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The aim of the present study was to compare in man the innervation pattern and the functional responses to neuronal messengers in medium sized lenticulostriate and branches of the posterior cerebral arteries (PCA). The majority of the nerve fibers found were sympathetic and displayed specific immunoreactivity for tyrosine hydroxylase (TH) and neuropeptide Y (NPY). Only few nerve fibers displayed vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP) and substance P (SP) immunoreactivity. In both arteries, the contractions induced by noradrenaline (NA), NPY and 5-hydroxytryptamine (5-HT) and the relaxant responses induced by acetylcholine (ACh), VIP and pituitary adenylate cyclase activating peptide-27 (PACAP) as well as CGRP and SP were compared in vitro. In conclusion, there was no major difference in innervation pattern or vasomotor sensitivity (pEC50 and pIC50 values) between the two vessels. However, the general pattern indicates stronger vasomotor responses (Emax and Imax) in the PCA branches as compared to the lenticulostriate arteries which may lend support for the clinical observation of a difference in stroke expression between the two vascular areas.

Endothelin-1 (ET-1), a 21 amino acid peptide exerts a wide range of biological activities including vasoconstriction, mitogenesis and inotropic effects on the heart. In this study, we examined whether endocardial endothelial cells express ET-1 and evaluated its functional properties. Using immunofluorescence localization method, we demonstrated cytoplasmic staining of ET-1 in the human endocardial endothelial cells from the right atrium and left ventricle. Employing reverse transcriptase polymerase chain reaction (RT-PCR) expression of ET-1 mRNA and its receptors ET(A) and ET(B) mRNAs were found in human myocardial as well as in endocardial endothelial cells. Biological activity of endocardial endothelial cells derived ET-1 was established as the conditioned media obtained from cultured porcine endocardial endothelial cells induced a slowly developing, strong and long-lasting contraction of circular rat aortic segments, with similar characteristics to that obtained with exogenous ET-1. Furthermore, the selective endothelin-A receptor antagonist, FR 139317, blocked the conditioned media induced contractions. Our results suggest that endocardial endothelial cells express and release biologically active ET-1 which could play a pivotal role in the regulation of myocardial contractility as well as a circulatory peptide may further act in other peripheral target organs.

Excessive hyperbilirubinemia can cause irreversible neurological damage in the neonatal period. However, the complete understanding of the pathogenesis of unconjugated bilirubin (UCB) encephalopathy remains a matter of debate. This study investigates whether UCB inhibits the endocytosis of cationized ferritin (CF) by cultured rat astrocytes. The relationship between endocytosis and MTT reduction, as well as changes on tubulin and glial fibrillary acidic protein (GFAP) assembly, were also evaluated. Inhibition of endocytosis was complete in the presence of 171 microM UCB, while a marked decrease of CF labeling was noticed for 86 microM UCB. In addition, MTT reduction was inhibited by 60 to 76% as UCB concentrations changed from 17 to 171 microM, while alterations on both GFAP and microtubule morphology were only achieved by cell exposure to 171 microM UCB. These findings indicate that inhibition of CF endocytosis in rat cortical astrocytes by UCB is a concentration-dependent process that appears to be primarily related to a direct effect on the cell membrane and not to any alteration of cytoskeletal microtubules and intermediate filaments.

In this study, we investigated the presence of vasoactive intestinal peptide (VIP) receptors in the epithelial cells of the hamster seminal vesicle, by using cell clusters isolated from the gland and cultivated in a serum-free bicameral culture system. An immunocytochemical approach and autoradiographic and biochemical binding experiments with 125I-VIP as radioligand were performed. The effect of this neuropeptide on cultured cell proliferation and protein secretory activity was also analysed. The release of trichloroacetic-acid-precipitable radioactive material by epithelial cells to the apical and basal compartments of the bi-chamber was estimated in absolute and relative terms. The results of this work indicate that: (1) VIP receptors are present in the membranes of clusters of epithelial cells from seminal vesicles and are further maintained in cultured cells; (2) VIP does not exert any mitogenic effect in these cells; (3) VIP affects the directionality of secretion, favouring the absolute and relative amounts of protein released to the apical compartment of the bi-chamber. The expression of VIP receptors in the epithelial cells of the hamster seminal vesicle and the direct secretagogue-like activity of this neuropeptide in these cells might be affected by other factors, namely, androgens.

We characterized the 3272-26A-->G mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, creating an alternative acceptor splice site in intron 17a, that competes with the normal one, although we predict from consensus values, with lower efficiency. We analyzed five Cystic Fibrosis (CF) Portuguese patients with the 3272-26A-->G/F508del genotype. Besides clinical and haplotype characterization of those patients, we report here results from CFTR transcript analysis in nasal brushings from all five patients. RT-PCR analysis supports alternative splicing in all patients and carriers, but not in controls. By sequencing, we determined that the alternative transcript includes 25 nucleotides from intron 17a, which predictively cause frameshift and a premature stop codon. The use of this alternative splice site causes a reduction in the levels of normal transcripts from the allele with this mutation and, most probably, of normal protein as well. By immunocytochemistry of both epithelial primary cell cultures and slices from CF polyps, CFTR protein is detected at the cell membrane, with three different antibodies. Ussing chamber analysis of one nasal polyp shows a high sodium absorption, characteristic of CF. Altogether, the results suggest that the main defect caused by the 3272-26A-->G mutation is a reduction in normal CFTR transcripts and protein and therefore this mutation should be included in class V, according to Zielenski and Tsui.

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.

Atrial natriuretic peptide has frequently been detected in the cardiac conduction system and has been shown to regulate some intracellular effects in Purkinje fibers. To determine if atrial natriuretic peptide works as an autocrine and/or paracrine hormone on cardiac Purkinje fibers, we examined the different parts of the conduction system in the bovine heart by use of in vitro receptor autoradiography. In no parts of the bovine conduction system were specific binding sites for [125I] atrial natriuretic peptide observed, whereas the ventricular myocardium exhibited a large number of [125I] atrial natriuretic peptide binding sites. This is the first morphologic study showing the presence of [125I] atrial natriuretic peptide binding sites in the ventricular myocardium and their absence in the conduction system. The present observations together with results obtained in studies using other methods strongly suggest that natriuretic peptide receptors are localized on ventricular myocytes.

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.

The participation of nitric oxide and vasoactive intestinal peptideThis work was supported by grant 94/0388 from the “Fondo de Investigaciones (VIP) in the neurogenic regulation of bovine cerebral arteries was investigated. Nitrergic nerve fibers and ganglion-like groups of neurons were revealed by NADPH-diaphorase staining in the adventitial layerrecipient of an ENP short-term fellowship from the European Science of bovine cerebral arteries. NADPH diaphorase also was present in endothelial cells but not in the smooth muscle layer. Double immunolabeling for neuronal nitric oxide synthase and VIP indicated that both molecules co-localized in the same nerve fibers in these vessels. Transmural nerve stimulation (200 mA, 0.2 milliseconds, 1 to 8 Hz) of endothelium-denuded bovine cerebral artery rings precontracted with prostaglandin F2α, produced tetrodotoxin-sensitive relaxations that were completely suppressed by NG-nitro-L-arginine methyl (L-NAME) and by the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline (ODQ), but were not affected by the adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22,536), nor by VIP tachyphylaxis induced by pretreatment with 1 µmol/L VIP. Transmural nerve stimulation also elicited increases in intracellular cyclic GMP concentration, which were prevented by L-NAME, and small decreases in intracellular cyclic AMP concentration. Addition of VIP to bovine cerebral artery rings without endothelium produced a concentration-dependent relaxation that was partially inhibited by L-NAME, ODQ, and SQ 22,536. The effects of L-NAME and SQ 22,536 were additive. VIP induced a transient increase in intracellular cyclic GMP concentration, which was maximal 1 minute after VIP addition, when the highest relaxation rate was observed, and which was blocked by L-NAME. It is concluded that nitric oxide produced by perivascular neurons and nerve fibers fully accounts for the experimental neurogenic relaxation of bovine cerebral arteries and that VIP, which also is present in the same perivascular fibers, acts as a neuromodulator by activating neuronal nitric oxide synthase.

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.

The distribution of calcitonin gene-related peptide (CGRP)-immunoreactive nerves and CGRP binding sites, as well as the effect of CGRP on the muscle tension, was studied in the hamster seminal vesicle and coagulating gland. The use of an immunofluorescence staining technique on cryostat sections revealed that in the hamster seminal vesicle and coagulating gland, CGRP-positive nerve fibers are found in the connective interstitium and in the muscular and mucosal layers. Using an in vitro receptor autoradiographic technique, CGRP binding sites were found associated with the muscular coat. CGRP (10 pM to 1 microM) relaxed the seminal vesicle and the coagulating gland precontracted by either noradrenaline (10-30 microM) or the alpha 1-agonist, phenylephrine (10 microM). In preparations contracted by carbachol (10 microM), CGRP relaxed the seminal vesicle but not the coagulating gland. In both preparations, CGRP (1 microM) did not affect the muscle resting tension. These results suggest that CGRP may act as an inhibitory modulator of the autonomic control of contractility in the male accessory sex glands of the hamster.

The midgut of Dysdercus peruvianus is divided into three main sections (V1–V3), and is linked through V4 to the hindgut. V1 cells have microvilli ensheathed by a lipoprotein membrane (perimicrovillar membrane) extending toward the lumen as narrow tubes, which may be dilated in some places forming spheres, and have dead ends. The cytoplasmic side of the microvillar membrane is studded with small particles (portasomes), putatively involved in creating ionic gradients used in the cotransport of organic compounds. The basal membrane invaginates forming narrow and ramified channels with associated mitochondria. V2, V3 and V4 cells are similar to V1 cells, except for less developed basal infoldings. V4 cells lack particles associated to the cytoplasmic side of the microvillar membranes and morphological features associated with protein secretion. V3 and V4 cells show bacteria associated to the microvilli. These morphological features agree with physiological data showing that V1 absorbs water, V2 and V3 absorb amino acids and no digestion occurs in V4. α-Glucosidase, a perimicrovillar membrane marker, was immunolocalized in V1 cells. The data suggest that perimicrovillar membranes are formed in Golgi areas, migrate as the internal membrane of double membrane vesicles, which finally fuse at the cell apex—the outer vesicle membrane with the microvillar membrane and the inner vesicle membrane with the perimicrovillar membrane.

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.

Primary cultures of epithelial cells from the hamster seminal vesicle were established in a chemically defined medium supplemented with hormones and growth factors. Epithelial cell clusters were prepared combining enzymatic dissociation and mechanical disaggregation and then seeded in bicameral systems equipped with collagen-membrane inserts. A growth curve was generated and the cells were characterized morphologically and morphometrically by light and electron microscopy. The immunocytochemical detection of cytokeratins and the measurement of transepithelial electrical resistance were also performed. The secretory activity was studied by fluorography using L-[35S]methionine as a precursor, and endocytosis was approached using horseradish peroxidase as a tracer. Our results show that epithelial cells of the seminal vesicle can be grown as a monolayer of morphological and functionally polarized cells which retain secretory and endocytic activities. These cell cultures might therefore prove useful to investigate further the regulation of secretion and endocytosis in the seminal vesicle and are a promising model to approach, in a broader scope, cell polarity and protein sorting and targeting.

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 α calcitonin gene related peptide (αCGRP), 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, αCGRP, substance P, or VIP. All segments precontracted with U46619 responded with potent relaxation to αCGRP, 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.

Objective: The endocardium contains an extensive neural plexus, the composition and function of which are unclear. The aim of this study was to characterise the innervation of the endocardium in terms of the relative density and distribution of its autonomic and sensory nerve subpopulations and to assess the relationship between these nerves and endocardial endothelial cells. Methods : Immunohistochemical, histochemical, confocal, and quantitative image processing techniques were applied to whole mount preparations of human postmortem endocardium obtained within 24 h of death. Results : The overall distribution of nerve fibres and fascicles was demonstrated using antisera to the general neural marker protein gene product 9.5 (PGP 9.5). Nerves displaying acetylcholinesterase activity represented the main nerve subpopulation, occupying 9- 18% of the quantified field area. Neuropeptide Y immunoreactive nerves formed the most numerous peptide containing nerve subpopulation identified, occupying 5-19% and 2-7% of the field area in the ventricle and atrial endocardium respectively and having similar distribution patterns to tyrosine hydroxylase immunoreactive nerves. Nerves showing immuno- reactivity for somatostatin. vasoactive intestinal polypeptide, and substance P were detected at a lower density, occurred more frequently in the venlricular than atrial endocardium, and showed a similar distribution in the right and left sides of the heart. Combined peptide immunofluorescence and acetylcholinesterase staining, of the same preparation, indicated that putative sympathetic and sensory nerve subpopulations could be distinguished from presumed parasympathetic, acetylcholinesterase positive, nerves. The relationship between immunostained nerves and endothelial cells was assessed using confocal microscopy. Varicose nerve fibres were detected within 0.2 μm of overlying endothelial cells in the right ventricle and between 0.4-0.6 μm in the left ventricle. Conclusions: The heterogeneous population of nerve fibres demonstrated in the human endocardium may influence the known interaction between endocardial endothelial cells and the myocardium. Cardiovascular Research 1994; 28 :1490-1499

* Corresponding author. Tel.: +46 46 171485; Fax: +46 46 110908. ropeptides in particular. There is now ample evidence that peptides such as neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), substance P (SP) and calcitonin gene-related peptide (CGRP) are present in nerve fibers supplying the cerebral vasculature of laboratory animals [2]. Only few reports exist on the occurence of peptide-containing nerve fibers around human cerebral vessels [3-5]. This is due to difficulties in obtaining good preparations. In the present article, we will give an overview of the distribution of cerebrovascular nerve fibers in man.

The peptidergic innervation of guinea-pig coronary arteries was investigated by means of immunohistochemical, ultrastructural and in vitro pharmacological techniques. A network of nerves was demonstrated in all major epicardial arteries by means of an antiserum to the neuronal marker protein gene product 9.5. The majority of nerve fibres possessed neuropeptide Y (NPY) and tyrosine hydroxylase (TH) immunoreactivity, the number and distribution of nerves immunoreactive for NPY being similar to that of nerves containing TH immunoreactivity. Numerous nerve fibres displaying immunoreactivity for substance P, neuropeptide K and calcitonin gene-related peptide (CGRP) were also found. In double-stained preparations substance P immunoreactivity was co-localized with CGRP and with neuropeptide K immunoreactivities in the same varicose nerve fibres. Ultrastructural studies revealed the presence of numerous axon varicosities at the adventitial-medial border. NPY immunoreactivity was localized in large granular vesicles in nerve varicosities which also contained numerous small granular vesicles. Large granular vesicle-containing nerves also displayed immunoreactivity for dopamine-β-hydroxylase. With an in vitro method, the vasomotor responses to perivascular peptides were characterized in epicardial and intramyocardial arteries. In epicardial arteries neither noradrenaline nor NPY elicited a contractile response. Only in some intramyocardial arteries was an NPY-mediated contraction demonstrated. No potentiating effect of noradrenaline and NPY was observed in either epicardial or intramyocardial arterial segments. In contrast, CGRP, substance P and vasoactive intestinal peptide (VIP) all produced a concentration-dependent relaxation of both epicardial and intramyocardial arteries. These results suggest that peptide-containing nerves associated with guinea-pig coronary arteries may predominantly be involved in mediating vasodilation.

Myocardial neural cell adhesion molecule (N-CAM) is temporally regulated, being expressed during cardiac morphogenesis and innervation and suppressed in the adult heart. We have investigated the plasticity of N-CAM expression in hypertrophic muscle using the rat model of chronic hypoxia to selectively induce right ventricular hypertrophy over a 14 day time course. Sarcolemmal and intercalated disc N-CAM immunostaining was more extensive in the ventricular myocardium of hypoxic rats compared to normoxic controls. Quantitative assessment of the immunoreactivity in tissue extracts demonstrated a selective increase in the amount of N-CAM immunoreactivity in the hypertrophic myocardium of the right ventricle of rats exposed to hypoxia and this was associated with an increase of the 125 kDa isoform. We conclude that myocardial hypertrophy may be a factor influencing N-CAM expression in the heart and adhesion molecules may have a role in cardiac remodelling.

The distribution of peptidergic nerves in canine mammary tissues was studied by immunohistochemical techniques. In addition, the general and the noradrenergic innervations were demonstrated using protein gene product 9.5 and tyrosine hydroxylase immunoreactivities as markers, respectively. Tissue specimens from the caudal mammary glands were obtained from adult, non-lactating, female dogs. The overall innervation of the mammary gland tissue was sparse and primarily associated with the arterial vasculature. Nerve fibres positive for protein gene product 9.5 were rarely found in the secretory parenchyma. The nipple was not richly innervated, although it displayed a greater amount of nerve fibres than the mammary parenchyma. Nerve fibres supplying nonvascular structures of the nipple expressed immunoreactivity for the sensory neuropeptides calcitonin gene-related peptide, substance P and neuropeptide K, but not for vasoactive intestinal peptide, peptide histidine isoleucine and C-flanking peptide of neuropeptide Y. Somatostatin immunoreactivity was not detected in mammary gland tissue. Our results indicate that the innervation of the canine mammary gland is mainly affiliated with the vasculature and comprises peptidergic nerves which may be involved in the regulation of local blood flow. The presence of sensory neuropeptides in nerves supplying the mammary nipple suggest that these peptides may play a role in the afferent pathway of the milk ejection reflex.

Endothelin-1 (ET-1), a 21 amino acid peptide exerts a wide range of biological activities including vasoconstriction, mitogenesis and inotropic effects on the heart. In this study, we examined whether endocardial endothelial cells express ET-1 and evaluated its functional properties. Using immunofluorescence localization method, we demonstrated cytoplasmic staining of ET-1 in the human endocardial endothelial cells from the right atrium and left ventricle. Employing reverse transcriptase polymerase chain reaction (RT-PCR) expression of ET-1 mRNA and its receptors ET(A) and ET(B) mRNAs were found in human myocardial as well as in endocardial endothelial cells. Biological activity of endocardial endothelial cells derived ET-1 was established as the conditioned media obtained from cultured porcine endocardial endothelial cells induced a slowly developing, strong and long-lasting contraction of circular rat aortic segments, with similar characteristics to that obtained with exogenous ET-1. Furthermore, the selective endothelin-A receptor antagonist, FR 139317, blocked the conditioned media induced contractions. Our results suggest that endocardial endothelial cells express and release biologically active ET-1 which could play a pivotal role in the regulation of myocardial contractility as well as a circulatory peptide may further act in other peripheral target organs.

Excessive hyperbilirubinemia can cause irreversible neurological damage in the neonatal period. However, the complete understanding of the pathogenesis of unconjugated bilirubin (UCB) encephalopathy remains a matter of debate. This study investigates whether UCB inhibits the endocytosis of cationized ferritin (CF) by cultured rat astrocytes. The relationship between endocytosis and MTT reduction, as well as changes on tubulin and glial fibrillary acidic protein (GFAP) assembly, were also evaluated. Inhibition of endocytosis was complete in the presence of 171 microM UCB, while a marked decrease of CF labeling was noticed for 86 microM UCB. In addition, MTT reduction was inhibited by 60 to 76% as UCB concentrations changed from 17 to 171 microM, while alterations on both GFAP and microtubule morphology were only achieved by cell exposure to 171 microM UCB. These findings indicate that inhibition of CF endocytosis in rat cortical astrocytes by UCB is a concentration-dependent process that appears to be primarily related to a direct effect on the cell membrane and not to any alteration of cytoskeletal microtubules and intermediate filaments.

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.

The distribution of vasoactive intestinal peptide (VIP)- and calcitonin gene-related peptide (CGRP)-immunoreactive nerves and 125I-labeled VIP- and CGRP-binding sites was studied in the hamster seminal vesicle of 12-, 30- and 60-day-old animals. In addition, the general innervation of the seminal vesicle was examined using the general neuronal marker synaptophysin. Our results show that the densities of the overall (synaptophysin immunoreactive) and CGRP-immunoreactive innervation is constant during the post-natal development of the gland. However, a significant decrease in VIP-containing nerves is observed at the end of puberty. The autoradiographic study revealed that in 12-day-old animals, the epithelium presents VIP binding sites. However, in 30-day-old animals, VIP binding sites are observed in the epithelium of only a few clumps of acini. In 60-day-old animals, the gland is composed of acini with dilated lumina where VIP binding sites are not detected. In all groups studied the epithelium does not exhibit CGRP binding sites. The seminal vesicle muscle layer displays specific binding sites for both VIP and CGRP at all post-natal developmental times, but the density of VIP binding sites is higher in 12- than in 30- and 60-day-old animals. Our results, showing the presence of specific VIP and CGRP binding sites during the development of the hamster seminal vesicle, suggest that these neuropeptides may be involved in the growth and differentiation of the gland.

Rats were given radiotherapy (total dose 30 Gy) over the abdomen. Seven days later specimens of the duodenum were prepared for in vitro receptor autoradiography using the radioligand [125I]VIP. The autoradiograms were quantitatively analyzed using a computer system. Histological examination revealed that a very marked reorganization of the mucosa had occurred in response to irradiation. Using receptor autoradiography, we found [125I]VIP-specific binding sites in the reorganizing mucosa, except where denudation had occurred. Such binding sites also occurred in the smooth muscle layer of the duodenal wall. The observations suggest that VIP has profound effects in radiation-induced enteropathy.

The molecular basis of bilirubin toxicity to nerve cell function is still unclear. Since astrocytes are the main transporters of synaptically released glutamate and impaired glutamate uptake results in neuronal death, we investigated the effect of unconjugated bilirubin (UCB) on [3Hlglutamate uptake in cultured rat astrocytes and the role of bilirubin ionization on toxicity. Astrocytes were incubated for 5-15 min with UCB concentrations from 17 to 342 pM and UCB/albumin molar ratios of 0.2-3.0, at pH 7.0, 7.4, and 8.0. Exposure of astrocytes for 15 min to 85.5 1M UCB and 28.5 pM albumin resulted in a 63.1% decrease of glutamate uptake (p < 0.01). Interestingly, the effect demonstrated to be correlated with the UCB/albumin molar ratio (r = -0.986, p < 0.01) and a significant decrease was observed for a UCB/albumin molar ratio as low as 0.8. Inhibition of glutamate transport was also pH-dependent as it occurred at 7.4 (p < 0.05) and 8.0 (p, The authors thank Rosa Santos and Ana Homem for their technical assistance and Lorelia Pascolo and Felicia Cupeili for their comments and help during the experimental approach. We also thank Professor FranÇois Trivin for bis expert advice on astrocyte culture. This work was supported by grants from FundacQ para a Ciência e Tecnologia (PRAXIS/PSAU/C/SAU/127/96), Fondo Studi Fegato, and the Italian Ministry for Research (MURST, Rome).

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).

The importance of neuronal factors in the normal physiology of the seminal vesicles has been traditionally underestimated when compared to the trophic role of androgens. Immunohistochemical, autoradiographical and pharmacological experiments have, however, raised the possibility that neuropeptides, such as vasoactive intestinal polypeptide (VIP), neuropeptide tyrosine (NPY) and calcitonin gene-related peptide (CGRP), are necessary for full seminal vesicle function and development. These neuropeptides may be involved in the regulation of secretion, smooth muscle tone and blood flow. Furthermore, neuropeptides may have functional interactions with androgens affecting, probably, androgen receptor-dependent gene expression in these glands. It is now timely to focus attention on the biological relevance of neuropeptides in the seminal vesicles.

Mechanical and biological heart valve substitutes have evolved in parallel, since, in 1951, Hufnagel implanted the first mechanical prosthesis in the descending aorta and attempted, although without success, to implant valve allografts in the same position (reviewed in 35). Five years later, Murray, following the experimental work by Lam and colleages (20), succeeded where Hufnagel had failed, when by placing an allograft in the descending aorta succeeded in reducing an aortic valve insufficiency (29).

In the present work we have investigated the effects of medium- (15 days) and long-term (2 months) castration on vasoactive intestinal peptide (VIP)-immunoreactive nerve fibres and 125I-labelled VIP binding sites in the adult hamster seminal vesicle. The density of VIP- and synaptophysin (general neuronal marker)-containing nerve fibres was determined in immunofluorescently stained cryostat sections using a computerised image analysis system. The morphological analysis of 125I-VIP binding sites in seminal vesicle cryostat sections was performed by quantitative receptor autoradiography. Our results show that the densities of the overall (synaptophysin immunoreactive) and VIPergic innervation increase in both medium and long-term castrated animals. In absolute terms, the quantity of VIP- and synaptophysin- containing nerves is not altered in medium-term castrates, but decreases for synaptophysin in long-term castrates. Medium-term castration does not affect the density of 125I-VIP binding sites in the gland muscular coat, but a significant decrease is observed after long-term castration. In conclusion, our results indicate that whereas VIP nerves are apparently unaffected by castration, 125I-VIP binding sites in the muscular coat of hamster seminal vesicle are sensitive to androgen levels.

The aim was to investigate the innervation and vasomotor responses to classical and putative transmitters of the coronary venous bed.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.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.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 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.

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.

Four major mechanisms are considered the prime regulators of the cerebral circulation: metabolic, myogenic, chemical and neurogenic. In addition, recent studies have also suggested that the endothelium may have an important role in the regulation of cerebral blood flow.’ For the past two decades, the neurogenic mecha-nisms that evoke vasomotor effects have been our main field of research. However, more recently, the putative influence of the endothelium has also attracted our attention.

The peptidergic innervation of proximal (internal diameter, > 0.8 mm) and distal (internal diameter, < 0.8 mm) regions of human epicardial coronary arteries was investigated by means of immunohistochemical, chromatographic, radioimmunological, and in vitro pharmacological techniques. The use of an antiserum to the general neuronal marker protein gene product 9.5 revealed that the proximal part of epicardial arteries possessed a relatively sparse supply of nerve fibers forming a loose network in the adventitia. The perivascular network increased in density as the vessels were followed distally. In both proximal and distal regions, the majority of nerve fibers possessed neuropeptide Y and tyrosine hydroxylase immunoreactivity. Calcitonin gene-related peptide (CGRP)- and substance P-immunoreactive nerve fibers were very sparse in the proximal region of the arteries and increased in number distally. Only a few scattered vasoactive intestinal peptide (VIP)-immunoreactive nerve fibers were detected in both arterial regions. The use of high-performance liquid chromatography and radioimmunoassay revealed that the immunoreactive material present in coronary artery extracts closely resembled synthetic peptides. An in vitro pharmacological method demonstrated that neuropeptide Y elicited no detectable response in either proximal or distal arterial segments. In contrast, CGRP, substance P, and VIP all produced a concentration-dependent relaxation of both arterial regions. CGRP and substance P were stronger and more potent than VIP. CGRP and substance P induced a more potent response in distal compared with proximal regions of the arteries. These results suggest that the peptidergic nerves supplying human large epicardial coronary arteries may be predominantly involved in mediating vasodilation.

Recently, we have demonstrated that guinea-pig epicardial coronary arteries are supplied by numerous nerve fibres containing neuropeptide Y (NPY) immunoreactivity. However, examination of vasomotor responses revealed that NPY did not elicit a contractile response in these arteries. In contrast, acetylcholine (ACh), calcitonin gene-related peptide (CGRP), substance P and vasoactive intestinal polypeptide (VIP) all relaxed precontracted arteries. In the present study, we have used histochemical, immunohistochemical and in vitro pharmacological techniques, in order to further investigate the possible role of NPY in guinea-pig epicardial coronary arteries. A double-immunofluorescence staining technique revealed that CGRP and substance P were co-localized in nerve fibres distinct from those displaying NPY immunoreactivity. Furthermore, using a method combining immunofluorescence and histochemical techniques, we observed that putative cholinergic nerve fibres (identified by their acetylcholinesterase content) and NPY-immunoreactive nerve fibres are two different nerve populations. An in vitro pharmacological method demonstrated that NPY markedly inhibited the relaxant responses mediated by ACh, VIP, substance P and isoprenaline but had no effect on CGRP. These results suggest that NPY-containing nerves associated with guinea-pig epicardial coronary arteries may be predominantly involved in modulating the action of vasodilator agents.

A simple method combining indirect immunofluorescence and histochemical techniques was developed in order to demonstrate the presence of both neuropeptide immunoreactivity and acetylcholinesterase activity in the same whole-mount preparation. It was found that the two methods can be combined without interfering with one another and may be viewed and photographed simultaneously. The guinea pig basilar artery was chosen as a model tissue. While vasoactive intestinal polypeptide immunoreactivity and acetylcholinesterase activity were found to occur in the same perivascular nerve fibres, tyrosine hydroxilase, neuropeptide tyrosine and calcitonin gene-related peptide immunoreactivity were present in distinct nerve subpopulations. It is possible using this double staining procedure, to analyse the interrelationship of putative cholinergic nerves with other components of the autonomic and sensory nervous system.

The peptidergic innervation of the human superficial temporal artery was investigated by means of immunohistochemical, ultrastructural, and in vitro pharmacological techniques. A dense network of nerve fibers was found in the adventitia. The majority of the nerve fibers displayed immunoreactivity for tyrosine hydroxylase and neuropeptide Y (NPY). A moderate supply of perivascular nerve fibers displayed either acetylcholinesterase activity or immunoreactivity for vasoactive intestinal peptide (VIP), peptide histidine methionine-27 (PHM), and calcitonin gene-related peptide (CGRP). Only a few nerve fibers displayed substance P (SP), neurokinin A (NKA), and neuropeptide K (NPK) immunoreactivity. In double immunostained preparations, SP immunoreactivity was co-localized with NPK and CGRP in the same nerve fibers. Ultrastructural studies revealed the presence of numerous axon variocosities at the adventitial—medial border. NPY, VIP, and CGRP immunoreactivities occurred in the same type of large granular vesicles, but in morphological distinct nerve profiles. NPY had, in general, no direct vasoconstrictor effect. However, at a low concentration of NPY contractile response induced by NA (10 −7 –10 −6 M ) was 9–15 times enhanced. The NPY-induced potentiation of the NA-induced contraction was not dependent on the presence of an intact endothelium. No significant difference was found between acetylcholine, VIP, and PHM in either potency or degree of relaxation. SP, NKA, and CGRP also acted as vasodilatory agents, with CGRP being more potent than the tachykinins. The response to SP, but not CGRP, was dependent on an intact endothelium. Pretreatment of the vessels with a low concentration of NPY did not change the responses to ACh, VIP, SP, or CGRP.

Neuropeptide tyrosine (NPY) is one of the most abundant and widespread peptides in the mammalian nervous system. Recent isolation and sequencing of the DNA encoding NPY has predicted the existence of a 97 amino acid precursor peptide. Proteolytic processing of this precursor could yield three separate peptide products, an N-terminal signal peptide, neuropeptide tyrosine and a 30 amino acid C-terminal flanking peptide (C-PON). Here, we present evidence that the predicted C-flanking peptide of NPY is widely distributed in both the central and peripheral nervous systems of several mammalian species including man, and has an identical distribution to NPY. It was also demonstrated, using correlative light microscopic immunostaining on serial sections and double electron microscopic immunocytochemistry, that C-PON and NPY immunoreactivities are co-localized in neuronal cell bodies of the brain cortex, sympathetic ganglion cells, norepinephrine-containing granules of the adrenal medulla and in human pheochromocytoma tumor cells.

The presence and distribution of neural cell adhesion molecule (N-CAM) was examined by light and electron microscopical immunocytochemistry in the descending thoracic aorta, the superior mesenteric artery and mesenteric arteries from fetal and adult rats (embryonic day 15 to post-natal day 90). In embryonic and early post-natal rats, N-CAM immunoreactivity was localized in perivascular nerves, in the smooth muscle cell plasma membrane and basal lamina. In nerves, N-CAM-immunoreactive sites were found associated with both the axon and Schwann cell membranes. N-CAM immunoreactivity was also found associated with the surface of adventitial fibroblast-like cells and with collagen fibrils, in regions where these fibrils were in contact with smooth muscle cells. In mature vessels N-CAM immunoreactivity was found to be restricted to the perivascular innervation and the surface of fibroblast-like cells. These observations indicate that N-CAM is expressed transiently in rat vascular tissues during development and is localized not only on the surface of smooth muscle cells but also in association with extracellular matrix components.

Peptide-containing nerves have been examined in the rat femoral artery and vein using immunocytochemical and vasomotor techniques. The general neuronal marker PGP 9.5 revealed a moderate supply of nerve fibres and fascicles forming a loose network in the adventitia and the adventitial-medial border of the artery and vein. The majority of the nerve fibres in both the artery and vein displayed immunoreactivity for neuropeptide Y (NPY) and tyrosine hydroxylase (TH). The distribution pattern and number of these two types correlated well. The artery had a slightly richer PGP 9.5- immunoreactive nerve supply compared to the vein, but the nerve plexus in the vein displayed a more uniform arrangement. In contrast, relatively few nerve fibres displayed calcitonin gene-related peptide, substance P, or vasoactive intestinal peptide immunoreactivity in either the artery or vein. The calcitonin gene-related peptide immunoreactive fibres had a similar distribution to that of the substance P containing fibres. Using a sensitive in vitro method the vasomotor responses to perivascular peptides were characterized. In the femoral artery NPY potentiated α1-adrenoceptor mediated contractions, and had very little effect by itself. In contrast, 10–7M NPY contracted femoral veins by up to 68% relative to 60 mM potassium induced conctraction, and there was no potentiation of α-adrenoceptor mediated contractions. Acetylcholine, peptide histidine isoleucine, vasoactive intestinal peptide, substance P and calcitonin gene-related peptide, all relaxed the contracted femoral artery and vein. Regarding the putative parasympathetic neurotransmitters, acetylcholine caused stronger relaxation of veins as compared to arteries whereas for vasoactive intestinal peptide and peptide histidine isoleucine the relaxations were stronger in the arterial preparation. These three agonists were more potent in the femoral vein. Substance P was more potent on the femoral vein, having the same maximum response in both preparations. On the other hand, the response induced by CGRP was some three times greater in the venous than in the arterial preparation. These data reveal that although there appear to be only minor differences in the peptidergic innervation of the rat femoral artery and vein pronounced differences occur in the peptide effector responses. The data support the concept that perivascular peptides play different roles in regulating various parts of the circulation.

Atrial natriuretic peptide is a peptide regulating salt and water balance, originally isolated from the cardiac atrium, where it is synthesised as part of a precursor molecule in specialised myocardial cells. The myocardium extends into the extrapulmonary part of the pulmonary veins in many species, including man. In some small mammals, however, such as the rat, mouse, and bat, it extends further to veins in the peripheral parts of the lung. Since this myocardial layer is continuous with that in the atrium, we have looked for the possible expression of the atrial natriuretic peptide gene in this tissue in rats. Strong immunoreactivity was seen for both the peptide and the N terminal sequence (cardiodilatin) of its precursor in extrapulmonary veins and in intrapulmonary veins extending into the lung as far as the second branching point, where it was localised in the dense cored granules by electron microscopy; in situ hybridisation showed atrial natriuretic peptide messenger RNA at identical sites. Chromatography and radioimmunoassay of extracts of extrapulmonary and intrapulmonary veins showed most of the atrial natriuretic peptide immunoreactivity to be in the uncleaved (precursor molecule) form. Thus the peptide is synthesised in veins both outside and inside the lung, and these extra-atrial sites may be an important additional source of circulating atrial natriuretic peptide.

We have demonstrated that calcitonin gene-related peptide (CGRP) immunoreactivity is widely distributed in cardiac and perivascular nerves of the guinea pig and rat. In the guinea pig the number and distribution of CGRP-immunoreactive nerve fibres closely paralleled that of fibres containing substance P, the two immunoreactivities being found invariably to coexist in the same perivascular networks and terminals. In the rat, CGRP-immunoreactive cardiovascular nerves had a similar distribution to those containing substance P, but in contrast to the guinea pig the former were far more numerous. Marked regional variations were observed in the density of the CGRP-immunoreactive innervation in both species. The CGRP-immunoreactive content of tissue extracts was in close agreement with the immunocytochemical findings, the highest levels of CGRP occurring in the mesenteric artery (guinea pig and rat) and inferior vena cava (guinea pig). Following capsaicin treatment of adult guinea pigs and neonatal rats, there was a significant loss of CGRP-immunoreactive nerves in the two species. In the guinea pig, substance P-and CGRP-immunostained fibres were depleted to a similar extent, throughout the cardiovascular system. However, the loss of rat CGRP-immunoreactive nerves was dose-dependent and displayed considerable variation, some perivascular nerve networks appearing less susceptible than others to the action of capsaicin. The results suggest that there may be species differences in the sensitivity of CGRP-containing nerves to capsaicin treatment, but at least the majority of CGRP-immunoreactive cardiovascular nerves may be presumed to be sensory in origin.

The various subpopulations of autonomic and sensory nerves supplying the mammalian cardiovascular system may be demonstrated using specific immunocytochemical and histochemical techniques, but no single marker has previously been available for the visualisation of the entire innervation. Protein gene product (PGP) 9.5 was first identified in extracts of human brain and found to represent a major protein component of the neuronal cytoplasm. We have demonstrated that PGP 9.5 immunoreactivity occurs in the guinea pig cardiovascular innervation and is present in more individual nerve fibres than other general neuronal markers (neuron-specific enolase and neurofilaments). PGP 9.5 immunoreactivity was localized to both intrinsic neurones and nerve fibres in the guinea pig heart. In the vascular system PGP 9.5-immunoreactivity occurred in an extensive plexus of fine perivascular nerve fibres and fascicles running around and along both arteries and veins, mainly at the adventitial-medial border. At the ultrastructural level, this immunoreactive material was localized to the axonal cytoplasm and did not appear to be associated with cytoskeletal elements or secretory vesicles. 6-Hydroxydopamine (6-OHDA) pretreatment resulted in the degeneration of noradrenergic axon terminals containing PGP 9.5, tyrosine hydroxylase (TH) and neuropeptide tyrosine (NPY) immunoreactivities. Most of the perivascular nerve fibres which remained displayed substance P- and calcitonin gene-related peptide (CGRP) immunoreactivity, as well as PGP 9.5 immunoreactivity. Capsaicin pretreatment resulted in a depletion of both substance P and CGRP immunoreactivity, but had no apparent effect on PGP 9.5 immunostaining. In the heart PGP 9.5 immunoreactivity also appeared to be present in presumed postganglionic cholinergic nerves. PGP 9.5 may be a useful marker when examining regional variations in cardiovascular innervation and for determining the relative proportions of nerve subpopulations.

Using double immunogold staining procedures, calcitonin gene-related peptide (CGRP)-like and substance P (SP)-like immunoreactivities were localized at the ultrastructural level to guinea pig trigeminal ganglia, dorsal root ganglia and peripheral nerve fibres associated with the vascular system. CGRP-like and SP-like immunoreactivities were found consistently in large granular secretory vesicles (70-100 nm in diameter), and both peptide immunoreactivities were co-localized to the same vesicle in both sensory ganglion cells and within axons and their terminals in the adventitia and adventitial-medial border of the superior mesenteric artery. These results suggest that CGRP and SP are co-stored and may be released together from peripheral axons in the guinea pig.

The innervation and myocardial cells of the human atrial appendage were investigated by means of immunocytochemical and ultrastructural techniques using both tissue sections and whole mount preparations. A dense innervation of the myocardium, blood vessels and endocardium was revealed with antisera to general neuronal (protein gene product 9.5 and synaptophysin) and Schwann cell markers (S-100). The majority of nerve fibres possessed neuropeptide Y immunoreactivity and were found associated with myocardial cells, around small arteries and arterioles at the adventitial-medial border and forming a plexus in the endocardium. Subpopulations of nerve fibres displayed immunoreactivity for vasoactive intestinal polypeptide, somatostatin, substance P and calcitonin gene-related peptide. In whole-mount preparations of endocardium, substance P and calcitonin gene-related peptide immunoreactivities were found to coexist in the same varicose nerve terminals. Ultrastructural studies revealed the presence of numerous varicose terminals associated with myocardial, vascular smooth muscle and endothelial cells. Neuropeptide Y immunoreactivity was localised to large electron-dense secretory vesicles in nerve terminals which also contained numerous small vesicles. Atrial natriuretic peptide immunoreactivity occurred exclusively in myocardial cells where it was localised to large secretory vesicles. The human atrial appendage comprises a neuroendocrine complex of peptide-containing nerves and myocardial cells producing ANP.