John Wharton

Protein gene product 9.5 (PGP 9.5)

SummaryThe guinea pig uterus is supplied by different populations of nerves which can be demonstrated by specific immunocytochemical and histochemical techniques. So far, there has been no single marker displaying entire peripheral innervation patterns. Recently, protein gene product (PGP) 9.5, a cytoplasmic protein in neurons and neuroendocrine cells, was found to visualize both different populations and subtypes of nerves. This prompted the present study of using PGP 9.5 for visualization of the whole uterine innervation. This was performed by the indirect immunofluorescence method using antiserum to PGP 9.5 raised in rabbits.PGP-immunoreactivity was present in all neuronal parts of the extrinsic and intrinsic uterine innervation, including different subpopulations of nerves. This was verified by chemical sympathectomy and sensory denervation with 6-hydroxydopamine and capsaicin-treatment respectively, and double immunostaining.By term a disappearance of uterine PGP-nerve-immunoreactivity was observed which was almost complete in fetus-bearing uterine tissue and further strengthens previous assumptions of a general, pregnancy-induced uterine neuronal degeneration.The developmental time-course and morphology of PGP-immunoreactive nerve structures was similar to that for other neuronal markers and support the suggestion of PGP-immunoreactivity as a general marker for the entire uterine innervation, and suggests that the presence of PGP 9.5-immunoreactivity may coincide with functional maturation of uterine innervation.

Sequential development of angiotensin receptors and angiotensin I converting enzyme during angiogenesis in the rat subcutaneous sponge granuloma.

The vasoconstrictor peptide antiotensin II (AII) can stimulate angiogenesis, an important process in wound healing, tumour growth and chronic inflammation. To elucidate mechanisms underlying AII‐enhanced angiogenesis, we have studied a subcutaneous sponge granuloma model in the rat by use of 133Xe clearance, morphometry and quantitative in vitro autoradiography. When injected directly into the sponge, AII (1 nmol day−1) increased 133Xe clearance from, and fibrovascular growth in sponge granulomas, indicating enhanced angiogenesis 6 to 12 days after implantation. This AII‐enhanced angiogenesis was inhibited by daily doses (100 nmol/sponge) of the specific but subtype non‐selective AII receptor antagonist (Sar1, Ile8)AII, and by the selective non‐peptide AT1 receptor antagonists losartan and DuP 532. In contrast, AII‐enhanced neovascularization was not inhibited by the AT2 receptor antagonist PD123319, nor was it mimicked by the AT2 receptor agonist CGP42112A (each at 100 nmol/sponge day−1). AI (1 nmol/sponge day−1), the angiotensin converting enzyme (ACE) inhibitors captopril (up to 100 μg/sponge day−1) and lisinopril (40 μg/sponge day−1), or AII receptor antagonists did not affect angiogenesis in the absence of exogenous AII. [125I]‐(Sar1, Ile8)AII binding sites with characteristics of AT1 receptors were localized to microvessels and to non‐vascular cells within the sponge stroma from 4 days after implantation, and were at higher density than in skin throughout the study. [125I]‐(Sar1, Ile8)AII binding sites with characteristics of AT2 receptors were localized to non‐vascular stromal cells, were of lower density and appeared later than did AT1 sites. The ACE inhibitor [125I]‐351A bound to sites with characteristics of ACE, 14 days after sponge implantation. [125I]‐351A bound less densely to sponge stroma than to skin. We propose that AII can stimulate angiogenesis, acting via AT1 receptors within the sponge granuloma. AT1 and AT2 receptors and ACE develop sequentially during microvascular maturation, and the role of the endogenous angiotensin system in angiogenesis will depend on the balanced local expression of its various components. Pharmacological modulation of this balance may provide novel therapeutic approaches in angiogenesis‐dependent diseases.

Localization of endothelinlike immunoreactivity and endothelin binding sites in human colon

The potent vasoconstrictor endothelin was originally isolated from vascular endothelial cells but has since been found in several other tissues. The aim of this study was to establish whether endothelinlike immunoreactivity occurs in human enteric nerves and to identify endothelin binding sites using immunocytochemical and in vitro autoradiographic techniques. Endothelinlike immunoreactivity was localized to nerve bundles throughout the colon and to most of the ganglion cells of the two major plexuses, many of which costored vasoactive intestinal polypeptide. High-affinity (dissociation constant = 0.35 +/- 0.014 nmol/L; mean +/- SEM) binding sites for endothelin 1, with an apparent binding capacity of 92 +/- 6.3 amol/mm2 (mean +/- SEM), were demonstrated in the myenteric plexus, with less dense binding being found in the submucous plexus, mucosa, muscle layers, and blood vessel walls. Competition data suggested two populations of binding sites, both showing high affinities for endothelins 1 and 2, vasoactive intestinal constrictor, and sarafatoxin b but differentiated by their affinity for endothelin 3 and sarafatoxin c. This study provides evidence that endothelin is a neuropeptide in the human intestine with binding sites on neural plexuses and mucosa, suggesting a role in the modulation of intestinal motility and secretion.

Nitric oxide synthase in human placenta and umbilical cord from normal, intrauterine growth-retarded and pre-eclamptic pregnancies.

1 It has been suggested that a deficiency of nitric oxide (NO) may explain many of the pathophysiological features of pre‐eclampsia (PE) and intra‐uterine (foetal) growth retardation (IUGR). To elucidate further the role of NO in the pathophysiology of pregnancy we have determined the relative amount and activity of NO synthase (NOS) in first trimester and normal‐term placental tissues, as well as in the placenta and umbilical cord in pregnancies complicated by PE and IUGR, using NG‐nitro‐L‐[2,3,4,5‐3H]‐arginine ([3H]‐L‐NOARG) binding, quantitative in vitro autoradiography, [3H]‐arginine to [3H]‐citrulline conversion and Western blotting. 2 Specific, high affinity (KD = 38 nM) [3H]‐L‐NOARG binding was demonstrated in the villous trophoblast of normal‐term placentae. Binding was calcium‐independent, stereoselective and exhibited a rank order of inhibition by NOS inhibitors and substrate (L‐NOARG ≥ L‐NMMA ≥ 7‐NI > L‐NAME > L‐Arg ≥ L‐NIO > ADMA). 3 [3H]‐L‐NOARG binding density and NOS activity were both significantly greater in placental tissues from first trimester and PE or IUGR complicated pregnancies compared to normal‐term placentae. 4 Western blotting, using an endothelial NOS peptide antiserum, demonstrated a ∼ 140 KDa protein band in placental extracts and indicated that the amount of immunoreactive material was significantly greater in first trimester compared to normal‐term placentae. 5 Specific [3H]‐L‐NOARG binding was also localized to the endothelial lining of umbilical arteries and veins, binding density being greater in the artery than the vein. [3H]‐L‐NOARG binding to the umbilical artery endothelium was significantly lower in PE and IUGR complicated pregnancies compared to normal‐term controls. 6 The role of trophoblast‐derived NO in human placental pathophysiology remains to be established, but differences in the amount of placental [3H]‐L‐NOARG binding, NOS activity and immunoreactive material indicate that expression of NOS in the villous trophoblast falls during pregnancy. Conversely, the apparent reduction in NOS in the umbilical artery endothelium in PE and IUGR complicated pregnancies may be indicative of endothelial dysfunction.

Profibrotic effects of endothelin-1 via the ETA receptor in cultured human cardiac fibroblasts.

Background/Aims: Endothelin-1 (ET-1) has been implicated in pathologic remodelling and tissue repair processes in the heart. We investigated the effects of ET-1 on growth and collagen synthesis responses in cardiac fibroblasts isolated from human hearts. We also studied the receptor subtype(s) mediating such responses and the factors regulating their expression. Methods: Fibroblasts were isolated from cardiac transplant recipient hearts and characterised by immunocytochemistry. Serum-starved cells were exposed to ET-1 and incorporation of [3H]proline and thymidine were measured as indexes of collagen and DNA synthesis respectively. Blocking experiments utilised the selective ETA receptor antagonist BQ123 and the ETB antagonist BQ788. Results: ET-1 elicited a potent collagen synthesis response in cardiac fibroblasts, with a maximum 29±5% increase that was abolished by BQ123. Cardiac fibroblasts responded to ET-1 with a concentration-dependent decrease in DNA synthesis rate. The effects of ET-1 were similar to those of TGF-β. Radioligand binding studies revealed the presence of high-affinity ET-1 binding sites on these cells, which were upregulated by treatment with the growth factors PDGF and EGF but downregulated by TGF-β. Conclusions: These results therefore implicate ET-1 as a trophic agent in the human heart with the ability to influence the development of cardiac fibrosis.

The differential distribution of vasoactive intestinal polypeptide in the normal human female genital tract

SummaryVIP-like immunoreactive material is present in the female reproductive tract, with a distinct pattern of distribution. The highest concentrations of extractable material and immunoreactive nerve fibres were found in the cervix and vagina. In the cervix these fibres were seen below the surface epithelium and around cervical glands as well as in association with blood vessels and smooth muscle bundles. In the vagina the nerve fibres were most abundant in the superficial regions of the lamina propria. Scattered fibres were also present in the rest of the uterus and in the fallopian tubes. Chromatographic evidence indicates that this VIP-like material is of a similar molecular size to that extracted from other organs. Possible roles for VIP in the regulation of myometrial activity and of cervical and vaginal dilation and secretion are proposed.

AT1 receptor characteristics of angiotensin analogue binding in human synovium.

1 Angiotensin II (AII) reduces blood flow, modulates vascular remodelling and is a growth factor. Human inflammatory arthritides are characterized by synovial hypoperfusion, hypoxia and proliferation. We aimed to localize and characterize receptors for AII in human synovium. 2 We used quantitative in vitro receptor autoradiography with [125I]‐(Sar1, Ile8)AII and [125I]‐AII on human synovium from patients with chondromalacia patellae, osteoarthritis and rheumatoid arthritis. 3 [125I]‐(Sar1, Ile8)AII and [125I]‐AII bound to similar sites on synovial blood vessels, lining cells and stroma. Binding to microvessels (< 100 μm diameter) was more dense than to arteriolar media, and vascular binding was more dense than that to lining cells and stroma. 4 Microvessels and arterioles which displayed angiotensin converting enzyme‐like immunoreactivity also displayed specific binding of [125I]‐(Sar1, Ile8)AII. 5 Specific binding of [125I]‐(Sar1, Ile8)AII to each structure was completely inhibited by 10 μm dithiothreitol and was inhibited by unlabelled ligands with the rank order of potency (Sar1, Ile8)AII > AII > losartan = SKF108566 > PD123319 indicating an AT1 subclass of angiotensin receptor. 6 GTPγS (1 μm) abolished specific binding of [125I]‐AII and abolished the high affinity component of the binding inhibition curve for AII against [125I]‐(Sar1, Ile8)AII, indicating G protein coupling. 7 The distribution of [125I]‐(Sar1, Ile8)AII binding sites was similar in all disease groups and no significant differences in binding densities, affinities or specificities were observed between disease groups. 8 Locally generated AII may act on synovial AT1 receptors to modulate synovial perfusion and growth. Specific AT1 receptor antagonists should help elucidate the role of angiotensins in human arthritis.

Localisation and characterisation of substance P binding to human synovial tissue in rheumatoid arthritis.

The neuropeptide substance P is found in perivascular and free unmyelinated nerve fibres in human synovial tissue. Quantitative receptor autoradiography was used to show specific, high affinity (Kd = 0.75 (0.21), nmol/l (mean (standard error of the mean)), low capacity (Bmax = 27.8 (7.9) amol/mm2) binding sites for substance P Bolton Hunter-labelled with iodine-125 localised to vascular endothelial cells in human synovial tissue. The binding could be saturated, was reversible, and was dependent on the magnesium concentration. Unlabelled substance P and neurokinin A competitively inhibited specific binding with 50% inhibition at concentrations of 1.25 (0.21) and 175 (29) nmol/l respectively. Neurokinin B (mumol/l) and calcitonin gene related peptide (1 mumol/l) did not inhibit binding. These binding sites show characteristics of the neurokinin 1 tachykinin receptor subtype. This provides further evidence that substance P may play a part in the vascular control of human synovium and may influence inflammatory processes in joints.

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 ≥ 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 [125I]‐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 (∼ 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 (∼ 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.

Organization of the guinea-pig uterine innervation. Distribution of immunoreactivities for different neuronal markers. Effects of chemical- and pregnancy-induced sympathectomy

SummaryThe structural organization of the guinea-pig uterine innervation was investigated by an immunofluorescence method using neurofibrillary protein (NF) and neuron-specific enolase (NSE) as general neuronal markers. NF- and NSE-immunoreactive nerve trunks and non-varicose nerves formed continuous networks similar to nerves with analogue morphology and with immunoreactivities for tyrosine hydroxylase (TH; adrenergic nerves) and neuropeptide Y (NPY). NF- and NSE-immunoreactive non-varicose nerves occurred in the myometrium and along vessels, where TH- and NPY-immunoreactive varicose nerves were also comparatively frequent. After chemical sympathectomy all TH- and NPY-immunoreactive varicose nerves and most NF- and NSE-immunoreactive non-varicose nerves disappeared, suggesting colocalization of TH, NPY, NF and NSE immunoreactivities. During pregnancy all NF-, NSE-, TH- and NPY-immunoreactive nerve structures disappeared in the foetus-bearing uterine horns whereas in the cervix and non-foetus-bearing uterine horns only the myometrial TH- and NPY-immunoreactive varicose nerves disappeared. After parturition there was a complete structural restoration of all types of immunoreactive nerves in previously non-foetus-related tissue. The reinnervation of this tissue followed a similar time-course to that after chemical sympathectomy. In contrast, the reinnervation of previously foetus-related tissue was much slower and incomplete.In conclusion, the whole autonomic uterine innervation undergoes overt structural changes during pregnancy and these changes are related to the foetus-bearing regions.