Suppression of 125I-vasoactive intestinal polypeptide binding sites in arteries of the hamster seminal vesicle following castration.

The presence and distribution of 125I-vasoactive intestinal polypeptide (VIP) binding sites in blood vessels supplying the hamster seminal vesicle was studied using a receptor autoradiographic technique before and following castration. 125I-VIP binding was studied in intact animals, in animals under a 15-day period of castration and in animals under the same period of castration but submitted to a further 15-day period of testosterone treatment. Our results show that, in the seminal vesicle, VIP-binding sites are localized in the gland smooth muscle coat and arterial smooth muscle. A 15-day castration period abolishes 125I-VIP binding to vascular smooth muscle but has no effect on 125I-VIP binding to the gland smooth muscle coat. Treatment with testosterone restores 125I-VIP binding to the vascular smooth muscle, completely reversing the effect of castration. Our results indicate that VIP-binding sites in the smooth muscle wall of arteries supplying the hamster seminal vesicle are under androgenic control and are more sensitive to androgen deprivation that VIP-binding sites associated to the gland smooth muscle coat.

Innervation of the human middle meningeal artery: immunohistochemistry, ultrastructure, and role of endothelium for vasomotility.

The majority of nerve fibers in the middle meningeal artery and branching arterioles are sympathetic, storing norepinephrine and neuropeptide Y (NPY). A sparse supply of fibers contain acetylcholinesterase activity and immunoreactivity toward vasoactive intestinal peptide (VIP), peptidine histidine methionine (PHM), and calcitonin gene-related peptide (CGRP). Only few substance P and neuropeptide K immunoreactive fibers are noted. Electronmicroscopy shows axons and terminals at the adventitial medial border of the human middle meningeal artery, with a fairly large distance to the smooth muscle cells (>500 nM). Several axon profiles contain vesicles of different types, including putative sensory profiles. The perivascularly stored signal substances, norepinephrine and NPY induced vasoconstrictor. Relaxations were induced by acetylcholine and substance P, and these were significantly reduced in arteries without endothelium, while the responses to norepinephrine, NPY, VIP, PHM, and CGRP were not changed by endothelium removal. Blockade experiments showed that the vasomotor responses to norepinephrine were blocked by prazosin, to NPY by BIBP 3226, acetylcholine by atropin, substance P by RP 67580, and the human alpha-CGRP response by human alpha-CGRP(8-37).

Innervation pattern of malformative cortical vessels in Sturge-Weber disease: an histochemical, immunohistochemical, and ultrastructural study.

OBJECTIVE: This study was undertaken to elucidate the pattern of vascular innervation in areas of pial angiomatosis in Sturge-Weber disease (SWD) and eventually correlating it with the pathophysiology of the disease, namely its chronic ischemic changes. METHODS: We processed part of a surgical specimen resected from a 3-year-old female patient who underwent functional hemispherectomy for SWD and characterized the pattern of innervation of the malformative cortical vessels using histochemical, immunohistochemical, and ultrastructural techniques. RESULTS: Cortical vessels were observed to be supplied with numerous varicose nerve fibers containing immunoreactivity for neuropeptide tyrosine and the catecholamine-synthesizing enzyme, tyrosine, tyrosine hydroxylase. In contrast, no nerve fibers containing acetylcholinesterase activity and immunoreactivity for Substance P, a calcitonin gene-related peptide and vasoactive intestinal peptide, were detected. Ultrastructural studies revealed the presence of numerous axon varicosities at the adventitial-medial border. Neuropeptide tyrosine immunoreactivity was localized in large granular vesicles in nerve varicosities that also contained numerous small granular vesicles. CONCLUSION: These results demonstrate that nerve supplying cortical vessels in SWD are arranged in a distribution pattern similar to the one observed in human normal cortical veins and suggest that these abnormal vessels are innervated only with noradrenergic sympathetic nerve fibers. This represents a clear difference from the pattern of innervation observed in both normal cortical arteries and veins, and is the consequence of the anatomic and functional dysangiogenic process characteristic of the affected cortical areas in SWD.

Nitroxidergic innervation of guinea pig cerebral arteries.

The presence and distribution of nitric oxide synthase (NOS)-immunoreactive nerve fibers associated with the guinea pig major cerebral arteries was studied by means of immunohistochemical, histochemical and ultrastructural techniques. Anterior arteries of the circle of Willis received a rich supply of perivascular nerve fibers containing NOS immunoreactivity while posteriorly localized arteries presented a moderate to sparse innervation. A double immunofluorescence staining technique revealed that NOS was localized in nerve fibers distinct from those displaying substance P or tyrosine hydroxylase. Combined immunofluorescence and histochemical staining of the same preparation indicated that NOS immunoreactivity was localized in putative cholinergic nerve fibers (identified by their acetylcholinesterase content) and that NADPH-diaphorase activity (a marker for NOS-containing neurons) was found in nerves which also possessed VIP immunoreactivity. The ultrastructural study revealed that NOS immunoreactivity was present in numerous nerve varicosities at the adventitial-medial border. These results suggest that NO and VIP co-exist in putative parasympathetic nerve fibers supplying the guinea pig cerebral arteries and may be release together in response to nervous stimulation.

Peptidergic innervation of guinea-pig brain vessels: comparison with immunohistochemistry and in vitro pharmacology in rostrally and caudally located arteries.

The peptidergic innervation of the guinea-pig basilar artery and the posterior, middle and anterior cerebral arteries were studied by means of immunohistochemical and image analysis techniques using whole mount preparations. An in vitro pharmacological study was performed to correlate the distribution of peptide-containing nerves and the action of neuropeptides on vessel segments from the same vascular regions. The overall distribution of perivascular nerve fibres was demonstrated using an antiserum to the general neuronal marker protein gene product 9.5 (PGP 9.5) and the percentage immunostained area of total vessel wall area occupied by PGP-containing nerves, in each of the basilar, posterior and middle cerebral arteries, was set at 100% and used to determine the relative density of specific populations of autonomic and sensory nerve fibres. In all four cerebral arteries, the majority of nerve fibres possessed neuropeptide Y (NPY) and tyrosine hydroxylase (TH) immunoreactivity, occupying 6.2-13.3% and 5.8-7.5% of the total vessel wall area, respectively. Vasoactive intestinal peptide (VIP), substance P (SP) and calcitonin-gene-related peptide (CGRP) were detected at lower densities. The pharmacological study performed on small circular segments with an intact endothelium revealed that, in all four cerebral arteries, NPY was a more potent constrictor than noradrenaline (NA). The rank order of potency for relaxant agents was CGRP = SP > VIP > ACh in the PCA and MCA, and SP = CGRP > VIP > ACh in the BA and ACA. The correlation between immunostained nerve area and the agonist potency suggested that the denser the peptidergic nerve-supply, the lower the sensitivity to the agonist.

Innervation of human epicardial coronary veins: immunohistochemistry and vasomotility.

OBJECTIVE: The aim was to investigate the innervation and vasomotor responses to classical and putative transmitters of the coronary venous bed. METHODS: The innervation of human epicardial coronary veins was investigated using acetylcholinesterase histochemistry and immunofluorescence staining, together with antisera against the general neuronal marker protein gene product 9.5 (PGP 9.5), the catecholamine synthesising enzyme tyrosine hydroxylase, and neuropeptides [neuropeptide Y, vasoactive intestinal peptide (VIP), substance P, and calcitonin gene related peptide (CGRP)]. The vasomotor responses to noradrenaline, acetylcholine, neuropeptide Y, substance P, human alpha calcitonin gene related peptide (alpha CGRP), and VIP were tested on isolated circular human epicardial coronary vein segments. RESULTS: A network of nerve fibres was shown in the major epicardial coronary veins by means of an antiserum to PGP 9.5. The majority of the perivascular nerve fibres possessed neuropeptide Y and tyrosine hydroxylase immunoreactivity. Only a few nerve fibres displayed substance P, CGRP, and VIP immunoreactivity and acetylcholinesterase activity. Noradrenaline and acetylcholine induced powerful contractions of all the tested segments, whereas no contraction was induced by neuropeptide Y, alpha CGRP, substance P, or VIP. All segments precontracted with U46619 responded with potent relaxation to alpha CGRP, substance P, and VIP, whereas noradrenaline and acetylcholine only in low concentrations induced weak relaxation of a few of the segments. No relaxation was induced by neuropeptide Y. CONCLUSIONS: This is the first study to demonstrate comprehensively the perivascular innervation of human coronary veins and corresponding vasomotor effects, suggesting a role in regulation of the coronary venous circulation.

The peptidergic innervation of human coronary and cerebral vessels.

It is now well established that in addition to nerves containing classical transmitters, the mammalian vascular system is also supplied by nerve fibre subpopulations containing several vasoactive peptides. The precise function of these peptides (neuropeptide Y, calcitonin gene-related peptide, vasoactive intestinal polypeptide, somatostatin and the tachykinins) is still unknown, however, their widespread occurrence in perivascular nerves indicates that they are likely candidates for a role in the neurogenic regulation of the vascular system. It has been suggested that they may exert a direct vasomotor action via their own receptors and/or modulate the release and action of other vascular transmitters. Recently, several studies have focused on the supply of nerve fibres storing neuropeptides in the coronary and cerebral vasculature of laboratory animals, however, little is known on the distribution of these putative transmitters in human coronary and cerebral vessels. In this paper, the immunocytochemical evidence that several neuropeptides are localized in subpopulations of afferent and efferent nerve fibres supplying the human coronary and cerebral vasculature is focused.

The innervation of guinea pig epicardial coronary veins: immunohistochemistry, ultrastructure and vasomotility.

The innervation and vasomotor responses to several vasoactive agents of guinea pig epicardial coronary veins were investigated by means of immunohistochemical, histochemical, ultrastructural and in vitro pharmacological techniques. The use of an antiserum to the general neuronal marker protein gene product 9.5 revealed that coronary veins are supplied by a network of fine varicose nerve fibres in the adventitia. The majority of the nerve fibres possessed neuropeptide Y (NPY) and tyrosine hydroxylase immunoreactivity. Only a few nerve fibres displayed substance P, neuropeptide K (NK) and calcitonin gene-related peptide (CGRP) immunoreactivity. In double stained preparations substance P immunoreactivity was co-localized with NK and CGRP in the same nerve fibres. Nerve fibres containing vasoactive intestinal peptide (VIP) immunoreactivity or acetylcholinesterase activity were not detected. Endothelin immunoreactivity was also found in the vein endothelial cells. Ultrastructural studies revealed the presence of axon varicosities at the adventitial-medial border. In vitro pharmacological studies showed that endothelin-1 and -2 elicited a significant contractile response of epicardial vein segments. Noradrenaline, NPY, serotonin and uridine 5'-triphosphate induced only a relatively weak contractile response in the vein segments. Although vasodilatory responses were difficult to examine in these preparations, it was found that substance P, CGRP and VIP elicited a relaxation of the vein segments. These results indicate that guinea pig epicardial coronary veins are innervated by several nerve populations, however, the control of vasomotor tone of coronary veins appears to be predominantly regulated by 'non-neuronal' vasoactive agents such as endothelin and 5-HT.

Vasoactive intestinal peptide in the hamster seminal vesicle: distribution, binding sites and possible functions.

The presence and functional role of vasoactive intestinal peptide in the hamster seminal vesicle were studied by a combination of structural and functional approaches. The use of an immunofluorescence staining technique in both cryostat sections and whole-mount preparations revealed that vasoactive intestinal peptide-immunoreactive nerve fibres were mainly localized in the lamina propria of the mucosal layer. In double-stained preparations, vasoactive intestinal peptide immunoreactivity was found to be localized in nerves also containing acetylcholinesterase activity. At the ultrastructural level, the use of an immunogold staining method showed that vasoactive intestinal peptide immunoreactivity occurred in large granular vesicles (80-150 nm in diameter) in nerve varicosities which also contained small pleomorphic agranular vesicles. In order to evaluate the anatomical distribution of vasoactive intestinal peptide binding sites in the seminal vesicle, we have utilized an in vitro receptor autoradiographic technique. Vasoactive intestinal peptide binding sites were localized in the basal region of the secretory epithelium, in the muscle layer and in the wall of blood vessels. In vitro incorporation of [3H]L-leucine into protein by tissue slices revealed that vasoactive intestinal peptide (1 microM) significantly increases the amount of released protein. Vasoactive intestinal peptide (0.1-1 microM) did not affect the resting tension of the muscle but significantly inhibited the increase in muscle tension induced by carbachol. Atropine prevented the effect of carbachol, indicating that the latter is mediated by muscarinic receptors. Our results suggest that in the hamster seminal vesicle, vasoactive intestinal peptide is involved in the modulation of muscarinic function and in the control of secretion.