The prepro-adrenomedullin (prepro-AM) gene encodes several biologically active peptides, including the potent vasodilator AM. At least part of the vasodilator action of AM appears to be mediated via interactions with receptors on vascular smooth muscle cells (VSMC); however, the specific receptors involved are not known. The aim of the present study was to identify putative AM receptor genes that are co-expressed with AM in cultured rat aortic VSMC that may mediate the actions of AM. AM mRNA was shown to be expressed in rat aortic VSMC cultures. In acute (4 h) secretion studies, only 20% of the total immunoreactive AM was intracellular, with the majority (80%) found in the medium, indicating active release of AM peptide from VSMC. Using highly specific ribonuclease protection analysis, mRNAs encoding three putative AM receptors [L1, calcitonin-receptor-like receptor (CRLR) and RDC1] were shown to be present at high concentrations in RNA extracts from lung. In cultured VSMC, however, whereas RDC1 mRNA was expressed at relatively high concentrations, transcripts encoding CRLR and L1 were not detected. The co-expression of prepro-AM mRNA with the RDC1 receptor implies that AM may act in a localized manner via this receptor to modulate VSMC function.

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.

1. Lumbar spinal substance P and calcitonin-gene-related peptide derive from spillage out of the dorsal horns associated with activity of small primary sensory afferents (C- and Aδ-fibres). Cerebrospinal fluid and sural nerve levels of substance P and calcitonin-gene-related peptide have been measured in patients with diabetic polyneuropathy to determine whether differences in small primary sensory afferent activity are related to the presence or absence of painful symptoms. 2. Calcitonin-gene-related peptide was undetectable in the cerebrospinal fluid of the majority of diabetic patients (14 out of 22); it was lower overall in diabetic patients as compared with control subjects ( P <0.01), it was lower in those diabetic patients with painless neuropathy (100% undetectable) as compared with those with painful neuropathy (50% undetectable; P <0.05) and it correlated conversely with warming threshold ( r = 0.50; P <0.01). 3. Substance P showed no overall numerical inter-group differences or correlation with other measured variables, but six diabetic patients as compared with one control subject had undetectable cerebrospinal fluid levels and the proportion of patients with undetectable levels was higher in the group with painless neuropathy than in the group with painful neuropathy ( P <0.05). 4. The levels of each peptide in cerebrospinal fluid correlated with its equivalent in sural nerve ( P <0.01 for calcitonin-gene-related peptide and P <0.03 for substance P). Calcitonin-gene-related peptide correlated with substance P in the sural nerve ( r = 0.84; P <0.002) and in the cerebrospinal fluid ( r = 0.30; P <0.03). 5. We conclude that in sensory diabetic polyneuropathy the lumbar cerebrospinal fluid levels of substance P and calcitonin-gene-related peptide relate directly to their levels in sural nerve and appear to be reduced in the painless compared with the painful syndrome, suggesting a relatively spared and/or more active primary sensory afferent fibre population in the latter.

1. Binding sites for calcitonin-gene-related peptide were localized and characterized in porcine coronary arteries using quantitative autoradiography, and the density of binding sites was compared between large epicardial and small intramyocardial coronary arteries. 2. A single class of binding sites for calcitonin-gene-related peptide with a dissociation constant of 2.1 ± 0.2 nmol/l was detected in both the large and small coronary arteries. The density of specific binding sites was higher (maximum binding site density 231 ± 14 fmol/mg of protein) in the small coronary arteries than in the large epicardial coronary arteries (maximum binding site density 108 ± 5 fmol/mg of protein). β-Human calcitonin-gene-related peptide showed higher affinity than α-human calcitonin-gene-related peptide for the binding sites. Most of the specific binding sites for both peptides in the large coronary artery were localized in the intima and media. 3. In coronary artery from patients with coronary heart disease, there were more binding sites for calcitonin-gene-related peptide in the smooth muscle layer of atheromatous segments (7.2 ± 0.7 amol/mm 2 ) than in that of normal segments (3.0 ± 0.3 amol/mm 2 , P < 0.002). 4. The present findings lend further support to the theory of regional variation in the vasodilator response to calcitonin-gene-related peptide in porcine coronary arteries, which seems to be due to different densities of a single type of receptor for calcitonin-gene-related peptide.

1. Immunohistochemical, immunoblotting and release experiments were performed on ileum from control rats, from 8-week streptozotocin-diabetic rats and from diabetic rats after acute application of insulin in vitro . 2. There was an increase in vasoactive-intestinal-polypeptide-like and a decrease in calcitonin-gene-related-peptide-like immunoreactivity in the myenteric plexus of the diabetic rat ileum, although electrically evoked release of both peptides from enteric nerves was defective. Acute application of insulin in vitro reversed the defective release and changes in immunoreactivity of vasoactive intestinal polypeptide and calcitonin-gene-related peptide seen in the enteric nerves of streptozotocin-diabetic rat ileum. 3. In addition, using a monoclonal neurofilament antibody RT 97 that recognizes a phosphorylated neurofilament epitope present in normal enteric nerves, it was shown that this phosphorylated neurofilament epitope was absent in diabetic nerves, even though a polyclonal neurofilament antibody revealed that neurofilaments were present in both axons and cell bodies of the myenteric plexus of diabetic rat ileum. After only 2 h of insulin incubation in vitro , the phosphorylated neurofilament epitope was again present in the nerves. 4. It is suggested that the abnormal distribution of phosphorylated neurofilaments and defective storage and release of vasoactive intestinal polypeptide and calcitonin-gene-related peptide in the present study may be a more general feature of diabetes. The restoration of these abnormalities by continuous acute insulin application in vitro shown here suggests that the availability of a steady level of insulin might prevent some of the changes which occur in early stages of diabetes. If so, this could influence the use of insulin in the treatment of diabetes, particularly in view of the recent report that short-term continuous subcutaneous insulin infusion restores the function of the autonomic and peripheral nerves in type I diabetic patients [Krönert, K., Hülsen, J., Luft, D., Stetter, T. & Eggstein, M. (1987) Journal of Clinical Endocrinology and Metabolism , 64 , 1219–1223].