Biomedical subjects

M Zaidi

Publications and source records attributed to M Zaidi.

At least 109 records · Page 6Linked to original sources

PubMed1990

Kidney: a target organ for calcitonin gene-related peptide.

In the present study we have evaluated the effect of calcitonin gene-related peptide (CGRP) on cyclic AMP levels in intact mouse kidneys. We have used an in vivo bioassay based on microwave irradiation as a means of rapid tissue fixation, followed by the determination of cyclic AMP concentration using a protein binding assay. CGRP was found to cause a dose-dependent elevation of renal cyclic AMP levels, and despite being slightly less potent than calcitonin (CT), the action of CGRP was significantly more prolonged. The extended time course of action of CGRP is in sharp contrast to its known effect on bone cyclic AMP levels and could be of physiological relevance. CGRP may act on a receptor different from that of CT to produce distinct functional effects.

Animals↗

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

Intracellular regulation of enzyme secretion from rat osteoclasts and evidence for a functional role in bone resorption.

1. Osteoclasts are known to secrete acid phosphatase, an iron-containing phosphohydrolase. We have investigated (a) the possibility that acid phosphatase has a functional role in bone resorption and (b) the factors controlling enzyme secretion from isolated rat osteoclasts. 2. Osteoclasts were freshly disaggregated from neonatal rat long bones and dispersed at low densities on devitalized cortical bone slices or on plastic substrate. The levels of acid phosphatase in culture medium were measured spectrophotometrically using 4-nitrophenyl phosphate as hydrolysable substrate. The total plan area of bone resorbed was quantified by scanning electron microscopy in combination with image processing and analysis. 3. Ninety-three per cent of the total enzyme activity detected in the supernatant exposed to bone-osteoclast preparations was resistant to inhibition by D-tartaric acid and was bound to an antibody known to be highly specific for the osteoclast-derived isoenzyme, showing that it originated from osteoclasts. 4. A diminution in the level of supernatant enzyme activity achieved by incubating bone-osteoclast preparations with an antiserum specifically binding the osteoclast isoenzyme, or with a non-competitive inhibitor, molybdate or with competitive inhibitors, disphosphonates, led to a marked reduction of osteoclastic bone resorption. 5. The rate of the enzyme released into the culture supernatant, whether from resorbing (cultured on bone) or non-resorbing (cultured on plastic) osteoclasts declined gradually over 22 h, but that from the former was significantly depressed within the first 30 min of incubation. The supernatant enzyme concentration increased linearly up to 3 h; the levels released from resorbing osteoclasts remained consistently lower than those from non-resorbing cells. 6. Exposure of osteoclasts for 18 h to elevated [Ca2+]o levels produced a concentration-dependent inhibition of supernatant acid phosphatase levels. In the presence of 20 mM [Ca2+]o enzyme secretion from resorbing osteoclasts was significantly lower than that from non-resorbing cells. 7. Exposure of bone-osteoclast preparations to pertussis toxin produced no significant change of acid phosphatase release, while cholera toxin, dibutyryl cyclic AMP and forskolin produced a marked elevation of enzyme secretion. Ionomycin was found to inhibit enzyme release and this was less marked when osteoclasts were incubated on plastic substrate.(ABSTRACT TRUNCATED AT 400 WORDS)

Acid Phosphatase↗

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 calcitonin gene peptides: biology and clinical relevance.

The calcitonin/CGRP multigene complex encodes a family of peptides: calcitonin, its C-terminal flanking peptide, katacalcin, and a third novel peptide, calcitonin gene-related peptide (CGRP). The 32-amino acid peptide calcitonin inhibits the osteoclast, thereby conserving skeletal mass during periods of potential calcium lack, such as pregnancy, growth, and lactation. This hormonal role is emphasized by observations that lower circulating calcitonin levels are associated with bone loss and that calcitonin replacement prevents further bone loss. Structurally, CGRP resembles calcitonin and has been implicated in neuromodulation and in the physiological regulation of blood flow. Here we review the molecular genetics, structure, and function of the calcitonin-gene peptides as analyzed in the laboratory and focus on more recent clinical studies relating to disorders and therapeutics.

Amino Acid Sequence↗

PubMed1990

Development and performance of a highly sensitive and specific two-site immunometric assay of calcitonin gene-related peptide.

Calcitonin gene-related peptide (CGRP), a potent endogenous circulating vasodilator, is produced by the alternative splicing of the calcitonin/CGRP gene and is expressed mainly in neural and cardiovascular tissues. We recently reported a highly sensitive radioimmunoassay of CGRP, based on an antiserum recognizing the C-terminus of the molecule. We have also found that circulating immunoreactive CGRP is heterogeneous; thus we are unable to measure selectively the intact molecule with our one-site competitive approach. We therefore attempted to construct a two-site immunometric assay involving two antibodies, one that detects the C-terminus and another that recognizes the midregion of the molecule. To enhance assay sensitivity, we applied a colorimetric amplification system to this assay. This rapid, robust, and reproducible assay provides more nearly accurate estimates of circulating CGRP and offers a sensitive and more specific alternative to the radioimmunoassay, with advantages in speed, simplicity, and convenience.

Amino Acid Sequence↗

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↗

PubMed1989

Inhibition of osteoclastic acid phosphatase abolishes bone resorption.

Osteoclastic acid phosphatase is a member of a widely-distributed class of iron-containing proteins with acid phosphatase activity. Antibodies raised against one member of this class cross-react with other members from the same or different species, but not with acid phosphatase isoenzymes of different types. When antibodies to one such protein, porcine uteroferrin, are added to medium in which rat osteoclasts are incubated on devitalised cortical bone, both bone resorption and acid phosphatase activity are markedly inhibited. Furthermore, addition of molybdate (an inhibitor of this class of acid phosphatases) also inhibits both bone resorption and enzyme activity. These observations strongly suggest a functional role for osteoclastic acid phosphatase in bone resorption.

Acid Phosphatase↗

PubMed1989

Production and characterisation of immunoreactive calcitonin gene-related peptide (CGRP) from a CGRP receptor-positive cloned osteosarcoma cell line (UMR 106.01).

A subclone of an osteoblast-like osteosarcoma cell line (UMR 106.01) has recently been shown to possess specific binding sites for calcitonin gene-related peptide (CGRP) linked to adenylate cyclase. The present study provides the first demonstration for the production of immunoreactive CGRP from CGRP-receptor positive osteosarcoma cells. Mean immunoreactive CGRP levels were 15 pmol/g and 1 pmol/l for acid extracts of cells and cell-exposed media respectively. On gel filtration and high performance liquid chromatography, a major proportion of immunoreactive CGRP was found to co-elute with synthetic rat CGRP(1-37). Only negligible quantities of calcitonin were detected in cell extracts or cell-exposed supernatant. The production of authentic CGRP from a CGRP-receptor positive tumour suggests that the peptide may have autocrine effects on its producer cell.

Animals↗

PubMed1989

The effect of extracellular calcium elevation on morphology and function of isolated rat osteoclasts.

Osteoclasts are large multinucleate cells unique in their capacity to resorb bone. These cells are exposed locally to high levels of ionised calcium during the process of resorption. We have therefore examined the effect of elevated extracellular calcium on the morphology and function of freshly disaggregated rat osteoclasts. Cell size and motility were quantitated by time-lapse video recording together with digitization and computer-centred image analysis. In order to assess the resorptive capacity of isolated osteoclasts, we measured the total area of resorption of devitalised cortical bone by means of scanning electron microscopy and computer-based morphometry. The results show that elevation of the extracellular calcium concentration causes a dramatic reduction of cell size, accompanied by a marked diminution of enzyme release and abolition of bone resorption. We propose that ionised calcium might play an important role in the local regulation of osteoclastic bone resorption.

Acid Phosphatase↗

PubMed1989

Calcitonin gene-related peptide: endocrine distribution and characterization of circulating forms.

This study reports the application of a highly sensitive and specific extraction-based radioimmunoassay for determining levels of calcitonin gene-related peptide (CGRP) in endocrine tissues and plasma of normal rats. In addition, we have characterized the immunoreactive material found in plasma using gel filtration and high performance liquid chromatography. The CGRP content of the thyroid was 100- to 1000-fold higher than that of the adrenals, pituitary or pancreas, while the gonads and kidneys contained appreciably smaller amounts of CGRP. There was no age- and sex-related difference in tissue content, with the exception of a subgroup of 450 g rats. These had a significantly raised thyroidal CGRP content compared to other rats of the same group. A significant correlation between thyroidal content and plasma levels was noted only in old (450 g) rats. CGRP-like immunoreactivity in plasma pooled from young adult (150 g) rats was found to be heterogeneous. Only 8% of the total immunoreactivity recovered from a Sephadex G50 column was found to coelute with synthetic rat CGRP when freeze-dried plasma was chromatographed and a significantly greater proportion (approximately 67%) eluted in the void volume. The void volume peak was markedly reduced when acid-methanol extracts of plasma were chromatographed under similar conditions. A major proportion of the immunoreactive material co-eluting with synthetic CGRP on gel filtration was also found to elute at a position corresponding to the monomer on HPLC: this was consistent with, though not proof of, structural identity.

Adrenal Glands↗

PubMed1989

Epidemic of Serratia marcescens bacteremia and meningitis in a neonatal unit in Mexico City.

A case-control study was conducted on an epidemic of bacteremia and meningitis caused by Serratia marcescens in the neonatal intensive care unit and special care nursery of a general hospital in Mexico City, Mexico. A 19.9% incidence of bacteremia and meningitis was recorded in contrast to 1.4% and 3.7% during preepidemic and post-epidemic periods; a 69% mortality rate was observed. Peripheral IV catheters and the use of mixed IV fluids prepared in the wards were the major risk factors (P less than 0.001). Rectal and nasopharyngeal cultures were positive in 68% of asymptomatic neonates and hand cultures were positive in 16.7% of personnel. Strains were resistant to all aminoglycosides and broad-spectrum penicillins, and belonged to the A5/8 biogroup. Containment of this outbreak was difficult because of failure to identify colonized infants early in the epidemic and because of persistent carriage of S marcescens by personnel. Comparisons between this hospital and tertiary care centers in Mexico suggest that in developing countries nosocomial infections could be of greater magnitude in secondary than in tertiary level centers.

Cross Infection↗

PubMed1989

Further evidence for the origin of circulating calcitonin gene-related peptide in the rat.

1. We have attempted to determine the origin of a potent circulating vasodilator, calcitonin gene-related peptide (CGRP), by assessing the effects of administration of capsaicin and colchicine, and of thyroidectomy on plasma levels. Plasma CGRP, neurokinin A and calcitonin levels were measured using highly sensitive and specific radioimmunoassays, and the circulating forms of CGRP characterized by size-exclusion gel and high-performance liquid chromatography. 2. Seven minutes following the intraperitoneal injection of capsaicin (10 mg/kg body weight) plasma CGRP and neurokinin A levels were found to rise by 15- and 4-fold respectively, while there was no change in circulating calcitonin levels. Chromatography revealed that the immunochemical forms of CGRP released into the circulation by capsaicin were similar to those found in normal plasma, which included a peak co-eluting with the intact CGRP molecule. 3. Six hours after the intraperitoneal administration of colchicine (10 mg/kg body weight), CGRP was not detectable in the circulation of 40-day-old rats and plasma levels were significantly lowered in the group of greater than or equal to 200-day-old rats. Chromatography revealed that peaks of circulating immunoreactivity corresponding to monomeric CGRP and its fragments were substantially reduced following colchicine treatment. 4. The magnitude of capsaicin-evoked elevation of plasma CGRP was only marginally (significant at 0.1 greater than P greater than 0.05) reduced by pre-treatment with colchicine. 5. Thyroidectomy alone caused a significant reduction of plasma CGRP levels in greater than or equal to 200-day-old rats; a greater reduction was observed when thyroidectomy was combined with colchicine administration. However, when 40-day-old rats were thyroidectomized, there was an unexplained elevation of plasma CGRP levels. 6. The results suggest that while CGRP is normally released into the circulation from nerve terminals, both in young adult (40-day-old) and old (greater than or equal to 200-day-old) animals, a thyroidal origin is obvious only in old rats.

Animals↗

PubMed1989

Production of calcitonin gene-related peptide from human cancer cells.

Calcitonin (CT) is produced ectopically from a variety of non-thyroidal cancers. The CT genes also encode another peptide, calcitonin gene-related peptide (CGRP) which is a potent vasodilator. In the present study we have used immunochemical and chromatographic methods to demonstrate the presence and characterize the molecular forms of CGRP in cultured human cancer cells. Using two highly sensitive and specific radioimmunoassays, we have detected immunoreactive CGRP (i-CGRP) in cell extracts and cell-exposed media of cultured promyelocytic leukaemia (HL60) and bronchogenic carcinoma (BEN) cells. The mean i-CGRP content of the HL60 and BEN cell extracts was 2 and 45 pmol/g wet weight respectively. On gel filtration and high performance liquid chromatography, the immunoreactive material was found to be heterogeneous, though a major proportion co-eluted with synthetic human CGRP(1-37), suggesting structural identity with the intact CGRP molecule. Finally, we have discussed some interesting features of CT-gene peptide expression in tumour cells.

Calcitonin↗

PubMed1989

Uncoupling of receptor-mediated cellular responses by ionic lithium.

The propensity of ionic lithium to interfere with the coupling of receptors to guanine nucleotide binding proteins (G-proteins) has only recently been investigated using rat cortical membranes. In the present study we have used intact isolated osteoclasts to investigate lithium-induced uncoupling of the receptor-mediated actions of calcitonin. All actions of calcitonin on the osteoclast were abolished by ionic lithium. We believe that the cation prevents signal transduction by inhibiting G protein-receptor interaction, the first step in intracellular signalling.

Animals↗