Mucosal modulation of contractility in bladder strips from normal and overactive rat models and the effect of botulinum toxin A on overactive bladder strips.

AIMS: To investigate the local, regulatory role of the mucosa on bladder strip contractility from normal and overactive bladders and to examine the effect of botulinum toxin A (BoNT-A). METHODS: Bladder strips from spontaneously hyperactive rat (SHR) or normal rats (Sprague Dawley, SD) were dissected for myography as intact or mucosa-free preparations. Spontaneous, neurogenic and agonist-evoked contractions were investigated. SHR strips were incubated in BoNT-A (3 h) to assess effects on contractility. RESULTS: Spontaneous contraction amplitude, force-integral or frequency were not significantly different in SHR mucosa-free strips compared with intacts. In contrast, spontaneous contraction amplitude and force-integral were smaller in SD mucosa-free strips than in intacts; frequency was not affected by the mucosa. Frequency of spontaneous contractions in SHR strips was significantly greater than in SD strips. Neurogenic contractions in mucosa-free SHR and SD strips at higher frequencies were smaller than in intact strips. The mucosa did not affect carbachol-evoked contractions in intact versus mucosa-free strips from SHR or SD bladders. BoNT-A reduced spontaneous contractions in SHR intact strips; this trend was also observed in mucosa-free strips but was not significant. Neurogenic and carbachol-evoked contractions were reduced by BoNT-A in mucosa-free but not intact strips. Depolarisation-induced contractions were smaller in BoNT-A-treated mucosa-free strips. CONCLUSIONS: The mucosal layer positively modulates spontaneous contractions in strips from normal SD but not overactive SHR bladder strips. The novel finding of BoNT-A reduction of contractions in SHR mucosa-free strips indicates actions on the detrusor, independent of its classical action on neuronal SNARE complexes.

Identification of PDGFRα positive populations of interstitial cells in human and guinea pig bladders.

PURPOSE: The bladder wall comprises a complex array of cells, including urothelium, smooth muscle, nerves and interstitial cells. Interstitial cells have several subtypes based on site, morphology and differential expression of markers such as anti-vimentin and anti-KIT. We examined whether a subpopulation of interstitial cells immunopositive for PDGFRα exists in human and guinea pig bladders. MATERIALS AND METHODS: Human and guinea pig bladder tissues were processed for immunohistochemistry and examined by bright field or confocal microscopy. Whole mount tissues and paraffin sections were labeled with antibodies to PDGFRα, vimentin, KIT and PGP9.5. Protein expression was assessed by Western blot. RESULTS: PDGFRα(+) cells were present in human and guinea pig bladders. In the guinea pig PDGFRα(+) cells had a branched stellate morphology and formed networks in the lamina propria. In human and guinea pig detrusors PDGFRα(+) cells were elongated on the boundary of smooth muscle bundles or were seen as groups of stellate cells in the interbundle spaces. PDGFRα(+) cells were located close to nerves labeled by PGP9.5. Double labeling revealed that PDGFRα(+) cells were a subgroup of the vimentin(+) population. A significant proportion of PDGFRα(+) cells were also KIT(+). Bands corresponding to PDGFRα, KIT and vimentin proteins were detected on Western blot. CONCLUSIONS: To our knowledge this study is the first to identify PDGFRα(+)/KIT(+) cells in the bladder lamina propria and detrusor layers. These cells are a subgroup of the vimentin(+) population, showing the complexity of bladder interstitial cells. PDGFRα(+) cells are apparently structurally associated with intramural nerves, indicating integration with bladder control mechanisms.

GABAergic control of arteriolar diameter in the rat retina.

PURPOSE: To investigate the role of γ-aminobutryic acid (GABA) in the regulation of arteriolar diameter in the rat retina. METHODS: The actions of GABA on arteriolar diameter were examined using ex vivo retinal whole-mount preparations and isolated vessel segments. In most experiments, arterioles were partially preconstricted with endothelin (Et)-1. The expression levels of GABAA and GABAB receptors on isolated rat retinal Müller cells were assessed by immunohistochemistry. RESULTS: GABA (0.1-1 mM) evoked vasodilation or vasoconstriction of arterioles in whole-mount preparations. No such effects were observed with isolated vessel segments. In whole mount samples, the GABAA receptor agonist muscimol caused vasomotor responses in only a small proportion of vessels. In contrast, arteriolar responses to the GABAB receptor agonists baclofen and SKF97541 more closely resembled those observed with GABA. No responses were seen with the GABAC receptor agonist 5-methylimidazoleacetic acid. GABA-induced vasodilator responses were, for the most part, repeatable in the presence of the GABAA receptor antagonist bicuculline. These responses, however, were completely blocked in the presence of the GABAB receptor inhibitor 2-hydroxysaclofen. Strong immunolabeling for both GABAA and GABAB receptors was detected in isolated Müller cells. In the absence of Et-1-induced preconstriction, most vessels were unresponsive to bicuculline or 2-hydroxysaclofen. CONCLUSIONS: GABA exerts complex effects on arteriolar diameter in the rat retina. These actions appear largely dependent upon the activation of GABAB receptors in the retinal neuropile, possibly those located on perivascular Müller cells. Despite these findings, endogenous GABA appears to contribute little to the regulation of basal arteriolar diameter in the rat retina.

Nucleotide regulation of the voltage-dependent nonselective cation conductance in murine colonic myocytes.

ATP is proposed to be a major inhibitory neurotransmitter in the gastrointestinal (GI) tract, causing hyperpolarization and smooth muscle relaxation. ATP activates small-conductance Ca(2+)-activated K(+) channels that are involved in setting the resting membrane potential and causing inhibitory junction potentials. No reports are available examining the effects of ATP on voltage-dependent inward currents in GI smooth muscle cells. We previously reported two types of voltage-dependent inward currents in murine proximal colonic myocytes: a low-threshold voltage-activated, nonselective cation current (I(VNSCC)) and a relatively high-threshold voltage-activated (L-type) Ca(2+) current (I(L)). Here we have investigated the effects of ATP on these currents. External application of ATP (1 mM) did not affect I(VNSCC) or I(L) in dialyzed cells. ATP (1 mM) increased I(VNSCC) and decreased I(L) in the perforated whole-cell configuration. UTP and UDP (1 mM) were more potent than ATP on I(VNSCC). ADP decreased I(L) but had no effect on I(VNSCC). The order of effectiveness was UTP = UDP > ATP > ADP. These effects were not blocked by pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) (PPADS), but the phospholipase C inhibitor U-73122 reversed the effects of ATP on I(VNSCC). ATP stimulation of I(VNSCC) was also reversed by protein kinase C (PKC) inhibitors chelerythrine chloride or bisindolylmaleimide I. Phorbol 12,13-dibutyrate mimicked the effects of ATP. RT-PCR showed that P2Y(4) is expressed by murine colonic myocytes, and this receptor is relatively insensitive to PPADS. Our data suggest that ATP activates I(VNSCC) and depresses I(L) via binding of P2Y(4) receptors and stimulation of the phospholipase C/PKC pathway.