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Biomedical subjects

J Wharton

Publications and source records attributed to J Wharton.

At least 55 records · Page 3Linked to original sources
PubMed1994

Angiotensin II (AT1) vascular binding sites in human placentae from normal-term, preeclamptic and growth retarded pregnancies.

Angiotensin II (ANG II) is a potent vasoconstrictor in isolated human placental cotyledons and may contribute to the regulation of fetoplacental perfusion. We have used quantitative in vitro receptor autoradiography to examine antagonist ((Sar1,Ile8)-[125I]ANG II) and agonist ligand ([125I]ANG II) binding sites in normal-term, preeclamptic and fetal (intrauterine) growth retarded pregnancies. A similar distribution of binding sites was demonstrated using both ligands, localized to blood vessels in placental villi. Binding density was inversely related to vessel size, being significantly greater on microvessels in distal regions of the villous tree than on proximal vessels in main stem villi. Binding sites exhibited the characteristics of the AT1 class of ANG II receptor, ligand binding being sensitive to dithiothreitol, completely inhibited by nonpeptide AT1 antagonists (Losartan, EXP3174 and SKF108566) and not inhibited by the AT2 antagonist (PD123319). Guanine nucleotides also inhibited [125I]ANG II binding and abolished the high affinity component of agonist inhibition of (Ser1,Ile8)-[125I]ANG II binding, indicating G protein coupling. The capacity and affinity of the binding sites were significantly lower in placentae from pregnancies complicated by preeclampsia and intrauterine growth retardation compared to that in normal-term controls. These differences were apparently not due to prior receptor occupancy by endogenous ligand, but may reflect activation of the placental renin-angiotensin system and receptor down-regulation. Locally generated ANG II, acting via AT1 receptors, may contribute to the regulation of fetoplacental blood flow and influence placental perfusion in preeclamptic and growth retarded pregnancies.

Angiotensin II↗
PubMed1993

Capsaicin induced afferent denervation and receptor-linked responses to CGRP in the rat.

We have examined the effect of neonatal capsaicin-induced destruction of primary afferent nerves in rats on the response to calcitonin gene-related peptide (CGRP) in vitro and in vivo. Denervation was confirmed by immunohistochemistry. Rat alpha-CGRP (rCGRP) activated adenylate cyclase in homogenates of rat spleen (basal activity 45 +/- 8.27, delta Vmax 75 +/- 16 pmol cyclic AMP min-1 protein; K(act) 2.04 +/- 0.44 nM). A single specific binding site for [125I]hCGRP was demonstrated in homogenates of spleen (Kd = 4.84 +/- 0.66 nM, Bmax = 1.43 +/- 0.35 pmol mg-1 protein). Neither adenylate cyclase activation nor binding site characteristics were affected by capsaicin-induced denervation. In addition, hypotensive responses to intravenous boluses of rCGRP were examined in anaesthetized rats. Neither the basal blood pressure nor the blood pressure fall in response to rCGRP were altered by neonatal denervation by capsaicin. In conclusion, there is no evidence of denervation hypersensitivity of receptor mediated responses to CGRP in vivo or in vitro, following capsaicin-induced denervation in the rat. This suggests that CGRP is unlikely to exert a sustained tonic influence on cardiovascular regulation in the rat.

Adenylyl Cyclases↗
PubMed1993

Expression of the human neuropeptide tyrosine Y1 receptor.

Neuropeptide tyrosine (NPY) is the predominant peptide in the innervation of many human tissues and is considered to play a role in the regulation of blood flow, gastrointestinal secretion and motility, and renal function. Three NPY receptors have been identified (Y1, Y2, and Y3) and the cDNAs encoding the human Y1 and bovine Y3 receptors have recently been cloned. We have demonstrated the expression of the Y1 receptor subtype in several fetal and adult human tissues, including the colon, kidney, adrenal gland, heart, and placenta. A single transcript was identified (approximately 2.2 kb) and localized in tissue sections by in situ hybridization. In the colon the receptor is expressed in the mucosa and basal glands, as well as the myenteric and submucous plexuses. Y1 receptor mRNA was detected in renal collecting ducts, loop of Henle, and juxtaglomerular apparatus and in the syncytiotrophoblast layer of placental villi. Fetal aorta and adult intramyocardial, colonic, and renal blood vessels also exhibited receptor expression, localized to the intima as well as the media. The distribution of Y1 receptor expression correlates with that of NPY-immunoreactive nerves and the apparent actions of NPY in the intestine, kidney, and heart. Although the placenta is devoid of nerves, an NPY-like transcript was detected in the villous trophoblast layer. The results indicate a tissue-specific regulation of NPY Y1 receptor expression.

Adult↗
PubMed1993

Quantitative immunohistochemical assessment of bovine myocardial innervation before and after cryosurgical cardiac denervation.

OBJECTIVE: The aim was to determine the relative distribution and possible origins of peptide containing nerves in the bovine heart before and after functional extrinsic denervation established by cryosurgery. METHODS: A quantitative immunohistochemical technique was used. RESULTS: In the intact heart, myocardial nerve fibres and fascicles displaying immunoreactivity for the general neural marker protein gene product 9.5 had an atrial to ventricular gradient in density. The right atrium was the most densely innervated region and a major proportion of the total myocardial innervation, visualised by protein gene product 9.5 immunoreactive nerves, showed neuropeptide tyrosine (45%) and tyrosine hydroxylase (20%) immunofluorescence staining, while nerves immunoreactive for vasoactive intestinal polypeptide and calcitonin gene related peptide formed relatively minor subpopulations (representing less than 2% and 0.5%, respectively, of the total fluorescent myocardial innervation). Following cryoablation there was a significant reduction in the percentage fluorescent area of protein gene product 9.5 immunoreactive nerves throughout the heart, of greater than 90% of the control values. There were highly significant reductions in the percentage fluorescent area of nerves showing immunoreactivity to neuropeptide tyrosine, tyrosine hydroxylase, and calcitonin gene related peptide, to 1.01, 0.92, and 0.05%, respectively, of the intact myocardial innervation. The distribution of vasoactive intestinal polypeptide immunoreactive nerves was more variable and displayed an equivocal response to cardiac cryoablation. CONCLUSIONS: The majority of nerves showing immunoreactivity to neuropeptide tyrosine, tyrosine hydroxylase, and calcitonin gene related peptide are of extrinsic origin, while vasoactive intestinal polypeptide immunoreactive nerves may have intrinsic as well as extrinsic origins. The distribution and apparent origins of immunohistochemically defined nerves in the bovine heart are similar to those observed in the human heart which suggests that the calf may be an appropriate model for comparative studies.

Animals↗
PubMed1993

Localization and characterization of neuropeptide Y binding sites in porcine and human colon.

1. We have used quantitative receptor autoradiography to investigate the localization and characteristics of binding sites for 125Iodine-Bolton Hunter-labelled human neuropeptide Y ([125I]-BH-NPY) in porcine and human colon, and compared the binding characteristics with those found in porcine spleen. 2. Saturable, specific, high affinity [125I]-BH-NPY binding was localized to myenteric ganglia in porcine and human colons, and to submucosal ganglia in porcine colon. 3. Specific [125I]-BH-NPY binding to porcine myenteric ganglia was reversible in the presence of guanosine 5'-O-(3-thiotriphosphate) and was inhibited by related peptides with the rank order of potency; porcine NPY = human NPY = peptide tyrosine tyrosine (PYY) >> pancreatic polypeptide. 4. The Y2 selective analogue, NPY (13-36), competed for [125I]-BH-NPY binding to porcine myenteric ganglia with greater potency than the Y1 selective analogue, [Leu31, Pro34] NPY, the difference being small, but significant. 5. The characteristics of [125I]-BH-NPY binding to porcine myenteric ganglia were similar to those observed concurrently to porcine splenic red pulp. 6. The small difference in inhibitory potencies between NPY(13-36) and [Leu31, Pro34]NPY observed in this study in comparison with previous studies was not explained by differential ligand depletion during incubations, but may be due to differences in methodology between binding studies performed on tissue sections and on membranes. 7. We conclude that specific [25I]-BH-NPY binding sites are present in the myenteric and submucosal ganglia of the colon and that these sites may act as functional receptors by which NPY and PYY modulate colonic motility and electrolyte transport.

Animals↗
PubMed1993

Differential localization of endothelin ETA and ETB binding sites in human placenta.

1. The localization and differential distribution of endothelin (ET) receptor subtypes (ETA and ETB) was investigated in sections of human placenta by use of quantitative in vitro autoradiography and receptor selective ligands. 2. Specific, high density [125I]-ET-1 binding sites were localized to the decidua and foetal membranes as well as to arteries and veins in the chorionic plate and throughout the villous tree. Moderate to low density binding was found in the extravillous and villous trophoblast respectively. 3. [125I]-ET-1 binding sites exhibited a rank order of inhibition by unlabelled peptide sequences (ET-1 > ET-3 > [Ala3,11,18Nle7]-ET-1 > BQ123 > or = sarafotoxin 6c). However, in contrast to the monophasic inhibition curve of ET-1, the other sequences produced a significantly better fit to a two component inhibition curve suggesting the presence of a heterogeneous population of ET binding sites. 4. ETA and ETB receptors were distinguished by competitive inhibition of [125]-ET-1 binding with increasing concentrations of unlabelled ET-3, [Ala3,11,18Nle7]-ET-1, sarafotoxin 6c and BQ123 and by incubating sections with the ETB agonist, [125I]-BQ3020. ET receptor subtypes exhibited a differential distribution in the placenta. ETA type binding sites predominated (approximately 80% of the total) on veins and arteries in the chorionic plate. Veins in stem villi, blood vessels in distal regions of the villous tree and decidual cells displayed a high density (approximately 60-70% of the total) of the ETB receptor subtype. 5. No difference was detected in either the relative density of [125I]-ET-1 binding sites or the proportion of ETA to ETB sites in placentae from pregnancies complicated by pre-eclampsia compared with normal term controls.6. ET may have a local autocrine or paracrine role in the placenta, acting via specific receptors to influence foetoplacental blood flow and other aspects of placental function.

Adult↗
PubMed1993

Intractable chest pain in cardiomyopathy: treatment by a novel technique of cardiac cryodenervation with quantitative immunohistochemical assessment of success.

A novel method of cardiac denervation by cryoablation has been developed experimentally. The technique uses liquid nitrogen delivered under pressure to ablate the principal sources of cardiac innervation--namely, the adventitia surrounding the aorta, pulmonary arteries, and veins. The technique has been verified experimentally both in vivo by physiological means and in vitro by quantitative immunohistochemistry and the measurement of myocardial noradrenaline concentrations. A 35 year old woman presented with intractable precordial pain, normal epicardial coronary arteries, and hypertrophic cardiomyopathy. Her symptoms were refractory to maximal medical treatment and she was thought to be unsuitable for either conventional myocardial revascularisation, autotransplantation, or allografting with the concomitant risk of transplant coronary artery disease. She therefore underwent cardiac denervation by the method developed in the laboratory. There was quantitative immunohistochemical evidence of extrinsic cardiac denervation associated with a considerable improvement in her symptoms. This improvement persisted during a follow up period of over 16 months.

Adult↗
PubMed1993

Peptidergic innervation of human epicardial coronary arteries.

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.

Adult↗
PubMed1993

Microvascular substance P binding to normal and inflamed rat and human synovium.

The regulatory peptide substance P has been implicated in the development and persistence of inflammatory synovitis. The authors used quantitative in vitro receptor autoradiography to compare synovial binding of 125Iodine-Bolton Hunter-labeled substance P ([125I]BH-SP) in rats and humans and between uniflamed and persistently inflamed synovium. [125I]BH-SP binding to microvascular endothelium paralleled the distribution of substance P-immunoreactive nerves and had characteristics of the neurokinin (NK) 1 class of tachykinin receptor. Specific binding was inhibited by the selective NK1 receptor antagonist, FK888, and the dual NK1/NK2 receptor antagonist FK224, with Hill coefficients near unity. FK888 was > 1000 times and FK224 > 10 times more potent at inhibiting binding in human compared with rat synovium. Synovium from patients and rats with chronic arthritis contained heterogeneously distributed inflammatory cell infiltrates. For the 10 microvessels with the densest [125I]BH-SP binding in each section, no significant differences in binding density, affinity, or Ki values for substance P, FK888 or FK224 were found between synovium from naive and monoarthritic rats, nor between that from patients with rheumatoid arthritis or osteoarthritis. However, in both rat and human specimens, microscopic examination suggested that microvascular [125I]BH-SP binding in intensely infiltrated regions of synovium was less dense than in adjacent, less infiltrated areas. It was concluded that NK1 receptors are similarly distributed in rat and human synovium but show major differences in selectivity for antagonists such as FK888. NK1 receptors in synovium may mediate proinflammatory actions of locally released substance P; defective neurovascular regulation may contribute to the persistence of chronic arthritis.

Adult↗
PubMed1993

Development of the peptidergic innervation of human heart.

The aim of the present investigation was to study the developing peptidergic innervation of the human fetal heart of 7-24 wk gestational age. An immunohistochemical approach was adopted and the total innervation visualised with antisera to general neuronal and Schwann cell markers, while the onset and development of specific neuropeptide-containing subpopulations were investigated using antisera to neuropeptide Y (NPY), somatostatin, vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP) and substance P (SP). Cardiac ganglia and nerves were demonstrated from 7 wk of gestation whereas peptide-immunoreactive nerves were not observed until the 10th week of gestation. NPY-immunoreactive nerve fibres constituted the major subpopulation of peptide-containing nerves identified in the fetal heart, exhibiting a descending atrial to ventricular density gradient, and were first identified during the 10th wk of gestation. Somatostatin- and VIP-immunoreactive nerves appeared at 10-12 wk of gestation and were mainly distributed in the atria. Somatostatin immunoreactivity was localised to cell bodies in cardiac ganglia, as well as to nerve fibres, indicating an intrinsic origin for this nerve subpopulation. Conversely, the other peptide-containing nerves appear to be of extrinsic origin, including those immunoreactive for VIP. Intracardiac neurons exhibit a transient expression of tyrosine hydroxylase immunoreactivity. Putative sympathetic nerve fibres, displaying tyrosine hydroxylase and NPY immunoreactivity, were demonstrated before the adrenergic innervation has previously been shown to be present by formaldehyde-induced fluorescence staining of catecholamines. The onset of the CGRP- and SP-immunoreactive innervation, at 18-24 wk of gestation, followed the appearance of other peptide-containing nerves, suggesting that the sensory, afferent innervation occurs later than the autonomic. The differential appearance and distribution of peptide-containing nerve subpopulations indicate that there is a chronological order to the development of the autonomic and sensory components of human cardiac innervation.

Calcitonin Gene-Related Peptide↗
PubMed1993

Neuropeptide degrading enzymes in normal and inflamed human synovium.

Regulatory peptides, including neuropeptides, are metabolized by membrane-bound peptidases. We have localized the membrane peptidases angiotensin-converting enzyme (ACE), dipeptidyl peptidase IV (DPPIV), and aminopeptidase M (APM) in normal and inflamed human synovium by immunohistochemistry and enzyme histochemistry. ACE was localized to endothelial cells of all vessels, whereas endothelial DPPIV and APM were restricted to veins and capillaries of some cases. Perivascular spindle-shaped cells stained positively for APM, but rarely for ACE and DPPIV. Synovial lining cells were universally positive for APM but rarely for ACE or DPPIV, whereas a subintimal layer of spindle-shaped cells was frequently positive for all three enzymes, particularly APM. Staining for each enzyme was also observed on some stromal cells. Inflamed synovium displayed increased cellularity with corresponding increases in membrane peptidase staining. APM-positive, perivascular spindle-shaped cells and DPPIV-positive lymphocytes frequently bore HLA class II antigens. This distribution of membrane peptidases supports the hypothesis of a functional compartmentalization of vascular peptidergic systems, with the activities of vasoactive peptides localized to their sites of release. Furthermore, metabolic pathways for regulatory peptides may vary between different structures within human synovium, and these enzymes may be of different relative importance in normal and inflamed tissues. The implied regional control of regulatory peptide activity by membrane peptidases suggests novel potential approaches to the pharmacological manipulation of inflammation by specific enzyme inhibitors.

Adolescent↗
PubMed1993

Species and tissue specificity of vasoactive regulatory peptides.

Peptide regulatory systems are increasingly being implicated in inflammatory processes through their actions on vascular function. Peptides released by sensory nerves (substance P and calcitonin gene-related peptide) and autonomic nerves (neuropeptide Y), or generated by endothelium (angiotensin II) or injured tissues (bradykinin), regulate vascular tone, permeability and proliferation. Peptide systems comprise multiple components responsible for generation, release and metabolism of peptides, as well as receptor expression, combinations of which act together to produce a particular biological effect. Each of these components may vary between species, between tissues and during disease, and the particular combination of these components contributes to the specificity of peptide regulation of tissue function. Understanding the species and tissue specificity of peptide-induced responses is important in developing animal models of human disease and in developing drugs for subsequent use in man. In this paper we review some of the evidence for and implications of species and tissue specificity of vasoactive peptide systems.

Animals↗
PubMed1993

Reexamination of gender differences in the source location of N1m.

Gender differences in the source location of the auditory evoked field (AEF) component N1m have been reported previously in a small group of subjects. The present study was conducted to evaluate further the existence of gender differences in a larger sample. Neuromagnetic recordings of AEFs were obtained from young normal hearing subjects using a 1000-Hz tone burst presented at 60 dB hearing level (HL). No significant gender-related differences were observed for the N1m peak latencies following left and right ear stimulation. The stimulating parameters and larger sample used in the present study size may account for the difference in gender effects observed between the two studies.

Acoustic Stimulation↗
PubMed1992

Regional distribution and regulation of [125I]calcitonin gene-related peptide binding sites in coronary arteries.

Quantitative in vitro autoradiographic techniques were used to localize and characterize 125I-labelled human calcitonin gene-related peptide ([125I]hCGRP) binding sites in sections of bovine left anterior descending coronary artery (LAD). Specific high affinity (Kd 0.4 nM) [125I]hCGRP binding sites were localized to the media of both epicardial and myocardial coronary arteries. Binding site density was greater in distal epicardial and myocardial arteries than in proximal epicardial regions of the LAD. Binding sites exhibited a significantly higher affinity for alpha-hCGRP (Ki 1.1 nM) than for hCGRP-(8-37) (Ki 7.0 nM) and [Cys(ACM)2,7]hCGRP (Ki 27.4 nM). Guanosine-5'-O-(3-thiotriphosphate) inhibited [125I]hCGRP binding in a concentration-dependent manner. Extrinsic denervation of the bovine heart resulted in a depletion of CGRP-like immunoreactive perivascular nerve fibres and an increase in the density of coronary artery [125I]hCGRP binding sites (P = 0.0092). The regional distribution of binding sites in human coronary arteries differed from that observed in bovine and porcine vessels. It is concluded that selective, G protein-coupled, CGRP receptors are present in the media of bovine coronary arteries; there are both regional and species differences in the distribution of CGRP binding sites in coronary arteries and endogenous CGRP may exert a tonic influence on coronary vasomotor tone.

Animals↗
PubMed1992

Neuropeptide Y modulates the action of vasodilator agents in guinea-pig epicardial coronary arteries.

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.

Acetylcholine↗
PubMed1992

Endocardial localization and characterization of natriuretic peptide binding sites in human fetal and adult heart.

Specific, high affinity binding sites for 125I-human-alpha-atrial natriuretic peptide-(1-28)) (125I-hANP-(1-28)) were identified in human fetal and adult heart and the binding characterized using quantitative in vitro autoradiography. Binding sites were localized to atrial and ventricular endocardium, aorta, pulmonary arteries and epicardial mesothelium. Kinetic studies indicated a Kd value of 32 pM for ventricular endocardial 125I-hANP-(1-28) binding. The binding was completely inhibited by an excess (1 microM) of unlabelled hANP-(1-28), human brain natriuretic peptide-(1-32) (hBNP-(1-32)) and by the 'clearance receptor' specific ring-deleted analogue, C-ANP-(4-23). Competitive inhibition studies indicated a relative inhibitory potency for hBNP-(1-32) and C-ANP-(4-23) of 6% and 3% respectively. The data suggest that a distinct natriuretic peptide receptor subtype is expressed in the endocardium and in addition to a possible clearance function, may represent a site for feedback regulation and peptide interaction.

Analysis of Variance↗