Distribution and Function of Platelet-derived Growth Factor Receptor Alpha-positive Cells and Purinergic Neurotransmission in the Human Colon: Is It Different Between the Right and Left Colon?

Background/Aims: Platelet-derived growth factor receptor alpha-positive (PDGFRα+) cells function in the purinergic regulation of gastrointestinal motility, and purines are reportedly inhibitory neurotransmitters in the enteric nervous system. We explore the distribution and function of PDGFRα+ cells related to purinergic inhibitory neurotransmission in human right and left colons. Methods: Human colonic segments were prepared with mucosa and submucosa intact, and the circular muscle tension and longitudinal muscle tension were recorded. Purinergic neurotransmitters were administered after recording the regular contractions. Immunohistochemistry was performed on the circular muscle layers. Intracellular recording was performed on the colonic muscular layer. SK3, P2RY1, and PDGFR-α mRNA expression was tested by quantitative real-time polymerase chain reaction (qPCR). Results: Adenosine triphosphate (ATP) treatment significantly decreased the frequency and area under the curve (AUC) of the segmental contraction in right and left colons. Beta-nicotinamide adenine dinucleotide (ß-NAD) decreased the frequency in the right colon and the amplitude, frequency and AUC in the left colon. Apamin significantly increased frequency and AUC in the left colon, and after apamin pretreatment, ATP and ß-NAD did not change segmental contractility. Through intracellular recordings, a resting membrane potential decrease occurred after ATP administration; however, the degree of decrease between the right and left colon was not different. PDGFRα+ cells were distributed evenly in the circular muscle layers of right and left colons. SK3, P2RY1, and PDGFRα expression was not different between the right and left colon. Conclusion: Purines reduce right and left colon contractility similarly, and purinergic inhibitory neurotransmission can be regulated by PDGFRα+ cells in the human colon.

Role of detrusor PDGFRα+ cells in mouse model of cyclophosphamide-induced detrusor overactivity.

Cyclophosphamide (CYP)-induced cystitis is a rodent model that shares many features common to the cystitis occurring in patients, including detrusor overactivity (DO). Platelet-derived growth factor receptor alpha positive (PDGFRα+) cells have been proposed to regulate muscle excitability in murine bladders during filling. PDGFRα+ cells express small conductance Ca2+-activated K+ channels (predominantly SK3) that provide stabilization of membrane potential during filling. We hypothesized that down-regulation of the regulatory functions of PDGFRα+ cells and/or loss of PDGFRα+ cells generates the DO in CYP-treated mice. After CYP treatment, transcripts of Pdgfrα and Kcnn3 and PDGFRα and SK3 protein were reduced in detrusor muscle extracts. The distribution of PDGFRα+ cells was also reduced. Inflammatory markers were increased in CYP-treated detrusor muscles. An SK channel agonist, CyPPA, increased outward current and hyperpolarization in PDGFRα+ cells. This response was significantly depressed in PDGFRα+ cells from CYP-treated bladders. Contractile experiments and ex vivo cystometry showed increased spontaneous contractions and transient contractions, respectively in CYP-treated bladders with a reduction of apamin sensitivity, that could be attributable to the reduction in the SK conductance expressed by PDGFRα+ cells. In summary, PDGFRα+ cells were reduced and the SK3 conductance was downregulated in CYP-treated bladders. These changes are consistent with the development of DO after CYP treatment.

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.

UTP activates small-conductance Ca2+-activated K+ channels in murine detrusor PDGFRα+ cells.

Purines induce transient contraction and prolonged relaxation of detrusor muscles. Transient contraction is likely due to activation of inward currents in smooth muscle cells, and prolonged relaxation may be due to activation of small-conductance Ca(2+)-activated K(+) (SK) channels via P2Y1 receptors expressed by detrusor PDGF receptor (PDGFR)α(+) cells. We investigated whether other subtypes of P2Y receptors are involved in the activation of SK channels in PDGFRα(+) cells of detrusor muscles. Quantitative analysis of transcripts revealed that P2ry2, P2ry4, and P2ry14 are expressed in PDGFRα(+) cells of P2ry1-deficient/enhanced green fluorescent protein (P2ry1(-/-)/eGFP) mice at similar levels as in wild-type mice. UTP, a P2Y2/P2Y4 agonist, activated large outward currents in detrusor PDGFRα(+) cells. SK channel blockers and an inhibitor of phospholipase C completely abolished currents activated by UTP. In contrast, UTP activated nonselective cation currents in smooth muscle cells. Under current-clamp (current = 0), UTP induced significant hyperpolarization of PDGFRα(+) cells. MRS2500, a selective P2Y1 antagonist, did not affect UTP-activated outward currents in PDGFRα(+) cells from wild-type mice, and activation of outward currents by UTP was retained in P2ry1(-/-)/eGFP mice. As a negative control, we tested the effect of MRS2693, a selective P2Y6 agonist. This compound did not activate outward currents in PDGFRα(+) cells, and currents activated by UTP were unaffected by MRS2578, a selective P2Y6 antagonist. The nonselective P2Y receptor blocker suramin inhibited UTP-activated outward currents in PDGFRα(+) cells. Our data demonstrate that P2Y2 and/or P2Y4 receptors function, in addition to P2Y1 receptors, in activating SK currents in PDGFRα(+) cells and possibly in mediating purinergic relaxation responses in detrusor muscles.

A novel class of interstitial cells in the mouse and monkey female reproductive tracts.

Growing evidence suggests important roles for specialized platelet-derived growth factor receptor alpha-positive (PDGFRalpha(+)) cells in regulating the behaviors of visceral smooth muscle organs. Examination of the female reproductive tracts of mice and monkeys showed that PDGFRalpha(+) cells form extensive networks in ovary, oviduct, and uterus. PDGFRalpha(+) cells were located in discrete locations within these organs, and their distribution and density were similar in rodents and primates. PDGFRalpha(+) cells were distinct from smooth muscle cells and interstitial cells of Cajal (ICC). This was demonstrated with immunohistochemical techniques and by performing molecular expression studies on PDGFRalpha(+) cells from mice with enhanced green fluorescent protein driven off of the endogenous promoter for Pdgfralpha. Significant differences in gene expression were found in PDGFRalpha(+) cells from ovary, oviduct, and uterus. Differences in gene expression were also detected in cells from different tissue regions within the same organ (e.g., uterine myometrium vs. endometrium). PDGFRalpha(+) cells are unlikely to provide pacemaker activity because they lack significant expression of key pacemaker genes found in ICC (Kit and Ano1). Gja1 encoding connexin 43 was expressed at relatively high levels in PDGFRalpha(+) cells (except in the ovary), suggesting these cells can form gap junctions to one another and neighboring smooth muscle cells. PDGFRalpha(+) cells also expressed the early response transcription factor and proto-oncogene Fos, particularly in the ovary. These data demonstrate extensive distribution of PDGFRalpha(+) cells throughout the female reproductive tract. These cells are a heterogeneous population of cells that are likely to contribute to different aspects of physiological regulation in the various anatomical niches they occupy.

Purinergic inhibitory regulation of murine detrusor muscles mediated by PDGFRα+ interstitial cells.

Purines induce transient contraction and prolonged relaxation of detrusor muscles. Transient contraction could be due to activation of inward currents in smooth muscle cells, but the mechanism of purinergic relaxation has not been determined. We recently reported a new class of interstitial cells in detrusor muscles and showed that these cells could be identified with antibodies against platelet-derived growth factor receptor-α (PDGFRα(+) cells). The current density of small conductance Ca(2+)-activated K(+) (SK) channels in these cells is far higher (∼100 times) than in smooth muscle cells. Thus, we examined purinergic receptor (P2Y) mediated SK channel activation as a mechanism for purinergic relaxation. P2Y receptors (mainly P2ry1 gene) were highly expressed in PDGFRα(+) cells. Under voltage clamp conditions, ATP activated large outward currents in PDGFRα(+) cells that were inhibited by blockers of SK channels. ATP also induced significant hyperpolarization under current clamp conditions. A P2Y1 agonist, MRS2365, mimicked the effects of ATP, and a P2Y1 antagonist, MRS2500, inhibited ATP-activated SK currents. Responses to ATP were largely abolished in PDGFRα(+) cells of P2ry1(-/-) mice, and no response was elicited by MRS2365 in these cells. A P2X receptor agonist had no effect on PDGFRα(+) cells but, like ATP, activated transient inward currents in smooth muscle cells (SMCs). A P2Y1 antagonist decreased nerve-evoked relaxation. These data suggest that purines activate SK currents via mainly P2Y1 receptors in PDGFRα(+) cells. Our findings provide an explanation for purinergic relaxation in detrusor muscles and show that there are no discrete inhibitory nerve fibres. A dual receptive field for purines provides the basis for inhibitory neural regulation of excitability.

Functional expression of SK channels in murine detrusor PDGFR+ cells.

We sought to characterize molecular expression and ionic conductances in a novel population of interstitial cells (PDGFRα(+) cells) in murine bladder to determine how these cells might participate in regulation of detrusor excitability. PDGFRα(+) cells and smooth muscle cells (SMCs) were isolated from detrusor muscles of PDGFRα(+)/eGFP and smMHC/Cre/eGFP mice and sorted by FACS. PDGFRα(+) cells were highly enriched in Pdgfra (12 fold vs. unsorted cell) and minimally positive for Mhc (SMC marker), Kit (ICC marker) and Pgp9.5 (neuronal marker). SK3 was dominantly expressed in PDGFRα(+) cells in comparison to SMCs. αSlo (BK marker) was more highly expressed in SMCs. SK3 protein was observed in PDGFRα(+) cells by immunohistochemistry but could not be resolved in SMCs. Depolarization evoked voltage-dependent Ca(2+) currents in SMCs, but inward current conductances were not activated in PDGFRα(+) cells under the same conditions. PDGFRα(+) cells displayed spontaneous transient outward currents (STOCs) at potentials positive to -60 mV that were inhibited by apamin. SK channel modulators, CyPPA and SKA-31, induced significant hyperpolarization of PDGFRα(+) cells and activated SK currents under voltage clamp. Similar responses were not resolved in SMCs at physiological potentials. Single channel measurements confirmed the presence of functional SK3 channels (i.e. single channel conductance of 10 pS and sensitivity to intracellular Ca(2+)) in PDGFRα(+) cells. The apamin-sensitive stabilizing factor regulating detrusor excitability is likely to be due to the expression of SK3 channels in PDGFRα(+) cells because SK agonists failed to elicit resolvable currents and hyperpolarization in SMCs at physiological potentials.

Platelet-derived growth factor receptor-α cells in mouse urinary bladder: a new class of interstitial cells.

Specific classes of interstitial cells exist in visceral organs and have been implicated in several physiological functions including pacemaking and mediators in neurotransmission. In the bladder, Kit(+) interstitial cells have been reported to exist and have been suggested to be neuromodulators. More recently a second interstitial cell, which is identified using antibodies against platelet-derived growth factor receptor-α (PDGFR-α) has been described in the gastrointestinal (GI) tract and has been implicated in enteric motor neurotransmission. In this study, we examined the distribution of PDGFR-α(+) cells in the murine urinary bladder and the relation that these cells may have with nerve fibres and smooth muscle cells. Platelet-derived growth factor receptor-α(+) cells had a spindle shape or stellate morphology and often possessed multiple processes that contacted one another forming a loose network. These cells were distributed throughout the bladder wall, being present in the lamina propria as well as throughout the muscularis of the detrusor. These cells surrounded and were located between smooth muscle bundles and often came into close morphological association with intramural nerve fibres. These data describe a new class of interstitial cells that express a specific receptor within the bladder wall and provide morphological evidence for a possible neuromodulatory role in bladder function.