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J A Fischer

Publications and source records attributed to J A Fischer.

At least 55 records · Page 3Linked to original sources
PubMed1996

Structure of a parathyroid hormone/parathyroid hormone-related peptide receptor of the human cerebellum and functional expression in human neuroblastoma SK-N-MC cells.

Cloning and functional expression of a cDNA from the human cerebellum revealed a parathyroid hormone/parathyroid hormone-related peptide (PTH/PTHrP) receptor protein of 593 amino acids, identical in sequence to the PTH/PTHrP receptor of the human kidney and an osteoblast-like cell line (Schipani et al., Endocrinology, 132 (1993) 2157-2165). Expression of mRNA hybridizing with the cloned cDNA, indistinguishable in size on Northern blots from a 2.3 kb transcript in kidney and liver, was detected in eight brain areas. In situ hybridization histochemistry in rat brain tissue sections revealed predominant signals in the Purkinje cell layer of the cerebellum and in the mesencephalic nucleus of the trigeminal nerve. In human neuroblastoma (SK-N-MC) cells, stably transfected with the cloned cDNA, hPTH(1-84) and hPTH(1-34) displaced binding of 125 pM [125I][Tyr36]chPTHrP(1-36) to the PTH/PTHrP receptor with IC50 values of 4.0 +/- 0.6 nM and 2.00 +/- 0.08 nM, and stimulated cyclic AMP accumulation with EC50 values of 0.19 +/- 0.06 nM and 0.09 +/- 0.01 nM, respectively. 16 out of 48 cells responded to 100 nM hPTH(1-34) with a 2-10-fold transient increase of cytosolic free calcium concentrations. In conclusion, a PTH/PTHrP receptor, identified in the human cerebellum, has the primary structure of the corresponding receptors of kidney and bone. Expression in human neuroblastoma SK-N-MC cells revealed functional properties indistinguishable from those of non-neuronal tissues. The widespread distribution of PTHrP and its receptor in brain implies biological functions remaining to be elucidated.

Amino Acid Sequence↗
PubMed1995

A human orphan calcitonin receptor-like structure.

A novel calcitonin receptor-like protein of 461 amino acids with seven putative transmembrane domains has been identified through molecular cloning in a cDNA library of the human cerebellum. 91% and 56% of the amino acids are identical in a rat orphan calcitonin receptor-like sequence and the human calcitonin receptor, respectively. 5.2 kb mRNA is predominantly expressed in the lung, heart and kidney. Specific binding of 125I-labeled salmon calcitonin and human calcitonin gene-related peptide-I to COS-7 cells transiently transfected with the receptor cDNA was less then 0.5%. Cellular cAMP accumulation was indistinguishable in cDNA transfected and non-transfected control COS-7 and renal tubular cells from the American opossum stimulated with human and salmon calcitonin, human calcitonin gene-related peptide-I and -II, human amylin, human adrenomedullin, lizard helodermin, salmon stanniocalcin and chicken parathyroid hormone-related protein. The receptor-like protein whose ligand remains to be discovered belongs to the family of receptors of calcitonin, parathyroid hormone, secretin, vasointestinal peptide and pituitary adenylate cyclase-activating polypeptide.

Amino Acid Sequence↗
PubMed1995

Adrenomedullin and calcitonin gene-related peptide interact with the same receptor in cultured human neuroblastoma SK-N-MC cells.

Inhibition of human [125I]calcitonin gene-related peptide-I ([125I]hCGRP-I) binding by human adrenomedullin (hADM), its N-terminal truncated fragments, CGRP and amylin, and cyclic AMP accumulation were examined in the human neuroblastoma cell line SK-N-MC. Binding of [125I]hCGRP-I (125 pM) was inhibited by hCGRP-I, hADM(1-52), hADM(13-52), and human amylin with IC50 of 0.32 +/- 0.06, 2.11 +/- 0.26, 3.45 +/- 0.54, and 68.8 +/- 6.6 nM, respectively. hCGRP-I(8-37) and hADM(22-52), which lack the N-terminal ring structure, inhibited [125I]hCGRP-I binding with IC50 of 2.35 +/- 0.45 and > 1000 nM. hCGRP-I, hADM(1-52), hADM(13-52) and human amylin stimulated cAMP accumulation with EC50 of 0.40 +/- 0.05, 18.1 +/- 2.6, 51.3 +/- 9.0 and 925 +/- 159 nM, respectively. hCGRP-I(8-37) (100 nM) antagonized hCGRP-I and hADM(1-52) stimulated cAMP production with the same Ki of 16.6 +/- 1.2 and 16.8 +/- 1.1 nM. In conclusion, human ADM, which is more distantly related to CGRP than amylin, interacts more potently with the CGRP receptor in SK-N-MC cells than amylin. The N-terminal ring structure of hADM, unlike that of hCGRP, is essential for binding to the CGRP receptor. Coupling of hADM binding to cAMP stimulation is less efficient than for hCGRP-I and is reduced by deletion of the unique 12 amino acid sequence of hADM N-terminal to the ring structure.

Adrenomedullin↗
PubMed1995

Receptors for calcitonin, calcitonin gene related peptide, amylin, and adrenomedullin.

Calcitonin, calcitonin gene related peptide, amylin, and adrenomedullin are structurally related polypeptides characterized by a six or seven amino acid ring structure linked by a disulfide bridge and an amidated C-terminus. They exhibit overlapping biological actions as a result of cross-reactivity between the different receptors. In this article, the respective receptors and G-protein-coupled postreceptor events are reviewed in relation to some of the biological actions of the peptides.

Animals↗
PubMed1995

Calcitonin, calcitonin gene-related peptide, adrenomedullin and amylin: homologous peptides, separate receptors and overlapping biological actions.

Calcitonin, calcitonin gene-related peptide, adrenomedullin and amylin are structurally related peptides with N-terminal 6-7 amino acid ring structures linked by a disulfide bridge and with amidated C-termini. Among the related bioactive peptides, the structures of the calcitonin receptor and subtypes thereof have been identified so far through molecular cloning. Cross-reaction between receptors of calcitonin, calcitonin gene-related peptide, adrenomedullin and amylin, as well as overlapping biological actions, anticipate that the respective receptors belong to a family of G-protein-coupled receptors that include those of parathyroid hormone, secretin and vasointestinal peptide.

Adrenomedullin↗
PubMed1994

Functional expression of a stably transfected parathyroid hormone/parathyroid hormone related protein receptor complementary DNA in CHO cells.

Chinese hamster ovary (CHO) cells were stably transfected with OK-O complementary DNA encoding the parathyroid hormone/parathyroid hormone related protein (PTH/PTHrP) receptor derived from opossum kidney (OK) cells (Jüppner et al., 1991). A subclone of transfected CHO cells, CHO-E2, presented high affinity binding of 125I-labeled [Tyr36]chickenPTHrP(1-36)amide ([125I]chPTHrP(1-36)) (Kd 1.28 +/- 0.10 nM) similar to that of wildtype OK cells (Kd 2.23 +/- 0.16 nM) (P < 0.01). Photoaffinity labeling of the PTH/PTHrP receptors using N-hydroxysuccinimidyl-4-azidobenzoate modified [125I]chPTHrP(1-36) revealed the same specifically labeled 90 kDa protein in CHO-E2 and OK cells. In CHO-cells, chPTHrP(1-36) stimulated cyclic AMP accumulation in dose-dependent fashion (EC50 0.15 +/- 0.04 nM) and raised peak cytosolic free calcium concentration (EC50 2.90 +/- 0.36 nM) independent of extracellular calcium, and stimulated phosphate uptake (EC50 0.21 +/- 0.07 nM). Both, chPTHrP(1-36) and 12-O-tetradecanoylphorbol-13-acetate stimulated phosphate uptake were suppressed by staurosporine. But, Sp-cyclic adenosine-3',5'-monophosphothioate did not affect phosphate uptake in CHO-E2 cells. In conclusion, a PTH/PTHrP receptor stably expressed in CHO cells is linked to stimulation of phosphate uptake. Receptor coupling presumably occurred through the protein kinase C rather than the protein kinase A pathway.

Alkaloids↗
PubMed1994

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.

Adenylyl Cyclases↗
PubMed1994

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.

Animals↗
PubMed1993

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.

Animals↗
PubMed1993

Stimulation of calcitonin/CGRP-I and CGRP-II gene expression by dibutyryl cAMP in a human medullary thyroid carcinoma (TT) cell line.

In a human medullary thyroid carcinoma (TT) cell line, expression of the calcitonin (CT)/CT gene-related peptide (CGRP-I) gene (CALC-I or alpha) at the level of mRNA and of encoded peptides is higher than that of the closely related CGRP-II gene (CALC-II or beta). In response to 1 mM dibutyryl cAMP ((Bu)2cAMP), mature CGRP-II mRNA and intact cellular CGRP-II were raised 65- and 10-fold, respectively, at 72 h. Also at 72 h, 1 mM sodium butyrate enhanced CGRP-II mRNA only 9-fold and cellular CGRP-II 2-fold; stimulation of CGRP-I and CT mRNA and of cellular CGRP-I and CT by both (Bu)2cAMP and sodium butyrate was similarly low. During the same incubation time period secreted CGRP-II was raised 44-fold in response to (Bu)2cAMP, and CGRP-I and CT 8- and 42-fold, respectively. In conclusion, gene products of CALC-I (CGRP-I and CT) are present in higher amounts in TT cells than those of CALC-II (CGRP II). Yet (Bu)2cAMP predominantly stimulates the expression of CALC-II.

Bucladesine↗
PubMed1993

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.

Adenylyl Cyclases↗
PubMed1992

Comparison of a calcitonin gene-related peptide receptor in a human neuroblastoma cell line (SK-N-MC) and a calcitonin receptor in a human breast carcinoma cell line (T47D).

A specific CGRP-binding protein of M(r) 60,000 has been identified in the human neuroblastoma cell line SK-N-MC. After N-deglycosylation a M(r) of 48,000 was found. The M(r) were indistinguishable from those determined in the human cerebellum. Receptor binding of CGRP is coupled to cyclic AMP formation. The latter is antagonized by hCGRP-I8-37. CT and DAPamide interact only minimally with the CGRP receptor, whereas CGRP and DAPamide are full agonists in T47D cells. The CT receptor on human breast cancer cell line T47D is clearly different from the human CGRP receptor.

Amino Acid Sequence↗
PubMed1992

Intracolonic bioavailability of human calcitonin in man.

Human calcitonin (hCT) injected into the lumen of the descending colon of normal human subjects was absorbed within minutes and could be recognized intact in plasma as shown by RIA in combination with reverse-phase HPLC. The absorption was low and variable, with bioavailabilities ranging from 0.01% to 2.7% relative to intravenously administered hCT (area under the concentration-time curve). With intravenous hCT serum calcium was lowered and the fractional urinary excretion of calcium, phosphorus, sodium and chloride was significantly stimulated. With the intracolonic hCT, the fractional urinary excretions of calcium, sodium and chloride were also marginally stimulated relative to intracolonic vehicle (placebo). In conclusion, hCT is absorbed intact from the colon, but the bioavailability is low and highly variable.

Adult↗
PubMed1992

Early diagnosis of the multiple endocrine neoplasia type 2 syndrome: consensus statement. European Community Concerted Action: Medullary Thyroid Carcinoma.

The diagnosis of medullary thyroid carcinoma by biochemical and genetic testing is possible in families with multiple endocrine neoplasia type 2. At an early stage total thyroidectomy usually cures the patient. As the clinical penetrance of the autosomal dominant, transmitted, multiple endocrine neoplasia type 2 gene is not complete, family screening is indicated for every new patient who presents with apparently sporadic medullary thyroid carcinoma. Problems related to a screening programme and early diagnosis have led the members of the European Community Concerted Action: Medullary Thyroid Carcinoma group to formulate a consensus on biochemical and genetic screening. For biochemical screening, measurement of basal and pentagastrin and/or calcium stimulated serum levels of calcitonin by radioimmunoassay are essential starting at the age of three and continuing annually until 35 years of age. Furthermore, annual screening for pheochromocytoma by measuring the urinary excretion of catecholamines and for hyperparathyroidism by serum calcium determination is indicated. Genetic screening using linked markers can be done with a 95% accuracy in informative families when DNA is available from at least two family members proven to be affected. Biochemical screening can thus be reserved for gene carriers, while those at low risk can be reassured. Combined biochemical and genetic screening for multiple endocrine neoplasia type 2 is important and effective for the cure of medullary thyroid carcinoma.

Adrenal Gland Neoplasms↗
PubMed1992

Parathyroid hormone receptors in control of proximal tubule function.

Parathyroid hormone action on renal proximal tubule function involves phospholipase C/protein kinase C as well as adenylate cyclase/protein kinase A mediated regulatory pathways. Tissue culture experiments suggest that low concentrations of PTH affect preferentially the phospholipase C/protein kinase C pathway. In vivo, both regulatory cascades are probably involved in the regulation of proximal tubule function. It is not clear at present whether the two intracellular pathways are linked to one or two PTH receptors. A polarized distribution of PTH receptor(s) involving different second messengers appears possible in proximal tubule epithelial cells. High-affinity (Kd 10(-11)-10(-12) M) PTH receptors in the range of circulating PTH concentrations in vivo remain to be identified. Structural and functional characterization of PTH receptors as well as of the PTH-sensitive intracellular mediators and transport systems form the basis for a better understanding of PTH-dependent regulation of proximal tubule function.

Absorption↗