A CD4-like molecule can be expressed in vivo in human parathyroid.

The expression of several epitopes of CD4, a molecule usually restricted to a subset of T-lymphocytes, was observed after immunofluorescent labeling of frozen-cut sections of human parathyroid. Seven samples obtained from three subjects presenting adenomas and from seven renal insufficiency patients with secondary or tertiary hyperplasia were found to express this molecule. Dot enzyme-linked immunosorbent assay, polyacrylamide gel electrophoresis, and Western blotting were used to characterize this peptide in cytosol and membrane fractions of these glands. These studies confirmed, in the parathyroids found positive in immunofluorescence, the presence of a protein with a mol wt similar to that of lymphocyte-derived CD4.

Parathyroid glands in chronic aluminum intoxication.

In chronic renal failure, aluminum overload may influence parathyroid function. In a study of possible aluminum-induced parathyroid abnormalities, parathyroid glands from nine parathyroidectomized patients on hemodialysis were examined by light and electron microscopy and by X-ray microanalysis. Aluminum overload was assessed by the presence of stainable aluminum (aluminum surface, 23.3% +/- 11% of total surface) in bone biopsy specimens. The mean plasma aluminum concentration was 7.7 +/- 1.9 mumol/L. All patients but one had elevated plasma concentrations of immunoreactive parathyroid hormone as well as osteitis fibrosa. The aluminum concentrations in bone and parathyroid gland from these patients were significantly higher than those in tissue from patients on hemodialysis without stainable bone aluminum. Abundant aluminum deposits were present in parathyroid chief cell cytoplasm in lipoid bodies, lipofuscin granules, and mitochondria. These cells exhibited features of active hormonal synthesis and contained numerous secretory granules. The data show that in the parathyroid glands of these aluminum-intoxicated patients the presence of aluminum deposits neither induced cellular damage or chief cell necrosis nor interfered with the production of parathyroid hormone.

DNA analysis and parathyroid pathology.

The nuclear DNA content of 85 parathyroid glands (4 carcinomas, 39 adenomas, 21 secondary parathyroid hyperplasias, and 21 normal parathyroid glands) were determined by flow cytometric analysis. All normal parathyroid glands, 85% of the adenomas, and 83.3% of the secondary hyperplastic glands had DNA indices within values of 0.85-1.1. Paraffin-embedded fixed glands showed less DNA staining than that found with fresh or normal glands. Glands from patients with carcinoma showed DNA indices outside the normal DNA index range. When the percent of nuclei within the G0/G1 phase of the cell cycle was compared between the study groups, highly significant results were found. While patients with secondary hyperplasia showed a similar distribution to the normal glands studied, only 48% of primary adenomas showed over 80% of cells within the G0/G1 region. A clear subgroup of adenomas was defined with more rapidly cycling tetraploid cells, and showing classical adenoma pathology. This group showed negative correlation with gland weight, plasma calcium, and ionized calcium. These findings suggested that a different etiology of the disease process occurs between secondary hyperplasia and parathyroid adenoma. Such abnormal adenomas may form a group worthy of long-term follow-up.

Immunohistochemical localization of parathyroid hormone-related protein in parathyroid adenoma and hyperplasia.

Parathyroid hormone-related protein (PTHrP) is invoked as the cause of humoral hypercalcaemia of malignancy (HHM); it is contained in the keratinocyte layer of normal skin; and there is evidence that is is produced by fetal parathyroids. Antibodies against synthetic PTHrP peptides have been raised in rabbits and sheep. This immunohistochemical study has found that primary parathyroid adenomata and hyperplastic glands from patients with chronic renal failure stain positively with antisera against PTHrP(1-34) and PTHrP(50-69). Primary hyperplastic glands are negative. No staining with anti-PTHrP(106-141) antiserum could be detected immunohistochemically in any of the parathyroid adenomata or hyperplasia.

Measurement of parathyroid hormone-related protein in extracts of fetal parathyroid glands and placental membranes.

A radioimmunoassay based on an antiserum to human parathyroid hormone-related protein PTHrP(1-16) was used with PTHrP(1-34) standard to measure the concentration of immunoreactive PTHrP in extracts of fetal parathyroid glands from lambs and calves and also placental membranes obtained from several species, including man. Dilution curves from these sources were parallel to those obtained for PTHrP(1-34) standard. It was demonstrated that this parallelism was not the result of tracer damage caused by enzymic activity in the tissue extracts. Extracts of human placental membranes were subjected to high-pressure liquid chromatography with a linear acetonitrile gradient. Co-elution of cytochemical biological activity with 125I-labelled PTHrP(1-34) was noted. These results provide further evidence for both the fetal parathyroid glands and the placenta containing material resembling PTHrP which may be responsible for sustaining the activity of the placental calcium pump which maintains the fetus hypercalcaemic relative to its mother.

Immunohistochemical localization of chromogranin A in normal tissues from laboratory animals.

To analyze the distribution of Chromogranin A in endocrine cells of various species of laboratory animals (dog, gerbil, guinea pig, hamster, monkey, mouse, and fetal, neonatal, and adult rats), normal tissues were stained immunohistochemically with polyclonal anti-bovine Chromogranin A antiserum (SP-1). Selected tissues (pituitary, adrenal, thyroid, parathyroid, pancreas, brain, peripheral nerve, stomach, small and large intestine, bone marrow, spleen, thymus, lymph node, and liver) from these species and from the rabbit were stained with two monoclonal anti-human Chromogranin A antibodies (LK2H10 and PHE5) to compare the immunoreactivities of the monoclonal antibodies and polyclonal antiserum. Staining with the polyclonal antiserum (SP-1) resulted in a broader spectrum of immunoreactivity but had more nonspecific background staining than either monoclonal antibody. Immunoreactivity and staining intensity with SP-1 varied between species, but most endocrine tissues (pituitary cells in the anterior and intermediate lobes, thyroid "C" cells, adrenal medulla, parathyroid, pancreatic islets, and enterochromaffin cells) from most species stained positively. In some species, pancreatic alpha cells stained more intensely, and two populations of adrenal medullary cells with different staining intensities were observed. Sciatic nerve (axonal area) was immunoreactive with monoclonal antibodies and/or the polyclonal antiserum in several species. The spectrum of immunoreactive tissues from fetal and neonatal rats increased with age. There was good cross-reactivity between species with SP-1, but not with either LK2H10 or PHE5. These results indicate that many endocrine cells with secretory granules in laboratory animals express Chromogranin A and that a polyclonal antiserum, such as SP-1, is more sensitive in detecting this protein in various species than monoclonal antibodies such as LK2H10 or PHE5.

Glycoprotein nature of dopamine D1 receptors in the brain and parathyroid gland.

Dopamine D1 receptors can be covalently labeled with the photo-affinity ligand (+-)-7-[125I]iodo-8-hydroxy-3-methyl-1-(4-azidophenyl)-2,3,4,5-tetrah yd ro-1H-3-benzazepine ([125I]IMAB) and visualized following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. In brain membranes, [125I]IMAB labels a polypeptide of apparent Mr approximately equal to 74,000 as the major ligand binding subunit of D1 receptors and two minor polypeptides of Mr approximately equal to 64,000 and 52,000. In contrast, [125I]IMAB labels a single polypeptide of apparent Mr approximately equal to 64,000 in bovine parathyroid glands. In this study, the carbohydrate nature of dopamine D1 receptors from the brain and parathyroid gland were examined using specific exo- and endoglycosidases and lectin affinity chromatography. [125I]IMAB-labeled brain and parathyroid D1 receptors were sensitive to treatment with the exoglycosidases neuraminidase or alpha-mannosidase, suggestive of the existence of terminal sialic acid and oligomannose residues. Photolabeled D1 receptor polypeptides are not however, associated with distinct populations of complex-type or high mannose-containing carbohydrate chains because 1) wheat germ agglutinin and concanavalin A lectin chromatography of solubilized and photolabeled neuronal D1 receptors followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed no differences in the electrophoretic mobility of column pass-through and specifically eluted [125I]IMAB-labeled polypeptides, and 2) [125I]IMAB-labeled D1 receptors specifically bound to and eluted from concanavalin A-Sepharose were neuraminidase sensitive, indicative of the colocalization of oligomannose- and complex-type glycans. Removal of these terminal glycan residues did not affect the binding of [3H]SCH 23390 to dopamine D1 receptors. Complete N-linked deglycosylation of photolabeled D1 receptors from both the brain and parathyroid with peptide N-glycosidase F resulted in the migration of a single major labeled polypeptide of apparent Mr approximately equal to 46,000. These data suggest that, despite differences observed in the electrophoretic mobility and glycosylation patterns of brain and parathyroid D1 receptor polypeptides, the protein backbones of central and peripheral dopamine D1 receptors display similar if not identical molecular weights.

Microfluorometric measurements of cytoplasmic calcium in chief and oxyphil parathyroid cells of adenomatous and hyperplastic glands and of normal-sized glands associated with adenomas.

The effects of extracellular calcium on the cytoplasmic Ca2+ concentration (Ca2+i) were studied by dual-wavelength microfluorometry in individual human parathyroid cells obtained from adenomatous glands and normal-sized glands associated with adenomas in hypercalcemic hyperparathyroidism (HPT), as well as from enlarged glands of patients with uremia with HPT. In comparison with the normal parathyroid tissue, chief cells of the adenomatous and hyperplastic glands showed significantly lower Ca2+, and also right-shifted responses of Ca2+i to increases in the extracellular calcium concentration within the 0.5 to 3.0 mmol/L range. This pathophysiologic disturbance apparently was independent of the cell size. Oxyphil cells of nodules from the hyperplastic glands had lower Ca2+i and responded less to increments in extracellular Ca2+ than the chief cells from the surrounding parts of the same glands. Also the chief cells from the normal-sized glands associated with single adenomas exhibited a disturbance of the regulation of Ca2+i, which was less pronounced than that in the cells of the adenomas. These findings support the presence of relative calcium insensitivity of Ca2+i in chief and oxyphil parathyroid cells from adenomatous and hyperplastic glands. This derangement may also be found in all parathyroid glands of individuals with adenomatous HPT.

Immunocytochemical localization of vitamin D-dependent calcium-binding protein (calbindin) in thyroid parafollicular cells of guinea pig.

The presence of the 28K vitamin D-dependent, calcium-binding protein (28K calbindin) was investigated by immunocytochemistry in normal thyroid glands and parathyroid glands of rats, guinea pigs, rabbits and men, as well as in human thyroid medullary carcinomas and human parathyroid adenomas. In addition, thyroid glands and parathyroid glands of rats and guinea pigs were studied after treatment with vitamin D3 injected intramuscularly at a total dose of 1.2 x 10(6) IU per 100 g body weight. 28K calbindin was found exclusively in parafollicular cells of guinea pigs and never in those of other species investigated. It was present predominantly in the cytoplasm and in lower concentration in nuclei. After vitamin D3 treatment, increased immunoreactivity of 28K calbindin was observed in the cytoplasm and, even more pronounced, in the nuclei. In normal parathyroid cells and in parathyroid tumors and medullary thyroid carcinomas, 28K calbindin was not demonstrable. Our findings suggest an important function of calbindin in the cellular calcium processing of parafollicular cells of guinea pigs.

Microinjected Xenopus oocytes secrete mature, biologically active parathyroid hormone.

Xenopus oocytes have been shown to faithfully translate, process, and secrete a number of secretory proteins after the injection of heterologous mRNAs. The oocyte has the capacity to perform a variety of posttranslational protein modifications but has been reported to be incapable of carrying out certain two-step cleavages which proceed via propeptide intermediates. We examined the ability of the oocyte to process preproPTH after the injection of parathyroid mRNA. Microinjected oocytes secreted material which could be detected in a sensitive cytochemical bioassay for PTH. This activity paralleled that of the PTH standard in the assay and was entirely eliminated by a competitive inhibitor of PTH binding, by preincubation with an anti-PTH antiserum, and by coinjecting oocytes with an oligonucleotide mixture complementary to PTH sequences. Immunoprecipitable proPTH and PTH were present in oocyte homogenates, but oocyte-conditioned medium contained only mature PTH(1-84). We conclude that the Xenopus oocyte is capable of accurately processing preproPTH to the mature secretory form of the peptide.

Hyperparathyroidism and abnormal calcitriol metabolism in the spontaneously hypertensive rat.

Abnormalities of calcium metabolism and of its two principal regulating hormones, parathyroid hormone and 1,25-dihydroxyvitamin D3 (calcitriol), have been reported in the spontaneously hypertensive rat (SHR). Reports of abnormal calcitriol metabolism in the SHR by several groups have not provided measurements of tissue calcitriol receptors. Similarly, few data are available as to the parathyroid status of the SHR. In the present study, circulating calcitriol levels and intestinal and parathyroid gland calcitriol receptor status were determined in male SHR and in Wistar-Kyoto (WKY) rats. Parathyroid status was investigated by determination of parathyroid gland mass together with tissue micromorphometry and by quantitative histology of bone as a measure of the biological action of parathyroid hormone. Circulating calcitriol levels were reduced in the 11-week-old SHR compared with the WKY rat (165 +/- 23 vs. 194 +/- 28 pmol/l, p less than 0.01, mean +/- SD). Calcitriol-free ratio was diminished and maximal specific binding capacity for calcitriol was increased in the SHR in parathyroid tissue (172 +/- 4.9 vs. 123 +/- 6.6 fmol/mg protein, p less than 0.01) and in intestinal mucosa with no change of receptor affinity. Plasma ionized calcium (1.29 +/- 0.05 vs. 1.45 +/- 0.35 mmol/l, p less than 0.05) and phosphate (1.5 +/- 0.26 vs. 2.4 +/- 0.03 mmol/l, p less than 0.05) were significantly lower in the SHR. Parathyroid gland mass was increased in the SHR (59 +/- 12 vs. 17 +/- 7 micrograms/100 g body wt, p less than 0.001) as a result of hyperplasia and not hypertrophy. Higher osteoclast numbers were observed in SHR bone (27.6 +/- 0.79 vs. 23.9 +/- 0.66 osteoclasts/mm2, p less than 0.01), suggesting increased parathyroid hormone activity. In summary, in the 11-week-old SHR we observed reduced circulating calcitriol levels together with increased tissue calcitriol receptor numbers, increased parathyroid gland mass, and histological evidence of hyperparathyroidism. It is possible that these abnormalities influence the development of hypertension in the SHR.

Effects of isoproterenol treatment on the parathyroid glands in golden hamsters of different ages, with special reference to the frequency of lipid droplets.

The frequency of lipid droplets in the parathyroid glands of young, adult and senile golden hamsters after treatment with isoproterenol was investigated. In the parathyroid glands of the young and senile animals the number of lipid droplets increased gradually by 1 h, and thereafter it remained almost unchanged at 3 h after administration. In the glands of adult animals it increased, at first rapidly and then gradually, by 3 h after administration. It is considered that in the parathyroid glands of the golden hamsters stimulated by isoproterenol there is a relationship between the number of lipid droplets and aging.

Different responses between the upper and the lower parathyroid gland in a state of secondary hyperfunction. A study on chronic renal failure by morphometry and nuclear DNA analysis.

The size of the parathyroid gland and the size, the numerical density and nuclear DNA-content of the parathyroid gland cells were evaluated in chronic renal failure (CRF) and revealed a difference between the upper and the lower glands in the manner of adaptation to a state of long-term hyperfunction, secondary to CRF. The parathyroid gland enlarged as a whole in CRF, an effect more marked in the lower gland, whereas individual parathyroid gland cell enlargement in CRF was mainly seen in the upper gland cells. The numerical density of the lower parathyroid gland cells was higher than that of the upper gland. Nuclear DNA-content of the parathyroid gland cells were increased in CRF and the lower gland tended to show hyperdiploid aneuploidy. These findings are probably related to the fact that parathyroid adenomas occur most often in the lower gland. The higher proliferative activity of the lower parathyroid gland in long-term hyperfunction may explain the higher risk for the lower gland in the occurrence of adenomas.

Further evidence for a parathyroid hormone-related protein in fetal parathyroid glands of sheep.

Extracts of ovine fetal parathyroid glands contained a substance in addition to parathyroid hormone (PTH) which could not be neutralized by antiserum against PTH in a cytochemical bioassay. This substance reacted similarly to human parathyroid hormone-related protein, the humoral hypercalcaemic factor associated with malignancy in man. The ovine fetal PTHrP may be responsible for maintaining the fetus hypercalcaemic relative to the mother.

Dopamine D1 receptors of the calf parathyroid gland: identification and characterization.

The dopamine D1 receptor was identified in the calf parathyroid gland. The binding of the selective D1 receptor antagonist [3H]SCH-23390 to membranes of calf parathyroid was specific, reversible, and saturable with a dissociation constant of approximately 200 pM and a receptor density of 30 fmol/mg of protein. Dopaminergic agonists and antagonists inhibited [3H]SCH-23390 binding in a concentration-dependent and stereoselective manner with an appropriate pharmacological specificity for D1 dopamine receptors. Moreover, potent dopaminergic agonists recognized two affinity forms of the receptor, one displaying high affinity for agonists, termed D1 High, and one with low affinity, D1 Low. The addition of the nonhydrolyzable guanine nucleotide guanyl 5'-yl-imidodiphosphate caused the complete transition of the agonist high affinity form (D1 High) of the receptor to one displaying only low affinity for agonists (D1 Low). Sodium ions, however, caused a approximately 5-fold decrease in the affinity of agonists at both D1 High and D1 Low. Virtually identical results were obtained on D1 receptor preparations of neural origin. The D1 receptor identified here appears to be the one responsible for the physiological effects on the parathyroid gland, because dopamine-stimulated cAMP accumulation is stereoselectively blocked by the D1 receptor antagonist SCH-23390 in dispersed cells of the parathyroid gland. Moreover, a series of nine dopaminergic antagonists and agonists shows an excellent correlation between their potency in [3H]SCH-23390 binding assays and their corresponding effects on cAMP accumulation. In the case of agonists, Ka for activation of cAMP accumulation agrees most closely with the agonist low affinity site in binding experiments. Specific [3H]spiperone binding to D2 dopamine receptors was not detected in this tissue and as such, the calf-parathyroid gland provides a model system in which to study the molecular characteristics of dopamine D1 receptor-mediated events.

A technique for the in vitro culture of human parathyroid gland tissue.

A technique for prolonged in vitro culture of human parathyroid tissue is described. Cells could be maintained in this monolayer system and were shown to continue releasing high levels of parathyroid hormone into their culture medium even after 140 days in culture. Furthermore, growth of fibroblasts, persistence of epithelial cells and parathyroid hormone release was demonstrated in cells derived from parathyroid tissue that had been cryopreserved for 2 years. The availability of viable and functioning human parathyroid tissue produced in this culture system may be of value in both auto- and allo-transplantation in patients with permanent hypoparathyroidism.