Conformational studies of a bicyclic,lactam‐constrained parathyroid hormone‐related protein‐derived agonist

The N‐terminal 1–34 segments of both parathyroid hormone (PTH) and parathyroid hormone‐related protein (PTHrP) bind and activate the same membrane receptor in spite of major differences in their amino acid sequence. The hypothesis was made that they share the same bioactive conformation when bound to the receptor. A common structural motif in all bioactive fragments of the hormone in water/trifluoroethanol mixtures or in aqueous solution containing detergent micelles is the presence of two helical segments at the N‐ and C‐termini of the sequence. In order to stabilize the helical structures, we have recently synthesized and studied the PTHrP(1–34) analog [(Lys13–As p¹⁷, Lys26–As p³⁰)]PTHrP(1–34)NH₂, which contains lactam‐constrained Lys‐Asp side chains at positions i, i+4. This very potent agonist exhibits enhanced helix stability with respect to the corresponding linear peptide and also two flexible sites at positions 12 and 19 in 1:1 trifluoroethanol/water. These structural elements have been suggested to play a critical role in bioactivity. In the present work we have extended our conformational studies on the bicyclic lactam‐constrained analog to aqueous solution. By CD, 2D‐NMR and structure calculations we have shown that in water two helical segments are present in the region of the lactam bridges (13–18, and 26–31) with high flexibility around Gly¹² and Arg¹⁹. Thus, the essential structural features observed in the aqueous‐organic medium are maintained in water even if, in this solvent, the overall structure is more flexible. Our findings confirm the stabilizing effect of side‐chain lactam constraints on the α‐helical structure. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Conformational studies of parathyroid hormone (PTH)/PTH‐related protein (PTHrP) point‐mutated hybrids

The N‐terminal 1–34 segments of both parathyroid hormone (PTH) and parathyroid hormone‐related protein (PTHrP) bind and activate the same membrane receptor in spite of major differences between the two hormones in their amino acid sequence. Recently, it was shown that in (1–34)PTH/PTHrP segmental hybrid peptides, the N‐terminal 1–14 segment of PTHrP is incompatible with the C‐terminal 15–34 region of PTH leading to substantial reduction in potency. The sites of incompatibility were identified as positions 5 in PTH and 19 in PTHrP. In the present paper we describe the synthesis, biological evaluation, and conformational characterization of two point‐mutated PTH/PTHrP 1–34 hybrids in which the arginine residues at positions 19 and 21 of the native sequence of PTHrP have been replaced by valine (hybrid V²¹) and glutamic acid (hybrid E¹⁹), respectively, taken from the PTH sequence. Hybrid V²¹ exhibits both high receptor affinity and biological potency, while hybrid E¹⁹ binds weakly and is poorly active. The conformational properties of the two hybrids were studied in aqueous solution containing dodecylphosphocholine (DPC) micelles and in water/2,2,2‐trifluoroethanol (TFE) mixtures. Upon addition of TFE or DPC micelles to the aqueous solution, both hybrids undergo a coil‐helix transition. The maximum helix content in 1 : 1 water/TFE, obtained by CD data for both hybrids, is ∼ 80%. In the presence of DPC micelles, the maximum helix content is ∼ 40%. The conformational properties of the two hybrids in the micellar system were further investigated by combined 2D‐nmr, distance geometry (DG), and molecular dynamics (MD) calculations. The common structural motif, consisting of two helical segments located at N‐ and C‐termini, was observed in both hybrids. However, the biologically potent hybrid V²¹ exhibits two flexible sites, centered at residues 12 and 19 and connecting helical segments, while the flexibility sites in the weakly active hybrid E¹⁹ are located at position 11 and in the sequence 20–26. Our findings support the hypothesis that the presence and location of flexibility points between helical segments are essential for enabling the active analogs to fold into the bioactive conformation upon interaction with the receptor. © 1999 John Wiley & Sons, Inc. Biopoly 50: 525–535, 1999     

Molecular cloning and functional characterization of the canine parathyroid hormone/parathyroid hormone related peptide receptor (PTH1)

Parathyroid hormone (PTH) is a major mediator of calcium and phosphate metabolism through its interactions with receptors in kidney and bone. PTH binds with high affinity to PTH1 and PTH2, members of the superfamily of G protein-coupled receptors. In order to clone the canine PTH1 receptor, a canine kidney cDNA library was screened using the human PTH1 receptor cDNA and two clones were further characterized. The longest clone was 2177 bp and contained a single open reading frame of 1785 bp, potentially encoding a protein of 595 amino acids with a predicted molecular weight of 66.4 kD. This open reading frame exhibits >91% identity to the human PTH1 receptor cDNA and >95% identity when the putative canine and human protein sequences are compared. Competition binding following transfection of the canine PTH1 receptor into CHO cells demonstrated specific displacement of ¹²⁵I-human PTH 1-34 by canine PTH 1-34, human PTH 1-34, and canine/human parathyroid hormone related peptide (PTHrP) 1-34. Treatment of canine PTH1 receptor transfected cells, but not mock transfected cells, with these ligands also resulted in increased levels of intracellular cAMP. In contrast, the non-related aldosterone secretion inhibiting factor 1-35 neither bound nor activated the canine PTH1 receptor. Northern blot analysis revealed high levels of PTH1 receptor mRNA in the kidney, with much lower, but detectable, levels in aorta, heart, lung, prostate, testis, and skeletal muscle. Together, these data indicate that we have cloned the canine PTH1 receptor and that it is very similar, both in sequence and in functional characteristics, to the other known PTH1 receptors.     

Elevation of PTH and PTHrp Induced by Excessive Fluoride in Rats on a Calcium-deficient Diet

Study on the role of parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrp) in the process of skeletal fluorosis, involved especially in calcium deficiency, is rare. We evaluated the level of serum PTH and mRNA expression of PTHrp in femur when rats were exposed to excessive fluoride with low-calcium diet. Wistar rats (n = 60) was divided into four groups, a control group, fluoride group, low-calcium group, and low-calcium fluoride group. The fluoride groups were treated with fluoride by drinking tap water containing 100 mg F-/L. The low-calcium diet contained 0.05% calcium. Serum was collected in the first, fourth, eighth, and 12th of phase for the detemination of PTH and Ca²⁺. RNA extraction from femora was used to analyze the mRNA express of PTHrp, osteopontin (OPN), and osteocalcin (OCN) after 12 weeks of fluoride dosing. Results showed that serum PTH increased gradually with the extension of fluoride exposure, but Ca²⁺ decreased, both of which embodied a time-dependent relationship. Cotreatment of excessive fluoride with low-calcium diet largely stimulated the secretion of PTH. The low dietary calcium markedly increased mRNA expression of PTHrp in animals with fluoride treatment. Expression of OPN and OCN significantly increased in the rats treated with excessive fluoride and low-calcium diet. We demonstrated that fluoride by itself affected the body's calcium metabolism and stimulate the secretion of PTH. PTH may play an important role in anabolic effect of excessive fluoride on bone turnover of skeletal fluorosis and calcium deficiency exacerbated the action of PTH and PTHrp on the characteristic bone lesion of fluorosis.     

Expression - Level Dependent Activation of Recombinant Human Parathyroid Hormone/Parathyroid Hormone-Related Peptide Receptor: Effect of Human Parathyroid Hormone (1-34), (1-31), and (28-48)

Abstract

A stable recombinant Chinese hamster ovary (CHO) cell model system expressing the human type-1 receptor for parathyroid hormone and parathyroid hormone-related peptide (hPTH-R) was established for the analysis of human PTH (hPTH) variants. The cell lines showed receptor expression in the range from 10⁵ to 1.9xl0⁶ receptors per cell. The affinity of the receptors for hPTH-(l-34) was independent of the receptor number per cell (K<j = 8 nmol/1). The induction of cAMP by hPTH-(l-34) is maximal in clones expressing >2xl0⁵ receptors per cell and Ca⁺⁺ signals were maximal in cell lines expressing >1.4xl0⁶ receptors per cell. Second messenger specific inhibitors demonstrated that PTH-induced increases in intracellular cAMP and Ca⁺⁺ are independent and Ca⁺⁺ ions are derived from intracellular stores. The cAMP-specific receptor activator hPTH-(l-31) showed also an increase in intracellular Ca⁺⁺. Even in cell lines expressing more than 10 receptors per cell the Ca⁺⁺/PKC specific activator hPTH-(28-48) did not activate hPTH-Rs. Based on these results, synthesis of further derivatives of PTH is required to identify pathway-specific ligands for the type-1 hPTH-R.     

Gene structure,transcripts and calciotropic effects of the PTH family of peptides in <Emphasis Type="Italic">Xenopus</Emphasis> and chicken

Background  

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) belong to a family of endocrine factors that share a highly conserved N-terminal region (amino acids 1-34) and play key roles in calcium homeostasis, bone formation and skeletal development. Recently, PTH-like peptide (PTH-L) was identified in teleost fish raising questions about the evolution of these proteins. Although PTH and PTHrP have been intensively studied in mammals their function in other vertebrates is poorly documented. Amphibians and birds occupy unique phylogenetic positions, the former at the transition of aquatic to terrestrial life and the latter at the transition to homeothermy. Moreover, both organisms have characteristics indicative of a complex system in calcium regulation. This study investigated PTH family evolution in vertebrates with special emphasis on Xenopus and chicken.     

Recombinant Human Parathyroid Hormone Therapy (1-34) In an Adult Patient with a Gain-of-Function Mutation in the Calcium-Sensing Receptor-a Case Report

ObjectiveTo describe a case of hypocalcemia in a patient with a gain-of-function mutation in the calcium-sensing receptor that was undetected until adulthood and successfully treated with recombinant parathyroid hormone.MethodsThe clinical findings, laboratory data, and a review of the pertinent literature are presented.ResultsA 55-year-old woman was hospitalized and seen by the endocrinology consult service for hypocalcemia that was refractory to repeated doses of intravenous calcium gluconate. She expressed concern about chronic leg muscle cramps and paresthesias of the lips and fingertips. In addition, she had no history of neck surgery, neck irradiation, or any autoimmune disease. She was a well-appearing female with no dysmorphic features or skin changes. Laboratory tests revealed hypocalcemia, hyperphosphatemia, hypomagnesemia, and hypovitamino-sis D. Her parathyroid hormone concentration (PTH) was low at 14.2 pg/mL. Her PTH and calcium concentrations remained low despite repletion of magnesium and treatment with calcitriol and oral calcium replacement. A 24-hour collection for urinary calcium showed inappropriate hypercalciuria. Medical records showed her hypocalcemia to be chronic. Additionally, several family members had also complained of muscle cramps. A congenital cause of her hypoparathyroidism was considered, and genetic testing confirmed heterozygosity for a gain-of-function mutation in the calcium-sensing receptor gene associated with autosomal dominant familial isolated hypoparathyroidism (ADH). Treatment with subcutaneous recombinant human parathyroid hormone teriparatide (rhPTH [1-34]) 20 mcg twice daily for three days normalized her calcium and phosphorus concentrations.ConclusionrhPTH (1-34) is an effective treatment for patients with hypoparathyroidism due to gain-of-function mutations in the calcium-sensing receptor. ADH can be insidious in presentation and the diagnosis can be missed unless there is a high index of suspicion. (Endocr Pract. 2013;19:e24-e28)     

Parathyroid hormone co-stimulation of hepatocyte proliferation is sensitive to protein kinase A and calcium channel inhibitors

Parathyroid hormone (PTH) mobilises calcium in the hepatocyte, an effect which is abolished by verapamil and staurosporine. In our study parathyroid hormone was shown to act additively to dHGF in inducing hepatocyte DNA synthesis. It is also shown that PTH induced the production of inositol 1,4,5 trisphosphate (IP₃) andc-fos expression at early times in culture. Co-incubation of PTH and dHGF with ac-fos antisense oligodeoxynucleotide inhibited hepatocyte DNA synthesis, indicating that the additive effect of PTH is correlated with the induction ofc-fos. H-89, a PKA specific inhibitor, inhibited the PTH effect on IP₃ production as well as the PTH effect on hepatocyte DNA synthesis. Verapamil and staurosporine also inhibited the PTH effect in dHGF-induced DNA synthesis. Therefore it is suggested that PKA mediated at a great extent the co-stimulatory effects of PTH on hepatocyte proliferation via IP₃ production.     

Binding specificity of the ectodomain of the parathyroid hormone receptor

The parathyroid hormone (PTH)1 receptor is a member of the class B G protein-coupled receptor (GPCR) family and regulates bone and mineral metabolism of vertebrates. A truncated highly active parathyroid hormone fragment PTH (1-34) exerts stimulatory effects on the receptor and is used for treatment of osteoporosis. To study the interacting amino acids of the natural peptide ligand PTH (1-84) with the ectodomain of its receptor we used peptide micro arrays on solid cellulose membranes. The amino acids Arg20 and Trp23 within the identified core binding stretch PTH (20-26) were found to be most important for affinity to the ectodomain of PTH1R. Isothermal titration calorimetry and NMR spectroscopy allowed peptide binding studies in solution and verified peptide positions required for high affinity. With this combination of biochemical and biophysical methods we extend former findings on this essential interaction and can now provide a strategy to screen for optimized therapeutic peptides.     

Defective regulation of the cytosolic Ca2+ activity in parathyroid cells from patients with hyperparathyroidism

The parathyroid hormone (PTH) release and cytosolic Ca²⁺ activity were determined in normal bovine parathyroid cells and parathyroid cells obtained from patients with hyperparathyroidism (HPT). There was a sigmoid relation between the cytosolic Ca²⁺ activity and the extracellular calcium concentration between 0.5 and 6.0 mmol/l. The PTH release was inhibited in parallel with the rise in the cytosolic Ca²⁺ activity. Both the hormone release and the cytosolic Ca²⁺ activity were lower in cells from human adenomas and hyperplastic glands~ and in comparison with the bovine preparations these ceils had higher set points for the cytosolic Ca²⁺ activity and PTH release. There was a close correlation between the individual set points for the cytosolic Ca²⁺ activity and PTH release in a material containing both normal and pathological cells. The results indicate that the abnormal PTH release characteristic of HPT is due to a defective regulation of the cytosolic Ca²⁺ activity.     

Parathyroid hormone induced stimulation of calcium uptake by renal microsomes

Cortical and papillary microsomes prepared from feline kidneys perfused with parathyroid hormone (PTH) showed an enhanced ability to accumulate calcium (Ca⁺²). PTH was unable to stimulate Ca⁺² uptake into microsomes prepared from outer medulla. These data suggest that renal microsomes may be a valid model system for studying the action of PTH on Ca⁺² transport in the kidney.     

Parathyroid hormone stimulates adenylate cyclase in rat cerebral microvessels

We have studied the effect of parathyroid hormone (PTH) on adenylate cyclase of microvessels isolated from rat cerebral cortex. Native bovine (b) PTH-(1–84), the synthetic amino-terminal fragment bPTH-(1–34) and the synthetic analog [Nle⁸, Nle¹⁸, Tyr³⁴]-bPTH- (1–34) amide stimulated adenylate cyclase in a dose-dependent manner with apparent ED₅₀ values of 16 nM, 6.3 nM and 15 nM respectively. The stimulation by bPTH was greatly enhanced by guanosine triphosphate. The PTH antagonist, [Nle⁸, Nle¹⁸, Tyr³⁴]-bPTH-(3–34) amide inhibited the action of bPTH-(1–84) and bPTH-(1–34). In summary, PTH stimulated adenylate cyclase in rat cerebral microvessels in a very similar manner to its stimulation in the renal cortex.     

Ca2+-Induced Increases in Steady-State Concentrations of Intracellular Calcium Are Not Required for Inhibition of Parathyroid Hormone Secretion

It has been well established that increases in extracellular calcium concentration ([Ca²⁺]) inhibit parathyroid hormone (PTH) secretion. The effects of [Ca²⁺] are mediated through a G-protein-coupled receptor that has been cloned and characterized. Additionally, it has been demonstrated in parathyroid cells that an increase in [Ca²⁺] results in an increase in steady-state levels of intracellular calcium ([Ca²⁺]_i). At present, it has not been fully resolved whether changes in [Ca²⁺]_i are related to changes in PTH secretion. In the current study, the effect of increased [Ca²⁺] on PTH secretion and the connection regarding changes in concentrations of intracellular calcium [Ca²⁺]_i have been examined in primary cultures of bovine parathyroid cells. PTH secretion was measured by radioimmunoassay and intracellular calcium was determined by single cell calcium imaging. Bovine parathyroid cells pre-incubated with either 0.5 or 1 mM calcium responded to rapid increases in [Ca²⁺] (≥0.5 mM) with an immediate and sustained increase in steady-state levels of [Ca²⁺]_i that persisted for time intervals greater than 15 minutes. Although the magnitude of the sustained increase in [Ca²⁺]_i varied among individual cells (∼40% to >300%), the overall pattern and course of time were similar in all cells examined (n = 142). In all trials, [Ca²⁺]_i immediately returned to baseline levels following the addition of the calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Additional control studies, however, suggest that sustained increases in [Ca²⁺]_i do not correlate with regulation of parathyroid hormone secretion. Sustained elevations of [Ca²⁺]_i were not observed when [Ca²⁺] was gradually increased by the addition of 0.1 mM increments at 1 minute intervals. Furthermore, the effect on inhibition of PTH secretion was the same regardless of whether [Ca²⁺] was increased by gradual or rapid addition.     

Structural Basis for Parathyroid Hormone-related Protein Binding to the Parathyroid Hormone Receptor and Design of Conformation-selective Peptides

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) are two related peptides that control calcium/phosphate homeostasis and bone development, respectively, through activation of the PTH/PTHrP receptor (PTH1R), a class B G protein-coupled receptor. Both peptides hold clinical interest for their capacities to stimulate bone formation. PTH and PTHrP display different selectivity for two distinct PTH1R conformations, but how their binding to the receptor differs is unclear. The high resolution crystal structure of PTHrP bound to the extracellular domain (ECD) of PTH1R reveals that PTHrP binds as an amphipathic α-helix to the same hydrophobic groove in the ECD as occupied by PTH, but in contrast to a straight, continuous PTH helix, the PTHrP helix is gently curved and C-terminally “unwound.” The receptor accommodates the altered binding modes by shifting the side chain conformations of two residues within the binding groove: Leu-41 and Ile-115, the former acting as a rotamer toggle switch to accommodate PTH/PTHrP sequence divergence, and the latter adapting to the PTHrP curvature. Binding studies performed with PTH/PTHrP hybrid ligands having reciprocal exchanges of residues involved in different contacts confirmed functional consequences for the altered interactions and enabled the design of altered PTH and PTHrP peptides that adopt the ECD-binding mode of the opposite peptide. Hybrid peptides that bound the ECD poorly were selective for the G protein-coupled PTH1R conformation. These results establish a molecular model for better understanding of how two biologically distinct ligands can act through a single receptor and provide a template for designing better PTH/PTHrP therapeutics.The parathyroid hormone receptor (PTH1R)³ is a class B G protein-coupled receptor (GPCR) that transduces signals from two related signaling molecules that have distinct functions in biology: parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) (Ref. 1; reviewed in Ref. 2). PTH is an 84-amino acid polypeptide endocrine hormone that is produced by the parathyroid glands and secreted into the circulation in response to low calcium levels (reviewed in Refs. 3–5), to act on bone and kidney cells and thus restore blood calcium to normal levels. In bone, PTH directly stimulates osteoblasts, resulting in bone formation (reviewed in Ref. 6), which in turn activate osteoclasts to induce bone resorption. In the kidney, PTH stimulates the reabsorption of filtered calcium, inhibits the reabsorption of phosphate, and stimulates the synthesis of 1,25-dihydroxyvitamin D3. The paradoxical anabolic/catabolic actions of PTH on bone can be modulated by exogenous PTH, and provide the molecular basis for the clinical use of PTH as an anabolic therapy for osteoporosis (7). Anabolic PTH therapy requires intermittent administration to minimize bone-resorptive effects, which predominate with sustained administration of PTH. PTHrP is a 141-amino acid polypeptide that was originally isolated as the factor responsible for humoral hypercalcemia of malignancy (8–11) and was subsequently shown to be a critical developmental paracrine factor that controls endochondral bone formation (Refs. 12, 13; reviewed in Ref. 14). PTHrP can also mediate bone-anabolic effects when administered to osteoporosis patients (15) and has been suggested to be more anabolic than PTH due to a differential effect on the coupled bone formation and resorptive responses (16).PTH and PTHrP are encoded by separate genes, each of which is found in vertebrate species ranging from fish to man. How PTH and PTHrP evolved to mediate distinct biological activities: calcium/phosphate homeostasis and tissue development, respectively, via actions upon a single receptor, remains unclear. Amino acid sequence homology is most apparent in the first 34-residue segments of the proteins, and N-terminal 34-residue peptide fragments of PTH and PTHrP are sufficient for high affinity binding to the PTH1R and are generally found to be equally potent for stimulating cAMP formation in PTH1R-expressing cells (1). The interaction of the (1–34)-length ligand with the PTH1R has been postulated to follow a “two-domain” model: residues within the approximate (1–14) segment interact with the 7-transmembrane (7-TM) helical domain embedded in the membrane, and residues within the approximate (15–34) segment interact with the N-terminal extracellular domain (ECD) of the receptor (17, 18). The 1–14 domains of PTH and PTHrP share eight amino acid sequence identities, reflecting a critical role in activating the receptor (18), while the 15–34 domains share only three amino acid identities, despite a critical role in imparting high affinity binding to the receptor.Recent studies suggest that PTH and PTHrP differ in their relative capacities to bind to two pharmacologically distinguishable high-affinity PTH1R conformations (19–22). One conformation, termed R⁰, is stable in the presence of GTPγS, but presumably in the absence of G protein coupling, correlates with prolonged signaling responses in vitro and in vivo, and is bound preferentially by PTH-(1–34). The other conformation, termed RG, is sensitive to GTPγS addition, promoted by the overexpression of a high affinity variant of Gα_s, and bound preferentially by PTHrP-(1–36). A mechanistic basis for the differing capacities of PTH and PTHrP ligands to bind to these altered PTH1R conformations is not clear at present, although, both the (1–14) and (15–34) portions of PTH contribute importantly to the capacity to bind stably to the proposed R⁰ conformation (19, 21, 22).We previously developed a method that allowed us to determine the high resolution crystal structure of recombinant PTH1R ECD in complex with the 15–34 synthetic fragment of PTH (23). The PTH1R ECD adopts a tertiary fold that is conserved among class B GPCR ECDs (24–26), and the PTH(15–34)NH₂ domain binds as a continuous and straight amphipathic α-helix to a hydrophobic groove in the ECD. Here we present the high resolution crystal structure of the PTHrP 12–34 fragment in complex with the PTH1R ECD, which reveals a distinct docking conformation toward the C terminus of the PTHrP peptide. Based on the structural differences, we designed hybrid PTH/PTHrP peptides exchanged for residues involved in altered ECD contacts; functional analyses of these peptides confirmed that the altered modes of binding indeed translate into functional consequences in terms of receptor affinity. These results provide critical insights into how PTH and PTHrP can act through a single receptor, and a structural model for designing better PTH/PTHrP analogs for treating osteoporosis.     

Peptide–peptoid hybrids based on (1–11)‐parathyroid hormone analogs

A series of peptide–peptoid hybrids, containing N‐substituted glycines, were synthesized based on the H‐Aib‐Val‐Aib‐Glu‐Ile‐Gln‐Leu‐Nle‐His‐Gln‐Har‐NH₂ (Har = Homoarginine) as the parent parathyroid hormone (1–11) analog. The compounds were pharmacologically characterized in their agonistic activity at the parathyroid hormone 1 receptor. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Parathyroid mRNA directs the synthesis of pre-proparathyroid hormone and proparathyroid hormone in the Krebs ascites cell-free system.

Translation in a cell-free extract of Krebs II ascites cells of a mRNA fraction prepared from bovine parathyroid glands results in the synthesis of two radioactive products that appear identical to pre-proparathyroid hormone (Pre-ProPTH) (M.W. ～ 14,000), the suspected earliest biosynthetic precursor of parathyroid hormone (PTH) (M.W. 9,500), and to proparathyroid hormone (ProPTH) (M.W. 10,200), the immediate biosynthetic precursor of PTH. The two products of synthesis in the ascites extract co-electrophoresed on both urea-acetate and urea-SDS acrylamide gels with Pre-ProPTH obtained from cell-free translation of parathyroid RNA in extracts of wheat-germ and with ProPTH isolated from parathyroid slices. Both products were precipitated with an antiserum to PTH. Partial analysis of the amino acid sequence of [³⁵S]methionine-labeled Pre-ProPTH synthesized by the ascites extract indicates that a substantial fraction of the product is lacking the two N-terminal methionines present in the Pre-ProPTH synthesized by the wheat-germ system. The results indicate that, ($\text{i}$), unlike the wheat-germ, ascites extracts contain enzymes that remove the initiator methionine from Pre-ProPTH and convert Pre-ProPTH into ProPTH (no ProPTH was observed in the wheat-germ system) and ($\text{ii}$) the cleavage processes appear to occur in association with synthesis, inasmuch as neither removal of NH₂-terminal methionine nor formation of ProPTH was observed upon incubation of Pre-ProPTH isolated from either the wheat-germ system or from the ascites system when put back into the ascites system.     

Thymus-associated parathyroid hormone has two cellular origins with distinct endocrine and immunological functions

In mammals, parathyroid hormone (PTH) is a key regulator of extracellular calcium and inorganic phosphorus homeostasis. Although the parathyroid glands were thought to be the only source of PTH, extra-parathyroid PTH production in the thymus, which shares a common origin with parathyroids during organogenesis, has been proposed to provide an auxiliary source of PTH, resulting in a higher than expected survival rate for aparathyroid Gcm2 ^−/− mutants. However, the developmental ontogeny and cellular identity of these “thymic” PTH–expressing cells is unknown. We found that the lethality of aparathyroid Gcm2 ^−/− mutants was affected by genetic background without relation to serum PTH levels, suggesting a need to reconsider the physiological function of thymic PTH. We identified two sources of extra-parathyroid PTH in wild-type mice. Incomplete separation of the parathyroid and thymus organs during organogenesis resulted in misplaced, isolated parathyroid cells that were often attached to the thymus; this was the major source of thymic PTH in normal mice. Analysis of thymus and parathyroid organogenesis in human embryos showed a broadly similar result, indicating that these results may provide insight into human parathyroid development. In addition, medullary thymic epithelial cells (mTECs) express PTH in a Gcm2-independent manner that requires TEC differentiation and is consistent with expression as a self-antigen for negative selection. Genetic or surgical removal of the thymus indicated that thymus-derived PTH in Gcm2 ^−/− mutants did not provide auxiliary endocrine function. Our data show conclusively that the thymus does not serve as an auxiliary source of either serum PTH or parathyroid function. We further show that the normal process of parathyroid organogenesis in both mice and humans leads to the generation of multiple small parathyroid clusters in addition to the main parathyroid glands, that are the likely source of physiologically relevant “thymic PTH.”     

Different secretory actions of pancreastatin in bovine and human parathyroid cells

Chromogranin A is an acidic protein that is costored and cosecreted with parathyroid hormone (PTH) from parathyroid cells. Pancreastatin (PST), is derived from chromogranin A, and inhibits secretion from several endocrine/neuroendocrine tissues. Effects of different pancreastatin peptides were investigated on dispersed cells from bovine and human parathyroid glands. Bovine PST(1–47) and bovine PST(32–47) inhibited PTH release from bovine cells in a dose-dependent manner. The former peptide was more potent and suppressed the secretion at 1–100 nM. This inhibition was evident in 0.5 and 1.25 mM, but not in 3.0 mM external Ca²⁺. Both peptides failed to alter the concentration of cytoplasmic Ca²⁺([Ca²⁺]_i) of bovine cells. Human PST(1–52) and PST(34–52) did not affect PTH release or [Ca²⁺]_i of parathyroid cells from patients with hyperparathyroidism, nor [Ca²⁺]_i of normal human parathyroid cells. Furthermore, bovine PST(1–47) and bovine PST(32–47) failed to alter the secretion of abnormal human parathyroid cells. The study indicates that PST exerts secretory inhibition on bovine but not human parathyroid cells, and that this action does not involve alterations of [Ca²⁺]_i.