NITRERGIC REACTIVITY IN NEURONS INNERVATING THE URINARY BLADDER IN NORMAL AND URETHRAL OBSTRUCTED GUINEA PIGS

Abstract

Nitrergic reactivity in neurons innervating the urinary bladder in guinea pigs and its alteration following complete urethral obstruction have been studied by using nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry and nitric oxide synthase (NOS) immunohistochemistry. NADPH-d positive and NOS immunoreactive neurons were localized in the intramural ganglia of stretched wholemount preparations of the urinary bladder which was divided into 3 regions: base, body and dome. The highest frequency both of NADPH-d and NOS positive neurons was observed in the bladder base. Cell counts showed that the number of NADPH-d positive neurons was significantly more than that of NOS immunoreactive neurons in the urinary bladder. Using neuron specific enolase (NSE) positive neurons as a reference (100%), NADPH-d positive neurons accounted for 84% while NOS immunoreactive neurons only made up 45% of the total neuronal population (3260±421) of the whole bladder. The significant difference in the number of NADPH-d positive and NOS immunoreactive neurons suggests that the localization of one enzyme does not necessarily reflect the presence of the other. These results suggest that nitric oxide (NO) may be involved in the relaxation activity in the bladder base during micturition. As a step to further determine the relationship between NOS and NADPH-d in the urinary bladder, double labelling study was then carried out to ascertain the colocalization of the two enzymes in the intramural ganglion cells. The localization of NADPH-d with tyrosine hydroxylase (TH), a marker for sympathetic postganglionic neurons, and acetylcholinesterace (AChE), a marker for parasympathetic postganglionic neurons was also studied; other neuronal markers such as vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP) and substance P (SP) were also examined. The results showed that NADPH-d and NOS were colocalized in the majority of the neurons in the urinary bladder. Some NADPH-d positive neurons were NOS negative. AChE reactivity was present in the majority of the intramural ganglion cells with 54% of them expressing NOS immunoreactivity (-IR). TH positive neurons were distributed mainly in the bladder base, especially in the trigone. It is noteworthy that TH-IR was never colocalized with NADPH-d reactivity. VIP-IR was detected in a quarter (28%) of intramural ganglion cells, some of them also exhibited NADPH-d reactivity. CGRP and SP were localized only in the nerve fibres either surrounding the neuronal soma stained for NADPH-d or extending along NADPH-d positive fibres to link with other ganglia. Positively stained CGRP and SP fibres often coursed along the wall of blood vessels. Present results have shown that while NADPH-d and NOS may be colocalized in the intramural ganglion cells, a one to one correlation between these two enzymes was not always present. In view of their contents of AChE, it is suggested that the nitrergic neurons are parasympathetic postganglionic neurons. They are clearly distinct from TH immunoreactive neurons which were consistently unreactive for NADPH-d and which were considered to be sympathetic postganglionic neurons. These results suggest that NO is involved in regulating the parasympathetic but not sympathetic neural activity in the urinary bladder. Nitrergic neurons also expressed VIP-IR