P. Milner

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.

Endothelial Cells Cultured from Human Umbilical Vein Release ATP, Substance P and Acetylcholine in Response to Increased Flow

Cultured human umbilical vein endothelial cells superfused with Krebs’ solution were used to investigate release of ATP, substance P and acetycholine with shear stress. ATP was consistently released when the cells were exposed to increased flow rate; release was rapid, had declined by 1 min and occurred upon a second exposure. Release of substance P and acetylcholine was more varied; increased shear stress led to release of substance P from 4 out of 16 endothelial-cell columns, whereas acetylcholine was released in 4 out of 12 columns. This is the first time that unequivocal evidence has been presented for release of these neurotransmitter substances from vascular endothelial cells. These findings have important implications about the mechanisms of local regulation of vascular tone.

Ultrastructural localisation of substance P and choline acetyltransferase in endothelial cells of rat coronary artery and release of substance P and acetylcholine during hypoxia.

Substance P and choline acetyltransferase have been localised in a small proportion of endothelial cells of rat coronary arteries using electron microscopic immunocytochemistry. During a hypoxic period of 1 min, coronary vasodilatation was produced in the Langendorff heart preparation and increased levels of substance P and acetylcholine were released into the perfusate. The possibility that these substances are released from endothelial cells during hypoxia and contribute to the hyperaemic response is discussed.

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.

Vasoactive intestinal polypeptide levels in sigmoid colon in idiopathic constipation and diverticular disease

The distribution in the bowel wall of vasoactive intestinal polypeptide-, neuropeptide Y-, and substance P-containing nerve cell bodies and nerve fibers has been described in human sigmoid colon by immunohistochemical examination. In patients with chronic idiopathic constipation, diverticular disease, and in controls (of tissue taken from patients with carcinoma, from a site distant from the tumor that appeared macroscopically normal), the concentrations of vasoactive intestinal polypeptide, neuropeptide Y, and substance P have been measured by immunoassay in the following preparations of sigmoid colon: mucosa, whole colonic wall with mucosa dissected away, circular muscle, and taenia coli. In idiopathic constipation, the vasoactive intestinal polypeptide content of the whole wall minus mucosa was reduced when compared with controls (P less than 0.05) but was unaltered in the mucosa, circular muscle, and taenia coli. In diverticular disease, the vasoactive intestinal polypeptide content of the mucosa and whole wall minus the mucosal layer was increased when compared with control tissue (P less than 0.05 and P less than 0.02, respectively) but was unaltered in the circular muscle and taenia coli. Substance P and neuropeptide Y levels in all layers of colonic wall were unaltered in these two diseases. The disturbances in the normal neural content of vasoactive intestinal polypeptide in the bowel wall in idiopathic constipation and diverticular disease may initiate or contribute to the functional changes seen in these disorders.

Progressive changes in adrenergic, serotonergic, and peptidergic nerves in proximal colon of streptozotocin-diabetic rats

The effect of progression of diabetes on adrenergic, serotonergic, and peptidergic innervation of the proximal colon of the rat at 8, 16, and 25 wk after induction of diabetes with streptozotocin was investigated using immunohistochemical, biochemical, and immunochemical methods. Two different responses to diabetes emerged from the present study. The first response, which involves noradrenaline and vasoactive intestinal peptide, was characterized by a sign of degeneration, where there was an initial increase in tissue level and immunoreactivity of the transmitters followed by a decrease in tissue level and density of nerve fibers at 16 and 25 wk after induction of diabetes. The second response, which involves 5-hydroxytryptamine, substance P, and calcitonin gene-related peptide, was characterized by changes in tissue level and immunoreactivity of the transmitters with no evidence of degeneration. The third feature was one of resistance to change due to diabetes, which was demonstrated by neuropeptide Y-containing nerves, where there was neither a change in tissue level of neuropeptide Y nor a change in immunoreactivity. It seems likely that the overall changes described will have profound implications in the function of the gut in the streptozotocin-diabetic rat model that may have some parallels in diabetic humans.

Marked increases in calcitonin gene-related peptide-containing nerves in the developing rat following long-term sympathectomy with guanethidine

Changes in the innervation of the cardiovascular system, urinogenital tract and sympathetic and non-sympathetic ganglia have been examined following long-term sympathectomy. Patterns of innervation were investigated using histochemical and immunohistochemical techniques, while levels of noradrenaline and neuropeptides were measured by neurochemical assays. Large doses of guanethidine (50 mg/kg) were given daily for 3 weeks to 8-day-old rat pups, which were killed at 6 or 20 weeks of age. In both age groups noradrenergic nerves were severely depleted or absent, while in some regions dramatic increases of calcitonin gene-related peptide levels were demonstrated. This was revealed by an increase in the density of nerve fibres and in calcitonin gene-related peptide content (up to 18-fold), most notably in the right atrium and superior cervical ganglion. No changes in substance P- or vasoactive intestinal polypeptide-immunolabelled nerves were seen. Conversely, short-term sympathectomy by 6-hydroxy-dopamine treatment caused a depletion of noradrenaline which was not accompanied by an increase in the number or content of calcitonin gene-related peptide-immunolabelled nerves. The possibility that nerve growth factor is involved in the mechanism of hyperinnervation by calcitonin gene-related peptide-containing sensory nerves following long-term sympathectomy is discussed.

Substance P is released from the endothelium of normal and capsaicin-treated rat hind-limb vasculature, in vivo, by increased flow.

The rat hind-limb vasculature releases substance P when subjected to a rapid increase in flow through the vascular bed. This release also occurs during high flow after rats have been capsaicinized, when loss of substance P-containing nerve fibers was verified by immunohistochemistry. Air treatment, a procedure shown by transmission electron microscopy to have removed endothelial cells from the arteries but not arterioles or capillaries of the hind-limb preparations, eliminated this release. Thus, the substance P released is unlikely to arise from perivascular nerves but rather from arterial endothelial cells.

Increased density of perivascular nerves to the major cerebral vessels of the spontaneously hypertensive rat: differential changes in noradrenaline and neuropeptide Y during development.

Fluorescence and immunohistochemical techniques were used to study the pattern and density of perivascular nerves containing noradrenaline (NA) and neuropeptide Y (NPY) supplying the major cerebral arteries of 4-, 6-, 8- and 12-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar (WIS) controls. Levels of NA and NPY in the superior cervical ganglia were measured. The density of nerves containing NA and NPY was greater in the hypertensive animals at all ages studied. However, the developmental changes in the density of innervation showed similar trends in both SHR and WIS groups. With few exceptions, there was a significant increase in the density of nerves containing NA from 4 to 6 weeks and from 8 to 12 weeks of age. This was in contrast to a low expression, and in some vessels a significant decrease in the number of NPY-containing nerves from 4 to 6 weeks. The density of nerve fibres containing NPY increased significantly in almost all vessels between 6 and 8 weeks of age and then stabilized. Thus there is a differential time course for the appearance of NA and NPY during development. Furthermore, the hyperinnervation of cerebral vessels in SHR by nerves containing NA and NPY precedes the onset of hypertension and associated medial hypertrophy. High-performance liquid chromatography and enzyme-linked immunosorbant assays show that the NA and NPY contents of the superior cervical ganglion do not reflect the changes in innervation pattern seen in the terminal fibres in the cerebral arteries. This tends to support the view that a local neurovascular mechanism is involved in the maintenance of hypertension. The possibility that increase in NPY as well as NA in cerebral perivascular nerves of hypertensive animals is involved in the protection of the blood-brain barrier against oedema and cerebral haemorrhage is raised.

Differential effect of streptozotocin-induced diabetes on the innervation of the ileum and distal colon

The effect of short-term and long-term streptozotocin-induced diabetes on the pattern of distribution and tissue content of adrenergic and peptidergic nerves in ileum and distal (descending) colon of the rat was examined using immunohistochemical, biochemical, and immunochemical techniques. The effect of short-term streptozotocin-induced diabetes on the level of noradrenaline compared with weight-restricted (starved) and untreated controls in the celiac (celiac-superior mesenteric ganglia complex) and inferior mesenteric ganglia, which supply the two regions of the intestine, was also compared. The pattern of change in the distribution of dopamine-beta-hydroxylase-, substance P-, calcitonin gene-related peptide-, and vasoactive intestinal polypeptide-like immunoreactive nerve fibres that was observed in the ileum from diabetic rats was not evident in the myenteric plexus of distal colon. In contrast to the ileum, there was no evidence of degenerative change in any of the nerve types investigated in the myenteric plexus of the distal colon. The level of vasoactive intestinal polypeptide in the diabetic rat ileum was significantly increased, whereas the level of noradrenaline was reduced; no such changes were observed in the distal colon. The tissue content of noradrenaline in the celiac ganglion, which projects to the ileum, was increased at 8-week diabetes compared with both weight-restricted and untreated controls, whereas the diabetic state had no effect on the levels of noradrenaline of the inferior mesenteric ganglion, which projects to the distal colon. It is concluded that there is a differential effect of streptozotocin-diabetes on different regions of the rat intestine. The adrenergic and peptidergic innervation of the distal colon were changed little compared with ileum. This may be explainable in terms of the different functional roles of these two regions of the intestine and/or by the difference in origin of the sympathetic nerves supplying the two regions of the intestine.