Effect of nitric oxide donor nitroglycerin on bone mineral density in a rat model of estrogen deficiency-induced osteopenia.

Nitric oxide (NO) may modulate estrogen's anabolic effects on bone homeostasis by restraining osteoclast-mediated bone resorption and stimulation of osteoblast activity. Accordingly, NO donated by organic nitrates, including nitroglycerin, is thought to protect against bone loss associated with estrogen deficiency. In this study, we have explored this phenomenon. Thirty-two 12-week-old female Wistar rats were divided into four groups prior to bilateral ovariectomy or a sham operation. The ovariectomised rats received (1). vehicle control (OVX control), (2). 17-beta-estradiol (OVX+E2), or (3). transdermal nitroglycerin (OVX+NG) for 4 weeks. Femoral and tibial bone mineral density (BMD), serum alkaline phosphatase and urine deoxypyridinoline and NO metabolites were analysed at the end of the study period together with failure torque and torsional rigidity of the tibiae and cellular localisation of the NO-synthase (NOS) isoforms. In OVX+E2 group, proximal and distal femoral and proximal tibial BMD exceeded that of the Sham controls. Nitroglycerin prevented BMD loss at these three sites at levels comparable to that of the Sham controls. Deoxypyridinoline excretion did not change except in the OVX-E2 group that showed an expected reduction when compared to the Sham and OVX controls. There were no treatment-related differences in total alkaline phosphatase or urinary NO metabolites. Tibial failure torque was comparable between the groups but both OVX+E2 and OVX+NG groups showed decreased torsional rigidity compared with the OVX controls. Endothelial and inducible NOS were found in osteoblast-like cells associated with calcifying cartilage spicules in the distal femoral metaphysis. These data confirm previous findings and show that nitroglycerin counteracts the estrogen deficiency-induced osteopenia in the ovariectomised rat model. Organic nitrates may thus be beneficial in conditions where bone turnover is compromised such as in osteoporosis.

Leptin prevents the fall in plasma osteocalcin during starvation in male mice.

Plasma osteocalcin, a marker of osteoblastic activity, is reduced in starvation, malnutrition, and anorexia nervosa, resulting in low bone turnover osteoporosis. Contradictory findings about the role of leptin as a link between nutritional status and bone physiology have been reported. We demonstrate that leptin-deficient ob/ob and leptin-resistant db/db male mice have increased plasma osteocalcin, and that in male ob/ob mice osteocalcin is not decreased by starvation, unlike control mice. Intraperitoneal leptin administration increased plasma osteocalcin in male ob/ob mice, and prevented its fall during 24h fasting and 5 days of food restriction in normal male mice. This effect may be mediated via actions on the hypothalamic-pituitary-testicular or -growth hormone axes, or a direct action on osteoblasts. These studies support the hypothesis that the fall in leptin during starvation and weight loss is responsible for the associated reduction in osteoblast activity, and suggest a role for leptin in regulating bone turnover.

Serum levels of bone-specific alkaline phosphatase and procollagen type I carboxyterminal peptide in vitamin D deficiency.

Vitamin D deficiency in adults causes osteomalacia where there is a defect in bone mineralization resulting in an excess of unmineralised osteoid in the bone matrix. The aim of this study was to evaluate the markers of bone formation: total (TALP), bone-specific alkaline phosphatase (BSALP) and procollagen type I carboxyterminal peptide (PICP) in vitamin D deficiency. We studied 100 vitamin D deficient subjects and 82 gender-matched controls. Vitamin D deficiency was defined as serum 25-hydroxyvitamin D level of less than 7 ng/ml, and greater than 10 ng/ml for normal controls. Serum TALP assay was performed by a standard automated method, BSALP and PICP were measured by enzyme immunoassays (Metra Biosystems) and vitamin D by radioimmunoassay. There was significant difference in the TALP between female vitamin D deficient and control subjects (mean +/- sem = 99.8 +/- 8.2 vs 70.5 +/- 2.8 iu/l, p<0.001). Elevated serum TALP (>130 iu/l) was found in 20% (20/100) of the vitamin D deficient patients. There were no significant differences in BSALP or PICP between vitamin D deficient patients and gender-matched control subjects. There was no correlation between vitamin D and PICP in patients but in control subjects, a significant negative correlation (r= -0.431, p<0.0001) was found. In conclusion, although elevated TALP was observed in a minority of vitamin D deficient patients, it is a better marker than PICP. The lack of PICP response in vitamin D deficient subjects suggests the possibility of vitamin D deficiency leading to a block in osteoblast differentiation.

Calcitonin gene related peptide in familial dysautonomia.

Familial dysautonomia (FD) patients have diminished sensory C-fibers. Calcitonin gene related peptide (CGRP) is a widely distributed neuropeptide and prominent neurotransmitter in C-fibers. We show that plasma CGRP levels measured by radioimmunoassay is significantly lower in 51 FD patients compared to controls (P<0.001). In 11/51 FD patients with FD crisis and in 19/51 FD patients with pneumonia, the mean CGRP levels rose significantly as compared to their baseline (P<0.003, P<0.001, respectively). The deficiency of CGRP in FD patients is consistent with their depletion of C-fibers, and may explain some of their symptoms, either directly or via modulation of sympathetic activity.

Magnesium deficiency in patients with chronic pancreatitis identified by an intravenous loading test.

Magnesium deficiency is a common clinical condition that may exist despite a normal serum magnesium concentration. Patients with chronic pancreatitis could develop magnesium deficiency due to either malabsorption, diabetes mellitus, or chronic alcoholism. Since serum levels of magnesium are a poor indicator of magnesium deficiency, the retention of a low-dose intravenous magnesium load (0.1 mmol/kg body weight) was determined in 13 patients with chronic pancreatitis (10 due to alcoholism) and eight healthy controls. Percentage magnesium retention was greater in patients with chronic pancreatitis than controls (59.8+/-37.3% S.D. versus 22.0+/-38.2% S. D.: P=0.038), and 10 of 13 patients showed evidence of magnesium deficiency. Routine evaluation of magnesium status could allow appropriate supplementation and conceivably symptomatic improvement in patients with severe chronic pancreatitis.

A new calcitonin-receptor-like sequence in rat pulmonary blood vessels.

1. Two rat clones have been isolated which are similar to known calcitonin-receptor sequences. One of these does not have the distribution expected of a calcitonin receptor. It is widely distributed, with extremely high levels of expression in the lung, where it is associated with the blood vessels. 2. This rat sequence may represent the receptor for calcitonin-gene-related peptide or islet amyloid polypeptide. Both have binding activity in the lung and are potent vasodilators. The gene represented by this sequence may therefore play an important role in the maintenance of vascular tone.

Extra-pancreatic expression of the rat islet amyloid polypeptide (amylin) gene.

Messenger RNA for rat islet amyloid polypeptide (IAPP) has been identified not only in the pancreas but also, in lesser amounts, in preparations from the stomach and dorsal root ganglia. In the stomach, insulin mRNA was not detectable, ruling out possible contamination by pancreatic tissue. Because IAPP and calcitonin gene-related peptide (CGRP) are related and CGRP is present in both stomach and dorsal root ganglia, it was possible that 'IAPP' signals were in fact due to cross-hybridization with CGRP mRNA. A second IAPP probe was constructed which does not cross-react. This probe also detected mRNA in both tissues, confirming the expression of IAPP in both tissues. The regional distribution of IAPP mRNA in the stomach did not parallel that of gastrin mRNA. IAPP mRNA was present in the antrum, centrum and pylorus and, like gastrin, the highest amounts were in the pylorus. However, the ratio between the pylorus and centrum was 3.6:1 for IAPP and 156:1 for gastrin. The effects of dietary manipulation were determined; a period of 48 h of starvation reduced pancreatic IAPP mRNA by approximately 60%, whereas in the stomach there was no significant reduction. If the action of IAPP was hormonal, pancreas and stomach would not be acting in concert. A paracrine role for gastric IAPP therefore seems more likely.

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.

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.

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.

Altered islet amyloid polypeptide (amylin) gene expression in rat models of diabetes.

The response of the islet amyloid polypeptide gene to chronic dexamethasone treatment in adult rats was investigated. After 12 daily injections, rats were severely underweight and fasting blood glucose levels were elevated. When pancreatic mRNA was analysed, a 16-fold elevation in islet amyloid polypeptide mRNA was observed with only a four-fold increase in insulin mRNA levels. Pancreatic islet amyloid polypeptide and insulin mRNA levels were also determined 12 days after streptozotocin treatment. In these rats, which were not severely diabetic, the reduction in islet amyloid polypeptide mRNA levels was sixfold less than the reduction in insulin mRNA levels. In both these models of diabetes the ratio of islet amyloid polypeptide to insulin mRNA levels was raised. This would not be expected if the physiological role of islet amyloid polypeptide is as a simple hyperglycaemic agent opposing insulin action or release.

Expression of the rat amylin (IAPP/DAP) gene.

We have used the polymerase chain reaction with mixed sequence primers to generate a probe for rat amylin and have used this to detect expression in various rat tissues. Amylin mRNA is found in greatest concentrations in the pancreas where a single mRNA species can be detected giving a hybridisation signal intensity approximately 10% that of insulin mRNA. When the beta cell population was depleted with streptozotocin, both amylin and insulin mRNAs were reduced to a similar extent. Consistent with its supposed role in the control of carbohydrate metabolism, amylin mRNA was also found in the stomach. Unlike the related peptide, CGRP, amylin mRNA is not present in the thyroid and is not widely distributed in the central nervous system. The only nervous tissue in which it could be detected was the dorsal root ganglion. Surprisingly, amylin mRNA was also found in the lung though only at very low levels.

Altered calcitonin gene in a young patient with osteoporosis.

To assess whether calcitonin is important in maintaining the integrity of bone the calcitonin gene of a young male patient with osteoporosis and no detectable plasma concentrations of calcitonin was studied. Genomic Southern blots with various restriction enzymes showed no large abnormalities in his calcitonin gene. Genomic clones representing his calcitonin gene were then analysed. His gene encoded normal precursor polypeptides for calcitonin and calcitonin gene related peptide; the only abnormality identified was a single base insertion in the intron separating exons IV and V of the gene. The affected sequence is homologous with an intron sequence from beta globin that is implicated in splicing and forming a crucial intermediate structure during the maturation of messenger RNA. The change observed may be responsible for the patient's calcitonin deficiency and consequently for his condition, suggesting that calcitonin is important in preventing bone loss.

Development and performance of a highly sensitive carboxyl-terminal-specific radioimmunoassay of calcitonin gene-related peptide.

The calcitonin genes encode a small family of peptides: the circulating hormone calcitonin; its flanking peptide, katacalcin; and a third novel peptide, calcitonin gene-related peptide (CGRP). CGRP is a potent vasodilator and a major circulating product from the calcitonin genes; it may be a physiologically important regulator of blood flow in humans. High concentrations of circulating CGRP are found in medullary thyroid carcinoma. We report the development and validation of a highly sensitive (detection limit 500 amol per tube) radioimmunoassay of CGRP involving a high-affinity antibody directed against the carboxyl terminus of the molecule and a highly pure tracer. The assay is precise, robust, and reproducible, and is therefore a potentially useful analytical method for studying the normal and abnormal physiology of this peptide.

Transient appearance of calcitonin gene-related peptide-like immunoreactive fibers in the developing cerebellum of the rat.

A plexus of calcitonin gene-related peptide (CGRP)-containing fibers were transiently found in the developing cerebellum of the rat by means of the indirect immunofluorescent method. CGRP-like immunoreactive fibers appeared in the cerebellum by embryonic day 22. Immunoreactive fibers rapidly increased and these made a dense plexus in the Purkinje cell layer by postnatal day 2. However, only a few if any immunoreactive fibers were seen in the Purkinje cell layer or molecular layer of adult rats.

Calcitonin gene-related peptide-like immunoreactive sensory fibers form synaptic contact with sympathetic neurons in the rat celiac ganglion.

The present study demonstrates synaptic contact between calcitonin gene-related peptide (CGRP)-like immunoreactive axon terminals and sympathetic neurons in the rat celiac ganglion. Our observations suggest that sensory ganglion neurons directly regulate the sympathetic activity via synapses, because CGRP immunoreactive (CGRPI) fibers in this ganglion are supplied by the sensory ganglia.

Distribution and origin of calcitonin gene-related peptide in the rat stomach and duodenum: an immunocytochemical analysis.

We studied the three-dimensional distribution of structures with calcitonin gene-related peptide-like immunoreactivity (CGRPI) in the rat stomach and duodenum, including the origins of these structures, using indirect immunofluorescence in both muscle strips and frozen sections. There was a very dense meshwork of CGRPI fibers in the circular and longitudinal muscle layers, and also in the myenteric and submucous plexuses of the stomach and duodenum. No CGRPI neurons were seen in the stomach, even in rats treated with colchicine; in the duodenum, there was a group of CGRPI cells in the myenteric and submucous ganglia. No regional differences were seen in the stomach and duodenum. We found by experimental manipulations that CGRPI fibers in the stomach were exclusively extrinsic in origin; some of such fibers in the duodenum were intrinsic in origin, though most were supplied by CGRPI cells outside the duodenum.

The origin of circulating calcitonin gene-related peptide in the rat.

It is known that in addition to the calcitonin precursor the calcitonin gene also encodes a novel peptide, calcitonin gene-related peptide (CGRP). This potent vasodilator has been found in the circulation of man. This present study demonstrates that CGRP is also found in the circulation of the rat and that plasma CGRP comes from two different sources: the thyroid, a major source in old rats, and the perivascular nerves probably at all ages.