NADPH diaphorase and nitric oxide synthase are expressed by the majority of intramural neurons in the neonatal guinea pig urinary bladder.

The distribution of neurons expressing NADPH diaphorase activity was examined histochemically in whole mount preparations of the neonatal guinea pig urinary bladder. NADPH diaphorase positive neurons were abundant in the intramural ganglia in both the detrusor and trigone regions of the bladder. Labelled nerve fibres were found in the ganglion interconnectives and in smooth muscle bundles. Mucosal epithelial cells and endothelial cells lining the blood vessels supplying the bladder were also found to express NADPH diaphorase activity. In order to verify that NADPH diaphorase activity represented the presence of nitric oxide synthase in bladder neurons, a well characterised tissue culture preparation was employed. This also provided an opportunity to estimate the proportion of the total population of bladder neurons which expressed NADPH diaphorase activity. Using a combination of histochemical and immunohistochemical techniques, NADPH diaphorase positive neurons were found to constitute approximately 90% of the total neuronal population, which was identified by labelling with an antiserum to the general neuronal marker protein gene product 9.5. Almost all neurons (99%) which expressed NADPH diaphorase activity in culture were also found to be immunoreactive for nitric oxide synthase. These findings indicate that nitric oxide may play a role in the neural control of bladder function, and this possibility is discussed.

NADPH-diaphorase is colocalized with nitric oxide synthase and vasoactive intestinal polypeptide in rat pancreatic neurons in culture.

NADPH-diaphorase activity together with nitric oxide synthase- and vasoactive intestinal polypeptide-immunoreactivities were examined in the neurons of the rat pancreas in culture. Nitric oxide synthase (NOS)- and vasoactive intestinal polypeptide (VIP)-immunoreactivities were localized in a subpopulation of neurons which were also NADPH-diaphorase positive. All neurons expressing colocalized activities for NOS/VIP and NADPH-diaphorase were solitary cells in culture. Each of these double-labelled neurons was characterized by a round or oval cell body, with short and long processes emanating from it. Some of these processes traversed several micrometres before terminating on the exocrine acinar and endocrine cells. The present study provides evidence that nitric oxide may act as a regulatory agent in the inhibitory neural control of the secretory functions in the pancreas, thereby maintaining the milieu interieur of the organism.

Secretion of atrial and brain natriuretic peptides from human cardiac atrial explants in culture: effect of dynorphin.

We have previously developed a method for maintaining human cardiac explants in culture under serum-free conditions, for the assessment of cardiac endocrine function and myocardial growth factors. In order to assess the local role of dynorphin in the human heart, we studied the effects of dynorphin on the secretion of atrial natriuretic peptide and brain natriuretic peptide by human cardiac atrial explants. Dynorphin did not affect the basal secretion of brain natriuretic peptide, but clearly enhanced the release of atrial natriuretic peptide from the human cardiac explants in culture. The atrial content of brain natriuretic peptide was not significantly reduced, whereas the atrial content of atrial natriuretic peptide in cultured explants was reduced two-fold in the presence of dynorphin. These findings indicate that dynorphin may have a direct stimulatory effect on the release of atrial natriuretic peptide, but not brain natriuretic peptide, from human cardiac atria.

Colocalisation of NADPH-diaphorase with nitric oxide synthase and vasoactive intestinal polypeptide in newborn pancreatic neurons.

Numerous NADPH-diaphorase positive neurons were localised in the pancreatic ganglia cultured from newborn guinea pigs. Most of these ganglia were found in the head region of the pancreas. A majority of the labelled neurons occurred in groups, although solitary neurons were also present. NADPH-diaphorase labelling was localised mainly in the cell cytoplasm and its processes, but not within the nucleus. There were more nitric oxide synthase (NOS)-immunoreactive neurons in culture as compared with those positive for NADPH-diaphorase. NADPH-diaphorase activity has been colocalised with NOS and vasoactive intestinal polypeptide (VIP) in a subset of neurons in culture. Colocalisation of NADPH-diaphorase with NOS and VIP in cultured neurons was not restricted to the cell body but also to the proximal and distal processes. Some of these labelled processes made contacts with exocrine acinar and endocrine cells. It is concluded that neuronal nitric oxide (NO) may be influencing the secretory roles of the pancreas, thereby participating in homeostasis.

Expression of insulin-like growth factor-1 by human ventricular explants in culture.

There is increasing evidence from animal studies for autocrine and paracrine systems in the heart. As the local role of these peptide systems in the human heart is not clear, we assessed cardiac explants in culture as a model for the study of growth factor expression by the heart in humans. Human right ventricular septal biopsies were maintained as explants for up to 9 days in serum-supplemented medium. Insulin-like growth factor-1 (IGF-1) expression was measured by radioimmunoassay and results corrected for tissue protein content. IGF-1 was synthesised by ventricular explants but not secreted into the culture medium. This culture model has the potential to provide insight into the regulation of and response to growth factor expression by the heart in humans.

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.