The purpose of this paper was to simultaneously examine changes in urothelial ATP and NO release in normal and spinal cord injured animals as well as in spinal cord injured animals treated with botulinum toxin type A (BoNT-A). Furthermore we correlated changes in transmitter release with functional changes in bladder contraction frequency, and determined the effects of BoNT-A on bladder efferent nerve function. Normal and spinal cord injured rat bladders were injected on day 0 with either vehicle (saline containing bovine serum albumin) or BoNT-A. On day 2, in vitro neurotransmitter release and bladder strip contractility studies as well as in vivo cystometrographic studies were conducted. Resting ATP release was significantly enhanced following spinal cord injury (i.e. 57% increase, p<0.05) and was unaffected by BoNT-A treatment. SCI increased hypoosmotic evoked urothelial ATP release by 377% (p<0.05). BoNT-A treatment reduced evoked ATP release in SCI bladders by 83% (p<0.05). In contrast, hypoosmotic stimulation induced NO release was significantly inhibited following SCI (i.e. 50%, p<0.05) but recovered in SCI rats treated with BoNT-A (i.e. 195% increase in NO release in SCI-BTX-treated rats compared to SCI controls, p<0.01). Changes in urothelial transmitter release coincided with a significant decrease in non-voiding bladder contraction frequency (i.e. 71%, p<0.05) in SCI-BTX rats compared to SCI rats. While no difference was measured between neurally evoked contractile amplitude between SCI and SCI-BTX animals, atropine (1 microM) inhibited contractile amplitude to a greater extent (i.e. 76%, p<0.05) in the SCI-BTX group compared to the SCI group. We hypothesize that alterations in the ratio of excitatory (i.e. ATP) and inhibitory (i.e. NO) urothelial transmitters promote bladder hyperactivity in rat bladders following SCI that can be reversed, to a large extent, by treatment with BoNT-A.

Withdrawal symptoms are a major deterrent when people try to quit smoking. The α7 subunit of the neuronal nicotinic acetylcholine receptor (nAChR) is highly expressed in the brain, and has been suspected to play a major role in nicotine addiction. We studied the influence of α7-containing nAChRs on nicotine withdrawal and tolerance, in wild type mice and mice null for the α 7 nAChR subunit (α7 −/−). For withdrawal experiments, animals were implanted with osmotic minipumps delivering nicotine for 13 days. A single intraperitoneal injection of the nAChR antagonists mecamylamine (MEC) or methyllycaconitine (MLA) was used to precipitate withdrawal. In wild type mice, both MEC and MLA precipitated somatic signs of withdrawal such as increased grooming, scratching and shaking. In α7 −/− mice, the somatic effects of MEC-precipitated nicotine withdrawal were significantly reduced. Interestingly, the presumed α7-specific antagonist MLA also precipitated withdrawal. Tolerance, which was measured as a decrease in nicotine-induced hypolocomotion after subchronic nicotine treatment, was normal in α7 −/− mice. Finally, because anxiety and withdrawal symptoms are highly correlated in humans, we studied anxiety-like behaviors in α7 −/− mice using a battery of anxiety-related tests. The behavior of α7 −/− mice was indistinguishable from that of control mice. Our results point to the α7 subunit as one of the players in nicotine withdrawal, but not in nicotine tolerance or basal anxiety-like behavior.

Smokers often report an anxiolytic effect of cigarettes. In addition, stress-related disorders such as anxiety, posttraumatic stress syndrome and depression are often associated with chronic nicotine use. To study the role of the a5 nicotinic acetylcholine receptor subunit in anxiety-related responses, control and a5 subunit null mice (a5 2/2 ) were subjected to the open field activity (OFA), light-dark box (LDB) and elevated plus maze (EPM) tests. In the OFA and LDB, a5 2/2 behaved like wild-type controls. In the EPM, female a5 2/2 mice displayed an anxiolytic-like phenotype, while male a5 2/2 mice were undistinguishable from littermate controls. We studied the hypothalamus-pituitary-adrenal axis by measuring plasma corticosterone and hypothalamic corticotropin-releasing factor. Consistent with an anxiolyticlike phenotype, female a5 2/2 mice displayed lower basal corticosterone levels. To test whether gonadal steroids regulate the expression of a5, we treated cultured NTera 2 cells with progesterone and found that a5 protein levels were upregulated. In addition, brain levels of a5 mRNA increased upon progesterone injection into ovariectomized wild-type females. Finally, we tested anxiety levels in the EPM during the estrous cycle. The estrus phase (when progesterone levels are low) is anxiolyticlike in wild-type mice, but no cycle-dependent fluctuations in anxiety levels were found in a5 2/2 females. Thus, a5-containing neuronal nicotinic acetylcholine receptors may be mediators of anxiogenic responses, and progesterone-dependent modulation of a5 expression may contribute to fluctuations in anxiety levels during the ovarian cycle.

BackgroundThe objective of our work was to investigate both the contractile function and the release of ATP and NO from strips of bladder tissue after removal of the urothelium.MethodsThe method of removal was a gentle swabbing motion rather than a sharp surgical cutting to separate the urothelium from the smooth muscle. The contractile response and ATP and NO release were measured in intact as well as on swabbed preparations. The removal of the urothelial layer was affirmed microscopically.ResultsAfter the swabbing, the smaller contractions were evoked by electrical as well as by chemical stimulation (50 μM carbachol or 50 μM α, β meATP). Electrical stimulation, carbachol and substance P (5 μM) evoked lower release of ATP in the swabbed strips than in intact strips. Although release of NO evoked by electrical stimulation or substance P was not changed, release of NO evoked by carbachol was significantly less in the swabbed preparations.ConclusionSince swabbing removes only the urothelium, the presence of the suburothelial layer may explain the difference between our findings and those of others who found an increase in contractility. Evoked release of ATP is reduced in swabbed strips, indicating that ATP derives solely from the urothelium. On the other hand, electrical stimulation and substance P evoke identical degrees of NO release in both intact and swabbed preparations, suggesting that NO can be released from the suburothelium. Conversely, carbachol-induced release of NO is lower in swabbed strips, implying that the cholinergic receptors (muscarinic or nicotinic) are located in the upper layer of the urothelium.

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