C. J. S. Hassall

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.

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.

Neuropeptide Y-like immunoreactivity in cultured intrinsic neurones of the heart

A novel culture preparation from the atria of newborn guinea-pig hearts was employed to study the intrinsic innervation of the heart under conditions of unequivocal extrinsic denervation. Using an indirect immunofluorescence technique, a subpopulation of intracardiac neurones grown in dissociated cell culture was demonstrated to contain neuropeptide Y (NPY)-like immunoreactivity. This shows that NPY is not confined to sympathetic nerves. Further, since no endogenous catecholamines could be demonstrated in neurone cell bodies in the culture preparation with fluorescence histochemistry, confirming previous studies in situ, the findings also suggest that NPY does not coexist with catecholamine in these intrinsic heart neurones.

Distribution of intracardiac neurones and nerve terminals that contain a marker for nitric oxide, NADPH-diaphorase, in the guinea-pig heart

There is strong evidence that NADPH-diaphorase can be used as a marker for neurones that employ nitric oxide as a messenger molecule. In the present study, the NADPH-diaphorase activity of intracardiac neurones and nerve terminals in whole-mount stretch preparations and sections of the newborn and adult guinea-pig atria and interatrial septum has been examined histochemically. Together with epicardial, endothelial and endocardial cells, which displayed some NADPH-diaphorase staining, a subpopulation of intracardiac neurones exhibited moderate-heavy labelling for NADPH-diaphorase, while the majority of neurones were only lightly stained or negative. Intracardiac ganglia containing positive neuronal cell bodies were located between the epicardial cells and atrial myocytes in four main regions: in association with the superior and inferior vena cavae, the points of entry of the pulmonary veins, and within the interatrial septum. Nerve terminals exhibiting NADPH-diaphorase activity were seen throughout the atrial tissue, forming basket-like endings around intracardiac neuronal cell bodies; varicose terminals were also observed on atrial myocytes and other non-neuronal structures. A proportion of the nerve fibres was clearly of intrinsic origin, other terminals may well have originated from neuronal cell bodies present outside the heart.

Intrinsic neurones and associated cells of the guinea-pig heart in culture

This paper describes a method for dissociation of intrinsic neurones from the atria and interatrial septum of newborn guinea-pig heart and their maintenance in culture. The appearance of the cultured intracardiac neurones, muscle and other non-neuronal cell types also present in the preparation has been observed by phase-contrast microscopy. Some of the neurochemical properties of the intracardiac neurones in culture have been investigated using histochemical methods. All the neurones studied were shown to contain acetylcholinesterase. No catecholamine-containing neurones were found. Using an indirect immunofluorescence technique, 20-50% of clearly identifiable neurones in culture contained neuropeptide Y-like immunoreactivity. Vasoactive intestinal polypeptide-like immunoreactive neurones were found in only one out of 15 culture preparations; no substance P-, neurotensin-, or enkephalin-like immunoreactivity was observed. These findings are consistent with those described for intracardiac neurones studied in situ, suggesting that the neurochemical differentiation of the intrinsic heart neurones is retained in culture. The culture preparation provides an opportunity to study the properties and role of intrinsic neurones of the heart. The characteristics of the intracardiac neurones may be distinguished from those of the extrinsic nerve fibres which degenerate in culture. Further, the intracardiac neurones are more accessible to experimental manipulation in culture than in situ.

Co-expression of four muscarinic receptor genes by the intrinsic neurons of the rat and guinea-pig heart

Expression of the messenger RNAs encoding the five different muscarinic acetylcholine receptor subtypes was examined in intracardiac neurons from the rat and guinea-pig heart by in situ hybridization techniques. Newborn guinea-pig intracardiac neurons were studied in dissociated cell culture preparations employing both 35S- and digoxigenin-labelled oligonucleotide probes specific for the m1, m2, m3, m4 or m5 muscarinic receptor messenger RNAs. When 35S-tailed oligonucleotides were used, all intracardiac neurons in culture were found to express m1, m2, m3 and m4, but not m5 messenger RNAs. However after hybridization with digoxigenin-tailed probes, only m1 and m2 transcripts were detected. This may reflect differences in the sensitivity of the two techniques. Further to these experiments, intracardiac ganglia in sections of adult rat heart were studied employing m1-, m2-, m3- or m4-specific, 35S-labelled oligonucleotides, and again, all intracardiac neurons expressed messenger RNA for each of these four muscarinic receptor subtypes. Atrial myocytes in culture were only labelled by [35S]- and digoxigenin-tailed m2 oligonucleotides. No other heart cell type seen expressed messenger RNA for any of the muscarinic receptors. The expression of four different muscarinic receptor transcripts by intrinsic neurons of the heart provides the molecular basis for the diverse muscarinic actions observed in these and other autonomic ganglia.

Immunocytochemical localisation of neuropeptide Y and 5-hydroxytryptamine in a subpopulation of amine-handling intracardiac neurones that do not contain dopamine β-hydroxylase in tissue culture

The colocalisation of neuropeptide Y (NPY)- and 5-hydroxytryptamine (5-HT)-immunoreactivities in intracardiac neurones in dissociated cultures from the atria and interatrial septum of newborn guinea pig heart was demonstrated by the sequential application of specific antisera which were visualised by two different fluorochromes. In this way it was observed that most 5-HT-immunoreactive neurones also contained NPY-immunoreactivity (approximately 40% of identified neurones), some neurones were 5-HT-immunoreactive alone (approximately 10%), while neurones that were NPY-immunoreactive only were rarely seen. No dopamine beta-hydroxilase (DBH)-immunoreactive intracardiac neurones were demonstrated in any of the culture preparations studied, although DBH-immunoreactive neurons could be detected in sections of the newborn guinea pig heart containing intracardiac ganglia. The possible implications of the colocalisation of 5-HT, that has been taken up from the culture medium, with NPY in a population of intracardiac neurones are discussed; and reasons for the loss of expression of DBH by these neurones under the conditions of culture are considered.

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.

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.