J.M. Allen

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.

NEUROPEPTIDE TYROSINE (NPY)—A MAJOR CARDIAC NEUROPEPTIDE

A newly discovered bioactive peptide, neuropeptide tyrosine (NPY), has been found in the human cardiac nervous system. Dense concentrations of NPY-immunoreactive nerve fibres were found in association with nodal tissue (atrioventricular node 22.1 +/- 3.7 pmol/g). NPY nerve fibres were seen in close contact with cardiac muscle fibres and were also found around the coronary vessels (19.6 +/- 6.2 pmol/g). Analysis of the peptide by high-performance liquid chromatography demonstrated that it was present in a single molecular form, closely similar or identical to that of the isolated bioactive peptide. Cardiac function in man has long been known to be influenced by cholinergic and adrenergic nerves. There now appears to be a further component of the nervous system in the human heart, involving peptidergic nerves containing NPY.

Neuropeptide Y (NPY) reduces myocardial perfusion and inhibits the force of contraction of the isolated perfused rabbit heart.

Isolated rabbit hearts, perfused under constant pressure (Langendorff technique) were used to study the effect of neuropeptide Y (NPY) on heart rate, force of heart contraction and rate of myocardial perfusion. No significant net change in heart rate was noted. A dose-dependent negative inotropic effect was consistently demonstrated which was characterised by slow onset and was often preceded by a transient positive inotropic response. Addition of small doses of NPY resulted in a prompt reduction in flow of the perfusate through the coronary vasculature. Since NPY is present locally in cardiac nerves, these effects may have physiological importance.

Neuropeptide Y in phaeochromocytomas and ganglioneuroblastomas.

Tumour tissue from nineteen patients with phaeochromocytomas and nine with ganglioneuroblastomas contained large numbers of neuropeptide Y (NPY) producing cells and extracts of these tumours had very high concentrations of immunoreactive NPY. Plasma NPY concentrations were also raised, averaging 460 pmol/l in patients with tumours of the sympathetic chain and 55 pmol/l in healthy controls. Since plasma NPY is straightforward to measure and relatively stable, its estimation may prove helpful as a screening tests for phaeochromocytoma.

Radioimmunoassay of neuropeptide Y

The development of a radioimmunoassay to the newly isolated peptide, neuropeptide Y is described. Four separate antisera have been developed using different immunisation schedules. Two of these antisera (YNI and YNIO) are directed to the C-terminal region of the peptide and cross-react with the related peptide PYY, whereas YN7 is specific being directed to the N-terminal region of NPY, YN6 is similarly specific for NPY, but is unable to bind the available fragments. These four antisera provide similar results for determination of NPY immunoreactivity within porcine brain extracts, however YN6 consistently undervalues all extracts from the other species examined (human, rat, guinea pig, cat and mouse). Chromatographic analysis by means of reverse phase high pressure liquid chromatography (HPLC) shows that NPY immunoreactivity of human extracts elutes in an earlier position than the porcine standard. It seems likely therefore that human and porcine NPY differ in their amino acid sequences.

Ontogeny of a novel peptide, neuropeptide Y (NPY) in rat brain

Changes in the concentration of a newly discovered peptide, neuropeptide Y (NPY) have been determined in the developing rat brain using a recently developed radioimmunoassay and chromatographic analysis. NPY was present in the brain stem (14.8 +/- 5.6 pmol/g) and diencephalon (12.1 +/- 12.1 pmol/g) in the earliest embryos studied (14 days postconception), but appeared only on the 19th day postconception in the cerebral cortex. The concentrations of NPY showed a rapid postnatal rise in all regions examined. The finding of NPY early in the development of the embryonic rat brain and particularly in caudal regions has some similarities to the pattern of development of the catecholaminergic system.

Peptide-containing nerves in human urinary bladder

Nerves containing immunoreactive vasoactive intestinal polypeptide (VIP), substance P and two newly discovered peptides, neuropeptide tyrosine (NPY) and PHI (peptide having N-terminal histidine and C-terminal isoleucine), have been found in the human urinary bladder by immunocytochemistry and radioimmunoassay. Somatostatin immunoreactivity was detected by radioimmunoassay. The VIP-immunoreactive nerves were widely distributed in all regions, but were particularly dense beneath the epithelium and in the muscle layer. Scattered intramural ganglia were found to be reactive to VIP antiserum. Higher concentrations of extractable VIP were detected in the trigone than in the dome. VIP- and PHI-immunoreactive nerves were similarly distributed, the latter being less numerous. NPY-immunoreactive nerves were seen mainly in the muscle layer, particularly in the trigonal area. The distribution patterns of VIP- and NPY-immunoreactive nerves resembled those of the previously reported cholinergic and adrenergic nerves, respectively. Many blood vessels were found to be innervated by both types of immunoreactive nerves. Scattered substance P-immunoreactive fibers were occasionally seen, being present in the submucosa and around the detrusor muscles. The significance of these nerves remains to be elucidated.

Neuropeptide Y innervation of the rodent pineal gland and cerebral blood vessels

Neuropeptide Y (NPY)-immunoreactivity has been shown to be present in sympathetic nerve fibres in the rat pineal gland and a dense network of NPY-containing nerve fibres demonstrated to innervate the rat circle of Willis. The NPY content of the major rabbit intracranial arteries was determined by radioimmunoassay and maximal levels found in the anterior cerebral arteries. After bilateral superior cervical ganglion (SCG) removal, no NPY was detectable in the rat pineal gland; however, significant NPY-immunoreactive nerve fibres remained throughout the rat vertebrobasilar arteries, and 47% of the assayable NPY was still present. Neither intraventricular 6-hydroxydopamine (6-OHDA) nor the combination of 6-OHDA treatment and SCG removal resulted in any further loss of NPY. In conclusion, the NPY innervation of the pineal gland originates exclusively from the peripheral sympathetic nervous system. In contrast the caudal portion of the rat circle of Willis contains NPY fibres which are resistant to sympathectomy.

Calcitonin gene-related peptide (CGRP) in the female rat urogenital tract

CGRP-immunoreactivity was found throughout the female rat urogenital tract by specific radioimmunoassay, and shown to be present in nerve fibres by immunocytochemistry. The highest concentrations of CGRP-like immunoreactivity were found in the urinary tract, with lower levels in regions of the genitalia. Chromatographic analysis of bladder and vaginal extracts on Sephadex G-50 columns and HPLC revealed at least three CGRP-immunoreactive peaks. The major peak emerged in the same position as synthetic rat CGRP. CGRP nerve fibres were associated mainly with blood vessels, non-vascular smooth muscle, squamous epithelium and uterine and cervical glands, and were particularly abundant in the ureter and bladder. CGRP-immunoreactivity was depleted by neonatal treatment with capsaicin and after surgical section of pelvic and/or hypogastric nerves. Immunocytochemistry demonstrated that depletion occurred predominantly in the mucosal layer of the urogenital tract. These findings indicate a sensory function for most of the CGRP-immunoreactive nerves in the rat urogenital tract.

Presence of neuropeptide Y in human circle of Willis and its possible role in cerebral vasospasm.

Neuropeptide Y (NPY) has been demonstrated in the human circle of Willis by specific radioimmunoassay. Concentrations were similar in cadaver and peroperative specimens. NPY concentrations were highest around the basilar bifurcation (2.9 +/- 1.0 pmol/g), anterior cerebral artery (2.7 +/- 0.5 pmol/g), and carotid trifurcation (2.4 +/- 0.6 pmol/g). Concentrations were lowest in the basilar artery (1.0 +/- 0.4 pmol/g). Intracarotid administration of NPY (50 pmol to 2 nmol) to rats reduced mean cerebral cortical blood flow by 40-98%. This vasoconstriction lasted for at least 2 h. These findings suggest that NPY may be involved in the cerebral vasospasm which follows subarachnoid haemorrhage and in the maintenance of normal vascular tone.