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I MacIntyre

Publications and source records attributed to I MacIntyre.

At least 37 records · Page 2Linked to original sources
PubMed1991

Predicted structure of the bovine calcitonin gene-related peptide and the carboxy-terminal flanking peptide of bovine calcitonin precursor.

We have isolated from a bovine genomic library a clone which contains the calcitonin (CT) and CT gene-related peptide (CGRP) sequences, using probes representing the human CT and CGRP sequences. Sequence analysis has identified the nucleotide sequence coding for bovine CT, its C-terminal flanking peptide and bovine CGRP. The deduced amino acid sequence of bovine CGRP revealed a significant homology with other CGRPs so far reported. It differs by only one amino acid from rat CGRP alpha and porcine CGRP, and by three and four amino acids from human CGRP beta and alpha respectively. Bovine CT has, however, only 14 out of 32 residues in common with human CT. As in the human CT precursor, the C-terminal flanking peptide of bovine CT precursor is a 21 amino acid peptide. It shares only 11 residues in common with its human counterpart. This study thus provides further evidence that CGRP, in contrast to CT and its C-terminal flanking peptide, is a highly conserved molecule.

Amino Acid Sequence↗
PubMed1990

Structure-activity relationship of human calcitonin-gene-related peptide.

The calcitonin-calcitonin-gene-related peptide (CGRP) gene complex encodes a small family of peptides: calcitonin, CGRP and katacalcin. Calcitonin is a circulating hormone that prevents skeletal breakdown by inhibiting the resorption of bone by osteoclasts. CGRP, a potent vasodilator, is involved in normal regulation of blood flow. The calcitonins structurally resemble the CGRP peptides, and both are known to cross-react at each others' receptors. The present study was undertaken to examine the structural prerequisites for biological activity of the intact CGRP molecule. We therefore prepared eight chymotryptic and tryptic fragments of CGRP and synthesized its acetylated and S-carboxyamidomethylcysteinyl analogues. The analogues were purified by h.p.l.c. and their structures were confirmed by fast-atom bombardment mass spectrometry. We have examined the effects of structurally modified analogues and fragments of human CGRP in a calcitonin-receptor-mediated assay, the osteoclast bone resorption assay, and in one or two CGRP-receptor-mediated assays, the rabbit skin blood flow assay and the oedema formation assay. The results showed that (1) in the osteoclast bone resorption assay, both CGRP peptides, alpha and beta, were equipotent, and were both at least 1000-fold were both approx. 1000-fold more potent than salmon calcitonin; human calcitonin had no effect; (3) the bis- and N-acetylated CGRP analogues retained reduced levels of biological activity in all assays, whereas S-carboxyamidomethylcysteinyl-human CGRP was without activity; and (4) all tryptic and chymotryptic fragments of CGRP were without biological activity, with the exception of hCGRP-(Ala1-Lys35): this fragment had much reduced activity compared with the intact peptide in inhibiting osteoclastic bone resorption and increasing blood flow in the rabbit skin. The results suggest that: (1) calcitonin and CGRP act at distinct receptors to mediate different physiological effects; (2) minor amino acid substitutions, as between the alpha and beta forms of CGRP (these two forms have 94% structural similarity) do not result in differences in biological activity; (3) the intact peptide is required for full biological activity of the CGRP molecule, and even the loss of two amino acids at the C-terminus of the molecule results in a marked decrease in activity; (4) the disulphide bridge appears to play an important role in the interaction of the intact CGRP molecule with its receptor; and (5) the C-terminal region is probably necessary for the peptide to assume the right conformation in the interaction with the receptor.

Amino Acid Sequence↗
PubMed1990

A dual effect of calcitonin gene-related peptide on plasma calcium levels in the chick.

Calcitonin gene-related peptide (CGRP) lowers plasma calcium in the rat and inhibits bone resorption by isolated rat osteoclasts. In our preliminary studies we found that rat CGRP elevates plasma calcium levels in the chick, a response that was somewhat similar to that of parathyroid hormone. Here, we report that human CGRP (alpha) produces a concentration-dependent elevation of plasma calcium levels. The two peptides did not follow precisely the same time course. Whereas at 15 minutes CGRP produced hypocalcaemia relative to the control plasma calcium levels, at 30 minutes both CGRP and PTH were found to be hypercalcaemic. These studies suggest that CGRP initially interacts with the calcitonin receptor to produce a calcitonin-like effect, which is followed by hypercalcaemia presumably by antagonising the action of endogenous circulating calcitonin.

Animals↗
PubMed1990

Isolation, purification and characterization of beta-hCGRP from human spinal cord.

Human beta-calcitonin gene-related peptide (beta-hCGRP) was isolated and purified from spinal cord. The complete characterization of this material was based on definitive mass analysis by fast atom bombardment mass spectrometry together with gas phase sequencing. Combining these data we have characterized the structure of beta-hCGRP including the C-terminal sequence, the presence of the S-S bridge and of phenylalanineamide as the C-terminal amino acid, and fully confirmed the amino acid sequence predicted from the nucleotide analysis.

Amino Acid Sequence↗
PubMed1990

Intracellular calcium in the control of osteoclast function. II. Paradoxical elevation of cytosolic free calcium by verapamil.

We report here for the first time that verapamil elevates cytosolic calcium. We have found that in the isolated rat osteoclast, verapamil at low micromolar concentrations did not block the elevation of cytosolic free calcium ([Ca++]i) in response to elevated extracellular calcium concentrations ([Ca++]e). However, high micromolar concentrations of verapamil (300 microM or above) led to a rapid sustained elevation of [Ca++]i. These concentrations of verapamil had effects on osteoclast morphology and resorptive activity that were similar to those produced by elevated [Ca++]e: there was a marked dose-dependent fall in cell spread area and osteoclastic bone resorption. The sensitivity of osteoclasts to high micromolar concentrations of verapamil is unique and could not be mimicked in macrophages and lymphocytes.

Animals↗
PubMed1990

Intracellular calcium in the control of osteoclast function. I. Voltage-insensitivity and lack of effects of nifedipine, BAYK8644 and diltiazem.

We have recently demonstrated that in the rat osteoclast, a rise in the ambient calcium concentration induces a rapid elevation of cytosolic calcium, and that this phenomenon is accompanied by a complete inhibition of osteoclastic bone resorption. Here, we have attempted to characterise the electrophysiological nature of the putative 'calcium-activated' calcium channel. We have established that calcium influx into the osteoclast that occurs on exposure to elevated extracellular calcium is independent of membrane voltage and is insensitive to modulation by organic calcium channel modulators, namely nifedipine, BAYK8644 and diltiazem. The latter compounds were also unable to block the reduction of cell spread area and the inhibition of bone resorption produced in response to elevated extracellular calcium levels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1990

A new approach for calcitonin determination based on target cell responsiveness.

We report the development and validation of three microbioassays for calcitonin based on calcitonin-induced inhibition of the activity of isolated osteoclasts. Having precisely quantified osteoclast motility, spreading and bone resorptive activity, we have applied stringent analytical procedures to define assay characteristics. We have found that the appropriately transformed responses significantly regress on log dose of the peptides. Furthermore, potency estimates obtained using calcitonins from three species (human, salmon and a synthetic analogue of eel calcitonin) have been found to be consistent with those obtained using conventional calcitonin bioassays. In addition, the assays are remarkably sensitive (detection limit 10(-15) M), highly specific and precise. We have determined plasma levels of bioactive calcitonin on samples from patients with medullary thyroid carcinoma; these are several-fold lower than those obtained using our routine calcitonin radioimmunoassay. Our study thus, forms the basis of an entirely new approach for the determination of 'biologically active' calcitonin, and we envisage that such target cell-specific assays could become useful microanalytical methods.

Animals↗
PubMed1990

Calcitonin gene-related peptide and calcitonin immunoreactivity in brain and spinal cord in Alzheimer-type dementia.

Calcitonin gene-related peptide (CGRP) and calcitonin (CT) immunoreactivity were measured in hypothalamus, parahippocampal gyrus, pituitary and grey matter of the posterior and anterior spinal cord from five to six cases of Alzheimer-type dementia (ATD) and from five to six controls. CGRP was slightly increased and choline acetyltransferase decreased in the anterior grey of ATD spinal cord. No other significant differences were observed between the levels of the two peptides in the ATD and control tissues, even in the parahippocampal gyrus and posterior grey of the spinal cord which had reduced choline acetyltransferase activity in the ATD cases. These results show that CGRP and CT are not affected in ATD, either as a consequence of a direct effect on peptidergic neurons or secondary to the loss of choline acetyltransferase activity.

Aged↗
PubMed1990

Biological activity of calcitonin and its assessment.

Radioimmunoassay has been the method of choice for the routine determination of calcitonin (CT) in biological fluids, mainly due to its precision, sensitivity, and ability to handle large numbers of samples. However, spuriously high results are often obtained due to undefined cross-reactivities of polyclonal antisera to precursors, polymers, metabolites, or fragments of CT, or to other physiological and pathological variants of the hormone. In routine practice, radioimmunoassays of CT are being replaced by immunometric two-site 'sandwich' assays utilizing monoclonal antibodies against defined antigenic epitopes. Hormone activity is, however, accurately quantifiable by biological assays based on the exquisite hormonal sensitivity of isolated osteoclasts. The assays are highly specific and remarkably sensitive. Their use in routine laboratories is nevertheless limited only to specific situations.

Antibodies, Monoclonal↗
PubMed1990

Amylin-amide: a new bone-conserving peptide from the pancreas.

Amylin-amide is a new member of the family of peptides encoded by the calcitonin multigene complex. In the present study, we have compared directly, the hypocalcaemic potency and duration of action of human amylin-amide and human calcitonin in an in vivo rat bioassay and an in vitro osteoclast bone resorption assay. Amylin-amide was found to have a potency approximately 40-fold lower than human calcitonin, whilst both peptides followed the same time course. This suggests that amylin-amide is the most potent non-calcitonin hypocalcaemic peptide so far reported. An important physiological implication follows. It would seem that amylin-amide can play a central role in the maintenance of the skeleton by virtue of its inhibitory influence on osteoclastic function.

Amyloid↗
PubMed1990

Amylin and amylin-amide lack an acute effect on blood glucose and insulin.

Amylin-amide has been implicated in the pathogenesis of type II diabetes due to its proposed inhibitory effect on insulin release from beta cells of the pancreatic islets, and on glucose uptake by the skeletal muscle. In experiments with rats and rabbits we failed to demonstrate these anti-insulin actions of amylin and amylin-amide. A single bolus dose of the two peptides (500 pmol) administered i.v. failed to suppress plasma insulin levels or to elevate blood glucose levels. The continuous infusion of amylin-amide into rabbits also failed to suppress the release of insulin in response to hyperglycaemia produced by an i.v. bolus injection of glucose. These in vivo observations imply that the amylin peptides may not have a primary physiological role in carbohydrate metabolism, but in view of our previous findings, we speculate that the peptide has a more prominent role in calcium homeostasis.

Amyloid↗
PubMed1990

Evidence that the action of calcitonin on rat osteoclasts is mediated by two G proteins acting via separate post-receptor pathways.

Calcitonin inhibits osteoclastic bone resorption and its action involves two separate acute effects on the osteoclast, both essential to the action of the hormone: abolition of cell motility (Q) and marked cellular retraction (R). The former was mimicked by dibutyryl cyclic AMP and cholera toxin and the latter by pertussis toxin, ionomycin and increases in ambient calcium. Aluminium fluoride ions produced both Q and R effects, while lithium prevented both. In addition, calcitonin elicited a biphasic elevation of cytosolic-free calcium in single isolated osteoclasts. We propose that the action of calcitonin is mediated by at least two G proteins, one responsible for the Q effect and the other for the R effect. In addition, two second messengers, cyclic AMP and calcium, are involved. These findings may help to explain the potency of calcitonin in inhibiting bone resorption, and may allow the rational design of new therapeutic agents designed to alter osteoclast behaviour.

Aluminum↗
PubMed1990

The demonstration of vasodilator activity of pancreatic amylin amide in the rabbit.

Amylin amide, a 37-amino acid peptide that is a major component of amyloid deposits in the diabetic pancreas, possesses vasodilator activity. Human synthetic amylin amide (30 to 300 pmol/site) stimulated a dose-dependent increase in blood flow after intradermal injection in rabbit skin. Amylin amide was 100 times less active than the structurally related potent vasodilator neuropeptide calcitonin gene-related peptide. Amylin amide did not induce edema formation; however, as a consequence of its vasodilator activity, amylin amide potentiated edema formation induced in rabbit skin by bradykinin. The intravenous injection of amylin amide (10 nmol) caused a systemic drop in blood pressure. This study demonstrates that amylin amide elicits vasodilator responses in vivo. It is possible that the release of amylin amide from the pancreas in type II diabetes could lead to changes in vascular tone.

Amyloid↗
PubMed1989

Amylinamide, bone conservation, and pancreatic beta cells.

Amylinamide is a potent osteoclast-inhibiting peptide that is co-secreted with insulin from the beta cell. It induces profound hypocalcaemia in rats and rabbits and abolishes bone resorption by isolated osteoclasts in vitro. The non-amidated human peptide forms an insoluble fibrillar amyloid deposit that may interfere with beta-cell function and precipitate type II diabetes.

Amyloid↗
PubMed1989

'Calcium-activated' intracellular calcium elevation: a novel mechanism of osteoclast regulation.

The osteoclast is unique in its capacity to resorb bone. An unbalanced increase in this activity causes osteoporosis, a crippling bone disease that poses a major public health problem. Despite this, our understanding of osteoclast regulation is very limited. Calcitonin is the only known physiological inhibitor of osteoclast function. We demonstrate here for the first time that the concentration of calcium ions at the resorptive site directly regulates osteoclast function by modulating the intracellular free calcium concentration. This represents an important feedback mechanism of osteoclast control.

Acid Phosphatase↗
PubMed1989

In vivo and in vitro effects of amylin and amylin-amide on calcium metabolism in the rat and rabbit.

Amylin is a new member of the calcitonin/CGRP family: it is a 37 amino acid polypeptide which was recently isolated from amyloid deposits in pancreatic islets obtained from type II diabetics. In the present study we investigated the effect of amylin and amylin-amide on calcium metabolism in the rat and rabbit. Two main methods were used: in vivo hypocalcaemic activity was assessed by measuring plasma calcium levels after injection of the peptide in 50 g rats; and in vitro resorption of cortical bone by disaggregated rat osteoclasts was quantified by scanning electron microscopy together with image analysis. We demonstrate that amylin and amylin-amide have calcitonin-like effects: both are powerful inhibitors of osteoclastic resorption and as a consequence lower plasma calcium in both rats and rabbits. We speculate that the peptide may exert systemic or local regulatory effects on bone cells.

Amyloid↗
PubMed1989

Transforming growth factor-beta-induced mitogenesis of human bone cancer cells.

We report for the first time the bifunctional effects of transforming growth factor-beta on the growth of cloned human osteosarcoma cells (Htb96). Cell growth was assessed by determining the cell number, replication index and [3H]-thymidine incorporation following 48 hours incubation of cultured Htb96 cells with the peptide. Exposure of cells to concentrations of TGF-beta upto 40 pM caused a mitogenic response, concentrations between 40 and 800 pM failed to stimulate cell growth and higher doses caused an inhibition of cell proliferation. The initial cell density was found to alter the responsiveness of Htb96 cells to TGF-beta; stimulation of proliferation was less profound at high and low cell densities. The observed cell density- and growth factor concentration-dependent effects of TGF-beta on the growth of tumour cells might suggest the existence of an autocrine regulatory mechanism. Furthermore, by demonstrating a sensitivity to inhibition by indomethacin, we conclude that the proliferative effect of TGF-beta is at least, in part, dependent on the de novo synthesis of prostaglandins.

Bone Neoplasms↗