Binding of [3H]-5-hydroxytryptamine to human coronary artery and bypass graft vessels.

OBJECTIVES: 5-Hydroxytryptamine (5-HT) has a wide range of vascular effects mediated via specific receptors and it has been suggested to be a mediator in ischemic heart disease. The aim of the present study was to localise the 5-HT receptors within the vessel wall. METHODS: Epicardial coronary arteries obtained from patients undergoing cardiac transplantation, internal mammary arteries from heart donors and saphenous veins from patients undergoing coronary bypass surgery, were sectioned and incubated with [3H]-5-HT for in vitro receptor autoradiography. RESULTS: Microscopic analysis of high resolution autoradiographic images revealed a similar pattern of [3H]-5-HT binding in epicardial coronary and internal mammary artery, where it predominated in the lamina muscularis. In the saphenous vein, binding increased towards the adventitia which showed dense, displaceable binding to the vasa vasorum as well as to nerve-like structures, from which binding was only partially displaced. Computer-assisted densitometric analysis of low resolution autoradiographs revealed a high degree of specific binding to all vessels examined. CONCLUSIONS: The distribution of the [3H]-5-HT binding is different in the saphenous vein compared to epicardial coronary and internal mammary artery. The dense binding to vasa vasorum in the saphenous vein suggests a role for 5-HT in closure of these nutrient vessels, which could contribute to the formation of atherosclerotic changes in saphenous vein grafts.

The effect of the ETA receptor antagonist, FR 139317, on [125I]-ET-1 binding to the atherosclerotic human coronary artery.

1. The distribution of [125I]-endothelin (ET-1) binding sites on atherosclerotic human epicardial coronary arteries has been studied by in vitro receptor autoradiography. 2. [125I]-ET-1 binding was to the tunica media and regions of neovascularization. 3. Competition studies were carried out in the presence of ET-1 and the ETA receptor antagonist, FR 139317. The IC50 values for ET-1 at the tunica media and regions of neovascularization were similar (mean +/- s.e.mean of n = 4 patients, 2.5 +/- 0.9 nM and 2.9 +/- 0.9 nM, respectively) whereas IC50 values for FR 139317 at regions of neovascularization (607 +/- 34 nM) were significantly higher than those of the tunica media (12.6 +/- 2.4 nM) (P < 0.0001). 4. These results indicate that ETA receptors are present on the tunica media of the diseased human coronary artery whereas a different ET receptor subtype exists at regions of neovascularization.

Distribution of 125I-labelled insulin-binding sites in peripheral tissues of the normal and diabetic rat: an autoradiographic study.

In-vitro autoradiography was used to demonstrate the regional distribution of 12I-labelled insulin-binding sites in the liver, kidney and heart of normal rats and rats made diabetic with streptozotocin. The distribution of insulin-binding sites in the liver of control rats was uniformly high, while in the kidney of control rats there was weak 125I-labelled insulin binding in the medulla and dense binding in the cortex. In the hearts of control rats a high density of 125I-labelled insulin-binding sites was evident both in the atrial and ventricular muscle. Non-ketotic diabetes mellitus caused a marked increase in 125I-labelled insulin-binding sites in both the liver and kidney with the former tissue exhibiting a time-dependent (7 to 62 days) increase. There was no apparent effect of diabetes on insulin-binding sites in the heart. Since experimental diabetes causes (1) a decrease in circulating insulin concentration and (2) impaired insulin action at many target tissues, the increase in 125I-labelled insulin-binding sites observed in the present study may represent a compensatory 'up regulation' of insulin receptors.

Regional variations in endothelin-1 and its receptor subtypes in human coronary vasculature: pathophysiological implications in coronary disease.

Endothelin-1 is a potent vasoconstrictor peptide and mitogen for vascular smooth muscle cells. Increased plasma or tissue levels of endothelin-1 have been described after myocardial infarction and in atherosclerosis, suggesting that this peptide may play a pathophysiological role in various coronary syndromes. Here, we have studied regional variations in ET-1 and its receptors in control and atherosclerotic human coronary vasculature using standard immunohistochemistry and in vitro autoradiography. ET-1 immunoreactivity was associated with luminal endothelial cells and smooth muscle cells at regions of atherosclerosis. ET(A) receptors were present on smooth muscle cells of coronary arteries and on cardiac myocytes. Medial ET(B) receptor binding at the proximal region of coronary arteries was weak, but increased significantly towards distal regions of this vessel (p<0.005 in control and p<0.0005 in ischaemic heart disease). Microvascular endothelial cells in the adventitia of coronary arteries, myocardial microvessels and the endocardial endothelium expressed the ET(B) receptor exclusively. The receptor variations revealed in this study provide supporting evidence that ET-1 is associated with (1) vascular smooth muscle and endothelial cell proliferation, including areas of intimal hyperplasia and regions of neovascularization (2) increased ET-1-induced reactivity of distal portions of the human coronary artery, (3) ET-1-mediated constriction of myocardial microvessels. These results provide new insights into different potential roles for this peptide in healthy and diseased human coronary vasculature.

Opiate receptor subtypes in the nucleus tractus solitarii of the cat: the effect of vagal section.

The distribution of mu, delta and kappa opioid receptors in the lower brainstem of the cat has been determined autoradiographically by studying the binding of tritiated [D-Ala2,MePhe4,Glyol5][tyrosyl-3,5-3H]enkephalin (DAGO), [D-Pen2,D-Pen5][tyrosyl-3,5-(n)-3H]enkephalin (DPDPE) and [9-3H]ethylketazocine (EKC), respectively. General opiate receptor binding was established using [3H]naloxone (NX). High densities of [3H]NX and DAGO binding sites were found most prominently in the nucleus tractus solitarii. There was no DPDPE and very weak EKC binding within this nucleus, although both these ligands bound to the cerebellum. The effect of unilateral vagotomy on receptor density was examined. Sectioning the cervical vagus had no effect on the density of mu receptors in the brainstem. Sectioning the vagus, accompanied by nodose ganglion excision, led to a marked depletion of mu receptors which was restricted to dorsal and medial regions of the ipsilateral nucleus tractus solitarii at, and rostral to, the obex. These results suggest that mu opiate receptors are located presynaptically on vagal afferents terminating within a restricted region of the nucleus tractus solitarii.

Correlated nerve conduction, somatosensory evoked potential and neuropathological studies in clioquinol and 2,5-hexanedione neurotoxicity in the baboon.

Observations have been made on 10 baboons receiving a high-dose regimen of clioquinol administered orally, 6 receiving a low-dose regimen and 6 treated with 2,5-hexanedione. The results were compared with those obtained from 10 control animals. Motor and sensory nerve conduction velocity was markedly reduced in the hexanedione-treated animals but only very minor abnormalities were detected in the clioquinol-treated baboons. Cervical and Rolandic somatosensory evoked potentials to lower and upper limb stimulation were delayed in both the high-dose clioquinol-treated and the hexanedione-treated animals, particularly in the latter. Histopathological studies in the low-dose clioquinol-treated group showed no abnormalities. In the high-dose group; axonal degeneration was confined to the spinal cord, cerebellar vermis and optic tract. It was most marked in the rostral portions of the dorsal spinal columns and the caudal parts of the direct and crossed corticospinal tracts. Occasional dorsal column fibres had degenerated back to the root entry zone in the cord. The distribution was that of a selective central distal axonopathy. There appeared to be no correlation with estimated blood levels of unaltered clioquinol. In hexanedione-treated animals there was also degeneration in the distal optic tracts and peripheral nerves in a pattern of central-peripheral distal axonopathy.

Abnormal Schwann cell/axon interactions in the Trembler-J mouse.

The Trembler-J (TrJ) mouse has a point mutation in the gene coding for peripheral myelin protein 22 (PMP22). Disturbances in PMP22 are associated with abnormal myelination in a range of inherited peripheral neuropathies both in mice and humans. PMP22 is produced mainly by Schwann cells in the peripheral nervous system where it is localised to compact myelin. The function of PMP22 is unclear but its low abundance (approximately 5% of total myelin protein) means that it is unlikely to play a structural role. Its inclusion in a recently discovered family of proteins suggests a function in cell proliferation/differentiation and possibly in adhesion. Nerves from TrJ and the allelic Trembler (Tr) mouse are characterised by abnormally thin myelin for the size of the axon and an increased number of Schwann cells. We report ultrastructural evidence of abnormal Schwann cell-axon interactions. Schwann cell nuclei have been found adjacent to the nodes of Ranvier whereas in normal animals they are located near the centre of the internodes. In some fibres the terminal myelin loops faced outwards into the extracellular space instead of turning inwards and terminating on the axon. In severely affected nerves many axons were only partially surrounded by Schwann cell cytoplasm. All these features suggest a failure of Schwann cell-axon recognition or interaction. In addition to abnormalities related to abnormal myelination there was significant axonal loss in the dorsal roots.

The extracellular matrix of peripheral nerve in diabetic polyneuropathy.

The pattern of collagenisation in peripheral nerve in diabetic polyneuropathy was examined in nerve biopsy specimens from patients with diabetic polyneuropathy in comparison with organ donor control nerves and disease controls (other neuropathies). There was increased endoneurial collagenisation both in the diabetic polyneuropathy cases and the disease controls, this predominantly involving types I and III. Type II collagen was not detected in organ donor control nerves or in the diabetic and the disease control nerves. There was a relative increase in type VI collagen in the endoneurium in the diabetic nerves immediately surrounding groups of Schwann cells. This was not a feature in the other neuropathies. The quantity of types IV, V and VI collagen was increased around the endoneurial microvessels in the diabetic patients and, to a lesser extent, in those with hereditary motor and sensory neuropathy (HMSN). Increased deposition of types IV and V collagen was observed in the perineurium in the diabetic nerves, the latter being most evident in the innermost lamellae where the amount of laminin was possibly also increased. The diameter of the general endoneurial collagen fibrils was greater in the diabetic nerves, although this was not more than in a disease control (HMSN). The collagen fibrils that were present within the basal laminal tubes that had surrounded degenerated myelinated fibres in the diabetic nerves, and those within the onion bulbs of the HMSN cases, were of the normal endoneurial calibre. The expression of laminin by Büngner bands in diabetic neuropathy did not differ from that in disease control nerves, nor were any differences detected for fibronectin. Whether the changes observed are important for the impaired regenerative capacity in diabetic neuropathy requires further investigation.

Lumbar and cortical somatosensory evoked potentials in rats with vitamin E deficiency.

Somatosensory evoked potentials (SEPs) from lumbar and cortical areas and electromyographic activity (EMG) were recorded in 40-42 week vitamin E deficient rats and in age matched controls. A significant increase in the latency (p less than 0.001) of the cortical SEP and a significant reduction in the lumbar to cortical conduction velocity (p less than 0.001) were observed in vitamin E deficient rats compared with controls. No significant differences were obtained in the latency of the lumbar SEP or in the peripheral conduction velocity from the ankle to lumbar region. All the vitamin E deficient rats had abnormal EMG findings (fibrillation potentials, positive sharp waves and polyphasic activity), whereas none of the controls showed any of these signs of dysfunction.

Pattern of myelinated fibre loss in the sural nerve in neuropathy related to type 1 (insulin-dependent) diabetes.

Sural nerve biopsies were obtained from 17 diabetic patients with neuropathy. All patients except three had both a symmetric distal sensory and autonomic polyneuropathy related to Type 1 (insulin-dependent) diabetes mellitus; 3 patients had a purely sensory polyneuropathy. Mean age was 34.5 years (range 18-53 years). The biopsies were compared with specimens from an age-matched control series. Myelinated fibre loss in the diabetic nerves was found to be nonuniform. Although patchy fibre loss has been considered to favour a vascular basis, an identical pattern of nonuniform loss was observed in a series of sural nerve biopsies from patients with Type I hereditary motor and sensory neuropathy, a subgroup within the spectrum of peroneal muscular atrophy, mainly of autosomal dominant inheritance, and a condition in which a vascular causation can be discounted. Possible reasons for nonuniform fibre loss other than vascular disease are discussed.

Peripheral neuropathy in the Chediak-Higashi syndrome.

The clinical features of a brother and sister with the Chediak-Higashi syndrome (CHS) are reported. Both showed evidence of a sensory neuropathy associated with central nervous system involvement. Nerve conduction studies indicated an "axonal" neuropathy. Sural nerve biopsy in the brother demonstrated a loss of myelinated nerve fibres, particularly those of larger size, and of unmyelinated axons. In contradistinction to some previous reports, giant lysosomes in Schwann cells were not observed and there were no inflammatory changes. Electron microscopy and teased-fibre studies showed no evidence of demyelination. It is concluded that the neuropathy of CHS is of axonal type. Its mechanism remains obscure.

Peripheral nerve regeneration in galactosaemic rats.

The use of galactosaemia as a model for some aspects of diabetic polyneuropathy allows the influence of glycation to be studied independently of other effects. There are well-studied abnormalities of the peripheral nerves in galactosaemic rats, one of which is that the efficiency of regeneration is initially reduced. One possible cause could be that glycated myelin debris in macrophages is less degradable and interferes with macrophage function. Macrophage recognition and ingestion of myelin glycosylated in vitro increases with the duration of incubation in a sugar-rich medium. This study was performed to investigate a possible correlation between galactosaemia and regeneration, together with the role of macrophages. Galactosaemia was induced by adding galactose to the rats' diet for 2 months before injury. Following a crush lesion to the sciatic nerve, regeneration was found to be delayed, demonstrated by a reduction in mean myelinated fibre size and density 1 month after crush, although, 2 and 3 months later, the differences did not reach statistical significance. There were also more macrophages in the galactosaemic rats than in the control animals at all time points. The initial delay in regeneration in galactosaemic rats was therefore only temporary and there was little evidence of long-term deleterious effects. In addition to the morphometric results, immunohistochemistry showed that there were more macrophages in the galactosaemic rats than in the control animals at all time points. Correlating macrophage and myelinated fibre counts suggests that the persistence of debris-containing macrophages does not appear to have a significant inhibitory effect on nerve regeneration. No evidence was found for persistent basal laminal tubes around the regenerating clusters.

Localization of [125I]endothelin binding sites in the region of the carotid bifurcation and brainstem of the cat: possible baro- and chemoreceptor involvement.

We have demonstrated by autoradiography, displaceable binding for [125I]endothelin-1 ([125I]ET-1), [125I]endothelin-2 ([125I]ET-2), and [125I]endothelin-3 ([125I]ET-3) in the cat carotid bifurcation as well as in the nucleus of the tractus solitarius, where baroreceptor and chemoreceptor afferents from the carotid body and sinus terminate. There was also significant binding in the nodose and superior cervical ganglia. Barosensory and chemosensory discharge was recorded from filaments of the carotid sinus nerve in cats anesthetized with pentobarbitone. Intra-carotid injection of ET-1 or ET-3 (4-402 pmoles) caused transient dose-related depression of baroreceptor discharge without any immediate effects on systemic blood pressure (BP) or heart rate; there was a delayed biphasic effect on BP. ET-1 had little effect on chemosensory discharge during the first 15 s post-injection, but there was a delayed (45-90 s) dose-related increase in discharge. The effects of all three ETs were qualitatively similar, and ET enhanced chemoexcitation evoked by either acetylcholine or sodium cyanide. Our results show that (a) ET binding sites are located in the baroreceptor and chemoreceptor afferent pathways and (b) ETs can influence afferent activity of baroreceptors and chemoreceptors. Further studies are needed to determine the significance of these findings, particularly with regard to reflex control of the cardiovascular system.

Influence of age on the late retrograde effects of sciatic nerve section in the rat.

The influence of age on the late retrograde effects of unilateral sciatic nerve section was investigated in rats. Operations were performed on young rats aged 3 months and older rats aged 15 and 18 months, with survival times ranging from 6 to 15 months depending upon age at the time of operation. As in previous studies, axonal atrophy was found in myelinated fibres proximal to nerve transection. This was observed to be greater in animals operated upon at 3 months of age than in those in which the sciatic nerve was transected at 15 and 18 months. In the sciatic nerve, focal intramyelinic oedema was present at a low frequency on the operated side just proximal to the section at all survival times but not on the unoperated side except in 1 old animal. Its frequency increased with age both in the dorsal and ventral roots on both sides but it was not more common on the operated side. Retrograde axonal atrophy is therefore unlikely to contribute to its occurrence. In the dorsal root ganglia the main abnormality was the presence of vacuolated neurons on the operated side. Nuclear eccentricity was also observed on the operated side in young animals in a proportion of the neurons; its frequency increased with age on the normal side and there was no difference in the older animals between operated and control sides. The possibility is discussed that growth factor deprivation secondary to axotomy is implicated in these changes. If so, there are age differences in its effect in giving rise to axonal atrophy and neuronal vacuolation.

The density and distribution of endothelin 1 and endothelin receptor subtypes in normal and diabetic rat corpus cavernosum.

OBJECTIVE: To investigate the density and distribution of endothelin 1(ET-1) and endothelin receptor subtypes in rat cavernosal tissue, and to assess any changes brought about by the onset of diabetes mellitus (DM). MATERIALS AND METHODS: Hyperglycaemic non-ketotic diabetes mellitus was induced in five rats using streptozotocin (STZ). Two months later the penises were excised and stored at -70 degrees C. Longitudinal serial sections (6 microns) were cut using a cryostat and thaw- mounted onto gelatinized microscope slides. Low and high resolution autoradiographs were taken using radioligands for ET-1 and endothelin A (ETA) and endothelin B (ETB) receptors and the autoradiographs analysed densitometrically. These results were compared with those from five age-matched control rats. RESULTS: ET-1 and ETA receptor binding was primarily localized to the endothelium lining the cavernosal lacunar spaces: trabecular corporeal smooth muscle was much less intensely stained. No ETB receptor binding could be demonstrated, but increased binding of ET-1 and ETA receptors was seen in the diabetic cavernosal tissue. CONCLUSION: ET-1 and ETA receptor binding is increased in diabetic rat cavernosal tissue. A reduction in receptor linked nitric oxide (NO) release has previously been reported in diabetic rats. This finding may provide an explanation for the upregulation of ET-1 and ETA receptor binding in the diabetic rat.

Calcitonin gene-related peptide in healthy and atheromatous human epicardial coronary arteries. Function and receptor characterization.

To examine the possible role of calcitonin gene-related peptide (CGRP) in the control of human coronary vascular tone, we have investigated the action of this peptide in healthy and atheromatous human epicardial coronary arteries and localised [125I]CGRP-binding sites in these vessels. Isolated arteries were obtained from 10 cardiomyopathic patients and 6 patients with ischaemic heart disease (IHD), who were undergoing heart transplantation. CGRP elicited concentration-dependent vasodilatation in preconstricted vessels. Both healthy and diseased coronary arteries exhibited similar maximum responses and sensitivity to this peptide. Removal of the endothelium did not diminish the vasodilator action of CGRP in non-atherosclerotic coronary arteries. [125I]CGRP bound to tissue sections in a concentration-dependent manner. Binding was similar in healthy and atheromatous vessels, with a Bmax value of 10.2 +/- 5.6 and 18.9 +/- 3.0 amol/mg protein, and dissociation constant (Kd) of 0.07 +/- 0.1 and 0.18 +/- 0.1 nM, respectively. Dense [125I]CGRP binding was mainly associated with vascular smooth muscle cells of both normal and diseased vessels with some patches of binding to regions of atherosclerotic plaque in vessels from patients with IHD. These data indicate that CGRP is a potent endothelium-independent vasodilator in the human coronary vasculature. The preservation of dilator function and CGRP receptor binding in atherosclerotic coronary arteries suggests that this peptide may play a role in the control or maintenance of vascular tone in certain disease states.

Sural nerve morphometry in diabetic autonomic and painful sensory neuropathy. A clinicopathological study.

Observations have been made on a selected series of insulin-dependent patients with neuropathy, subdivided into three groups: (1) severe autonomic neuropathy with an accompanying painless sensory neuropathy; (2) severe autonomic neuropathy with a chronic painful sensory neuropathy; and (3) chronic or acute painful sensory neuropathy with no autonomic neuropathy. All three groups showed a loss of large and small myelinated nerve fibres in sural nerve biopsy specimens which was greater in Groups 1 and 2. Regenerative activity was prominent in all three groups, but least in Group 3. Teased fibre studies showed evidence both of axonal regeneration and remyelination. Active fibre degeneration was rare. Measurements of g ratio (axon diameter:total fibre diameter) gave no indication of axonal atrophy. The density of unmyelinated axons was reduced in all three groups, as was their median diameter. Vibration sense threshold was positively correlated with the total number of myelinated fibres and thermal sensory threshold with median unmyelinated axon diameter but not with total unmyelinated axon numbers. No correlation between the occurrence of pain and active degeneration of myelinated fibres or with regenerative activity either in myelinated or unmyelinated axons was detectable. Assessment of differential loss of large or small myelinated nerve fibres was difficult because of the presence of large numbers of small regenerating myelinated axons. The results are discussed in relation to the concept of 'diabetic small fibre neuropathy' and the causation of pain in diabetic neuropathy.

Alterations in endothelin B receptor sites in cavernosal tissue of diabetic rabbits: potential relevance to the pathogenesis of erectile dysfunction.

PURPOSE: Diabetes Mellitus (DM) is a major risk factor for erectile dysfunction in both patients and animal models. The pathogenesis of this dysfunction has not been fully elucidated. However, alterations in the synthesis of a number of vasoactive compounds, such as nitric oxide (NO) and prostacyclin (PGI2), have been reported in various diabetic tissues. The interaction between NO, PGI2 and endothelin-1 (ET-1), a powerful vasoconstrictor and smooth muscle cell mitogen, is thought to be important in maintaining vascular tone and the erectile process. We investigated the density and distribution of ET-1 and endothelin receptor subtypes in cavernosal tissue and assessed any changes brought about by DM in a rabbit model. MATERIALS AND METHODS: DM was induced in New Zealand White rabbits using alloxan. Penises were excised from the diabetic rabbits three months (n = 6) and six months (n = 6) after the induction of DM. Low and high resolution autoradiography was performed using radioligands for ET-1, endothelin A (ETA) and endothelin B (ETB) receptors and were analyzed densitometrically. The results were compared with those from six age-matched healthy control rabbits for each group. Immunohistochemical localization of ET-1 immunoreactivity was also performed, together with ultrastructural evaluation of the corpus cavernosum. RESULTS: ET-1, ETA and ETB receptor binding sites were primarily localized to the smooth muscle cells of the corpus cavernosum and the endothelium lining the cavernosal spaces. A significant increase in ETB receptor binding sites was found only in cavernosal tissue six months after induction of DM, when compared with age-matched healthy controls. These receptor changes were accompanied by ultrastructural changes in the corpus cavernosum indicative of an early, atherosclerosis-like process. CONCLUSIONS: The autoradiographic and immunohistochemical findings in this study suggest that ET-1 may have a role in the pathophysiology of diabetic ED. This peptide may be released in an autocrine fashion causing cavernosal smooth muscle cell (CSMC) contraction and/or proliferation.

Neural architecture in transected rabbit sciatic nerve after prolonged nonreinnervation.

Observations have been made on the rabbit sciatic nerve distal to a transection, with survival periods of up to 26 mo and prevention of reinnervation. It was confirmed that the nerve becomes compartmented by fibroblast processes and that a zone of fine collagen fibrils develops around the Schwann cell columns that constitute the Büngner bands. The Schwann cells become progressively more atrophic but after 6 mo of denervation still expressed low affinity p75 nerve growth factor receptor (NGFR), the latest stage at which this was examined. NGFR was also expressed by the processes of the fibroblasts producing the endoneurial compartmentation. By 26 mo after transection the site of previous nerve fibres was indicated by sharply demarcated domains of approximately circular outline in transverse section consisting of densely packed longitudinally oriented collagen fibrils. Some of these domains still possessed centrally situated Schwann cells or residual basal lamina but many were acellular. The central collagen fibrils in these domains were of smaller diameter than those situated peripherally but were of larger size than those that form around the Büngner bands during wallerian degeneration. The peripherally located fibrils in the domains were of the same calibre as for normal endoneurial collagen. The collagen domains were encircled by fibroblast processes or at times enclosed in a perineurial cell ensheathment. Long-standing axonal loss therefore leads to a striking reorganisation of the internal architecture of peripheral nerve trunks. The findings may be relevant for the interpretation of the appearances in chronic peripheral neuropathies in man.