M.J. Saffrey

Colocalization of nitric oxide synthase and NADPH-diaphorase in the myenteric plexus of the rat gut

The pattern of distribution and colocalization of nitric oxide-synthase (NOS) and NADPH-diaphorase in the myenteric plexus of whole-mount preparations of the antrum, duodenum, ileum, caecum, proximal colon and distal colon of the rat were investigated using immunohistochemical and histochemical staining techniques. Almost all the myenteric neurons that were NOS-positive in all regions of the gut examined were also stained for NADPH-diaphorase. However, in the stomach, duodenum and ileum, only a few of the NOS-positive nerve fibres in the tertiary and secondary plexuses and circular muscle layer were also stained for NADPH-diaphorase, whereas in the caecum and distal colon almost all the NOS-positive nerve fibres were also stained for NADPH-diaphorase. The results in the present study are consistent with the view that nitric oxide (NO) has a mediating role in gastrointestinal neurotransmission.

Modulation of astroglial cell proliferation by analogues of adenosine and ATP in primary cultures of rat striatum.

We have studied the possible purinoceptor-mediated modulation of astroglial cell proliferation in neuron-glia primary cultures obtained from rat corpus striatum. Cultures were grown for three days in the presence of either 2-chloro-adenosine or alpha beta-methylene-ATP (which behave as agonists of adenosine/P1 and ATP/P2 purinoceptors, respectively), and then immunostained with an antibody to glial fibrillary acidic protein. 2-Chloro-adenosine decreased and alpha beta-methylene-ATP increased the number of astroglial cells in culture. For both derivatives, the effect was dose-dependent. The effect of alpha beta-methylene-ATP was antagonized by the trypanoside suramin, suggesting the involvement of a suramin-sensitive P2 purinoceptor, whereas the effect of 2-chloro-adenosine was not reversed by the P1 purinoceptor antagonist p-sulphonyl-phenyl-theophylline, implying the activation of a xanthine-insensitive adenosine purinoceptor subtype. In order to evaluate the extent of astrocyte proliferation in the presence of these two analogues, some cultures were incubated with bromodeoxyuridine for 24 h before fixing, and then double-immunostained for glial fibrillary acidic protein and bromodeoxyuridine. The percentage of bromodeoxyuridine positive astrocytes was significantly increased after exposure to both agents. It is therefore concluded that purines can modulate astroglial cells in opposite ways, inducing decreases or increases of cell number by activation of P1 and P2 purinoceptors, respectively. For the P2 purinoceptor-mediated effect, there was a quantitative correlation between the percentage of bromodeoxyuridine positive astrocytes and the cell number. For the P1 purinoceptor-mediated effect, no apparent correlation between these two parameters was found. This suggests the activation of independent effects, which involve other mechanisms besides the stimulation of DNA synthesis, and which eventually result in a reduction of cell number. The possible relevance of these findings to in vivo regulation of astrocyte cell function as well as in trauma- and ischaemia-associated hypergliosis is discussed.

Nitric oxide synthase immunoreactivity and NADPH-diaphorase activity in a subpopulation of intrinsic neurones of the guinea-pig heart

This is the first report of the presence of nitric oxide synthase (NOS) immunoreactivity and NADPH-diaphorase (NADPH-d) activity in a subpopulation of the intrinsic neurones that innervate the heart. A cytochemical technique to detect NADPH-d and antisera raised against purified rat cerebellar NOS were employed to examine the expression of these enzymes by cells in a dissociated cell culture preparation from newborn guinea-pig atria and interatrial septum. Comparison of the results obtained by these two techniques and double-labelling experiments indicate that a subpopulation of intracardiac neurones contain both NADPH-d and NOS. These results indicate that some intracardiac neurones are capable of synthesizing nitric oxide. This raises the possibility that nitric oxide plays a role in the neural control of the heart.

Colocalization of nitric oxide synthase and NADPH-diaphorase in cultured myenteric neurones.

Nitric oxide synthase immunoreactivity and NADPH-diaphorase activity were examined in explant culture preparations of the myenteric plexus from beneath the taenia coli of the guinea-pig caecum. Nitric oxide synthase immunoreactive neurones formed approximately one third of the total neuronal population. NADPH-diaphorase positive neurones, demonstrated histochemically, constituted a similar proportion of the total number of neurones. Immunocytochemistry and NADPH-diaphorase histochemistry performed on the same preparations revealed that all nitric oxide synthase immunoreactive neurones expressed NADPH-diaphorase activity. This histochemical evidence is consistent with the view that nitric oxide may act as a regulatory agent in the guinea-pig caecum.

Increase in SP-like immunoreactivity in nerve fibres of rabbit iris and ciliary body one to four months following sympathetic denervation

Substance P (SP) levels in rabbit iris-ciliary body preparations were found to be significantly elevated 30 days after removal of the superior cervical ganglion when compared with the contralateral unoperated side. These elevated levels were still apparent after two and three months. Immunohistochemical studies, using a monoclonal antibody to SP, revealed an increase in the density of SP-immunoreactive nerve fibres in the region of the iris dilator muscle and in the ciliary processes one to four months after sympathectomy, although the density of these fibres in other regions of the iris was not altered.

Distribution of vasoactive intestinal polypeptide-, substance P-, enkephalin-and neurotensin-like immunoreactive nerves in the chicken gut during development

The ontogeny and distribution of nerve cell bodies and fibres which contain vasoactive intestinal polypeptide-, substance P-, enkephalin- and neurotensin-like immunoreactivity have been studied in the chicken gastrointestinal tract, using immunocytochemistry. All four peptides were found in nerve fibres, with characteristic distribution patterns, which, in the cases of vasoactive intestinal polypeptide, substance P and methionine enkephalin were similar to those described for the mammalian gut. In addition, many of these fibres were shown to arise from intrinsic neurons, since immunoreactive nerve cell bodies for each of the peptides studied were observed. Neurotensin-immunoreactive nerves were confined to the upper part of the tract and neurotensin immunoreactive cell bodies were only observed in embryonic and newly hatched chicken gut. All four peptides were first observed at 11 days of incubation, or Hamburger-Hamilton stage 37, 20 in the upper part of the tract, particularly in the gizzard. Substance P and methionine enkephalin were subsequently seen in more caudal regions, while vasoactive intestinal polypeptide developed from each end of the tract. Adult patterns of immunoreactivity in nerve fibres were achieved during the first week after hatching. A striking observation was that immunoreactive neuronal cell bodies were much more abundant in the gut of young chickens and chicken embryos than in that of adult birds.

EFFECTS OF ATP ANALOGUES AND BASIC FIBROBLAST GROWTH FACTOR ON ASTROGLIAL CELL DIFFERENTIATION IN PRIMARY CULTURES OF RAT STRIATUM

We have used primary cultures of rat striatum to study the effects of ATP analogues on the elongation of astrocytic processes, a parameter of astroglial cell differentiation. Parallel studies were performed with basic fibroblast growth factor, a known regulator of astroglial cell function. After three days in culture, both the growth factor and αβ‐methylene‐ATP induced dramatic increases in the mean length of astrocytic processes/cell. For both agents, effects were dose‐dependent. The effect of αβ‐methylene‐ATP was antagonized by the trypanoside suramin and mimicked by 2‐methyl‐thio‐ATP, suggesting the involvement of a suramin‐sensitive P2‐purinoceptor. Neither an additive nor a synergistic effect between αβ‐methylene‐ATP and basic fibroblast growth factor on the elongation of processes was detected in cultures exposed to both agents. Indeed, an inhibition with respect to the effects induced by either agent alone was recorded, suggesting that the growth factor and the purine analogue can modulate astrocytic differentiation by activation of common intracellular pathways.

Trophic actions of 2-chloroadenosine and bFGF on cultured myenteric neurones.

The effects of the stable adenosine analogue, 2-chloroadenosine (2-CA) and basic fibroblast growth factor (bFGF) on myenteric neurones in dissociated cell culture were examined. 2-CA had no effect on neuronal numbers, but increased neurite length, in a dose-dependent manner. bFGF increased both the number of myenteric neurones and neurite length. When 2-CA was applied together with bFGF, an enhanced increase in neurite outgrowth, but no additional increase in neuronal numbers was observed. 2-CA-induced effects were blocked by the adenosine antagonist 8-(p-sulphophenyl)theophylline. These results show, for the first time, that both purines and bFGF may have trophic actions on myenteric neurones and also indicate that purines enhance some effects of bFGF, in a synergistic manner.

Distribution of neurons with high-affinity uptake sites for GABA in the myenteric plexus of the guinea-pig, rat and chicken

SummaryThe occurrence of neurons possessing high-affinity uptake sites for GABA was studied in the myenteric plexus of the guinea-pig ileum, caecum, and proximal and distal colon, the rat proximal colon, and the chicken gizzard with the use of 3H-GABA and autoradiography. Experiments were carried out on plexuses that had been freshly isolated from the gut wall or on isolated plexuses that had been maintained as expiant cultures for 7 to 14 days. Scattered neurons selectively labelled with 3H-GABA were found in the myenteric plexuses from all the areas examined. The results suggest that GABAergic neurons are widely distributed in the enteric nervous system.

Expression of NADPH-diaphorase activity by guinea-pig paratracheal neurones.

Pharmacological evidence suggests that nitric oxide (NO) is involved in non-adrenergic, non-cholinergic, nerve mediated responses seen in guinea-pig trachealis muscle. The synthetic enzyme for NO (NO synthase) has recently been shown to be responsible for neuronal NADPH-diaphorase activity. Therefore, to determine whether intrinsic paratracheal neurones could be a source of NO in the trachea, expression of NADPH-diaphorase activity was examined histochemically using whole mount preparations of the tracheal plexus. Many paratracheal neurones were found to express moderate to high levels of NADPH-diaphorase activity and are thus likely to be a source of NO in this tissue. This observation provides further evidence that NO is involved in the regulation of relaxation in airway smooth muscle.