Dopamine D1 receptors in human parathyroid gland: In vitro and in vivo studies

Dopamine receptors in human parathyroid were studied in vitro using ligand binding techniques. With 3H-piflutixol as ligand, binding characteristic of the dopamine D1 receptor was observed. Administration of apomorphine, flupenthixol or metoclopramide to normal controls or acute schizophrenic patients at doses producing significant alterations in serum prolactin concentrations did not alter serum parathyroid hormone (PTH) concentrations. Whilst D1 binding sites are present in human parathyroid, the measurement of PTH after administration of dopaminergic drugs is unlikely to provide a test of D1 receptor function in man.     

Endothelin receptors in human and guinea-pig gallbladder muscle

We measured contraction of muscle strips caused by endothelin (ET) isopeptides and binding of (125)I-ET-1 to muscle cell membranes prepared from human and guinea-pig gallbladders. Visualization of (125)I-ET-1 binding sites in tissue was performed by autoradiography. Results in human were similar to those in guinea-pig. ET-1 caused tetrodotoxin and atropine-insensitive contraction. The relative potencies for ET isopeptides to cause contraction were ET-1=ET-2>ET-3. ET-1 caused contraction was only slightly inhibited by BQ-123 (potent ET(A) receptor antagonist) and not by BQ-788 (potent ET(B) receptor antagonist). It was inhibited by the combination of both. Autoradiography localized (125)I-ET-1 binding to the smooth muscle layer. Binding of (125)I-ET-1 to muscle cell membranes was saturable and specific. Analysis of dose-inhibition curves demonstrated the presence of two classes of receptors. One class (ET(A) receptor) had a high affinity for ET-1 and ET-2 but a low affinity for ET-3, and the other (ET(B) receptor) a high affinity for ET-1, ET-2 and ET-3. These results demonstrate that similar to guinea-pig, human gallbladder possesses both ET(A) and ET(B) receptors cooperating to mediate muscle contraction.     

Calcium-binding proteins in the parathyroid gland. Detailed studies of parathyroid secretory protein

In order to identify calcium (Ca2+)-binding proteins in the parathyroid gland, we used electrophoretic blots of proteins separated by a two-dimensional nondenaturing/denaturing gel system and incubated them with 45Ca2+. Parathyroid secretory protein (PSP) and proteins with approximate molecular weights of 98,000, 88,000, 58,000, and 30,000 were noted to bind Ca2+ in cytosolic fractions from bovine parathyroid, adrenal, and pituitary glands. However, differences in the binding affinity and capacity of the various proteins were observed. PSP displayed a low affinity and high binding capacity for Ca2+. In the presence of 5 mM MgCl2 and 60 mM KCl, native PSP (immobilized on nitrocellulose filters) bound 7.5 mol of Ca2+/mol of protein monomer with an apparent Kd of 1.1 mM. Immunoblotting identified the association of PSP with parathyroid cell membranes in a Ca2+-dependent manner. This property, together with its heat stability, distinguished PSP from other cytosolic Ca2+-binding proteins which were identified. There was also evidence for a Ca2+-dependent protein-protein interaction (aggregation) of PSP present in a Nonidet P-40 extract of cell membranes. The high Ca2+ binding capacity of PSP and its Ca2+-dependent membrane association may be features that make PSP a potentially important protein in secretory cells.     

Endothelin receptors in light-induced retinal degeneration

Excessive light exposure leads to retinal degeneration in albino animals and exacerbates the rate of photoreceptor apoptosis in several retinal diseases. In previous studies we have described the presence of endothelin-1 (ET-1) and its receptors (ET-A and ET-B) in different sites of the mouse retina, including the retinal pigment epithelium, the outer plexiform layer (OPL), astrocytes, the ganglion cell layer (GCL), and vascular endothelia. After light-induced degeneration of photoreceptors, endothelinergic structures disappear from the OPL, but ET-1 and ET-B immunoreactivities increase in astrocytes. Here, we present novel observations about the course of light-induced retinal degeneration in BALB-c mice exposed to 1500 lux during 4 days with or without treatment with tezosentan, a mixed endothelinergic antagonist. Retinal whole mounts were immunostained with anticleaved caspase-3 (CC-3) serum to identify apoptotic photoreceptor cells within the outer nuclear layer (ONL). Glial activation was measured as glial fibrillary acidic protein (GFAP) immunoreactivity in retinal whole mounts and in Western blots from retinal extracts. Tezosentan treatment significantly reduced both the number of CC3-immunoreactive cells and GFAP levels, suggesting that inhibition of endothelinergic receptors could play a role in photoreceptor survival. Using confocal double immunofluorescence, we have observed that ET-A seems to be localized in bipolar cell dendrites, whereas ET-B is localized in horizontal cells. Our observations suggest the existence of an endothelinergic mechanism modulating synaptic transmission in the OPL. This mechanism could perhaps explain the effects of tezosentan treatment on photoreceptor survival.     

Immunocytochemical localization of parathyroid hormone in hamster parathyroid gland

The immunocytochemical localization of parathyroid hormone was examined in the hamster parathyroid gland by using the protein A-gold technique. Protein A-gold particles were concentrated over secretory granules, large secretory granules thought to be storage granules and Golgi vacuoles. No protein A-gold particles were detected over large vacuolar bodies and cisternae of the granular endoplasmic reticulum.     

Interaction of human parathyroid hormone-related peptide with parathyroid hormone receptors in clonal rat osteosarcoma cells

Synthetic peptides corresponding to the amino-terminal region of the human parathyroid hormone-related peptide (hPTHrp) were used to characterize the interaction of hPTHrp with parathyroid hormone (PTH) receptors in clonal rat osteosarcoma cells (ROS 17/2.8). Both hPTHrp-(1-34) and [Tyr40]hPTHrp-(1-40) showed full agonist activity in stimulating cyclic AMP accumulation in ROS cells; human PTHrp-(1-34) was approximately 2.5-fold as potent as hPTH-(1-34). Both [Tyr-40]hPTHrp-(3-40) and hPTH-(3-34) inhibited the cyclic AMP increase induced by either hPTHrp or PTH with parallel dose-inhibition curves. Binding to intact ROS cells of a 125I-labeled [Tyr40]hPTHrp-(1-40) (125I-[Tyr40]hPTHrp-(1-40)) which retains full biological activity was time- and temperature-dependent and reversible. Binding of 125I-[Tyr40]hPTHrp-(1-40) and 125I-labeled [Nle8, Nle18, Tyr34]bovine PTH-(1-34)NH2 to ROS cells was competed for, to the same extent and with the comparable potency, by either unlabeled hPTHrp or PTH peptides. The binding capacity and affinity of receptors in ROS cells were strikingly similar for hPTHrp and PTH. Affinity cross-linking with either radioligand resulted in high affinity, specific labeling of an apparently identical macromolecule centering at Mr = 80,000, which was detected in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in both reducing and nonreducing conditions. The data indicate that hPTHrp and PTH, their amino-terminal fragments at least, interact with the identical receptors with regard to affinity, capacity, specificity, and physicochemical characteristics in osteoblastic ROS 17/2.8 cells.     

Crystalline bodies in parathyroid gland cells ofRana temporaria L.

Summary Cytoplasmic and nuclear crystalline inclusions are described in parathyroid secretory cells of adult frogs (Rana temporaria L.) and their possible significance is discussed.We are indebted to Mr. Raynor L. Jones for his expert technical assistance and to the Science Research Council and the Central Research Fund of London University for awards enabling this work to be performed.     

Isolation and characterization of stem cells from the human parathyroid gland

Objectives: Somatic stem cells can be obtained from a variety of adult human tissues. However, it was not clear whether human parathyroid glands, which secrete parathyroid hormones and are essential in maintaining homeostasis levels of calcium ions in the circulation, contained stem cells. We aimed to investigate the possibility of isolating such parathyroid‐derived stem cells (PDSC). Materials and methods: Surgically removed parathyroid glands were obtained with informed consent. Cell cytogenetics was used to observe chromosomal abnormalities. Surface phenotypes were characterized by flow cytometry. Telomerase repeat amplification protocol (TRAP) assay was performed to observe the telomerase activity. RT‐PCR and real‐time PCR was was used to detect gene expressions. Real‐time calcium uptake imaging was performed for extent of calcium uptake and transmission electron microscopy and immunofluorecent staining for smooth muscle actin. Results: After enzymatic digestion and primary culture, plastic‐adherent, fibroblast‐like cells appeared in culture and a morphologically homogeneous population was derived from subsequent limiting dilution and clonal expansion. Karyotyping was normal and doubling time of clonal cell growth was estimated to be 70.7 ± 14.5 h (mean ± standard deviation). The surface phenotype of the cells was positive for CD73, CD166, CD29, CD49a, CD49b, CD49d, CD44, CD105, and MHC class I, and negative for CD34, CD133, CD117, CD114, CD31, CD62P, EGF‐R, ICAM‐3, CD26, CXCR4, CD106, CD90 and MHC class II, similar to mesenchymal stem cells (MSC). Detectable levels of telomerase activity along with pluripotency Sall4 gene expression were observed from the isolated PDSCs. Expression of calcium‐sensing receptor gene along with alpha‐smooth muscle actin was induced and cellular uptake of extracellular calcium ions was observed. Furthermore, PDSCs possessed osteogenic, chondrogenic and adipogenic differentiation potentials. Conclusions: Our results reveal that PDSCs were similar phenotypically to MSCs and further studies are needed to formulate induction conditions to differentiate PDSCs into parathyroid hormone‐secreting chief cells.     

Catalase-like crystals in parathyroid gland cells of Rana temporaria L.

Summary Crystalline inclusions in parathyroid gland cell nuclei of Rana temporaria were studied by electron microscopy using a specimen tilting stage. Images were analysed by optical diffraction. Results were compared with X-ray and electron microscopic data of trigonal bovine liver catalase to which a striking resemblance of the inclusions was found.We are grateful to Professor R. Mosebach (Giessen) for discussions, to the Deutsche Forschungsgemeinschaft for a grant (La 229/4) and instruments and to Messrs. Spindler & Hoyer, Göttingen and Messrs. Rank Precision Instruments, Nürnberg for putting apparatus at our disposal and performing diffraction photographs.     

Differential distribution of endothelin peptides and receptors in human adrenal gland

Summary Sub-type selective ligands revealed a differential distribution of endothelin (ET) receptors within human adrenal glands. High densities of ET_A receptors were localized, using [¹²⁵I]-PD151242, to the smooth muscle layer of the arteries, smaller vessels within the capsular plexus and to the secretory cells of zona glomerulosa (K _D=139.8±39.7,B _max=69.7±9.1 fmol mg⁻¹ protein, mean of 3 individuals±sem). ET_B receptors were present in the medulla (K _D=145.2±16.4,B _max=75.5±12.3), zona glomerulosa (K_D=100.6±35.1,B _max=63.1±10.0), fasiculata (K _D 145.1±162.,B _max=67.9±6.9) and reticularis (K_D=118.2±18.6,B _max=71.9±6.5). ET_B receptors were not detected within the smooth muscle of the vasculature. Messenger RNA encoding both sub-types was present in adrenals. ET-like immunoreactivity was localized to the cytoplasm of the endothelial cells from arteries supplying the gland and resistance vessels within the capsular plexus. Staining was also detected in these cells using anti-big ET-1 and less intensely with anti-big ET-2 antisera but not within cells within the cortex or medulla. Big ET-3-like immunoreactivity was localized to secretory cells of the medulla. Staining was not found using antiserum that could detect ET-3, suggesting further processing of big ET-3 may occur within the plasma, and that the cdrenals could be a source of ET-3. The presence of ET-1 was confirmed by high performance liquid chromatography and radioimmunoassay although ET-3 was not detected. The results suggest that ET-1 is the predominant mature isoform, which is localized mainly to adrenal vasculature, particularly the capsular plexus, and may contribute to blood flow regulation in the gland.     

Expression of adenylate cyclase-coupled osseous parathyroid hormone and parathyroid hormone-like peptide receptors in Xenopus oocytes.

Functional parathyroid hormone (PTH) and PTH-like peptide receptors were expressed in Xenopus laevis oocytes after injection of poly(A)+ RNA isolated from the rat osteogenic sarcoma cell line, UMR 106. Increases in cAMP were seen in individual oocytes in response to added bovine (b) PTH-(1-34) (10(-6) M), human (h) PLP-(1-34) (hPLP-(1-34), 10(-6) M), isoproterenol (10(-4) M), and forskolin (10(-4) M). Although both intracellular and extracellular cAMP levels were stimulated approximately 1.5-2-fold by these agonists, intracellular concentrations of cAMP were substantially higher than extracellular concentrations. Peak increases with bPTH-(1-34) occurred after a 30-min incubation with the hormone 48 h after oocyte injection. bPTH-(1-34) caused a concentration-dependent augmentation of cAMP in injected oocytes, and the in vitro antagonist hPLP-(3-34) produced dose-dependent inhibition of both bPTH-(1-34)- and hPLP-(1-34)-stimulated cAMP accumulation. Specific binding of PTH to oocyte membranes was also demonstrated 48 h after oocyte injection with UMR 106 cell mRNA. Following size fractionation of isolated UMR 106 poly(A)+ RNA by sucrose density gradients, mRNA directing the expression of both PTH- and PLP-stimulated cAMP in oocytes appeared in the 3.5-4.9-kilobase fraction. These results demonstrate that adenylate cyclase-coupled osseous PTH and PLP receptors can be expressed after injection of naturally occurring mRNA into Xenopus oocytes, that PTH- and PLP-stimulated increases in cAMP concentrations can be detected in individual oocytes injected with bone cell-derived mRNA, that PTH and PLP appear to cross-react at a common receptor after injection of UMR 106 cell mRNA into oocytes, and that size selection of mRNA encoding the PTH and PLP receptors can be achieved by density gradient centrifugation. These studies, therefore, indicate the potential usefulness of the Xenopus oocyte system in expression cloning of PTH and PLP receptor cDNAs and illustrate the feasibility of employing this system to examine the biology of PTH and PLP receptors.