Robert D. Corrigan

Important role of mucosal serotonin in colonic propulsion and peristaltic reflexes: in vitro analyses in mice lacking tryptophan hydroxylase 1

•  Previous studies have indicated that neither neuronal nor mucosal 5‐hydroxytryptamine (5‐HT) are important for colonic migrating motor complexes (CMMCs) or faecal pellet propulsion. Therefore, tryptophan hydroxylase 1 knockout (TPH1KO) mice were used to examine the role of mucosal 5‐HT in generating CMMCs and faecal pellet propulsion, as TPH1 is the regulatory enzyme necessary for the synthesis of 5‐HT in enterochromaffin cells in the mucosa. •  Control mice generated a robust CMMC when the mucosa was mechanically stimulated, which was blocked by ondansetron (5‐HT3 antagonist), and could propagate faecal pellets that did not significantly distend the bowel, suggesting that they were propelled by mucosal reflexes in the absence of stretch reflexes. •  TPH1KO mice exhibited no mucosal reflexes, reduced responses to intraluminal distension and propelled only larger faecal pellets, suggesting that they relied upon stretch reflexes alone. •  In control mice, CMMCs, which can propel a faecal pellet, propagated in an oral to anal direction, whereas, in TPH1KO mice, they rarely propagated. •  Both the propagation and amplitude of CMMCs were reduced by ondansetron in control mice, whereas this drug did not affect CMMCs in TPH1KO mice. •  This suggests that 5‐HT release from the mucosa and stretch reflexes are important for normal colonic propulsion.

Extensive projections of myenteric serotonergic neurons suggest they comprise the central processing unit in the colon

5‐Hydroxytryptamine (5‐HT, serotonin) is an important regulator of colonic motility and secretion; yet the role of serotonergic neurons in the colon is controversial.

Use of Genetically Encoded Calcium Indicators (GECIs) Combined with Advanced Motion Tracking Techniques to Examine the Behavior of Neurons and Glia in the Enteric Nervous System of the Intact Murine Colon.

Genetically encoded Ca2+ indicators (GECIs) have been used extensively in many body systems to detect Ca2+ transients associated with neuronal activity. Their adoption in enteric neurobiology has been slower, although they offer many advantages in terms of selectivity, signal-to-noise and non-invasiveness. Our aims were to utilize a number of cell-specific promoters to express the Ca2+ indicator GCaMP3 in different classes of neurons and glia to determine their effectiveness in measuring activity in enteric neural networks during colonic motor behaviors. We bred several GCaMP3 mice: (1) Wnt1-GCaMP3, all enteric neurons and glia; (2) GFAP-GCaMP3, enteric glia; (3) nNOS-GaMP3, enteric nitrergic neurons; and (4) ChAT-GCaMP3, enteric cholinergic neurons. These mice allowed us to study the behavior of the enteric neurons in the intact colon maintained at a physiological temperature, especially during the colonic migrating motor complex (CMMC), using low power Ca2+ imaging. In this preliminary study, we observed neuronal and glial cell Ca2+ transients in specific cells in both the myenteric and submucous plexus in all of the transgenic mice variants. The number of cells that could be simultaneously imaged at low power (100–1000 active cells) through the undissected gut required advanced motion tracking and analysis routines. The pattern of Ca2+ transients in myenteric neurons showed significant differences in response to spontaneous, oral or anal stimulation. Brief anal elongation or mucosal stimulation, which evokes a CMMC, were the most effective stimuli and elicited a powerful synchronized and prolonged burst of Ca2+ transients in many myenteric neurons, especially when compared with the same neurons during a spontaneous CMMC. In contrast, oral elongation, which normally inhibits CMMCs, appeared to suppress Ca2+ transients in some of the neurons active during a spontaneous or an anally evoked CMMC. The activity in glial networks appeared to follow neural activity but continued long after neural activity had waned. With these new tools an unprecedented level of detail can be recorded from the enteric nervous system (ENS) with minimal manipulation of tissue. These techniques can be extended in order to better understand the roles of particular enteric neurons and glia during normal and disordered motility.

Urothelial purine release during filling of murine and primate bladders.

During urinary bladder filling the bladder urothelium releases chemical mediators that in turn transmit information to the nervous and muscular systems to regulate sensory sensation and detrusor muscle activity. Defects in release of urothelial mediators may cause bladder dysfunctions that are characterized with aberrant bladder sensation during bladder filling. Previous studies have demonstrated release of ATP from the bladder urothelium during bladder filling, and ATP remains the most studied purine mediator that is released from the urothelium. However, the micturition cycle is likely regulated by multiple purine mediators, since various purine receptors are found present in many cell types in the bladder wall, including urothelial cells, afferent nerves, interstitial cells in lamina propria, and detrusor smooth muscle cells. Information about the release of other biologically active purines during bladder filling is still lacking. Decentralized bladders from C57BL/6 mice and Cynomolgus monkeys (Macaca fascicularis) were filled with physiological solution at different rates. Intraluminal fluid was analyzed by high-performance liquid chromatography with fluorescence detection for simultaneous evaluation of ATP, ADP, AMP, adenosine, nicotinamide adenine dinucleotide (NAD+), ADP-ribose, and cADP-ribose content. We also measured ex vivo bladder filling pressures and performed cystometry in conscious unrestrained mice at different filling rates. ATP, ADP, AMP, NAD+, ADPR, cADPR, and adenosine were detected released intravesically at different ratios during bladder filling. Purine release increased with increased volumes and rates of filling. Our results support the concept that multiple urothelium-derived purines likely contribute to the complex regulation of bladder sensation during bladder filling.

Premature contractions of the bladder are suppressed by interactions between TRPV4 and SK3 channels in murine detrusor PDGFRα cells.

During filling, urinary bladder volume increases dramatically with little change in pressure. This is accomplished by suppressing contractions of the detrusor muscle that lines the bladder wall. Mechanisms responsible for regulating detrusor contraction during filling are poorly understood. Here we describe a novel pathway to stabilize detrusor excitability involving platelet-derived growth factor receptor-α positive (PDGFRα+) interstitial cells. PDGFRα+ cells express small conductance Ca2+-activated K+ (SK) and TRPV4 channels. We found that Ca2+ entry through mechanosensitive TRPV4 channels during bladder filling stabilizes detrusor excitability. GSK1016790A (GSK), a TRPV4 channel agonist, activated a non-selective cation conductance that coupled to activation of SK channels. GSK induced hyperpolarization of PDGFRα+ cells and decreased detrusor contractions. Contractions were also inhibited by activation of SK channels. Blockers of TRPV4 or SK channels inhibited currents activated by GSK and increased detrusor contractions. TRPV4 and SK channel blockers also increased contractions of intact bladders during filling. Similar enhancement of contractions occurred in bladders of Trpv4−/− mice during filling. An SK channel activator (SKA-31) decreased contractions during filling, and rescued the overactivity of Trpv4−/− bladders. Our findings demonstrate how Ca2+ influx through TRPV4 channels can activate SK channels in PDGFRα+ cells and prevent bladder overactivity during filling.

Serum response factor regulates smooth muscle contractility via myotonic dystrophy protein kinases and L-type calcium channels

Serum response factor (SRF) transcriptionally regulates expression of contractile genes in smooth muscle cells (SMC). Lack or decrease of SRF is directly linked to a phenotypic change of SMC, leading to hypomotility of smooth muscle in the gastrointestinal (GI) tract. However, the molecular mechanism behind SRF-induced hypomotility in GI smooth muscle is largely unknown. We describe here how SRF plays a functional role in the regulation of the SMC contractility via myotonic dystrophy protein kinase (DMPK) and L-type calcium channel CACNA1C. GI SMC expressed Dmpk and Cacna1c genes into multiple alternative transcriptional isoforms. Deficiency of SRF in SMC of Srf knockout (KO) mice led to reduction of SRF-dependent DMPK, which down-regulated the expression of CACNA1C. Reduction of CACNA1C in KO SMC not only decreased intracellular Ca2+ spikes but also disrupted their coupling between cells resulting in decreased contractility. The role of SRF in the regulation of SMC phenotype and function provides new insight into how SMC lose their contractility leading to hypomotility in pathophysiological conditions within the GI tract.

AB297. SPR-24 Spinal cord injury and detrusor PDGFRα+ cells

Objective Neurogenic bladder dysfunction due to spinal cord injury (SCI) poses a significant threat to the well-being of patients. The complications of this condition include but not are limited to incontinence, renal impairment, urinary tract infection, stones, and poor quality of life. Clinical manifestations of SCI involve combination of storage and voiding bladder problems. Although a number of clinical studies have reported overactive bladder (OAB) after SCI, the pathophysiological mechanisms remain unclear. SCI is widely used to induce neurogenic bladder in rodent models. These animals exhibited dysfunctional condition results in different symptoms, ranging from acute urinary retention to an OAB or a combination of both. There is an abundance of PDGFRα+ cells in detrusor muscles. This cell involves the membrane stabilization via activation of SK channels in detrusor PDGFRα+ cells during filling. Thus we investigate the molecular and protein expression of PDGFRα+ cells from SCI mice to characterize the role of these cells that contributes to development of OAB in SCI. Methods SCI was induced by complete compression of T12-L1 spinal cord. Experiments were performed on 24, 48 and 72 hr after surgery. We employed molecular approaches and ex vivo cystometry. Pdgfrα and Kccn1−3 transcripts were analyzed for molecular expression. Ex vivo compliance was used for testing SK channel sensitivity in control and SCI mice. Results In quantitative analysis of transcripts, Pdgfrα and Kcnn3 transcripts in SCI detrusor were significantly decreased in a time-dependent manner after SCI surgery compared with control detrusor. In ex vivo cystometry, SCI bladder revealed an increase in the amplitude and frequency of non−voiding pressure responses during filling. Effects of a SK blocker (apamin) and a SK channel activator (SKA-31) were reduced in non-voiding contractions in SCI mice compared to control. Conclusions These findings support that downregulation of PDGFRα+ cells and SK channels in SCI detrusors might involve the development of OAB in SCI. Funding Source(s) NIDDK, RO1 DK098388

AB315. SPR-42 Cyclophosphamide-induced overactive bladder via downregulation of relaxation factors in detrusor PDGFRα+ cells

Objective Morphology and functional role of PDGFRα+ cells have been recently characterized in the detrusor muscle layer. Detrusor relaxation is caused by activation of small conductance Ca2+ activated-K+ (SK) channels and purinergic inhibitory responses in detrusor PDGFRα+ cells. Loss of PDGFRα+ cells or alteration of P2Y receptors and SK channels will affect detrusor excitability. Cyclophosphamide (CYP)-treated animals exhibited overactive bladder (OAB). We hypothesized that the downregulation of P2Y receptors and/or SK channels in PDGFRα+ cells will display the phenotype of CYP-induced OAB. Methods CYP was injected intraperitoneally in PDGFRα+/eGFP and SMC/eGFP mice. We harvested the detrusor muscle without urothelium and disperse the cells for the fluorescence activated cell sorting (FACS). Sorted PDGFRα+ cells and smooth muscle cells (SMCs) were used for molecular study to compare the changes in transcripts between CYP-injected and control group. Transcripts were examined included; Pdgfrα, P2ry1, P2ry2, P2ry4, Kcnn1, Kcnn2, Kcnn3 and inflammation marker (Il-6). Immunohistochemistry, mechanical contractility and ex vivo cystometry were also performed. Results Quantitative analysis of PCR revealed that CYP-injected detrusor muscle increased transcriptional expression of Il-6, but decreased the expression of Pdgfrα. Transcriptional changes in CYP-injected sorted PDGFRα+ cells from PDGFRα+/eGFP mice showed Pdgfα, Kccn3 (SK3), P2ry1, P2ry2 and P2ry4 genes were decreased compared with saline-injected control. Sorted SMCs from SMC/eGFP mice did not show significant expression of those genes and no detectable changes. Immunohistochemistry showed SK3 in PDGFRα immunoreactivity was downregulated in CYP-injected detrusor muscle. Apamin (a SK blocker) sensitivity on spontaneous contractile activity was decreased in CYP-injected mice compared to saline-injected mice. In ex vivo cystometry, increased spontaneous non-voiding contractions and less apamin sensitivity were observed in CYP-injected mice. Conclusions These findings are the first report to investigate the role of PDGFRα+ cells in relation to OAB mechanisms. In conclusion, we found that CYP-induced OAB is resulted from down regulation of PDGFRα, P2Y receptors and SK channels in CYP-injected bladder. These results provide novel mechanisms of functional role of PDGFRα+ cells on OAB. Funding Source(s) NIDDK, RO1 DK098388 and Urology Care Foundation Research Scholar Award (Interstitial Cystitis Association)