Innervation of the canine mammary gland: an immunohistochemical study.

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.

Endocardial expression and functional characterization of endothelin-1.

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.

Endocytosis in rat cultured astrocytes is inhibited by unconjugated bilirubin.

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.

Neuronal messengers in the human cerebral circulation.

In recent years our knowledge of the nervous control of the cerebral circulation has increased. The use of denervations and retrograde tracing in combination with immunohistochemical techniques has demonstrated that cerebral vessels are supplied with sympathetic, parasympathetic, and sensory nerve fibers and possibly central pathways containing a multiplicity of new transmitter substances in addition to the classical transmitters. The majority of these transmitters are neuropeptides. More recently it has been suggested that a gaseous transmitter, nitric oxide (NO) also could participate in the neuronal regulation of cerebral blood flow. Although little is known about the physiological actions and inter-relationships among all these putative neurotransmitters, their presence within cerebrovascular nerve fibers will make it necessary to revise our view on the mechanisms of cerebrovascular neurotransmission.

Vasoactive intestinal peptide stimulates apical secretion in hamster seminal vesicle epithelial cells in culture.

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.

Inhibition of glutamate uptake by unconjugated bilirubin in cultured cortical rat astrocytes: role of concentration and pH.

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 [(3)H]glutamate 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 microM 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 microM UCB and 28.5 microM 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 < 0.01), but not at 7.0, suggesting that the monoanionic species of UCB accounted for the inhibition. These findings indicate that UCB, and more precisely the monoanionic species, impairs a crucial function of astrocytes such as glutamate transport and support a potential role of astrocyte function in the pathogenesis of UCB-related brain damage (kernicterus).

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.

Distribution of vasoactive intestinal peptide and calcitonin gene-related peptide immunoreactive nerve fibers and binding sites in the hamster seminal vesicle during post-natal development.

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.

Cystic fibrosis patients with the 3272-26A-->G mutation have mild disease, leaky alternative mRNA splicing, and CFTR protein at the cell membrane.

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.

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).

Binding sites for VIP in the reorganizing mucosa of the irradiated bowel.

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.

Neuronal nitric oxide synthase activation by vasoactive intestinal peptide in bovine cerebral arteries.

The participation of nitric oxide and vasoactive intestinal peptide (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 layer 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 F2 alpha, produced tetrodotoxin-sensitive relaxations that were completely suppressed by NG-nitro-L-arginine methyl ester (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 mumol/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.

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.

Mechanisms underlying degeneration of cryopreserved vascular homografts.

OBJECTIVE: To analyze the mechanism(s) underlying homograft degeneration, we designed an experimental model in which the behavior of cryopreserved autografts and homografts, as well as fresh autografts, implanted in the same animal was compared. METHODS: A cryopreserved homograft was implanted in the aorta of 14 sheep. The excised aortic autologous segment was then subjected to cryopreservation, and 1 to 8 weeks later it was implanted 1 to 2 cm below the cryopreserved homograft. The intermediate segment of the native aorta, the fresh autograft, was dissected at this point. Animals were put to death at different times and the implanted segments were harvested together with a portion of native aorta. Histologic and immunohistochemical analyses, as well as cell viability assessments, were then performed on the explanted segments. Similar studies were also conducted on fragments of cryopreserved autografts and homografts before implantation. RESULTS: With the exception of a partial loss of the endothelium, cryopreserved specimens retained cell viability and morphologic integrity before implantation. Explanted cryopreserved homografts showed profound changes affecting all strata, as well as a decline in cell viability. Lymphocyte infiltrates were found up to 12 months after implantation. Endothelium was always absent in cryopreserved homografts. However, a reendothelialization of the cryopreserved autografts was observed. After an initial period of neuronal degeneration, reenervation of the cryopreserved autograft segment occurred 6 to 12 months after the operation. Findings regarding the fresh autografts were similar to those of the cryopreserved autografts. CONCLUSION: Our results suggest that the immunologic reaction rather than the cryopreservation process is responsible for the degenerative process occurring in cryopreserved homografts.

Neuropeptides in the seminal vesicles: locations, binding sites and functional implications.

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.

Occurrence of binding sites for [125I] ANP in the myocardium but not in Purkinje fibers of the bovine heart.

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.

Effect of castration on the VIPergic innervation and 125I-labelled vasoactive intestinal peptide (VIP) binding sites in the hamster seminal vesicle. A quantitative immunohistochemical and receptor autoradiographic study.

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.

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.

Calcitonin gene-related peptide in the hamster seminal vesicle and coagulating gland: an immunohistochemical, autoradiographical, and pharmacological study.

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.