In the urinary bladder, the capsaicin-gated ion channel TRPV1 is expressed both within afferent nerve terminals and within the epithelial cells that line the bladder lumen. To determine the significance of this expression pattern, we analyzed bladder function in mice lacking TRPV1. Compared with wild-type littermates, trpv1(-/-) mice had a higher frequency of low-amplitude, non-voiding bladder contractions. This alteration was accompanied by reductions in both spinal cord signaling and reflex voiding during bladder filling (under anesthesia). In vitro, stretch-evoked ATP release and membrane capacitance changes were diminished in bladders excised from trpv1(-/-) mice, as was hypoosmolality-evoked ATP release from cultured trpv1(-/-) urothelial cells. These findings indicate that TRPV1 participates in normal bladder function and is essential for normal mechanically evoked purinergic signaling by the urothelium.

P ainful sensations induced by capsaicin, the pungent substance in hot peppers, are caused by stimulation of vanilloid receptor 1 (VR1), an ion channel protein expressed by nociceptive primary afferent neurons. VR1 also participates in the detection of at least two additional noxious stimuli, acid (pH Ͻ 6) and heat (Ͼ43°C). The urinary bladder is rich with capsaicinsensitive afferent fibers that detect bladder distension or the presence of irritant chemicals and in turn trigger reflex bladder activity. Here, we demonstrate that VR1 is expressed not only by afferent nerves that form close contacts with bladder epithelial (urothelial) cells but also by the urothelial cells themselves. We further show that exogenously applied vanilloids increase intracellular Ca 2ϩ and evoke NO release in urothelial cells and that these responses require VR1. These and other data suggest that urothelial cells work in concert with underlying afferent nerves to detect the presence of irritant stimuli.

Mitochondrial nitric oxide synthase (mtNOS), its cellular NOS isoform, and the effects of mitochondrially produced NO on bioenergetics have been controversial since mtNOS was first proposed in 1995. Here we functionally demonstrate the presence of a NOS in cardiac mitochondria. This was accomplished by direct porphyrinic microsensor measurement of Ca 2؉ -dependent NO production in individual mitochondria isolated from wild-type mouse hearts. This NO production could be inhibited by NOS antagonists or protonophore collapse of the mitochondrial membrane potential. The similarity of mtNOS to the neuronal isoform was deduced by the absence of NO production in the mitochondria of knockout mice for the neuronal, but not the endothelial or inducible, isoforms. The effects of mitochondrially produced NO on bioenergetics were studied in intact cardiomyocytes isolated from dystrophindeficient (mdx) mice. mdx cardiomyocytes are also deficient in cellular endothelial NOS, but overexpress mtNOS, which allowed us to study the mitochondrial enzyme in intact cells free of its cytosolic counterpart. In these cardiomyocytes, which produce NO beat-to-beat, inhibition of mtNOS increased myocyte shortening by approximately one-fourth. Beat-to-beat NO production and altered shortening by NOS inhibition were not observed in wildtype cells. A plausible mechanism for the reversible NO inhibition of contractility in these cells involves the reaction of NO with cytochrome c oxidase. This suggests a modulatory role for NO in oxidative phosphorylation and, in turn, myocardial contractility.cardiomyocytes ͉ cytochrome oxidase ͉ oxidative phosphorylation ͉ respiration ͉ chronoamperometry T he existence of a nitric oxide synthase (NOS) that is localized in the mitochondria (mtNOS) was originally described in a series of immunohistochemical studies published between 1995 (1, 2) and 1996 (3). Because all of the known NOS isoforms are encoded by nuclear DNA and NOS is not encoded by mitochondrial DNA, this finding implied that one of the recognized NOS isoforms was targeted to the mitochondria after protein synthesis in the cytosol. In these early studies, it was reported that the endothelial NOS (eNOS) isoform was localized to the inner mitochondrial membrane in all tissues that were tested, which included brain, kidney, liver, and skeletal and cardiac muscle. Between 1997 and 1998, more in-depth studies using a variety of NO detection techniques with isolated rat liver mitochondria (4), submitochondrial particles (SMPs; ref. 4), and purified NOS enzyme (5-7) added further support for the existence of a mtNOS. However, these studies were unable to determine whether the enzyme was novel or related to the neuronal (nNOS), inducible (iNOS), or eNOS isoforms. In the last three years, some laboratories have extended their studies to the investigation of the functional implications of mtNOS (8-11), while others have used a NO-sensitive dye to stain the mitochondria in intact cells and demonstrate the presence of NO within these organelles (12). Howeve...