Calcitonin gene-related peptide and the lung: neuronal coexistence with substance P, release by capsaicin and vasodilatory effect

The occurrence and distribution of calcitonin gene-related peptide (CGRP) in the lower airways was studied by means of immunohistochemistry and radioimmunoassay (RIA) in combination with high performance liquid chromatography (HPLC). CGRP-like immunoreactivity (-LI) was observed in nerves from the epiglottis down to peripheral bronchi in rat, cat and guinea pig and also in human bronchi. Double staining revealed colocalization of CGRP-LI and substance P (SP)-LI in cell bodies of nodose and jugular ganglia as well as in axons and nerve terminals of the airways. Systemic capsaicin pretreatment induced a marked loss of the CGRP- and SP-immunoreactive (-IR) nerves in the lower airways. CGRP-IR was also present in epithelial endocrine cells and neuroepithelial bodies. The content of CGRP-LI as measured with RIA in guinea pig bronchi was significantly lower after capsaicin pretreatment. Analysis of human bronchial extracts revealed that CGRP-LI coeluted with synthetic human CGRP on HPLC. In the isolated perfused guinea pig lung capsaicin exposure caused overflow of CGRP-LI suggesting release from peripheral branches of sensory nerves. Both in vivo experiments in the guinea pig measuring insufflation pressure as well as in vitro studies on isolated guinea pig and human bronchi showed that whereas tachykinins contracted bronchial smooth muscle no contractile or relaxing effect was elicited by human or rat CGRP. However, CGRP caused relaxation of serotonin precontracted guinea pig and human pulmonary arteries. In conclusion, the presence and release of CGRP-LI from capsaicin sensitive nerves in the lower airways adds another possible mediator, in addition to tachykinins, of vascular reactions upon sensory nerve irritation.     

Calcitonin gene-related peptide in human fetal lung and in neonatal lung disease

The ontogeny of calcitonin gene-related peptide immunoreactivity (CGRP-IR) was evaluated immunohistochemically in 67 human fetal or newborn lungs previously analyzed for calcitonin immunoreactivity (CT-IR). CGRP-IR was present by 10 weeks of gestation in rare, solitary neuroendocrine (NE) cells of developing conducting airways in two of eight first-trimester lungs. During the second trimester, cells with CGRP-IR were found consistently (21/23 fetuses). However, the numbers of positively staining cells did not appear to increase in these fetuses or in third-trimester infants dying of non-pulmonary causes. The highest concentrations of CGRP-IR cells were seen in lungs of premature infants with advancing chronic lung disease associated with bronchopulmonary dysplasia (BPD). CGRP-IR was seen earlier in gestation and in greater numbers of NE cells than was calcitonin immunoreactivity (CT-IR) reported previously in these same fetal lungs (Lab Invest 52:52, 1985). Its presence paralleled that of CT-IR in postnatal chronic lung disease.     

Calcitonin gene-related peptide and its receptor in the thymus

Calcitonin gene-related peptide (CGRP), a 37-amino acid residue neuropeptide, was immunostained in rat thymus at two sites: a subpopulation of thymic epithelial cells, namely subcapsular/perivascular cells, were heavily stained besides some nerve fibers surrounding arteries and arterioles. The administration of nanomolar concentrations of rat -CGRP dose-dependently raised intracellular cyclic adenosine monophosphate (cAMP) levels in isolated rat thymocytes (half-maximum stimulation 1 nM) but not in cultured rat thymic epithelial cells. Peptides structurally related to CGRP (i.e., rat calcitonin or amylin) had no effect. CGRP(8–37), an N-terminally truncated form, acted as an antagonist. Peripheral blood lymphocytes did not respond to CGRP, suggesting that receptors are present only on a subpopulation of thymocytes but not on mature T cells. This was substantiated by visualization of CGRP receptors on single cells by use of CGRP-gold and -biotin conjugates of established biological activity: only a small proportion of isolated thymocytes was surface labeled. In situ, the CGRP conjugates labeled receptors on large thymocytes residing in the outer cortical region of rat thymus pseudolobules. Thus, immunoreactive CGRP is found in subcapsular/perivascular thymic epithelial cells and acts via specific CGRP receptors on thymocytes by raising their intracellular cAMP level. It is suggested that CGRP is a paracrine thymic mediator that might influence the differentiation, maturation, and proliferation of thymocytes.     

Calcitonin gene-related peptide and hypertension

Capsaicin-sensitive sensory nerves participate in the regulation of cardiovascular functions both in the normal state and the pathophysiology of hypertension through the actions of potent vasodilator neuropeptides, including calcitonin gene-related peptide (CGRP). CGRP, a very potent vasodilator, is the predominant neurotransmitter in capsaicin-sensitive sensory nerves, and plays an important role in the initiation, progression and maintenance of hypertension via: (1) the alterations in its synthesis and release and/or in vascular sensitivity response to it; (2) interactions with pro-hypertensive systems, including renin-angiotensin-aldosterone system, sympathetic nervous system and endothelin system; and (3) anti-hypertrophy and anti-proliferation of vascular smooth muscle cells. The decrease in CGRP synthesis and release contributes to the elevated blood pressure, as shown in the spontaneously hypertensive rats, alpha-CGRP knockout mice, Dahl-salt or phenol-induced hypertensive rats. In contrast, the increase in CGRP levels or the enhancement of vascular sensitivity response to CGRP plays a beneficial compensatory depressor role in the development of hypertension, as shown in deoxycorticosterone-salt, sub-total nephrectomy-salt, N(omega)-nitro-L-arginine methyl ester or two-kidney, one-clip models of hypertension in rats. We found that rutaecarpine causes a sustained depressor action by stimulation of CGRP synthesis and release via activation of vanilloid receptor subtype 1 (VR1) in hypertensive rats, which reveals the therapeutic implications of VR1 agonists for treatment of hypertension.     

Calcitonin gene-related peptide: novel neuropeptide

Calcitonin gene-related peptide (CGRP) is a 37 amino acid peptide encoded in the calcitonin gene. Its expression is dependent on tissue-specific alternative RNA processing: mRNA for CGRP predominates in the brain, whilst calcitonin (CT) mRNA predominates in thyroid C cells. The existence of this hitherto unsuspected peptide was predicted by mRNA analysis and demonstrated using antibodies raised against a synthetic peptide corresponding to the predicted C-terminal sequence of CGRP. The distribution of CGRP in the central and peripheral nervous system and its co-localization in some neurons with substance P (SP) or acetylcholine suggests several possible roles in autonomic, sensory and motor functions. Its actions appear to depend on the existence of specific CGRP receptors in target tissues, distinct from the receptors for CT but bearing some resemblance to them.     

Calcitonin and calcitonin gene-related peptide immunoreactivity and colocalization in newborn cat lung.

Calcitonin (CT) and calcitonin gene-related peptide (CGRP) are generated by alternate RNA processing from alpha and beta CT/CGRP genes. In this report, an immunocytochemical investigation was undertaken on the occurrence and distribution of immunoreactive CT as well as its colocalization with CGRP in newborn cat bronchopulmonary endocrine cells. A widespread distribution of solitary endocrine cells and neuroepithelial bodies immunostained for CT was recorded within the lung. In all animals studied, CT immunoreactivity represents a subpopulation of CGRP positive cells, while the intrapulmonary nerve fibers contain only CGRP. To the best of our knowledge, this is the first time that CT and its colocalization with CGRP have been demonstrated immunocytochemically in the cat lung. Our results indicate, that different molecular processing of both CT/CGRP genes may be represented by different patterns in the cellular immunoreactivity of the synthetized peptides.     

Calcitonin gene-related peptide and its mRNA in pulmonary neuroendocrine cells and ganglia

Summary The occurrence of calcitonin gene-related peptide (CGRP) and it's mRNA was studied in lungs of rats and piglets using in situ hybridization with two synthetic oligonucleotide probes followed by immunocytochemistry (ICC). CGRP mRNA was present in pulmonary neuroendocrine cells (PNEC) of both the solitary type and cluster type (neuroepithelial body; NEB) at all levels of the airway epithelium from bronchi to alveoli. The distribution of labelled cells was similar to that previously described with ICC. The 44-mer probe provided stronger hybridization signal than the 34-mer and the two combined increased labelling slightly. Formalin fixation reduced labelling and tended to increase background. Labelling for CGRP mRNA was evenly distributed over the cytoplasm, whereas CGRP-like immunoreactivity (LI) usually was of highest intensity toward the base of the PNEC, suggesting basal accumulation of synthesized peptide. CGRP-LI was also observed in occasional rat ganglia and in some, but not all, piglet ganglia. These local neurons may contribute to the CGRP fibers of airways and vasculature, and could theoretically bridge their dendrites and axons between NEB and the effector organ (e.g. artery or arteriole) thus accomplishing a function similar to the postulated axon reflex.     

Calcitonin gene-related peptide in the human hypothalamus

Calcitonin gene-related peptide (CGRP)-like immunoreactivity (LI) in the human hypothalamus was investigated by radioimmunoassay and by immunocytochemistry. CGRP-LI was detected from two hypothalami obtained at autopsy (2.1 and 7.0 ng/g wet tissue) by radioimmunoassay. Reverse phase high performance liquid chromatography revealed that most of the CGRP-LI in the human hypothalamus was eluted in an identical position with synthetic human CGRP. For immunocytochemistry, human hypothalami obtained at autopsy were fixed and cryostat-sectioned at 40 microns. Free floating sections were immunostained with antibody to CGRP. CGRP-immunoreactive cell bodies were found in the supraoptic nucleus, paraventricular nucleus and infundibular nucleus. These findings indicate that CGRP exists in the cell bodies of the supraoptic nucleus, paraventricular nucleus and infundibular nucleus in the human hypothalamus and CGRP may play some roles in the endocrine and other functions of the human hypothalamus.     

Calcitonin gene-related peptide in human obesity.

We studied plasma calcitonin gene-related peptide (CGRP) levels in obese women before (n = 24) and after (n = 13) weight loss, and in normal weight controls (n = 15). Furthermore, the influence of two isocaloric meals (high carbohydrate vs. high fat) on plasma CGRP concentrations was studied. The CGRP concentration in the obese group (32.26 +/- 2.01 pg/ml) was significantly (p less than 0.0001) higher than in the control group (21.64 +/- 0.15 pg/ml). After weight loss (14.3 +/- 0.72% of original weight) CGRP concentrations remained unchanged. Only the high-fat meal caused a significant (p less than 0.02) rise in CGRP levels. Our results indicate that elevated plasma CGRP levels may constitute a primary phenomenon in obese women, and that fat intake may be associated with increased CGRP secretion.     

Calcitonin gene-related peptide and its mRNA in pulmonary neuroendocrine cells and ganglia.

The occurrence of calcitonin gene-related peptide (CGRP) and it's mRNA was studied in lungs of rats and piglets using in situ hybridization with two synthetic oligonucleotide probes followed by immunocytochemistry (ICC). CGRP mRNA was present in pulmonary neuroendocrine cells (PNEC) of both the solitary type and cluster type (neuroepithelial body; NEB) at all levels of the airway epithelium from bronchi to alveoli. The distribution of labelled cells was similar to that previously described with ICC. The 44-mer probe provided stronger hybridization signal than the 34-mer and the two combined increased labelling slightly. Formalin fixation reduced labelling and tended to increase background. Labelling for CGRP mRNA was evenly distributed over the cytoplasm, whereas CGRP-like immunoreactivity (LI) usually was of highest intensity toward the base of the PNEC, suggesting basal accumulation of synthesized peptide. CGRP-LI was also observed in occasional rat ganglia and in some, but not all, piglet ganglia. These local neurons may contribute to the CGRP fibers of airways and vasculature, and could theoretically bridge their dendrites and axons between NEB and the effector organ (e.g. artery or arteriole) thus accomplishing a function similar to the postulated axon reflex.     

Calcitonin gene-related peptide: antinociceptive activity in rats, comparison with calcitonin

The effects of synthetic human calcitonin gene-related peptide (CGRP) on nociceptive response were evaluated in rats by two behavioral tests (tail-flick and hot-plate) and by electrophysiological recording of the firing of thalamic neurons evoked by peripheral noxious mechanical stimuli. CGRP was administered intracerebroventricularly (i.c.v.) and its effects were compared with that of salmon calcitonin (sCT). In the tail-flick test, CGRP (0.25, 2.5 and 5 micrograms/rat) dose-dependently increased response latencies, whereas sCT (0.125, 2.5, 5 and 10 micrograms/rat) did not. Conversely, in the hot-plate test CGRP was effective in enhancing response latencies only at the highest dose of 10 micrograms/rat, while sCT (0.125, 0.25 and 2.5 micrograms/rat) inhibited the hot-plate response dose-dependently. In electrophysiological studies, CGRP (2.5 micrograms/rat, i.c.v.) completely inhibited the evoked neuronal thalamic firing and the same dose of sCT induced only a partial reduction. Furthermore, the antinociceptive effects of CGRP in the tail-flick test and in the electrophysiological studies were not prevented by naloxone. These results demonstrate that central administration of CGRP is effective in inhibiting nociceptive responses and its action like that of sCT does not involve an opioid mechanism. The differences in the antinociceptive profiles of CGRP and sCT suggest that the inhibitory effects of these peptides may involve different neuronal pathways.     

Calcitonin gene-related peptide: Brain and spinal action on intestinal motility

The effects of intracerebroventricular (ICV) and intrathecal (IT) administration of calcitonin gene-related peptide (CGRP) on intestinal motility were examined in conscious rats chronically fitted with intraparietal electrodes in the duodeno-jejunum and a cannula in a cerebral lateral ventricle or catheter in the subarachnoid space. ICV administration of CGRP (0.5–10 μg) restores the fasted pattern of intestinal motility in fed rats in a dose-related manner. Intrathecal administration of CGRP or calcitonin also induces fasted pattern but after a 30 min delay. These effects persisted after transection of the spinal cord and no change in intestinal motility appeared after intravenous administration of CGRP at a dose effective when given IT. This study suggests that CGRP, as calcitonin, has a neuromodulatory role in the control of intestinal motility at both brain and spinal cord levels.     

Evidence for the coexistence of serotonin and calcitonin gene-related peptide at the subcellular level in neuroepithelial bodies in the lung of a marsupial,Isoodon macrourus

Summary The coexistence of serotonin and calcitonin gene-related peptide (CGRP) in neuroepithelial bodies of the bandicoot, Isoodon macrourus, has been examined using immunocytochemistry at the light- and electronmicroscope levels. The avidin-biotin technique of antigen localisation was used initially to identify serotonin-like and CGRP-like immunoreactivity (-LI). Serotonin-LI and CGRP-LI were found in neuroepithelial cells in the lungs of 30-day-old bandicoots. CGRP-LI could also be demonstrated in nerve fibres associated with some neuroepithelial bodies. The protein A-gold technique of antigen localisation was used to label neuroepithelial cells and nerve fibres at the subcellular level. Serotonin-LI and CGRP-LI were observed in the same dense-cored vesicles of most neuroepithelial cells; however, some neuroepithelial cells were shown to possess serotonin-LI without CGRP-LI. Nerve fibres immediately adjacent to neuroepithelial bodies exhibited mainly CGRP-LI. These results show that serotonin-LI and CGRP-LI are present in neuroepithelial cells of the bandicoot in the same secretory vesicles. This pattern of co-localisation may reflect co-ordinated or synergistic actions of these two neuroactive substances.     

Calcitonin gene-related peptide: a sensory and motor neurotransmitter in the feline lower esophageal sphincter

The effect of calcitonin gene-related peptide (CGRP) on the feline lower esophageal sphincter (LES) was determined and correlated with its anatomic distribution as determined by immunohistochemistry. Intraluminal pressures of the esophagus and LES were recorded in anesthetized cats. In separate cats, gastroesophageal junctions were removed after locating the LES manometrically and stained for CGRP-like immunoreactivity (LI) and substance P-LI (SP-LI) by indirect immunohistochemistry. CGRP-LI in the LES was most prominent in large nerve fascicles between the circular and longitudinal muscle layers and only rarely seen in nerve fibers within the circular muscle. The myenteric plexus contained numerous CGRP-LI nerve fibers but cell bodies were not seen. Many CGRP-LI nerve fibers in the myenteric plexus and occasional varicose nerves in the circular muscle demonstrated colocalization with SP-LI. Colocalization of CGRP-LI with SP-LI was also seen in the perivascular nerves of the submucosal and intramural blood vessels and in varicose fibers in the lamina propria of the gastric fundic mucosa. In the esophagus, CGRP-LI nerves extended through the muscularis mucosa and penetrated the squamous epithelium to the lumen. CGRP, given intra-arterially caused a dose-dependent fall in basal LES pressure, with a threshold dose of 10(-8) g/kg (2.63 pmol/kg). At the maximal effective dose, 5 x 10(-6) g/kg (1.31 x 10(3) pmol/kg), CGRP produced 61.0 +/- 6.0% decrease in basal LES pressure. At this dose, mean systemic blood pressure fell by 40.9 +/- 7.8%. The LES relaxation induced by a submaximal dose of CGRP (10(-6) g/kg, 262.7 pmol/kg), 50.3 +/- 3.2% relaxation was partially inhibited by tetrodotoxin (26.9 +/- 10.8% relaxation, P less than 0.025). The inhibitory effect of CGRP was not affected by cervical vagotomy, hexamethonium, atropine, propranolol, or naloxone. The LES contractile response to the D90 of SP (5 x 10(-8) g/kg, 37.1 pmol/kg) was not altered by CGRP 10(-8) or 10(-6) g/kg and the CGRP relaxation effect was not altered by the threshold dose of substance P (5 X 10(-9) g/kg, 3.71 pmol/kg). CONCLUSIONS: (1) CGRP-LI is present at the feline LES and is primarily seen in large nerve fascicles which pass from the intermuscular plane and through the circular muscle layer to the submucosa and in mucosal nerves. (2) CGRP colocalizes with SP-LI in some varicose nerve fibers of the circular muscle of the esophagus, LES and fundus, in perivascular nerves of the submucosal and intramucosal blood vessels, and in nerves of the lamina propria of the gastric fundus. (3) The luminal penetration of CGRP-LI nerves in the squamous mucosa of the esophagus suggests a sensory func     

Calcitonin gene-related peptide: an autocrine growth factor with regulatory activity in vitro.

We show that an autocrine system for calcitonin gene-related peptide (CGRP) exists in F9 teratocarcinoma cells. Synthesis of CGRP by F9 cells was demonstrated by measuring the peptide concentration in cells and medium and by determining specific mRNA in cells. During six days of culture, CGRP secretion did not vary significantly in the medium, while intracellular CGRP and CGRP mRNA levels increased. F9 cells contained a CGRP-sensitive adenylate cyclase system and CGRP increases the accumulation of cAMP in the culture medium. Interestingly affinity purified antibodies against CGRP specifically inhibited growth of F9 cells by 50%. CGRP therefore stimulates F9 cell growth by an autocrine process, suggesting that CGRP may be a growth factor during early embryogenesis.     

Calcitonin gene-related peptide does not interact with sympathetic activity in myocardial ischemia

The aim of the present study was to assess whether calcitonin gene-related peptide (CGRP) modulates exocytotic norepinephrine release in ischemic myocardium. In isolated rat hearts subjected to 30 min of low flow ischemia, CGRP release increased 2.8-fold whereas stimulation-induced norepinephrine release decreased 4.1-fold. Pretreatment of rats with capsaicin almost completely depleted cardiac CGRP stores; however, suppression of norepinephrine release by 30 min of low flow ischemia was not affected. At normal flow, exogenous CGRP (5 micromol l-1) had no effect on norepinephrine release. These findings suggest that CGRP release from sensory neurons does not interact with the cardiac sympathetic system during myocardial ischemia.     

Calcitonin gene-related peptide and substance P in the pharynx and lung of the bullfrog,Rana catesbeiana

Indirect double immunofluorescence labelling in the pharynx and lung of the bullfrog, Rana catesbeiana, demonstrated the occurrence, distribution, and coexistence of two neuropeptides. In the pharynx, immunoreactive calcitonin gene-related peptide (CGRP) and substance P (SP) were localized in nerve fibers distributed within and just beneath the ciliated epithelium. In the lung, CGRP and SP were localized in nerve fibers in five principal locations: 1) within the smooth muscle layer in the interfaveolar septa; 2) in the luminal thickened edges of the septa; 3) around the pulmonary vasculature; 4) within, and 5) under the ciliated epithelium. Within the smooth muscle layer in the septa, luminal thickened septa, and around blood vessels, almost all fibers showed coexistence of CGRP and SP. Within and just beneath the ciliated epithelium in the thickened septa, all fibers showed coexistence of CGRP and SP. No immunoreactivity for vasoactive intestinal polypeptide, neuropeptide Y, galanin, somatostatin, FMRFamide, and leucine-and methionine-enkephalins was detected in the nerve fibers within the larynx and the lung. Together with our previous data, the present findings suggest that peptidergic mechanisms are involved in the regulation of amphibian respiratory systems throughout their life.     

Calcitonin gene-related peptide (CGRP): perivascular distribution and vasodilatory effects

The distribution of perivascular nerve fibers displaying calcitonin gene-related peptide (CGRP) immunoreactivity and the effect of CGRP on vascular smooth muscle were studied in the guinea-pig. Perivascular CGRP fibers were seen in all vascular beds. Generally, they were more numerous around arteries than veins. Small arteries in the respiratory tract, gastrointestinal tract and genitourinary tract had numerous CGRP fibers. The gastroepiploic artery in particular received a rich supply of such fibers. Coronary blood vessels had a moderate supply of CGRP fibers. In the heart, a moderate number of CGRP fibers was seen running close to myocardial fibers. The atria had a richer supply than the ventricles. Numerous CGRP immunoreactive nerve cell bodies and nerve fibers were seen in sensory (trigeminal, jugular and spinal dorsal root) ganglia. Sequential or double immunostaining with antibodies against substance P and CGRP suggested co-existence of the two peptides in nerve cell bodies in the ganglia and in perivascular fibers. In agreement with previous findings CGRP turned out to be a strong vasodilator in vitro as tested on several blood vessels (e.g. basilar, gastroepiploic and mesenteric arteries). Conceivably, perivascular CGRP/SP fibers have a dual role as regulator of local blood flow and as carrier of sensory information.