M.A. Ghatei

GLUCAGON-LIKE PEPTIDE-1 7-36: A PHYSIOLOGICAL INCRETIN IN MAN

The physiological role of glucagon-like peptide-1 7-36 amide (GLP-1 7-36) in man was investigated. GLP-1 7-36-like immunoreactivity was found in the human bowel; its circulating level rose after oral glucose and after a test breakfast. When it was infused into seven volunteers at a rate to mimic its postprandial plasma concentration in the fasting state, plasma insulin levels rose significantly and glucose and glucagon concentrations fell. During an intravenous glucose load, it greatly enhanced insulin release and significantly reduced peak plasma glucose concentrations, compared with a control saline infusion, even inducing postinfusion reactive hypoglycaemia. By comparison, infusion of glucose-dependent insulinotropic peptide (GIP) to physiological levels was less effective in stimulating insulin release. These observations suggest that GLP-1 7-36 is a physiological incretin and that it is more powerful than GIP. The observation of greatly increased postprandial plasma GLP-1 7-36 levels in patients with postgastrectomy dumping syndrome suggests that it may mediate the hyperinsulinaemia and reactive hypoglycaemia of this disorder.

Elevated plasma endothelin in patients with diabetes mellitus

SummaryPlasma concentrations of endothelin, a vasoconstrictor peptide released from vascular endothelial cells, have been measured by radioimmunoassay in 100 patients with diabetes mellitus and 19 healthy subjects. The plasma immunoreactive-endothelin concentrations were found to be greatly raised in the patients with diabetes (1,880±120 fmol/l, mean±SEM) compared with the healthy subjects (540±50 fmol/l, p<0.005). The elevation of immunoreactive-endothelin could not be explained by secondary changes in blood pressure or renal disease and did not correlate with the presence of diabetic retinopathy, duration of diabetes mellitus, fasting blood glucose or serum fructosamine. Fast protein liquid chromatographic analysis of the diabetic plasma immunoreactive-endothelin showed three forms, one in a very big molecular weight position, one intermediate and one in the position of endothelin-1 itself. No material appeared in the positions of endothelin-2 and 3. Chromatographic analysis of normal plasma showed only the big molecular weight peak while material in the endothelin-1,2 or 3 positions was below detection. The elevation of endothelin in diabetic patients may be a marker of, and further exacerbate, their vascular disease.

Calcitonin gene-related peptide in cardiovascular tissues of the rat.

The distribution of calcitonin gene-related peptide immunoreactivity in the cardiovascular system of the rat was investigated by radioimmunoassay and immunocytochemistry. The nature of the immunoreactivity was studied by gel permeation and high performance liquid chromatography. Immunocytochemistry demonstrated the existence of calcitonin gene-related peptide-containing nerve fibres throughout the cardiovascular system. These were present in all regions of the heart, particularly in association with the coronary arteries, within the papillary muscles and within the sinoatrial and atrioventricular nodes. Calcitonin gene-related peptide-containing fibres were found mainly in the adventitia of the arteries and veins. Calcitonin gene-related peptide concentrations were high in major arteries and veins but comparatively low in the heart, aortic arch and thoracic aorta. Chromatography showed that approximately 70% of the total immunoreactivity was identical to synthetic calcitonin gene-related peptide. Calcitonin gene-related peptide concentrations in the blood vessels of rats treated neonatally with capsaicin were not found to be significantly different from those in control animals although capsaicin caused significant reductions of calcitonin gene-related peptide levels in certain other tissues. The results of this study suggest that calcitonin gene-related peptide-containing fibres are likely to be of importance in the innervation of vascular tissues and raise the possibility that these fibres are different in character from calcitonin gene-related peptide-containing fibres found in other tissues.

Occurrence, distribution and ontogeny of CGRP immunoreactivity in the rat lower respiratory tract: Effect of capsaicin treatment and surgical denervations

The occurrence and distribution of calcitonin gene-related peptide (CGRP) immunoreactivity in the rat respiratory tract were investigated by means of immunocytochemistry and radioimmunoassay using antibodies raised in rabbits to synthetic rat CGRP. Substantial amounts of CGRP immunoreactivity (range 5-37 pmol/g) were detected in all parts of the respiratory tract, the highest being in the stem bronchus. Gel filtration chromatography of extractable CGRP immunoreactivity revealed one single peak, eluting at the position of synthetic rat CGRP. CGRP immunoreactivity was localized both in mucosal endocrine cells and nerve fibres from the larynx down to the peripheral lung. CGRP-immunoreactive endocrine cells were found singly in trachea and stem bronchi and in groups in intrapulmonary airways. They appeared at a late stage of gestation (17 days), reached a maximum number near term and decreased after birth to maintain a population similar to that of the adult animals by postnatal day 21. Similarly, CGRP-immunoreactive nerve fibres were first identified by day 18 of the gestation period and reached the adult distribution by postnatal day 21. CGRP-immunoreactive nerve fibres were localized among smooth muscle, seromucous glands, beneath and within the epithelium of the airways and around blood vessels. CGRP was also found in sensory ganglia and in motor end plates of the larynx musculature. Neonatal pretreatment with capsaicin caused a marked reduction in CGRP immunoreactivity of nerve fibres in the respiratory tracts as well as a less marked decrease in the population of CGRP-containing endocrine cells of the lung. No change was seen in motor end plates immunostaining. Vagal ligation experiments revealed that CGRP-immunoreactive nerve fibres travelling in the vagus originate mainly from neurons located in the jugular ganglion. Infranodosal right vagal ligation induced a marked loss in CGRP-immunoreactive nerves of the trachea, and of the ipsilateral stem bronchus, but no changes were observed in peripheral lung. By contrast infranodosal left side vagal ligation caused a decrease in CGRP-immunoreactive nerves of the ipsilateral lung and bronchus without affecting the peptide content in the trachea. Left vagal ligation also induced a marked increase in both the intensity of staining and number of CGRP-immunoreactive endocrine cells in the lung. We conclude that CGRP immunoreactivity is localized in both nerve fibres and endocrine cells and is associated principally with the afferent (sensory) innervation of the respiratory tract.(ABSTRACT TRUNCATED AT 400 WORDS)

The central melanocortin system affects the hypothalamo-pituitary thyroid axis and may mediate the effect of leptin.

Prolonged fasting is associated with a downregulation of the hypothalamo-pituitary thyroid (H-P-T) axis, which is reversed by administration of leptin. The hypothalamic melanocortin system regulates energy balance and mediates a number of central effects of leptin. In this study, we show that hypothalamic melanocortins can stimulate the thyroid axis and that their antagonist, agouti-related peptide (Agrp), can inhibit it. Intracerebroventricular (ICV) administration of Agrp (83-132) decreased plasma thyroid stimulating hormone (TSH) in fed male rats. Intraparaventricular nuclear administration of Agrp (83-132) produced a long-lasting suppression of plasma TSH, and plasma T4. ICV administration of a stable alpha-MSH analogue increased plasma TSH in 24-hour-fasted rats. In vitro, alpha-MSH increased thyrotropin releasing hormone (TRH) release from hypothalamic explants. Agrp (83-132) alone caused no change in TRH release but antagonized the effect of alpha-MSH on TRH release. Leptin increased TRH release from hypothalami harvested from 48-hour-fasted rats. Agrp (83-132) blocked this effect. These data suggest a role for the hypothalamic melanocortin system in the fasting-induced suppression of the H-P-T axis.

Calcitonin gene-related peptide immunoreactivity in afferent neurons supplying the urinary tract: Combined retrograde tracing and immunohistochemistry

The innervation of rat and guinea pig urinary tract was examined using immunohistochemistry, radioimmunoassay and True Blue retrograde tracing techniques and was further assessed following both surgical and chemical denervation experiments. Substantial amounts of calcitonin gene-related peptide-like immunoreactivity (range 20-150 pmol/g) were detected in tissue extracts and localised to nerve fibres distributed throughout the urinary tract of both species, these being concentrated in the ureter and base of the bladder. In the guinea pig, the number and distribution pattern of calcitonin gene-related peptide-like immunoreactive nerves appeared to be identical to that of substance P-containing nerves, whereas in the rat the former predominated. Seven days after injection of the fluorescent dye True Blue into tissues of the urinary tract, retrogradely labelled cells were found in the dorsal root ganglia. These cells had a segmental distribution pattern which was specific for each of the injection sites. Thus, after injection of True Blue into the left kidney hilum a single group of labelled cells were found in the ipsilateral T10-L2 dorsal root ganglia. In contrast, injection into the left ureter produced labelled cells in two separate groups of ipsilateral ganglia (T11-L3 and L6-S1). Injection into the wall of the bladder and upper urethra resulted in bilateral labelling, with most labelled cells occurring in L6 and S1 ganglia. Approximately 90% of labelled cells in T10-L3 dorsal root ganglia displayed calcitonin gene-related peptide-like immunoreactivity, but only 60% of retrogradely labelled bladder neurons in L6-S1 ganglia were immunoreactive for this peptide. Adult guinea pigs and neonatal rats injected systemically with capsaicin subsequently exhibited a marked reduction both in the amount of calcitonin gene-related peptide immunostaining and the concentration of immunoreactive material in the urinary tract, dorsal root ganglia and spinal cord. In rats treated neonatally with capsaicin, there was a significant reduction in the number of retrogradely labelled cells and a hypertrophy of the bladder. Sectioning of the pelvic and hypogastric nerves in the rat also resulted in a depletion of calcitonin gene-related peptide-like immunoreactive nerves in the bladder, whereas chemical sympathectomy appeared to have no effect. The results indicate that calcitonin gene-related peptide immunoreactivity occurs in a major proportion of afferent neurons supplying the urinary tract of the rat and guinea pig.(ABSTRACT TRUNCATED AT 400 WORDS)

Formation of endothelin by cultured airway epithelial cells.

Immunoreactivity to endothelin was detected in conditioned culture medium from both canine and porcine tracheal epithelial cells. Gel permeation chromatography and fast protein liquid chromatography were used to confirm the identity of the endothelin. The two peaks demonstrated on fast protein liquid chromatography co‐eluted with endothelin 1 and endothelin 3 respectively.

Calcitonin gene-related peptide immunoreactive sensory and motor nerves of the rat, cat, and monkey esophagus

In the mammalian esophagus calcitonin gene-related peptide (CGRP)-immunoreactive nerves form abundant subepithelial plexuses and penetrate the mucosa. The levels of extractable CGRP in separated epithelial layers are 15.8 +/- 2.4 pmol/g wet wt of tissue (n = 8, mean +/- SEM). Treatment of neonatal rats with capsaicin and ablation of the central portion of the feline nodose ganglion led to a marked reduction in the numbers of CGRP-immunoreactive nerve fibers. The loss of CGRP nerves demonstrated by immunocytochemistry was accompanied by a parallel reduction in the tissue content of CGRP, as measured by radioimmunoassay (1.5 +/- 0.5 pmol/g in capsaicin-treated animals compared with 9.4 +/- 1.9 pmol/g in vehicle-treated controls; p less than 0.0025). These findings indicate the sensory nature of the CGRP-immunoreactive nerves. Substance P-immunoreactive nerve fibers innervated in particular the blood vessels of the lamina propria; very few penetrated the esophageal epithelium and these were only partially depleted after removal of the central portion of the nodose ganglion. The esophageal muscle contained nerves immunoreactive for substance P and, in particular, for CGRP which was also found in the motor end plates of the striated muscle. No changes in the CGRP-containing motor end plates were observed either after treatment of neonatal rats with capsaicin or ablation of cell bodies from the central portion of the nodose ganglion. These nerve fibers may originate from rostral areas of the nucleus ambiguus, where CGRP-immunoreactive motor neurons have previously been described. Thus, our findings reveal dual components, motor and sensory, of the CGRP-containing innervation of the esophagus.

Developmental changes in bombesin, substance P, somatostatin and vasoactive intestinal polypeptide in the rat brain.

The regional distribution of the 4 neuropeptides, bombesin, substance P, somatostatin and vasoactive intestinal polypeptide (VIP) were investigated in the developing rat brain. Specific radioimmunoassay and immunocytochemistry were employed. VIP and bombesin were undetectable in the foetal brain whereas substance P and somatostatin were shown to be present in all regions as early as 14 days postcoitus. There was a dramatic postnatal increase in all 4 peptides in most regions. These results are discussed and compared with results of previous investigations of the ontogeny of the classic neurotransmitters.

Dose-response comparisons of canine plasma gastroenteropancreatic hormone responses to bombesin and the porcine gastrin-releasing peptide (GRP)

The effect on plasma gastroenteropancreatic hormone levels on infusing the porcine gastrin-releasing peptide and bombesin into dogs demonstrated no qualitative difference in the spectrum of activity of the two peptides. Sustained elevation in plasma immunoreactive gastrin, pancreatic polypeptide, enteroglucagon, gastric inhibitory polypeptide, pancreatic glucagon and transient elevations in plasma insulin were seen during infusions of both peptides. The similar spectrum of activities and the structural homology between the two peptides suggests that the porcine gastrin releasing peptide is the porcine counterpart of the amphibian peptide bombesin.