraction or analysis. These properties are consistent with a larger molecular weight, non-aggregated form of hPL. In comparison with the native hormone, the idsplacement curves for the larger form were parallel in radioimmunoassay studies. Sera obtained from pregnant women during various stages of gestation also showed consistent evidence for a large molecular weight form of the hormone. These observations provide direct evidence, both in placental tissue and in serum for "big" hPL.

1,25-Dihydroxyvitamin D3 suppresses parathyroid hormone secretion from bovine parathyroid cells in tissue culture.

To determine whether 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] regulates PTH secretion, we have tested its effects in both short term incubations (30-120 min) and long term primary cell cultures (24-96 h) of bovine parathyroid cells. In short term incubations, 10(-11)-10(-7) M 1,25-(OH)2D3 had no consistent effect on PTH secretion. In primary cultures of bovine parathyroid cells, significant suppression of PTH secretion occurred, as measured by both N-terminal and C-terminal PTH assays. Suppression of PTH secretion was dose dependent when 10(-11), 10(-9), and 10(-7) M 1,25-(OH)2D3 were tested for 48 h in culture, and the effects of 10(-7) M, 1,25-(OH)2D3 were noted as early as 24 h. Reversal of suppression of PTH secretion was observed after an additional 48 h in the absence of 1,25-(OH)2D3. Other studies from our laboratory have demonstrated that 1,25-(OH)2D3 suppresses levels of pre-pro-PTH mRNA in cultured bovine parathyroid cells, and we found a strong correlation at 48 h between the decrease in PTH release and that in mRNA. We conclude that 1) 1,25-(OH)2D3 suppresses PTH secretion rates in a dose-dependent manner in cells grown for 24-48 h in culture, but does not have a significant effect on short term PTH release (30-120 min); 2) cultured cells exhibiting suppression by 1,25-(OH)2D3 demonstrate nearly full recovery of PTH secretion after an additional 48 h in the absence of added 1,25-(OH)2D3; and 3) PTH secretion closely parallels levels of pre-pro-PTH mRNA in cultured cells, suggesting that the observed effects of PTH secretion reflect, at least in part, suppression of synthesis of PTH by 1,25-(OH)2D3.

Interaction between calcium and 1,25-dihydroxyvitamin D3 in the regulation of preproparathyroid hormone and vitamin D receptor messenger ribonucleic acid in avian parathyroids.

Regulation of prepro-PTH and vitamin D receptor (VDR) mRNAs in the parathyroid glands was studied in chickens in vivo. The birds were raised to 21 days of age on a vitamin D-deficient diet with 1% calcium and 0.65% phosphorous. At the end of this period, the chicks exhibited marked hypocalcemia and enlarged parathyroid glands. In three separate trials, the birds were repleted for 6 days with vitamin D and different dietary calcium and phosphate concentrations, with 2 micrograms/kg 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and different dietary calcium concentrations (0.5%, 1.0%, or 1.8%), or with 2 or 10 micrograms/kg 1,25-(OH)2D3 and 0.6% or 1.9% calcium or were kept vitamin D3 deficient and fed 0.5%, 1.0%, or 1.8% dietary calcium. Vitamin D treatment when combined with a high level of dietary calcium resulted in an increase in plasma calcium from 6 mg/dl to greater than 10 mg/dl, a decrease in PTH mRNA of 65%, and a 6- to 8-fold increase in VDR mRNA. In another experiment in which no vitamin D source was given and the diets contained increasing levels of dietary calcium, plasma calcium increased significantly (5.5 vs. 7 mg/dl), while PTH mRNA decreased by 40% and VDR mRNA increased by 60%. Neither parathyroid gland weight nor total RNA was significantly affected. When chicks were repleted with 1,25-(OH)2D3, the increase in plasma calcium and VDR mRNA and the decrease in PTH mRNA were considerably more pronounced than those in the absence of the vitamin D source. Furthermore, in the presence of the hormone, parathyroid weight and total RNA decreased significantly with increasing concentrations of dietary calcium. When the chicks were repleted, respectively, with the two levels of 1,25-(OH)2D3, a marked positive interaction was evident between the hormone and dietary calcium in affecting levels of PTH and VDR mRNA. These results suggest that both 1,25-(OH)2D3 and calcium participate in the regulation of PTH and VDR gene transcription in the avian parathyroid gland. Whereas the action of 1,25-(OH)2D3 requires a minimal level of dietary calcium, calcium affects PTH and VDR gene transcription even in the absence of any vitamin D source.

Renal and adrenal adenosine 3',5'-monophosphate production and corticosteroid secretion in response to synthetic chicken parathyroid hormone-(1-34).

The activity of synthetic chicken (c) PTH-(1-34) amide was tested in dispersed chicken and rat kidney and adrenocortical cells. In the adrenal cells the effect of intact cPTH was also evaluated. In chicken kidney cells, the time- and dose-response patterns of cAMP production were similar for cPTH-(1-34) amide and human (h) PTH-(1-34), whereas rat kidney cells were considerably more sensitive to hPTH-(1-34) than to cPTH-(1-34) amide. The agonist effects of both hPTH-(1-34) and cPTH-(1-34) amide in kidney cells were inhibited by the bovine PTH-(3-34) analog. In chicken adrenocortical cells, cPTH-(1-34) amide stimulated cAMP production and steroid secretion. This action of the peptide was inhibited by bovine PTH-(3-34) and hPTH-(1-34), which by themselves showed no agonist effects. The maximal response of steroid secretion to cPTH-(1-34) amide was significantly lower than that to ACTH, but intact cPTH (supplied as a semipurified parathyroid extract) stimulated steriodogenesis to the same extent as ACTH. In rat adrenocortical cells, intact cPTH stimulated both cAMP formation and steriodogenesis, but cPTH-(1-34) amine showed no agonist effect. The action of the intact hormone in the rat adrenal could be inhibited by cPTH-(1-34) amide. The present results demonstrate the interaction of cPTH-(1-34) with kidney and adrenocortical cells of either chicken or rat. The cAMP and steroidogenic responses of the adrenocortical cells to PTH appear to be dependent (completely in the rat and partially in the chicken) on some sequence beyond the 1-34 region.

Secretion of parathyroid hormone by abnormal human parathyroid glands in vitro.

The secretory response of abnormal parathyroid glands obtained surgically from eleven patients with primary and secondary hyperparathyroidsm was tested in vitro. Short term flask studies were used to measure release of parathyroid hormone (PTH) at high (3.0 mM) and low (0.5 mM) calcium. Of eight adenomas, all but one showed increased release of hormone when exposed to low calcium ("responsive to calcium"), the degree of stimulation at three hours ranging from 15 to 209%. By comparison, two normal human glands were stimulated an average of 180%. Glands from three patients with secondary hyperplasia were also responsive to calcium. Thus, parathyroid glands from patients with primary and secondary hyperparathyroidism were characterized by a spectrum of responsiveness to calcium, and absolute "autonomy" was unusual. Even at high calcium concentrations, hormone release persisted at a low but definite level ("basal secretion"). The total number of functioning parathyroid cells is therefore a principal determinant in the oversecretion of PTH in hyperparathyroidism.

Regulation of function of transplanted parathyroid glands in man.

The results of studies performed in nine patients who had undergone successful parathyroidectomy and gland transplantation are presented. Transplantation of parathyroid tissue to the forearm, performed for therapeutic reasons, provided a unique opportunity to sample parathyroid gland effluent and to assess secretory function in vivo. The relationship of calcium to immunoreactive parathyroid hormone (iPTH) release was studied during calcium and ethylenediamine tetraacetic acid (EDTA) infusions as well as dialysis against a low calcium bath (low calcium dialysis) in patients with chronic renal failure. Calcium infusions caused an abrupt decrease in hormone release down to a persistent base line within 30 minutes, whereas EDTA infusion caused a sharp increase which peaked between 30 to 60 minutes and returned towards base line despite continuation of the hypocalcemic stimulus. Low calcium dialysis caused an irregular release of hormone which appeared to deplete gland reserves during the period of the stimulus. Ready access to the venous effluent of the transplanted tissue makes this an excellent model for studying parathyroid physiology in man.

Effects of low-calcium diet on urine calcium excretion, parathyroid function and serum 1,25(OH)2D3 levels in patients with idiopathic hypercalciuria and in normal subjects.

We have used a low-calcium diet providing only 2 mg/kg (body weight) per 24 hours of calcium to distinguish between "renal" and "absorptive" idiopathic hypercalciuria. Sixteen of 27 hypercalciuric subjects excreted calcium in excess of intake during days seven, eight and nine of he diet, suggesting some element of renal hypercalciuria; however, all patients had low or normal serum PTH and urine cAMP levels. In general, fasting urine calcium was elevated in these 16 subjects and normal in the remaining 11, who conserved calcium more normally. SErum 1,25(OH)2D3 levels were the same in patients and normal subjects, even though PTH levels of the patients were below those of he normal subjects. Urine magnesium excretion and phosphorus excretion were both increased in the patients who excreted calcium in excess of intake. Our findings suggest that renal and absorptive hypercalciuria may not be distinct entities but rather the two extremes of a continuum of behavior. A uniform elevation of intestinal calcium absorption and a variable defect of renal calcium reabsorption could explain our results far better than the hypothesis of distinct absorptive and renal forms of hypercalciuria.

Postoperative chest percussion with postural drainage in obese patients following gastric stapling.

Vigorous respiratory therapy can prevent the development of postoperative pulmonary complications which occur with increased frequency after upper abdominal surgery. Obesity poses an additional risk factor. To study the effects of postoperative chest percussion with postural drainage (CPT), 53 consecutive patients undergoing Roux-en-Y gastric stapling procedures for treatment of morbid obesity were randomized to two groups. Both received identical postoperative respiratory care, except the study group received additional CPT. It was concluded that the addition of CPT to patients without prior chronic lung disease undergoing upper abdominal surgery caused patient discomfort, increased hospital cost, and failed to affect the incidence of postoperative pulmonary complications.

Transplantation of the parathyroid glands in man: clinical indications and results.

The physiologic function of human parathyroid autografts and allografts has not been demonstrated conclusively. During the past 30 months, we have transplanted parathyroid glands in 29 patients and tested their functional status. One immunosuppressed aparathyroid patient received a parathyroid allograft from a parent who previously had been his renal transplant donor. Twenty-seven patients with renal failure and secondary hyperparathyroidism received parathyroid autografts immediately after total parathyroidectomy, and one patient received a parathyroid autograft at the time of total parathyroidectomy for primary chief cell hyperplasia. At transplantation 1 times 1 mm. parathyroid pieces were grafted into the forearm musculature. Of 11 transplanted patients (one allograft and ten autografts) followed for 1 year, ten are normocalcemic; only two (autografted patients) are on supplemental calcium. Ten of the 29 patients have had biopsies performed, and all have had intact parathyroid architecture and intracellular secretory granules demonstrated by light and electron microscopy. Parathyroid hormone content in the grafted tissue of five patients was 179 plus or minus 118.8 ng. per milligram. In 11 random patients in whom bilateral measurements have been made, the parathyroid hormone content in the antecubital vein blood draining the grafted tissue has been markedly higher than that in the simultaneously sampled antecubital vein blood of the nongrafted arm. These data demonstrate that parathyroid autografts or allografts secret hormone and maintain a normal serum calcium in the host.

The successful transplantation of frozen parathyroid tissue in man.

Autologous parathyroid tissue frozen in liquid nitrogen for 6 weeks was transplanted successfully into the forearm muscle of an aparathyroid, uremic patient with renal osteodystrophy. Two years later the parathyroid graft was biopsied. Histologic evaluation revealed normal architecture, and there was ultrastructural demonstration of secretory granules within parathyroid cells. Physiologic function of the patient's graft was documented by the presence of a normal serum calcium concentration in the absence of replacement therapy and by a high level of parathyroid hormone in the venous blood draining the grafted muscle bed.

Pathogenesis and management of hypoparathyroidism and other hypocalcemic disorders.

Hypocalcemia frequently presents as an acute medical emergency or a chronic disorder which is difficult to control. Occasionally, it is found in routine blood screening tests when it is not anticipated. Recent developments in basic endocrine science have contributed greatly to our understanding and treatment of hypocalcemic disorders. The maintanance of a normal serum calcium concentration depends on the balanced actions of parathyroid hormone (PTH), vitamin D, and, to a lesser extent, calcitonin, Recent work on PTH secretion has defined the factors controlling its secretion in normal and abnormal states. In primary hypoparathyroidism, hormone secretion is decreased or absent, while in most other forms of hypocalcemia, secretion is stimulated secondarily by the hypocalcemia. However, acute or chronci disorders associated with hypomagnesemia may also decrease effective PTH secretion. Patients with the rare disorder, pseudohypoparathyroidism, have defects of hormone action and usually have elevated levels of PTH prior to therapy. Several forms of pseudohypoparathyroidism have been recognized, each representing a defect as a different site of PTH action. Calcitonin excess, as noted in medullary carcinoma of the thyroid, could theorectically cause hypocalcemia, but rarely does so. Vitamin D undergoes a series of two carefully controlled hydroxylation reactions leading to the final active metabolite, 1,25-hihyroxycholecalciferol. Chronic ingestion of certain drugs can lead to osteomalacia and hypocalcemia by potentiating the metabolism of vitamin D to inactive compounds. At least one form of rickets has been shown to result from a specific enzyme defect in the vitamin D pathway. Severe renal damage limits the conversion of vitamin D to its active form and contributes to vitamin D resistance. Current progress in the area depends on the development of procedures for the measurement of the metabolites in plasma and assessing the role of the vitamin (hormone) in normal and abnormal physiology. Although the therapy of acute hypocalcemia is usually readily accomplished, chronic hypocalcemia remains a very difficult therapeutic problem. Vitamin D, the hallmark of therapy, is a long-acting drug with a narrow therapeutic range. The complications of the disease and therapy are sometimes irreversible. The unraveling of vitamin D metabolism has led to the development of new therapeutic agents which might provide better relief of chronic hypocalcemic states. This review related new information about calcium homeostasis to the clinical situation encountered in the patient with hypocalcemia.

Secondary hyperparathyroidism in chronic renal failure. Pathophysiology and treatment.

The secondary hyperparathyroidism of chronic renal failure is a result of many factors which result in chronic stimulation of parathyroid hormone secretion and secondary hyperplasia of the parathyroid glands. The secretion and metabolism of parathyroid hormone and its fragments in chronic renal failure are complex and only partially understood. Constant elevated levels of PTH contribute to bone disease and other clinical features of chronic renal failure. Calcium supplementation, high calcium dialysis, control of plasma phosphate and judicious use of the vitamin D metabolites can, to a large extent, prevent or control the development of secondary hyperparathyroidism. Subtotal parathyroidectomy or total parathyroidectomy with autotransplantation is indicated in certain cases, sometimes on an emergency basis. Prevention of postoperative hypocalcemia requires careful management. Successful renal transplantation is usually associated with gradual healing of the bone disease and slow, but sometimes incomplete involution of the parathyroid hyperplasia.

The challenges of peripheral bone density testing: which patients need additional central density skeletal measurements?

Lower cost, portable, peripheral bone mass measurement devices are being increasingly utilized for widespread bone mass testing. These devices are being placed in traditional medical settings as well as nontraditional settings, such as pharmacies and grocery stores. Increased bone mass testing is appropriate at menopause in women who are undecided whether to begin systemic estrogen replacement. Women may decide to begin estrogen replacement if they are aware they have low bone mass and understand that bone mass will predictably decline after the menopause (1). With the approval of alendronate and raloxifene for the prevention of osteoporosis, even women who cannot or will not utilize estrogen replacement may be offered preventive interventions if they are identified as having low bone mass. More accessible bone mass measurements and more approved pharmacologic interventions will shift the focus of osteoporosis management to strategies that emphasize the reduction of lifetime fracture risk as well as current fracture risk. It will also be an impetus to focus on earlier identification and intervention (2-4).