Pathogenic SCN2A variants cause early-stage dysfunction in patient-derived neurons.

Pathogenic heterozygous variants in SCN2A, which encodes the neuronal sodium channel NaV1.2, cause different types of epilepsy or intellectual disability (ID)/autism without seizures. Previous studies using mouse models or heterologous systems suggest that NaV1.2 channel gain-of-function typically causes epilepsy, whereas loss-of-function leads to ID/autism. How altered channel biophysics translate into patient neurons remains unknown. Here, we investigated iPSC-derived early-stage cortical neurons from ID patients harboring diverse pathogenic SCN2A variants [p.(Leu611Valfs*35); p.(Arg937Cys); p.(Trp1716*)] and compared them with neurons from an epileptic encephalopathy (EE) patient [p.(Glu1803Gly)] and controls. ID neurons consistently expressed lower NaV1.2 protein levels. In neurons with the frameshift variant, NaV1.2 mRNA and protein levels were reduced by ~ 50%, suggesting nonsense-mediated decay and haploinsufficiency. In other ID neurons, only protein levels were reduced implying NaV1.2 instability. Electrophysiological analysis revealed decreased sodium current density and impaired action potential (AP) firing in ID neurons, consistent with reduced NaV1.2 levels. In contrast, epilepsy neurons displayed no change in NaV1.2 levels or sodium current density, but impaired sodium channel inactivation. Single-cell transcriptomics identified dysregulation of distinct molecular pathways including inhibition of oxidative phosphorylation in neurons with SCN2A haploinsufficiency and activation of calcium signaling and neurotransmission in epilepsy neurons. Together, our patient iPSC-derived neurons reveal characteristic sodium channel dysfunction consistent with biophysical changes previously observed in heterologous systems. Additionally, our model links the channel dysfunction in ID to reduced NaV1.2 levels and uncovers impaired AP firing in early-stage neurons. The altered molecular pathways may reflect a homeostatic response to NaV1.2 dysfunction and can guide further investigations.

2D Raman, ATR-FTIR, WAXD, SAXS and DSC data of PET mono- and PET/PA6 bicomponent filaments.

This data in brief article summarizes structural data obtained from monocomponent melt-spun and offline drawn poly(ethylene terephthalate) (PET) monofilaments, as well as from melt-spun bicomponent core-sheath PET-polyamide 6 (PA6) filaments. The diameters of the single filaments range from 27 µm to 79 µm. Presented analysis techniques and results thereof are (i) Raman mapping of filament cross-sections: 2D maps of peak positions, widths, peak area ratios; (ii) attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR): ATR-FTIR spectra and extraction of surface crystallinity; (iii) wide-angle x-ray diffraction (WAXD): WAXD patterns and extraction of average crystallinity; (iv) small-angle x-ray scattering (SAXS): SAXS patterns and determined crystallite sizes and long-spacings; (v) differential scanning calorimetry (DSC): thermograms and extracted average crystallinity as well as thermal properties; (vi) atomic force microscopy (AFM): AFM image of the surface of an embedded fiber cross-section. For more information, see the publication by E. Perret et al. 'High-resolution 2D Raman mapping of mono- and bicomponent filament cross-sections' [1].

Calcitonin gene-related peptide is a stimulator of renin secretion.

Calcitonin gene-related peptide (CGRP) was found to stimulate renin secretion in vivo in normal human volunteers. Moreover, CGRP stimulated the release of renin in vitro from isolated rat renal juxtaglomerular cells (half-maximal effective concentration [EC50] 100 nM) concomitant with stimulation of cAMP production (EC50 60 nM). Immunoreactive CGRP was recognized in rat renal cortical nerve fibers, and intact rat CGRP was identified in extracts of the rat renal cortex. Because CGRP containing sensory nerve fibers are seen in the region of the juxtaglomerular apparatus, it would seem that the release of CGRP from these afferent nerves may be involved in the physiological control of renin secretion.

Comparison of parathyroid hormone receptors in rat osteosarcoma cells and kidney.

Parathyroid hormone/parathyroid-hormone-related peptide (PTH/PTHrP) receptors have been characterized with chicken parathyroid hormone related protein [Tyr36]chPTHrP(1-36)amide (chPTHrP(1-36)) as radioligand in rat UMR-106 osteosarcoma (UMR) cells and in rat renal cortical membranes (RCM). Binding of 125 pM [125I][Tyr36]chPTHrP(1-36) was displaced by chPTHrP(1-36) with ID50 values of 2.6 +/- 0.22 nM (mean +/- S.E.) and 0.9 +/- 0.03 nM in UMR cells and RCM, respectively. ID50 values in membranes from UMR cells and RCM were the same in the presence and absence of 10 microM guanosine-5'-O-(3-thiotriphosphate). Rat [Nle8,18] PTH(1-34) was 5-fold more potent than chPTHrP(1-36) in RCM, but not in UMR cells. Hill coefficients derived from binding inhibition were 0.93 and 0.35 in UMR and RCM, respectively. For affinity labeling, N-hydroxysuccinimidyl-4-azidobenzoate-modified [125I]chPTHrP(1-36) was used. Specifically-labeled PTH/PTHrP-binding proteins had a molecular mass of 83 kDa in UMR cells and RCM. Treatment with N-endoglycosidases lowered the molecular mass of chPTHrP binding proteins to 54 kDa in UMR and RCM. In conclusion, skeletal UMR-106 cells and renal cortical membranes of the rat reveal PTH/PTHrP receptors with no apparent tissue specific differences in molecular mass of the polypeptide backbone and polysaccharide chains. Higher affinity of rat PTH(1-34) binding and lower Hill coefficients in kidney compared to bone are consistent with tissue specific receptor-ligand interactions.

Effect of dexamethasone on parathyroid hormone (PTH) and PTH-related protein regulated phosphate uptake in opossum kidney cells.

Effects of dexamethasone (DEX) on receptor binding of bovine PTH (1-34) [bPTH (1-34)] and human PTH-related protein (1-34) [hPTHrP(1-34)] and cAMP accumulation, and on the inhibition of Na(+)-dependent phosphate uptake were studied in opossum kidney (OK) cells. Maximal specific binding of [125I]hPTHrP(1-34) was reduced by 40% in cells treated with 100 nM DEX, but half-maximal inhibition of binding of [125I]hPTHrP(1-34) by bPTH(1-34) or hPTHrP(1-34) was unaffected by DEX (0.5 nM and 1.4 nM, in the absence and presence of DEX, respectively). Moreover, the EC50 of bPTH(1-34) and hPTHrP(1-34)-evoked cAMP accumulation ranged from 3-7 nM and was the same with and without DEX. The EC50 of the inhibition of phosphate uptake by bPTH(1-34) and hPTHrP(1-34) ranged from 0.2-0.5 nM. Treatment with 100 nM DEX increased phosphate uptake 1.5-fold (EC50 3 nM), and the inhibition of phosphate uptake by bPTH(1-34) and hPTHrP(1-34) was suppressed. The inhibition of phosphate uptake by forskolin was also suppressed in cells treated with 100 nM DEX. DEX, on the other hand, enhanced forskolin-stimulated cAMP accumulation 1.6- to 1.8-fold. 12-O-Tetradecanoylphorbol-13-acetate did not affect cAMP production, but phosphate uptake was inhibited both in the absence and the presence of 100 nM DEX (EC50 3 nM). In conclusion, treatment of OK cells with DEX only minimally affected PTH receptor binding, and cAMP accumulation evoked by bPTH(1-34) and hPTHrP(1-34) remained unaltered. The inhibition of phosphate uptake by PTH, however, was suppressed.

Calcitonin inhibits phosphate uptake in opossum kidney cells stably transfected with a porcine calcitonin receptor.

Calcitonin (CT), and PTH and PTH-related protein (PTHrP) stimulated urinary excretion of phosphate is brought about through inhibition of Na/P04 cotransport in proximal renal tubules. PTH/PTHrP receptors linked to inhibition of phosphate uptake have been characterized in a renal tubular cell line from the American opossum (OK). Specific binding of [125I]salmon CT (sCT) to OK cells was not recognized, but 1 microM sCT stimulated cAMP accumulation 10-fold and reduced phosphate uptake by 7 +/- 2% (P < 0.05). The responses were amplified in OK cells stably transfected with a cloned CT receptor from a porcine LLC-PK1 kidney cell line. The transfected cells expressed 20,000 CT receptors per cell with a Kd of 0.05 nM and an EC50 of cAMP accumulation of 6.2 nM; maximal cAMP stimulation in response to 1 microM sCT was 259-fold (P < 0.01). Phosphate uptake was inhibited by 35 +/- 4% in response to 1 microM sCT and by 34 +/- 3% to 1 microM chicken PTHrP(1-36) (P < 0.01). Half-maximal inhibition was obtained with 0.63 +/- 0.30 nM sCT and with 1.39 +/- 0.67 nM chicken PTHrP(1-36). The inhibition of [125I]sCT binding by nonlabeled human amylin required about 5000-fold higher concentrations than those of sCT, and human calcitonin gene-related peptide-I (CGRP) at up to 1 microM did not affect [125I]sCT binding. The rank order of potencies with respect to stimulation of cAMP accumulation and inhibition of phosphate transport of sCT, amylin and CGRP was the same. This is the first report linking a cloned CT receptor to inhibition of phosphate transport in a renal proximal tubular cell.

Photoaffinity labeling of rat calcitonin gene-related peptide receptors and adenylate cyclase activation: identification of receptor subtypes.

Different biological effects of calcitonin gene-related peptide (CGRP) analogs have suggested receptor subtypes. Here we have investigated molecular forms of rat CGRP receptors, ligand binding, and activation of adenylate cyclase. A single class of [125I]alpha-human (h)CGRP binding sites was identified in rat cerebellum, liver, and spleen, with dissociation constants of 206 +/- 70 pM, 128 +/- 23 pM, and 229 +/- 64 pM (mean +/- SEM), respectively. Competition experiments showed the same rank order of displacement of [125I]alpha-hCGRP binding in all the tissues examined with rat alpha-CGRP approximately alpha-hCGRP approximately beta-hCGRP > alpha-hCGRP(8-37) > [acetamidomethyl-Cys2,7]alpha-hCGRP > human amylin > salmon calcitonin. Photoaffinity labeling of CGRP receptors using [125I][C gamma-(4-azidoanilino)Asp3]alpha-hCGRP revealed specifically labeled 71-kilodalton (kDa) binding proteins in the cerebellum, brainstem, and spinal cord, of 74 kDa and 68 kDa in the liver, and of 75-90 kDa in the spleen. Enzymatic N-deglycosylation converted the labeled binding proteins into a common 48-kDa form (44 kDa with the molecular mass of the photoligand subtracted). In the presence of 100 microM guanosine-5'-O-(3-thiotriphosphate), the dissociation constant of [125I]alpha-hCGRP binding remained unchanged in the cerebellum but was increased 3-fold in the liver and spleen, suggesting interaction with GTP-binding proteins. In accordance with these results, adenylate cyclase was stimulated by CGRP in the liver and spleen, but not in the cerebellum and brainstem. Furthermore, the linear analog [acetamidomethyl-Cys2,7]alpha-hCGRP enhanced cAMP formation in the liver but not in the spleen. In conclusion, rat CGRP receptors with tissue-specific N-glycosylation but indistinguishable protein molecular mass have been identified in the cerebellum, brainstem, spinal cord, liver, and spleen. Activation of adenylate cyclase by CGRP in the liver and spleen, but not in the central nervous system, and by the linear analog [acetamidomethyl-Cys2,7]alpha-hCGRP in the liver alone provide evidence for CGRP receptor subtypes.

Differential expression of calcitonin and parathyroid hormone/parathyroid hormone-related protein receptors in P19 embryonic carcinoma cells treated with retinoic acid.

Mouse embryonic carcinoma P19 cell aggregates treated with retinoic acid (RA) sequentially differentiate into neurons and astrocytes, whereas attached cells develop a mesodermal phenotype. The expression of calcitonin (CT) and PTH/PTH-related protein (PTHrP) receptors was investigated in embryonic cells, and during neural and mesodermal differentiation. In embryonic P19 cells, specific binding of [125I]salmon (s) CT(1-32) ([125I]sCT(1-32)) was 56 fmol/mg protein, and of [125I]chicken (ch) [Tyr36]PTHrP(1-36) amide ([125I]chPTHrP(1-36)) < 0.5 fmol/mg protein. Correspondingly, cAMP was maximally stimulated 47-fold by sCT(1-32) (EC50 0.05 nM) and 3-fold by chPTHrP(1-36) (EC50 1.3 nM). Receptor autoradiography revealed specific binding of [125I]sCT(1-32) to the undifferentiated P19 cells, but not to RA induced neurons and astrocytes. At the same time, [125I]sCT(1-32) binding and cAMP accumulation by sCT were gradually decreased. But, specific binding of [125I]chPTHrP(1-36) was raised at least 6-fold compared with embryonic cells to 3 fmol/mg protein, in parallel with a 10-fold higher maximal cAMP accumulation. A similar, but delayed suppression of CT and stimulation of PTH/PTHrP receptor expression was observed during mesodermal cell differentiation. The results indicate that CT receptors are associated with undifferentiated P19 cells, whereas PTH/PTHrP receptors are expressed in RA induced neural and mesodermal cells.

A receptor activity modifying protein (RAMP)2-dependent adrenomedullin receptor is a calcitonin gene-related peptide receptor when coexpressed with human RAMP1.

Adrenomedullin (ADM) and alpha- and beta-calcitonin (CT) gene-related peptide (alpha-, betaCGRP) are structurally related vasodilatory peptides with homology to CT and amylin. An originally orphan human CT receptor-like receptor (hCRLR) is a Gs protein-coupled CGRP or ADM receptor when coexpressed with recently identified human single transmembrane domain receptor activity modifying proteins 1 (hRAMP1) or -2 (hRAMP2), respectively. Here, the function of the rat CRLR homologue (rCRLR) has been investigated in rat osteoblast-like UMR-106 cells and in COS-7 cells, in the absence and presence of hRAMP1 and -2 and combinations thereof. Transient expression of rCRLR in UMR-106 cells revealed an ADM receptor, and [125I]rat (r) ADM binding was enhanced with hRAMP2 and inhibited by 50% when hRAMP1 was coexpressed. Detectable [125I]h alphaCGRP binding required the presence of hRAMP1, and the expression of CGRP binding sites was unaffected by coexpressed hRAMP2. Specificity of ADM binding sites in [125I]rADM binding inhibition experiments was reflected by an over 1000-fold higher potency of rADM [half-maximal effective concentration = 0.19 +/- 0.05 nM (mean +/- SEM, n = 4)], compared with r alphaCGRP and r betaGRP, to induce a cAMP-responsive luciferase reporting gene (CRE-luc). In rCRLR and hRAMP1 cotransfected cells, expressing predominantly CGRP binding sites, r betaCGRP, r alphaCGRP, and rADM induced CRE-luc with half-maximal effective concentration of 0.27 +/- 0.17 nM, 0.37 +/- 0.27 nM, and 1.4 +/- 0.9 nM, respectively. In COS-7 cells, the results were comparable, but rCRLR required coexpressed hRAMP2 for ADM receptor function. This is consistent with higher levels of endogenous RAMP2 encoding messenger RNA in UMR-106, compared with COS-7 cells. In conclusion, the recognition of RAMP1 and -2 as mediators of CRLR expression as a CGRP or ADM receptor has been extended, with evidence that endogenous RAMP2 is sufficient to reveal an ADM receptor in UMR-106 cells. Inhibition of RAMP2-evoked ADM receptor expression by RAMP1 and generation of a CGRP receptor is consistent with competitive interactions of the different RAMPs with rCRLR.

An amylin receptor is revealed following co-transfection of a calcitonin receptor with receptor activity modifying proteins-1 or -3.

Human receptor activity modifying proteins (RAMP) regulate the ligand specificity of the calcitonin-receptor-like-receptor (McLatchie et al., Nature 393:333-339 (1998)). Here we have investigated binding of [125I]-labeled human (h) calcitonin ([125I]hCT) and rat amylin ([125I]amylin) to rabbit aortic endothelial cells (RAEC) co-transfected with the hCT receptor isotype 2 (hCTR2) and RAMP1, -2 or -3. Specific binding of 125 pM [125I]hCT to cells transfected with hCTR2 alone was 6.7 +/- 0.7 fmol/50,000 cells (n=5), and was reduced by 45 +/- 2% and 86 +/- 3% (P < 0.001) in the presence of RAMP1 and -3, but remained unchanged with RAMP2. In the absence and presence of individual RAMPs [125I]hCT binding inhibition occured with similar IC50 of between 6 nM and 11 nM hCT, and human amylin was 24- to 54-fold less potent. Specific binding of 125 pM [125I]amylin to cells transfected with hCTR2 alone was 0.9 +/- 0.2 fmol/50,000 cells (n=6), and was increased by 262 +/- 48% (P < 0.005), 73 +/- 26% (P < 0.05) and 338 +/- 57% (P < 0.005) with RAMP1, -2 or -3, respectively. [125I]amylin binding was inhibited with IC50 of 3.1 +/- 0.5 nM and 4.0 +/- 0.8 nM human amylin in cells co-transfected with RAMP1 or -3, respectively, and hCT was 45 +/- 2- and 126 +/- 3-fold less potent. In conclusion, RAMP1 and -3 decrease calcitonin receptor expression in RAEC transfected with hCTR2 encoding cDNA and simultanously reveal an amylin receptor.

Apical and basolateral parathyroid hormone receptors in rat renal cortical membranes.

Brush border (BBM) and basolateral membranes (BLM) of rat renal cortical cells separated by free flow electrophoresis revealed two distinct peaks of BBM-specific leucine aminopeptidase and Na+/K(+)-ATPase for BLM. PTH/PTH-related protein (PTHrP) receptors were identified in BBM and BLM. Specific binding of 125 pM [125I]chicken [Tyr36]-PTHrP-(1-36)amide [chPTHrP-(1-36)] to individual fractions of membranes separated by free flow electrophoresis overlapped with the leucine aminopeptidase and Na+/K(+)-ATPase profiles. Binding to pooled BBM was 53 +/- 5% (mean +/- SEM) of that to BLM (P < 0.01). In BBM and BLM, half-maximal inhibition of binding was obtained with 0.4-0.9 nM chPTHrP-(1-36) and 0.2-0.6 nM rat PTH-(1-34). Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S; 100 microM) lowered chPTHrP-(1-36) binding to 50% of control levels, and half-maximal inhibition of binding was obtained with 480 and 8 nM GTP gamma S in BBM and BLM, respectively. Cross-linking of the PTH/PTHrP receptors with [125I]chPTHrP-(1-36) modified with N-hydroxysuccinimidyl-4-azidobenzoate revealed indistinguishable doublets of 83 and 73 kilodaltons in both BBM and BLM. Adenylyl cyclase was stimulated 6- and 10-fold by chPTHrP-(1-36) and GTP gamma S, respectively, in BLM and 1.3- and 1.9-fold in BBM. In conclusion, PTH receptors were recognized in both the basolateral and brush border membranes. Different receptor coupling to G-proteins and minimal cAMP stimulation in BBM provide evidence for PTH/PTHrP receptor isotypes and/or different postreceptor activation in BBM and BLM.

Specific receptor and cardiovascular effects of calcitonin gene-related peptide.

Specific binding sites for calcitonin gene-related peptide (CGRP) were demonstrated in the rat heart and spleen. Autoradiography revealed rat [125I]iodo CGRP binding associated with the intima and media of the aorta, the coronary arteries and the heart valves, and the red pulp of the spleen. Half-maximal inhibition of rat [125I]iodo-CGRP binding to membranes of the rat atria and the spleen was obtained with, respectively, 5 and 0.35 nM unlabeled rat CGRP; these values correspond to EC50 values of 3 and 0.14 nM for activation of adenylate cyclase by CGRP. In the isolated, spontaneously beating right atrium, the EC50 values of stimulation of the force and rate of contraction by rat CGRP were 120 and 70 nM, respectively. Rat CGRP caused relaxation of splenic strips, precontracted with noradrenaline; the EC50 was 50 nM. The beta-adrenergic blocking agent metoprolol, while obliterating the increase in the force and rate of contraction evoked by noradrenaline in the right atrium, did not significantly change the action of CGRP. Similarly, preserved action of CGRP in the presence of indomethacin as well as mepyramine and cimetidine argues against a role of prostaglandins or histamine in the functional responses of CGRP. Much like CGRP, capsaicin, which releases mediators from sensory neurons, caused stimulation of the force and rate of contraction of the isolated right rat atrium. After tachyphylaxis to CGRP, the response to noradrenaline was intact, while the positive chronotropic and inotropic effects of capsaicin were suppressed. The results indicate that the cardiac effects of capsaicin may be due to the release of endogenous CGRP through a local mode of action.

Role of cytosolic free calcium concentration in the secretion of calcitonin gene-related peptide and calcitonin from medullary thyroid carcinoma cells.

Calcitonin gene-related peptide (CGRP) and calcitonin (CT) are secreted by medullary thyroid carcinoma (MTC). Relationships between extracellular calcium ([Ca2+]e), cytosolic free calcium ([Ca2+]i) (as measured with fura-2), and secretion of immunoreactive CGRP and CT have been investigated in rat and human MTC cell lines. Rat MTC 6-23 cells responded to a rise in [Ca2+]e from 0.5 to 3.0 mM with a transient increase of [Ca2+]i, and the secretion of CGRP and CT was raised from 19 +/- 2 (mean +/- SE) to 122 +/- 28 pg rat CGRP/mg protein . min and from 33 +/- 8 to 155 +/- 42 pg rat CT/mg protein . min (P less than 0.01). In the human MTC (TT) cell line, a rise of [Ca2+]e from 0.5 to 3.0 mM did not affect [Ca2+]i, and the secretion of CGRP and CT remained unchanged at 7.0 +/- 1.1 ng CGRP/mg protein . min and 1.0 +/- 0.1 ng CT/mg protein . min. However, when the plasma membrane was bypassed by electropermeabilization, the release of CGRP and CT was stimulated by calcium with an ED50 of 0.5 microM and 0.3 microM, respectively. With ionomycin, the secretion of CGRP and CT was also stimulated up to 17-fold in [Ca2+]i-dependent manner. The results indicate a role of [Ca2+]i in the secretion of CGRP and CT and provide evidence for a defect in Ca2+ signal transduction in the human MTC cell line.

Formation of neutralizing antibodies during intranasal synthetic salmon calcitonin treatment of Paget's disease.

Nine patients with Paget's disease were treated with 200 U (15 nmol) synthetic salmon calcitonin (sCT) intranasally (in)/day for 12 months. Five of them had received im or in sCT therapy for 1-4 yr up to 0.5-5 yr before this study. Low titer antibodies to sCT were detected in the serum of three of these five patients, but not in the four patients who had not received prior sCT therapy. After 2 months of in sCT administration, four of the former group, but none of the latter group, had antibodies to sCT. After 12 months of treatment, antibodies to sCT were found in all patients who had received sCT earlier and in three of the four patients who had not. The half-maximal inhibition of [125I]sCT binding ranged from 44-284 pmol/L sCT. In a cultured human breast cancer cell line (T47D) cAMP production was stimulated by sCT (EC50, 70 pmol/L). cAMP production stimulated by sCT (5 pmol/L) was reduced to 6-20% of the control value in the presence of serum from patients which inhibited [125I]sCT binding by more than 50% in a dilution of 1:50 or greater. In patients with lower titer antibodies cAMP production was not inhibited. Serum alkaline phosphatase activity was transiently lowered to 79 +/- 6% (+/- SE) of basal levels in the patients who had earlier received sCT (P greater than 0.1), while sustained reduction to between 66 +/- 2% and 84 +/- 6% of basal levels (P less than 0.05) occurred in the patients who had not been treated with sCT previously. In conclusion, reexposure to sCT of five patients with Paget's disease caused secondary antibody responses and clinical resistance.

Stimulation of parathyroid hormone secretion by phorbol esters is associated with a decrease of cytosolic calcium.

Unlike in other endocrine systems calcium inhibits parathyroid hormone (PTH) secretion and this inhibition is paralleled by a rise of cytosolic calcium concentration ([Ca]i). Because of evidence that diglyceride levels and protein kinase C activity are also decreased by high extracellular calcium we have investigated the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, on [Ca]i and PTH secretion using dispersed bovine parathyroid cells. At 1.5 mM medium calcium TPA enhanced PTH secretion and caused reduction of [Ca]i from 639 +/- 36 nM (SE) to 335 +/- 21 nM (P less than 0.001); at 0.5 mM calcium TPA was ineffective. Moreover, TPA suppressed the rise of [Ca]i evoked by high extracellular calcium. Thus TPA presumably stimulates PTH secretion via activation of protein kinase C, and the lowering of [Ca]i may TPA presumably stimulates PTH secretion via activation of protein kinase C, and the lowering of [Ca]i may be a secondary event related to diglyceride availability.

Glycosylation of the calcitonin receptor-like receptor at Asn(60) or Asn(112) is important for cell surface expression.

The human calcitonin (CT) receptor-like receptor (hCRLR) of the B family of G protein-coupled receptors is N-glycosylated and associates with receptor-activity-modifying proteins for functional interaction with CT gene-related peptide (CGRP) or adrenomedullin (ADM), respectively. Three putative N-glycosylation sites Asn(60), Asn(112) and Asn(117) are present in the amino-terminal extracellular domain of the hCRLR. Tunicamycin dose-dependently inhibited the glycosylation of a myc-tagged hCRLR and in parallel specific [(125)I]CGRP and -ADM binding. Similarly, the double mutant myc-hCRLR(N60,112T) exhibited minimal N-glycosidase F sensitive glycosylation, presumably at the third Asn(117), and the cell surface expression and specific radioligand binding were impaired. Substitution of the Asn(117) by Thr abolished CGRP and ADM binding in the face of intact N-glycosylation and cell surface expression.

Receptor activity modifying proteins regulate the activity of a calcitonin gene-related peptide receptor in rabbit aortic endothelial cells.

In Xenopus oocytes with an endogenous calcitonin gene-related peptide (CGRP) receptor, a receptor activity modifying protein (RAMP1) enhancing CGRP stimulated chloride currents of the cystic fibrosis transmembrane regulator was recently cloned [McLatchie, L.M. et al. (1998) Nature 393, 333-339]. Here, transient expression of RAMP1 in rabbit aortic endothelial cells (RAEC) brought about stimulation of cAMP accumulation by human (h) alphaCGRP with an EC50 of 0.41 nM. This was antagonized by a CGRP receptor antagonist alphaCGRP(8-37). Co-expression of RAMP3 together with RAMP1 reduced the maximal cAMP response to h alphaCGRP by 47% (P < 0.05). The cells also express RAMP2 encoding mRNA and an adrenomedullin (ADM) receptor coupled to stimulation of cAMP formation by hADM (EC50 0.18 nM). The latter was antagonized by an ADM receptor antagonist hADM(22-52). In conclusion, expression of a CGRP receptor in RAEC requires RAMP1. The same receptor presumably recognizes ADM making use of endogenous RAMP2. The results reveal competition between the different RAMPs in the regulation of CGRP/ADM receptor activity.

Efficacy of intranasal human calcitonin in patients with Paget's disease refractory to salmon calcitonin.

PURPOSE: A cause for the resistance to intranasal salmon calcitonin (sCT) therapy in patients with Paget's disease is the occurrence of neutralizing antibodies to sCT. As a result, a new formulation of intranasal human calcitonin (hCT) was developed, and the efficacy investigated in patients treated earlier with sCT. PATIENTS AND METHODS: Twelve patients with Paget's disease were treated twice daily for 6 months with 1 mg synthetic hCT administered intranasally. Five patients demonstrated low-titer antibodies to sCT, and four of the patients did not respond previously to 1-year therapy with intranasal sCT. The hypocalcemic effect of 3 mg hCT was compared to that of 0.1 mg sCT before and after the intranasal hCT therapy. Serum alkaline phosphatase and the ratio between the urinary excretion of hydroxyproline and creatinine were measured before and during intranasal hCT treatment. RESULTS: The hypocalcemic response to 3 mg intranasal hCT (-6.60 +/- 0.67%, mean +/- standard error) was similar before and at the end of intranasal hCT therapy (-5.92 +/- 0.80%, p greater than 0.1). Intranasal sCT (0.1 mg) lowered serum calcium less effectively (-2.86 +/- 0.76%) than 3 mg intranasal hCT (p less than 0.05). The presence of low-titer antibodies to sCT did not affect the hypocalcemic response to sCT or hCT. As a result of the 6-month intranasal hCT regimen, serum alkaline phosphatase and urinary hydroxyproline/creatinine ratio were reduced to 62 +/- 5% (p less than 0.001) and 80 +/- 7% (p less than 0.05) respectively, of pretreatment levels. In four patients previously resistant to intranasal sCT therapy because of neutralizing antibodies to sCT, serum alkaline phosphatase was similarly lowered by intranasal hCT to 66 +/- 6% of pretreatment levels (p less than 0.05). CONCLUSION: A new formulation of intranasal hCT effectively lowered serum calcium levels, alkaline phosphatase concentrations, and urinary hydroxyproline excretion in patients with Paget's disease, some of whom were previously resistant to intranasal sCT because of neutralizing antibodies.

Trypsinization of bovine parathyroid cells abolishes Ca2+-regulated parathyroid hormone secretion.

The secretion of parathyroid hormone (PTH) is inversely related to the extracellular Ca2+ concentration (Ca2e+). To test the hypothesis that a Ca2+ sensor on the surface of parathyroid cells is involved in Ca2+-regulated PTH secretion, limited trypsinization of bovine parathyroid cells was carried out. Treatment with trypsin (1.1-10 mg/ml) inhibited, in a dose-dependent manner, PTH secretion stimulated by lowering Ca2e+ from 2.0 to 0.5 mmol/l. In control cells, activation of protein kinase C with 12-O-tetradecanoylphorbol-13-acetate (TPA) enhanced PTH secretion at 2.0 mmol Ca2e+/l but not at 0.5 mmol Ca2e+/l. In trypsinized cells, however, TPA enhanced PTH secretion at both 0.5 and 2.0 mmol Ca2e+/l. Isoproterenol-stimulated PTH secretion was maintained in trypsinized cells, but reduced cyclic AMP production revealed that some beta-adrenergic receptors were destroyed. The cytosolic free Ca2+ concentration (Ca2i+), as measured with fura-2, was raised within seconds in response to increasing Ca2e+ from 0.5 to 2.0 mmol/l and was then lowered within 1 min to a sustained plateau; the changes were the same in trypsinized and control cells. In conclusion, trypsinization of parathyroid cells abolished Ca2+-regulated PTH secretion without affecting Ca2i+.

Calcitonin gene-related peptide and calcitonin secretion from a human medullary thyroid carcinoma cell line: effects of ionomycin, phorbol ester and forskolin.

Calcitonin gene-related peptide (CGRP) and calcitonin are secreted together from medullary thyroid carcinoma (MTC) cells. Interactions of cytosolic free calcium concentration (Cai2+) and the protein kinase C and A pathways on the secretion of immunoreactive CGRP and calcitonin have been investigated in a human MTC cell line. Ionomycin (10 mumol/l) raised the concentration of Cai2+, concomitant with a transient stimulation of the secretion of CGRP and calcitonin. 12-O-tetradecanoylphorbol-13-acetate (TPA; 16 nmol/l) did not affect the concentration of Cai2+, but caused a gradual rise of the secretion of CGRP and calcitonin. Combined addition of 10 mumol ionomycin/l and 16 nmol TPA/l resulted in additive stimulation of CGRP and calcitonin secretory responses. Forskolin (10 mumol/l) alone did not change the concentration of Cai2+, marginally enhanced (P greater than 0.1) the release of CGRP and calcitonin and increased by 23-fold the cellular levels of cyclic AMP (cAMP). Ionomycin and TPA did not change cellular cAMP. Forskolin synergistically enhanced (P less than 0.01) the ionomycin-induced early phase as well as the TPA-induced late phase of the CGRP and calcitonin secretory responses. In conclusion, increased concentrations of Cai2+ together with protein kinase C and A activation mediate the secretion of CGRP and calcitonin in MTC cells.