A. Loesch

Rapid release of endothelin and ATP from isolated aortic endothelial cells exposed to increased flow

Freshly harvested rabbit aortic endothelial cells on filters were exposed to two 3 min periods of a sixfold increase in flow rate of the perfusion buffer. This led to an increase in the levels of endothelin and ATP in the perfusate; arginine vasopressin remained at the basal level. Less ATP was released on the second exposure to high flow; however, endothelin release was not diminished. Using immunohistochemical techniques, endothelin and arginine vasopressin were localised in the same population of endothelial cells; endothelin and vasopressin were present in approximately 90% and 70% of endothelial cells, respectively, which suggests that there is some co-localisation. This is the first time that a stimulation has been shown to produce rapid release of endothelin.

Enteric nerves in diabetic rats: Increase in vasoactive intestinal polypeptide but not substance P

The distribution of vasoactive intestinal polypeptide (VIP) and substance P-like immunoreactivities was studied by immunohistochemistry in the myenteric plexus and circular muscle layer of the ileum and proximal colon of rats 8 wk after induction of diabetes with streptozotocin. A consistent increase was observed in fluorescence intensity of VIP-like immunoreactivity in the nerve fibers, and intensely stained cell bodies were significantly more frequent in the myenteric plexus of the ileum (p less than 0.001) from diabetic animals. Some varicosities of VIP-like immunoreactive fibers in the myenteric plexus appeared to be enlarged. Vasoactive intestinal polypeptide-like immunoreactivity was increased and VIP-like immunoreactive nerves appeared thicker in the circular muscle layer of both diabetic ileum and proximal colon. The VIP levels were measured biochemically in tissue consisting of the smooth muscle layers and myenteric plexus. A significant increase in the VIP content per centimeter of intestine was found in both the ileum (p less than and proximal colon (p less than 0.01) from diabetic rats. In contrast, no apparent change in substance P innervation was observed immunohistochemically in the myenteric plexus and circular muscle layer of either diabetic ileum or proximal colon when compared with controls. The results are discussed in relation to the symptoms of autonomic neuropathy of the gut in diabetes.

Ultrastructural localisation of serotonin and substance P in vascular endothelial cells of rat femoral and mesenteric arteries

SummaryAn electron microscopic-immunocytochemical study has been made on the localisation of serotonin and substance P in endothelial cells of femoral and mesenteric arteries of the rat. In the femoral and mesenteric arteries, less than 10% of the endothelial cells showed positive immunolabelling for these substances. The distribution of serotonin and substance P in endothelial cells was similar in both arteries. The immunoprecipitate was confined to the cytoplasm, including subcellular organelles. The results are discussed in terms of endothelial mechanisms of local control of blood flow.

An ultrastructural study of NADPH-diaphorase and nitric oxide synthase in the perivascular nerves and vascular endothelium of the rat basilar artery

SummaryThis is the first report on the ultra structural distribution of nicotinamide adenine dinucleotide phosphate-diaphorase activity and neuronal isoform (Type I) of nitric oxide synthase immunoreactivity in perivascular nerves (axons) and vascular endothelial cells. In the Sprague-Dawley rat cerebral basilar artery, positive labelling for nicotinamide adenine dinucleotide phosphate-diaphorase and nitric oxide synthase was localized in axons and the endothelium. Over half (∼ 53%) of the axon profiles examined were positive for nicotinamide adenine dinucleotide phosphate-diaphorase. Labelling of nicotinamide adenine dinucleotide phosphate-diaphorase activity in the axons and endothelial cells was mostly distributed in patches within the cytoplasm. In endothelial cells, a relation between the nicotinamide adenine dinucleotide phosphate-diaphorase-labelling and cytoplasmic vesicle-like structures was seen. In both axons and the endothelium, nitric oxide synthase immunoreactivity was seen throughout the cell cytoplasm and in association with the membranes of mitochondria, endoplasmic reticulum and cytoplasmic/synaptic vesicles (the lumen/content of the vesicles was negative for nitric oxide synthase). Also, microtubules were labelled in nitric oxide synthase positive axon profiles. The nitric oxide synthase-positive axon varicosities were characterized by the presence of spherical agranular vesicles with a diameter of 40–50 nm. Approximately 30% of the axon profiles examined were positive for nitric oxide synthase. The nicotinamide adenine dinucleotide phosphate-diaphorase-positive endothelial cells (approximately 20% of all observed endothelial cell profiles) were more frequently seen than those positive for nitric oxide synthase (approximately 7%). It is suggested that nitric oxide released from both perivascular nerves and endothelial cells may be involved in vasomotor control of cerebral circulation.

Enteric nerves in diabetic rats: Electron microscopic evidence for neuropathy of vasoactive intestinal polypeptide-containing fibres

SummaryEnteric nerves in the ileum of rats 8 weeks after streptozotocin-induction of diabetes were examined under the electron microscope before and after immunolabeling for vasoactive intestinal polypeptide (VIP). These studies have provided evidence of degenerative changes in the myenteric nerve fibres of diabetic rats, many of which were shown to contain VIP. It is suggested that VIP-ergic nerves in the gut may play a role in the developent of gastrointestinal dysfunction in diabetes.

Nitric oxide synthase-containing neurones and nerve fibres within cardiac ganglia of rat and guinea-pig: an electron-microscopic immunocytochemical study.

Abstract.The nitric oxide synthase-immunoreactivity and NADPH-diaphorase activity of intracardiac neurones in the rat and guinea-pig was studied at the ultrastructural level. While some nitric oxide synthase-containing intracardiac neurones were very heavily labelled, with electron-dense immunoprecipitate distributed throughout the neuronal cell bodies and their processes, most of the labelled neurones exhibited a lighter and more patchy distribution of nitric oxide synthase-immunoreactive material. Synapses made by nitric oxide synthase-negative nerve fibres with labelled intracardiac neurones were seen. Conversely, many nitric oxide synthase-containing nerve fibres that made synaptic contacts with unlabelled intracardiac neurones were also observed. Some small granule-containing cells were nitric oxide synthase-immunoreactive and were associated with unlabelled nerve terminals, while non-immunoreactive small granule-containing cells that were innervated by nitric oxide synthase-immunoreactive nerves were also seen. Small patches of osmiophilic electron-dense material were observed in the cytoplasm of NADPH-diaphorase-positive intracardiac neurones. This is the first description of the ultrastructural distribution of nitric oxide synthase-immunoreactivity and NADPH-diaphorase activity in a subpopulation of intracardiac neurones of rat and guinea-pig heart and provides further evidence in support of a role for nitric oxide in the local control of the heart by intrinsic neurones.

Endothelin immunoreactivity and mRNA expression in sensory and sympathetic neurones following selective denervation

The localization of endothelin (ET) in perivascular nerve varicosities supports pharmacological evidence that ET is a neurotransmitter in the autonomic nervous system. To examine the potential source of ET previously localized in cerebrovascular nerves, ganglia which send projections to these vessels were immunolabelled for ET and examined at the ultrastructural level. The trigeminal (TG) and superior cervical ganglia (SCG) were examined in control rats and following either sensory denervation or sympathectomy. In control TG, ET immunolabelling was detected throughout the cytoplasm of a subpopulation of neurones whereas in the SCG only the occasional ET‐positive neurone was seen. Following sensory denervation with capsaicin, very few ET‐immunoreactive nerve cell bodies or nerve fibres were detected in the TG compared with control ganglia, suggesting that ET is predominantly localized in primary afferent neurones, although some remaining myelinated nerve fibres stained positively. ET labelling of neurones in the SCG was unaffected by sensory denervation. Following selective damage to sympathetic nerves with 6‐hydroxydopamine, there was a marked increase in intensity of ET‐labelling of nerve fibres in the TG, probably due to increased availability of nerve growth factor for sensory nerves. There was no effect on ET immunoreactivity in the nerve cell bodies and nerve fibres within the SCG. However, in situ hybridization techniques demonstrated that 6‐hydroxydopamine sympathectomy resulted in a marked increase in ET‐1 mRNA expression in the SCG neurones. In conclusion, sensory nerves projecting from the TG are a more likely source of ET‐positive perivascular nerves in cerebral arteries than sympathetic nerves from the SCG. Damaged sympathetic neurones markedly increase ET mRNA expression. In view of the neuroprotective properties of ET, this may represent a compensatory mechanism to promote repair.

Ultrastructural localization of nitric oxide synthase and endothelin in rat pulmonary artery and vein during postnatal development and ageing

Abstract.The ultrastructural distribution of nitric oxide synthase (neuronal isoform, type I) and endothelin immunoreactivity was examined in the developing and ageing male Wistar rat pulmonary artery and vein. This study demonstrates that from birth to old age (24 months) nitric oxide synthase and endothelin are localized within subpopulations of endothelial cells in the pulmonary vasculature. In the pulmonary artery and vein of newborn rats, and pulmonary vein of 24-month-old rats, positive labelling for nitric oxide synthase was also observed in the vascular smooth muscle. During development and ageing there were ultrastructural and immunocytochemical alterations in the intima of the pulmonary artery and vein. In older animals, damaged endothelial cells were seen alongside healthy-appearing cells, rich in cytoplasmic vesicles and endoplasmic reticulum. In contrast to damaged cells, the healthy-appearing endothelial cells displayed positive cytoplasmic labelling for nitric oxide synthase or endothelin. These immunopositive cells also appeared in the altered regions of the vessels where substantial enlargement of subendothelial extracellular matrix and the presence of various forms of degenerating macrophages and large bundles of collagen fibres were evident. Damage to the pulmonary artery was particularly evident at the ages of 12 and 24 months; various forms of macrophages, some of which displayed positive labelling for nitric oxide synthase and endothelin, were present in the altered intimal subendothelial zone. In conclusion, this study on the pulmonary vasculature suggests that endothelin and NOS in endothelial cells play a role in the local control of vascular tone throughout the lifespan of rats, even in older animals when there is intimal thickening and some endothelial damage. NOS and endothelin was also seen in smooth muscle and macrophages at certain stages in postnatal development and ageing.

Ultrastructural investigation of nitric oxide synthase-immunoreactive nerves associated with coronary blood vessels of rat and guinea-pig

Ultrastructural investigation of nitrix oxide synthase-immunoreactive nerves closely associated with blood vessels in rat and guinea-pig hearts revealed many labelled nerve fibres in the walls of the main branches of the coronary arteries, and in arterioles, capillaries and post-capillary venules. The number of nitric oxide synthase-containing nerve fibres associated with different vessels, even those of the same calibre, varied. Terminal regions of nitric oxide synthase-immunoreactive fibres were observed in the endocardium and myocardium. Nitric oxide synthase-labelled fibres displayed electrondense immunoproduct in both varicose and intervaricose regions. Immunoreactive axonal varicosities contained both small and large synaptic vesicles. The characteristics of the nitric oxide synthase-immunoreactive nerve fibres observed in the heart and the possibility that these fibres represent the processes of intracardiac neurones and/or sensory neurones of extrinsic origin are discussed.

Ultrastructural localization of nitric oxide synthase and endothelin in coronary and pulmonary arteries of newborn rats

This is the first report on the ultrastructural distribution of nitric oxide synthase and endothelin immunoreactivities in the coronary and pulmonary arteries of newborn Wistar rats. The distribution of nitric oxide synthase and endothelin was investigated using pre-embedding peroxidase-antiperoxidase immunocytochemistry. In both arteries examined, positive labelling for nitric oxide synthase was localized both in the endothelium and smooth muscle, whereas positive labelling for endothelin was localized in the endothelium exclusively. In the coronary artery, approximately 80% and 55% of the endothelial cells examined were positive for nitric oxide synthase and endothelin, respectively, whereas in the pulmonary artery, 77% and 60% of the endothelial cells were positive for nitric oxide synthase and endothelin, respectively. These findings indicate that nitric oxide synthase and endothelin are colocalized in some of the endothelial cells of the newborn rat. In the endothelium, nitric oxide synthase and endothelin immunoreactivities were distributed throughout the cell cytoplasm and in association with the membranes of intracellular organelles. In smooth muscle, a relationship of nitric oxide synthase immunoreactivity to endoplasmic reticulum was observed in the pulmonary artery. In summary, in the newborn rat, endothelial cells of the coronary and pulmonary artery are rich in nitric oxide synthase (neuronal isoform) and endothelin, and it is suggested therefore that they may be substantially involved in vasomotor control of the cardiac and pulmonary circulation during early stages of postnatal development.