Immunoreactive Calcitonin Research Articles

Small cell lung carcinoma cell lines express mRNA for calcitonin and alpha- and beta-calcitonin gene related peptides

Calcitonin (CT) and calcitonin gene related peptide (CGRP) are derived from preprohormones encoded by three mRNAs (CT, α-CGRP and β-CGRP) from two genes (CALC1 and CALC2) on chromosome 11. Among 16 small cell lung cancer cell lines examined by RNase protection assay, 9 (56%) had detectable CT mRNA, 8 (50%) had α-CGRP mRNA, and 13 (81%) had β-CGRP mRNA. At least one CALC1 transcript (CT or α-CGRP) was found in 11 (69%) cell lines with three having only CT mRNA, two having only α-CGRP mRNA, and six having both. β-CGRP mRNA was detected in all of these 11 cell lines expressing a CALC1 transcript. Immunoreactive CT was detected by radioimmunoassay in eight of nine SCLC cell lines expressing CT mRNA, and immunoreactive CGRP was detected in 12 of 13 cell lines expressing a CGRP mRNA. The variety of expression of these three peptides in different cell lines of the same cell type should provide a useful system for further study of the control of expression of these peptides.

Effects of Humoral Hypercalcemia of Malignancy and Gallium Nitrate on Thyroid C Cells in Nude Mice: Immunohistochemical and Ultrastructural Investigations

Immunohistochemical and ultrastructural investigations of thyroid C cells were conducted in male nude (athymic) mice bearing a serially transplantable canine adenocarcinoma (CAC-8) model of humoral hypercalcemia of malignancy following subcutaneous administration of gallium nitrate. The following four groups were investigated: 1) vehicle-treated non-tumor-bearing control mice; 2) non-tumor-bearing mice treated with gallium nitrate; 3) vehicle-treated hypercalcemic mice bearing CAC-8; and 4) CAC-8 tumor-bearing mice treated with gallium nitrate. Gallium nitrate-treated tumor-bearing mice had a significant decrease in serum calcium as compared with tumor-bearing controls. C cells of non-tumor-bearing mice stained intensely for calcitonin and calcitonin gene-related peptide and weakly for chromogranin A and neuron-specific enolase. In C cells of both vehicle- and gallium-treated tumor-bearing mice, immunoreactive staining was decreased for calcitonin, calcitonin gene-related peptide, and chromogranin A, whereas there was a moderate increase in staining for neuron-specific enolase. Ultrastructurally, thyroid C cells in hypercalcemic tumor-bearing control and gallium-treated mice were hypertrophic and markedly degranulated as compared with those of non-tumor-bearing controls. Hypertrophic C cells contained few mature secretory granules, a well-developed Golgi apparatus, and lamellar arrays of rough endoplasmic reticulum. There was no evidence of C-cell hyperplasia. Immunohistochemical and ultrastructural findings revealed that C cells in mice with cancer-associated hypercalcemia were primarily in the actively synthesizing phase of the secretory cycle and had diminished immunoreactivity for calcitonin, calcitonin gene-related peptide, and chromogranin A.(ABSTRACT TRUNCATED AT 250 WORDS)

Disappearance rate of serum calcitonin after total thyroidectomy for medullary thyroid carcinoma.

We studied the half-life of serum calcitonin (CT) in patients subjected to total thyroidectomy for medullary thyroid carcinoma (MTC). One patient showed a rapid serum CT component with a half-life of 3 hours and a slow component with a half-life of 30 hours; in another case only the 30-hour component was found. By chromatography of tumor extracts, we found that all the immunoreactive CT had a molecular weight of 3,600. After surgery, normalization of serum CT was achieved within 15 days in 4 patients, at 3 months and at 6 months in 2 other patients, while 1 patient never normalized. Normalization of serum CT after surgery is not an index of definitive cure in MTC, as demonstrated by one patient who relapsed 3 months after normalization of serum CT. However, as a general rule, patients who reach undetectable serum CT levels soon after surgery, are those having the best prognosis.

Ontogeny of endocrine cells in the respiratory system of Syrian golden hamsters. I. Larynx and trachea.

The ontogeny of protein gene product 9.5 (PGP 9.5), serotonin (5-HT), calcitonin gene-related peptide (CGRP), and calcitonin (CT) immunoreactivity was evaluated in small-granule endocrine cells of hamster laryngotracheal epithelium from fetal day 11 to adulthood. Two centrifugal (proximal-to-distal) patterns of differentiation occur. The first pattern begins during fetal life. Endocrine cells, single and clustered in groups (presumptive- or protoneuroepithelial bodies, pNEBs), initially colocalize immunostaining for PGP 9.5, 5-HT, and CGRP in the larynx and proximal 2/3 of the trachea on day 12 and spread to the caudal trachea on day 13. 5-HT disappears fleetingly during the 24 h preceding birth; otherwise immunoreactivity for all three substances persists into adulthood. The clusters of endocrine cells survive beyond birth but are so diluted by expansion of the nonendocrine epithelium as to become inconspicuous. Since innervation was not actually observed, these clusters may persist as pNEBs, without developing connections to afferent or efferent nerve fibers. The second pattern concerns single small-granule cells stainable for CGRP but not for 5-HT. These cells first appear in the larynx and cartilaginous part of the cranial trachea on postnatal day 3, and in the middle and caudal trachea, on day 5. The cells increase in number on day 7. In adults, they predominate among endocrine cells of the cartilaginous region. A subset of these cells begins to co-express CT proximally on postnatal day 10, reaching the caudal end of the trachea by 3 weeks. A few elements of the older 5-HT-positive population may also become immunoreactive for CT in juvenile hamsters.

Expression of calcitonin and somatostatin peptide and mRNA in medullary thyroid carcinoma.

We have studied a series of 22 human medullary carcinomas (MCTs), both primary and metastatic, using immunocytochemistry (ICC) to localize calcitonin and somatostatin peptide and in situ hybridization (ISH) to localize calcitonin and somatostatin mRNA. All tumors were positive for calcitonin peptide with ICC, which often showed considerable intercellular heterogeneity, with many cells having undetectable levels of calcitonin. However, calcitonin mRNA localized by ISH was much more uniformly distributed, indicating that MCT tumor cells may retain the capacity to both synthesize and store calcitonin, whereas others lose their storage but not their synthetic capacity. Somatostatin peptide and mRNA were found in tumors from 15 patients. In contrast to the pattern seen with calcitonin, somatostatin mRNA and peptide were usually found in single scattered cells. When correlation was possible, the same cell showed positivity for somatostatin mRNA on ISH and positivity for somatostatin peptide on ICC. However, in one tumor many more cells were positive for mRNA than for peptide, suggesting that only a proportion of cells retained the ability to store the peptide. The variation in cellular content of immunoreactive calcitonin is interpreted as resulting from either an increased tumor growth rate or reduced ability to store peptide in a less differentiated tumor. With somatostatin there was good correlation between mRNA and peptide content, but it occurred in single widely scattered cells, most tumor cells being negative for both peptide and mRNA. It is suggested that somatostatin production might be associated with a reduction in the growth of the cell concerned, either through a differentiation step or through a direct effect of the hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

Immuno-electron-microscopic localization of enkephalin in the secretory granules of C cells in the chicken ultimobranchial glands

In the chicken, enkephalin-immunoreactive cells and nerve fibers are distributed in the ultimobranchial glands, which consist of C-cell groups and cyst structures. Ultrastructural features of the enkephalin cells and nerve fibers were examined by immuno-electron microscopy using both the streptavidin-biotin-peroxidase method and the protein A-colloidal gold method. Immunoreactivity for enkephalin was located on the secretory granules of C cells. In 1-day-old chickens, three types of C cells were distinguished on the basis of their granule size. Type-I cells were filled with large secretory granules (200-600 nm in diameter). These elements represented a majority of the C-cell population. Type-II cells contained medium-sized granules (100-280 nm in diameter). Type-III cells displayed small secretory granules (60-200 nm in diameter). The latter cells were elongate or irregular in shape and frequently extended cytoplasmic processes into the connective tissue stroma or contacted other C cells. Enkephalin-immunoactivity was revealed by dense deposits of immunogold particles on the secretory granules of type-II and type-III cells. There were only a few type-I cells showing immunoreactivity for enkephalin. A double immunogold labeling procedure demonstrated that calcitonin and enkephalin were colocalized in the same secretory granules of type-I and type-II cells. Type-III cells were devoid of immunoreactivity for calcitonin. Enkephalin-immunoreactive nerve fibers were characterized by the presence of granular vesicles, 60-160 nm in diameter, and frequently established direct contact with the surface of C cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcitonin gene expression in normal human liver

Immunoreactive calcitonin (CT) is present in liver. This could represent hormone synthesized by liver cells, degraded or bound to specific receptors reported in this organ. We report here that the calcitonin gene is expressed in liver. We proved this by demonstrating, by PCR amplification using specific primers, the presence of calcitonin messenger in human liver and in primary cultures of human hepatocytes and detected by radioimmunoassay CT in hepatic tissues and cells. The synthesis of hormone by liver that also possesses specific receptors for CT favors the presence of an autocrine or paracrine system involving calcitonin in this organ.

Chronic hyperoxia and hamster pulmonary neuroendocrine cell bombesin and calcitonin.

Hamsters were exposed to 60% hyperoxia for 1 week, 3 weeks, or 3 months. The exposed animals gradually failed to gain body weight as controls. The pulmonary neuroendocrine (PNE) cell peptides, mammalian bombesin (MB) and immunoreactive calcitonin (iCT), were determined in the lung and the serum. At 1 week and 3 weeks, lung MB was unchanged while the iCT levels were markedly depleted. In contrast, the lung levels of both MB and iCT were significantly elevated at 3 months. Serum levels of MB showed an initial decline at 1 week, which was followed by augmented levels at 3 weeks and at 3 months. In contrast, serum iCT showed considerable depletion at 1 week, and also at 3 weeks, followed by increased levels at 3 months. Thus, chronic exposure to hyperoxia causes profound perturbation of PNE cell peptides. In particular, the early depletion of lung and serum iCT appears to be a unique feature of the response to hyperoxia. The principal difference between the MB and the iCT responses was the lack of an initial depletion of lung MB, and the earlier rise of serum MB to supranormal levels. It seems likely that the early peptide effects of hyperoxia are related to oxygen toxicity upon the PNE cells, while the changes noted at 3 months reflect a hyperplastic accommodation of PNE cells to the prolonged oxygen exposure with resultant increases in MB and iCT. This response is distinctly different from that we have seen previously in hamsters exposed to hyperoxia combined with a nitrosamine, or a nitrosamine alone.

Heterogeneity studies of hamster calcitonin following acute exposure to cigarette smoke: Evidence for monomeric secretion

Various acute stimuli, including cigarette smoke, induce hypercalcitonemia in man and hamsters. We have shown that this occurs also in thyroidectomized subjects. In the present study we have further explored this phenomenon of secretion from the lungs by studying, simultaneously, the HPLC characteristics of pulmonary tissue and serum in control hamsters and in animals immediately following short-term exposure to cigarette smoke. In addition, we have studied the immunoheterogeneity of lung calcitonin 24 hours following the acute exposure. Control lungs contained monomeric immunoreactive calcitonin (M-iCT), high molecular mass iCT (H-iCT), and CT fragments. Immediately following smoke exposure, there was an acute decrease of lung iCT by radioimmunoassay (RIA) which consisted primarily of a decrease in M-iCT by HPLC. Simultaneously, the iCT increase in the serum by RIA was shown by HPLC to involve M-iCT. Twenty-four hours after smoke inhalation, the lung iCT by RIA and M-iCT by HPLC had returned towards control levels. These findings document the molecular characteristics of lung iCT following acute cigarette smoke stimulation, and suggest that under certain circumstances M-iCT may be actively secreted by the lung. It remains to be determined whether this type of secretion reflects hemocrine or paracrine release and what the physiological role for such a secretion may be.

Cholinergic‐nicotinic control of growth and secretion of cultured pulmonary neuroendocrine cells

Dispersed newborn hamster lung cells were established in vitro in a defined, low-serum growth medium. Neuroendocrine markers (immunohistochemistry for bombesin/gastrin-releasing peptide and calcitonin) revealed a cellular predominance of pulmonary neuroendocrine (PNE) cells. While the supernatant concentration remained stable, the concentration of PNE cell immunoreactive calcitonin (iCT) gradually declined over 4 weeks. Supplementation of the medium with nicotine for 3 weeks prevented this decline in cellular iCT. Concurrently, the number of cells and [3H]thymidine incorporation were significantly increased. The stimulatory effect of chronic nicotine was reversed by the coadministration of the nicotinic antagonist hexamethonium. In another set of experiments, prior multiple transplacental nicotine pretreatments resulted in a significant increase in iCT in the lungs of newborns; when these lungs were subsequently placed in cell culture without nicotine, despite the higher concentration of iCT, there was a drop in iCT similar to that observed in the control culture. In contrast, in vivo, the lung iCT remained significantly elevated at 1 week post-parturition. Cell culture supernatants were analyzed at week 4 for the evoked release of iCT; cholinergic-nicotinic agonists promptly increased the supernatant iCT, which was blocked by nicotinic but not by muscarinic antagonists. We suggest that this in vitro system provides a useful tool to study directly the PNE cell. The acute and chronic effects of nicotine are most likely related to stimulation of cholinergic-nicotinic receptors on iCT-containing PNE cells.

Localization of cholecystokinin-like and calcitonin-like peptides in infant carotid bodies: a light- and electron-microscopic immunohistochemical study

Previous immunohistochemical studies have identified several regulatory peptides in the carotid body chief cells in both humans and animals. These peptides, together with amines, may be important in the modulation of the chemoreflex by the carotid body. We report the localization and distribution of calcitonin and cholecystokinin-like (CCK) immunoreactivity in chief cells of human infant carotid body by light- and electron-microscopic immunohistochemical techniques. Consecutive sections immunostained with calcitonin and/or CCK antibodies revealed positively stained chief cells, both alone and in clusters, scattered throughout the carotid body lobule. Generally more chief cells were positive for calcitonin than for CCK. This was confirmed by quantitative analysis showing that the ratio of calcitonin to CCK immunoreactive cells was consistently > 2:1 in all cases studied. There was no apparent correlation between the immunoreactivity for the two peptides and the age, sex, or postmortem interval. Calcitonin-like and CCK-like immunoreactivities were localized electron-microscopically over the dense core granules of the chief cells. Calcitonin and CCK-like peptides in carotid body chief cells may act as neurotransmitters or neuromodulators involved in chemoreception.

CGRP radioreceptor assay: a new diagnostic tool for medullary thyroid carcinoma.

The quantitative analysis of immunoreactive calcitonin (iCT) is the assay of choice for diagnosis and follow-up of patients with medullary thyroid carcinoma (MTC). However, in a small percentage of patients with MTC or C cell hyperplasia (CCH), basal and stimulated iCT levels may not be significantly elevated. In these patients, calcitonin gene-related peptide (CGRP) radioreceptor assay (RRA) can be used complementary to immunoassay for CT (or katacalcin) for prompt diagnosis of MTC and CCH. CGRP RRA is a robust, rapid, sensitive, and specific determinant of "receptor-recognized" CGRP (RR-CGRP; intact molecule of CGRP) either in plasma or in tissue extracts. Plasma RR-CGRP levels rose > 100% 2-5 minutes after stimulation with intravenous pentagastrin (calcium or oral alcohol) (p < 0.001), whereas iCGRP levels were raised to a lesser degree (p < 0.01). In six patients who had a false positive iCT response after pentagastrin or had raised basal iCT levels measured with a two-site immunoradiometric assay, RR-CGRP showed only a minimal change. On the other hand, in patients with CCH (true positive, n = 8), iCT was increased by only 40% after pentagastrin but RR-CGRP levels rose by 140% (p < 0.001). No change in iCT or RR-CGRP levels in plasma were detected in healthy normal volunteers after administration of pentagastrin. Therefore, in addition to the plasma iCT levels, RR-CGRP would resolve some of the difficult diagnostic problems associated with MTC and likely improve the specificity and sensitivity of identifying CCH.

A new highly sensitive radioimmunoassay for human calcitonin useful for physiological studies

A highly sensitive and specific radioimmunoassay (RIA) for human calcitonin-like immunoreactivity (CT-LI) has been developed. The assay is performed directly without extraction and may be used for measurements in plasma or serum as well as buffer solutions. The detection limit of the assay is < 0.4 pmol/l , intra- and interassay variations 5 and 10%, respectively when calculated from a control sample within the range of normal concentrations. The recovery of calcitonin (CT) varied between 87 and 118% in the concentration range 10–130 pmol/l. Measurements of CT-LI in sera from 70 normal individuals, showed significantly lower concentrations in women as compared to men, 7.4 pmol/l ± 2.1 pmol/l and 9.1 pmol/l ± 2.2 pmol/l , respectively ( mean ± S.D. ), P < 0.005. The chromatographic profile of immunoreactive CT in normal serum was investigated by gel chromatography and high pressure liquid chromatography (HPLC) and showed heterogeneity of circulating CT-LI, but with the majority eluting corresponding to the elution position of synthetic human CT. The study of physiological variations in CT levels has so far been hindered by the limitations in assay sensitivity. It is hoped that the significantly improved sensitivity of the presently described assay may contribute towards the elucidation of the physiological functions of CT.

Synthesis and release of calcitonin-like immunoreactivity by anterior pituitary cells: evidence for a role in paracrine regulation of prolactin secretion.

Calcitonin (CT) is a potent and specific inhibitor of basal and TRH-induced PRL release and PRL mRNA levels in rat anterior pituitary (AP) cells, an action mediated through specific inhibition of the Ca(2+)-inositol phosphate messenger system. Because CT and CT-like peptides have been reported to be present in the AP, the present studies investigated 1) whether 35S-labeled CT-like substances can be precipitated from rat AP cell lysates, 2) whether immunoreactive CT is secreted from rat AP cells, as assessed from a cell blot assay and from RIA of spent medium from cultured AP cells, and 3) whether the release of PRL from cultured rat AP cells can be influenced by immunoneutralization of endogenous CT. Dispersed rat AP cells were labeled with [35S]cysteine, CT-like substances were immunoprecipitated from the lysate with antihuman CT (anti-hCT) and antisalmon CT (anti-sCT) immunoglobulin G conjugated to protein-A-Sepharose beads, and the immunoprecipitates were fractionated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by fluorography. The results revealed that both anti-sCT and anti-hCT sera precipitated three major bands of 35S-labeled material from AP cell lysates, with approximate molecular masses of 23, 6.5, and 3.5 kilodaltons. Synthetic sCT completely displaced anti-sCT-precipitable bands and partially displaced anti-hCT precipitates. The cell blot assay revealed the presence of CT-immunopositive AP cells, and secretion of this substance was indicated by zones of secretion surrounding these cells. Immunoreactive CT was also detected in spent medium from cultured rat AP cells, and the average rate of release over 4 days of culture was approximately 1 ng sCT eq/3 million AP cells.24 h. Both anti-sCT and anti-hCT sera significantly stimulated PRL release from rat AP cells, and the stimulatory effect of anti-sCT serum was dose dependent up to a concentration of 1:50. The present findings demonstrate that CT-like peptide(s) is synthesized and released from cultured AP cells and suggest that this peptide may participate in the regulation of PRL secretion via paracrine or autocrine actions.

Detection of calcitonin and calcitonin gene‐related peptide mRNA in human medullary thyroid carcinoma. A retrospective study

In situ hybridization finds many applications in modern pathology. In many cases, special attention is paid to the processing of the tissues prior to in situ hybridization. In order to investigate the value of RNA in situ hybridization (RISH) in retrospective studies, we performed RISH for calcitonin and calcitonin gene-related peptide (CGRP)-I and -II mRNA in eight medullary thyroid carcinomas processed in 1981-1983. RISH was successful with radioactive calcitonin and CGRP-I probes. With biotinylated probes, only calcitonin-specific probes gave adequate results. The concentrations of CGRP mRNA were probably too low to be detected by non-radioactive RISH. The results of RISH were correlated with the immunohistochemical localization of the polypeptides. The results matched in all cases except one, where hybridization for calcitonin mRNA was found, but no immunoreactive calcitonin polypeptide. We conclude that RISH can be successfully used for retrospective analysis, even after long storage of tissue embedded in paraffin.

Immunoreactive "calcitonin-like" material in heroin addicts: varying reactivity with different antibodies.

High levels of immunoreactive calcitonin (iCT) in the blood of heroin addicts were previously reported. As it is well known that multiple forms of calcitonin exist in the blood and in tissues, the purpose of the present study was to investigate the immunological nature of the CT-like immunoreactive material found in the blood of these subjects. We investigated 25 addicts, who had been using heroin for more than one year and were hospitalized for a 2 week detoxication program. Blood samples were drawn at the start of the program (when the subjects were still on heroin) and after 5 and 12 days of abstinence from heroin. Twenty-five healthy subjects served as controls. We used 2 commercial RIA kits, calibrated against the same reference material (WHO 70-234), but employing different antisera. One antiserum substantially confirmed the previous findings of increased levels of calcitonin during heroin use, but the other seemed to exclude any change in the hormone concentrations. This suggests that the "calcitonin like" material found in heroin addicts contains some epitopes similar to those found in the calcitonin standard which are detected by the first antiserum. However it lacks other epitopes which are also present in calcitonin standard and which are recognized by the second antiserum. Therefore, this substance seems to be different from the standard human calcitonin 1-32. A possible involvement of a calcitonin analogue (precursor or metabolite) in the biochemical changes occurring during chronic opiate use is suggested.

Effect of injury on pulpal levels of immunoreactive substance P and immunoreactive calcitonin gene-related peptide

Neuropeptides such as calcitonin gene-related peptide (CGRP) and substance P are present in dental pulp in relatively high concentrations. Previous studies have demonstrated that the staining density of immunoreactive CGRP (iCGRP) changes in dental pulp after tissue injury. This study evaluated injury-related changes in levels of both immunoreactive CGRP (iCGRP) and immunoreactive substance P (iSP) in dental pulp using radioimmunoassays. After pulpal exposure, iSP levels decreased to about 10% of baseline values, while iCGRP levels decreased to about 45% of baseline measures. After dentin exposure with acid etch, iSP levels decreased to about 10 to 20% of baseline measures, while iCGRP levels decreased to 60% of baseline values. For both forms of injury, iSP decreased to a greater extent than did iCGRP levels. Collectively, these findings indicate that pulpal neuropeptides undergo dynamic, injury-specific, and peptide-specific responses following trauma to dental pulp.

Serum Calcitonin May Be a Marker for Inhalation Injury in Burns

One of the principal causes of death from burns is inhalation injury. The pulmonary neuroendocrine cell contains and secretes immunoreactive calcitonin (iCT), and, under the influence of various irritative stimuli, can be induced to secrete iCT in excess. A prospective study of serum iCT levels in 41 patients with burns was undertaken. Mean serum iCT levels were four times normal values at the time of admission and reached 31 times normal values by 24 hours after injury. These levels did not correlate specifically with burn size. However, serum iCT had a very strong positive correlation with mortality, and in addition, was highest in patients who died early after injury compared with those who died late after injury. Patients who were clinically suspected to have pulmonary injury and who died had markedly higher levels of iCT than those who survived. In addition, serum iCT correlated positively with the need for mechanical ventilation and the amount of pulmonary shunting. Although other factors may also play a role in hypercalcitoninemia, serum iCT may be an important marker for the presence of inhalation injury, as well as a prognostic indicator.

Serum procalcitonin as an index of inhalation injury in burns.

The molecular heterogeneity of serum immunoreactive calcitonin (iCT) was analyzed from a prospective study of 41 burn patients. Using different region-specific anticalcitonin antisera, the ratio of mid-region-recognizing to carboxyl terminal-region-recognizing iCT was found to increase acutely in those who subsequently died. The highest ratios occurred in those who died early of respiratory complications. Sephadex chromatography and reversed-phase HPLC demonstrated that the serum iCT circulated predominantly in the large molecular mass prohormone form (16 kDa). In comparison, iCT of normal human lung and of normal thyroid was shown to consist primarily of smaller monomeric mass forms. Furthermore, in 12 normal volunteers who were evaluated with a calcium-pentagastrin infusion, the ratio of iCT levels did not differ from the baseline ratio despite a 50% increase in serum iCT. These results suggest that in burns, the inhalational injury-associated hypercalcitonemia is characterized by a preferential release of procalcitonin; a form of constitutive secretion. The measurement of serum procalcitonin levels would appear to be a useful prognostic indicator of the severity of inhalational injury occurring in burn patients.

Age-related changes in tissue contents of immunoreactive calcitonin gene-related peptide

Age-related changes in calcitonin gene-related peptide (CGRP) content were found in a number of rat tissues, and most can be explained by the physiological and pathological changes associated with advancing age. In some specific areas of the brain, and some cardiovascular tissues such as atria and arteries, the CGRP content declined with age (p < 0.01), while the contents were increased in the thyroid gland, plasma and in veins (p < 0.01). In the substantia nigra and striatum, the immunoreactive-CGRP (i-CGRP) levels found in older rats were about 50% of those found in adolescent rats (p < 0.01). The i-CGRP levels found in the penis, bladder, kidney, testis and adrenal gland gradually increased up to maturity, and then rapidly declined in the aging rats. However, in the lungs and sensory ganglia (dorsal root and trigeminal), the highest content of i-CGRP was found in immature 3-week-old rats, and then declined with age. Changes of i-CGRP observed with age may support multiple physiological roles for CGRP at various stages of development. The changes described here, particularly in the lungs, substantia nigra, striatum and the cardiovascular system, should help in further understanding the physiological and pathophysiological role of CGRP in health and in age-related disorders.