Masaaki Kurahashi

A functional role for the 'fibroblast-like cells' in gastrointestinal smooth muscles.

Smooth muscles, as in the gastrointestinal tract, are composed of several types of cells. Gastrointestinal muscles contain smooth muscle cells, enteric neurons, glial cells, immune cells, and various classes of interstitial cells. One type of interstitial cell, referred to as ‘fibroblast‐like cells’ by morphologists, are common, but their function is unknown. These cells are found near the terminals of enteric motor neurons, suggesting they could have a role in generating neural responses that help control gastrointestinal movements. We used a novel mouse with bright green fluorescent protein expressed specifically in the fibroblast‐like cells to help us identify these cells in the mixture of cells obtained when whole muscles are dispersed with enzymes. We isolated these cells and found they respond to a major class of inhibitory neurotransmitters – purines. We characterized these responses, and our results provide a new hypothesis about the role of fibroblast‐like cells in smooth muscle tissues.

Platelet-derived growth factor receptor α-positive cells in the tunica muscularis of human colon.

An obstacle to understanding motor pathologies of the gastrointestinal (GI) tract is that the physiology of some of the cellular components of the gut wall is not understood. Morphologists identified fibroblast‐like cells in the tunica muscularis many years ago, but little is known about these interstitial cells because of inadequate techniques to identify these cells. Recent findings have shown that fibroblast‐like cells express platelet‐derived growth factor receptor α (PDGFRα) in mice and that antibodies for these receptors can be used to label the cells. We used immunohistochemical techniques to study the phenotype and intercellular relationships of fibroblast‐like cells in the human colon. Fibroblast‐like cells are labelled specifically with antibodies to PDGFRα and widely distributed through the tunica muscularis of human colon. These cells form discrete networks in the region of the myenteric plexus and within the circular and longitudinal muscle layers. Platelet‐derived growth factor receptor α+ cells are distinct from c‐Kit+ interstitial cells of Cajal and closely associated with varicose processes of neurons expressing substance P (excitatory motor neurons) or neuronal nitric oxide synthase (nNOS) (inhibitory motor neurons). Platelet‐derived growth factor receptor α+ cells express small conductance Ca2+‐activated K+ channels (SK3), which are likely to mediate purinergic neural regulation of colonic muscles. Our data suggest that PDGFRα+ cells may have an important role in transducing inputs from enteric motor neurons. This study identifies reagents and techniques that will allow investigation of this class of interstitial cells and help develop an understanding of the role of PDGFRα+ cells in the human GI tract in health and disease.

Relationship between interstitial cells of Cajal, fibroblast-like cells and inhibitory motor nerves in the internal anal sphincter

Interstitial cells of Cajal (ICC) have been shown to participate in nitrergic neurotransmission in various regions of the gastrointestinal (GI) tract. Recently, fibroblast-like cells, which are positive for platelet-derived growth factor receptor α (PDGFRα+), have been suggested to participate additionally in inhibitory neurotransmission in the GI tract. The distribution of ICC and PDGFRα+ cell populations and their relationship to inhibitory nerves within the mouse internal anal sphincter (IAS) are unknown. Immunohistochemical techniques and confocal microscopy were therefore used to examine the density and arrangement of ICC, PDGFRα+ cells and neuronal nitric-oxide-synthase-positive (nNOS+) nerve fibers in the IAS of wild-type (WT) and W/Wv mice. Of the total tissue volume within the IAS circular muscle layer, 18% consisted in highly branched PDGFRα+ cells (PDGFRα+-IM). Other populations of PDGFRα+ cells were observed within the submucosa and along the serosal and myenteric surfaces. Spindle-shaped intramuscular ICC (ICC-IM) were present in the WT mouse IAS but were largely absent from the W/Wv IAS. The ICC-IM volume (5% of tissue volume) in the WT mouse IAS was significantly smaller than that of PDGFRα+-IM. Stellate-shaped submucosal ICC (ICC-SM) were observed in the WT and W/Wv IAS. Minimum surface distance analysis revealed that nNOS+ nerve fibers were closely aligned with both ICC-IM and PDGFRα+-IM. An even closer association was seen between ICC-IM and PDGFRα+-IM. Thus, a close morphological arrangement exists between inhibitory motor neurons, ICC-IM and PDGFRα+-IM suggesting that some functional interaction occurs between them contributing to inhibitory neurotransmission in the IAS.

Platelet-derived growth factor receptor-α-positive cells and not smooth muscle cells mediate purinergic hyperpolarization in murine colonic muscles.

Enteric inhibitory neurotransmission is an important feature of the neural regulation of gastrointestinal motility. Purinergic neurotransmission, via P2Y1 receptors, mediates one phase of inhibitory neural control. For decades, ATP has been assumed to be the purinergic neurotransmitter and smooth muscle cells (SMCs) have been considered the primary targets for inhibitory neurotransmission. Recent experiments have cast doubt on both of these assumptions and suggested that another cell type, platelet-derived growth factor receptor-α-positive (PDGFRα(+)) cells, is the target for purinergic neurotransmission. We compared responses of PDGFRα(+) cells and SMCs to several purine compounds to determine if these cells responded in a manner consistent with enteric inhibitory neurotransmission. ATP hyperpolarized PDGFRα(+) cells but depolarized SMCs. Only part of the ATP response in PDGFRα(+) cells was blocked by MRS 2500, a P2Y1 antagonist. ADP, MRS 2365, β-NAD, and adenosine 5-diphosphate-ribose, P2Y1 agonists, hyperpolarized PDGFRα(+) cells, and these responses were blocked by MRS 2500. Adenosine 5-diphosphate-ribose was more potent in eliciting hyperpolarization responses than β-NAD. P2Y1 agonists failed to elicit responses in SMCs. Small hyperpolarization responses were elicited in SMCs by a small-conductance Ca(2+)-activated K(+) channel agonist, cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine, consistent with the low expression and current density of small-conductance Ca(2+)-activated K(+) channels in these cells. Large-amplitude hyperpolarization responses, elicited in PDGFRα(+) cells, but not SMCs, by P2Y1 agonists are consistent with the generation of inhibitory junction potentials in intact muscles in response to purinergic neurotransmission. The responses of PDGFRα(+) cells and SMCs to purines suggest that SMCs are unlikely targets for purinergic neurotransmission in colonic muscles.

Uridine adenosine tetraphosphate is a novel neurogenic P2Y1 receptor activator in the gut.

Significance Millions of people suffer of gastrointestinal (GI) motility disorders. P2Y1 purine receptors and Ca2+-activated small-conductance K+ (SK) channels are established as key mediators of enteric inhibitory neurotransmission in the distal GI tract. However, the identity of the purinergic neurotransmitter in the bowel is controversial. We describe uridine adenosine tetraphosphate (Up4A) as a highly potent native activator of purinergic P2Y1 receptors and SK channels that is released spontaneously and during nerve stimulation in the human and mouse colons. We characterized potential sites of release, mimicry of the endogenous neurotransmitter, action on postjunctional targets, and metabolic pathways for Up4A. Our data identify Up4A as a novel factor in the purinergic signaling in the gut, including enteric inhibitory motor neurotransmission. Enteric purinergic motor neurotransmission, acting through P2Y1 receptors (P2Y1R), mediates inhibitory neural control of the intestines. Recent studies have shown that NAD+ and ADP ribose better meet criteria for enteric inhibitory neurotransmitters in colon than ATP or ADP. Here we report that human and murine colon muscles also release uridine adenosine tetraphosphate (Up4A) spontaneously and upon stimulation of enteric neurons. Release of Up4A was reduced by tetrodotoxin, suggesting that at least a portion of Up4A is of neural origin. Up4A caused relaxation (human and murine colons) and hyperpolarization (murine colon) that was blocked by the P2Y1R antagonist, MRS 2500, and by apamin, an inhibitor of Ca2+-activated small-conductance K+ (SK) channels. Up4A responses were greatly reduced or absent in colons of P2ry1−/− mice. Up4A induced P2Y1R–SK-channel–mediated hyperpolarization in isolated PDGFRα+ cells, which are postjunctional targets for purinergic neurotransmission. Up4A caused MRS 2500-sensitive Ca2+ transients in human 1321N1 astrocytoma cells expressing human P2Y1R. Up4A was more potent than ATP, ADP, NAD+, or ADP ribose in colonic muscles. In murine distal colon Up4A elicited transient P2Y1R-mediated relaxation followed by a suramin-sensitive contraction. HPLC analysis of Up4A degradation suggests that exogenous Up4A first forms UMP and ATP in the human colon and UDP and ADP in the murine colon. Adenosine then is generated by extracellular catabolism of ATP and ADP. However, the relaxation and hyperpolarization responses to Up4A are not mediated by its metabolites. This study shows that Up4A is a potent native agonist for P2Y1R and SK-channel activation in human and mouse colon.

Spontaneous transient hyperpolarizations in the rabbit small intestine

Recently, it was shown that fibroblast‐like cells (FLCs) possess the apparatus to mediate purinergic motor neurotransmission in the gastrointestinal tract. However, the electrophysiological properties of FLCs in situ have not been determined. We recorded two patterns of slow waves from longitudinal smooth muscle cells and circular smooth muscle cells, large amplitude slow waves from interstitial cells of Cajal, and spontaneous transient hyperpolarizations (STHs) from FLCs in the rabbit small intestine using intracellular recording combined with dye injection to identify the cellular morphology of impaled cells. Drugs that inhibit the signalling pathway involved in purinergic neurotransmission inhibited STHs in FLCs. Small amplitude STHs were recorded in smooth muscle cells but not in interstitial cells of Cajal, suggesting that STHs from FLCs were conducted passively to smooth muscle cells. We conclude that FLCs display the molecular apparatus necessary to mediate purinergic neurotransmission and may tonically dampen smooth muscle excitability in the rabbit small intestine by an ongoing discharge of STHs.

Na+-K+-Cl− cotransporter (NKCC) maintains the chloride gradient to sustain pacemaker activity in interstitial cells of Cajal

Interstitial cells of Cajal (ICC) generate electrical slow waves by coordinated openings of ANO1 channels, a Ca2+-activated Cl- (CaCC) conductance. Efflux of Cl- during slow waves must be significant, as there is high current density during slow-wave currents and slow waves are of sufficient magnitude to depolarize the syncytium of smooth muscle cells and PDGFRα+ cells to which they are electrically coupled. We investigated how the driving force for Cl- current is maintained in ICC. We found robust expression of Slc12a2 (which encodes an Na+-K+-Cl- cotransporter, NKCC1) and immunohistochemical confirmation that NKCC1 is expressed in ICC. With the use of the gramicidin permeabilized-patch technique, which is reported to not disturb [Cl-]i, the reversal potential for spontaneous transient inward currents (ESTICs) was -10.5 mV. This value corresponds to the peak of slow waves when they are recorded directly from ICC in situ. Inhibition of NKCC1 with bumetanide shifted ESTICs to more negative potentials within a few minutes and reduced pacemaker activity. Bumetanide had no direct effects on ANO1 or CaV3.2 channels expressed in HEK293 cells or L-type Ca2+ currents. Reducing extracellular Cl- to 10 mM shifted ESTICs to positive potentials as predicted by the Nernst equation. The relatively rapid shift in ESTICs when NKCC1 was blocked suggests that significant changes in the transmembrane Cl- gradient occur during the slow-wave cycle, possibly within microdomains formed between endoplasmic reticulum and the plasma membrane in ICC. Recovery of Cl- via NKCC1 might have additional consequences on shaping the waveforms of slow waves via Na+ entry into microdomains.

Transcriptome analysis of PDGFRα+ cells identifies T-type Ca2+ channel CACNA1G as a new pathological marker for PDGFRα+ cell hyperplasia.

Platelet-derived growth factor receptor alpha (PDGFRα)+ cells are distributed into distinct morphological groups within the serosal, muscular, and submucosal layers as well as the myenteric and deep muscular plexi. PDGFRα+ cells directly interact with interstitial cells of Cajal (ICC) and smooth muscle cells (SMC) in gastrointestinal smooth muscle tissue. These three cell types, SMC, ICC, and PDGFRα+ cells (SIP cells), form an electrical syncytium, which dynamically regulates gastrointestinal motility. We have previously reported the transcriptomes of SMC and ICC. To complete the SIP cell transcriptome project, we obtained transcriptome data from jejunal and colonic PDGFRα+ cells. The PDGFRα+ cell transcriptome data were added to the Smooth Muscle Genome Browser that we previously built for the genome-scale gene expression data of ICC and SMC. This browser provides a comprehensive reference for all transcripts expressed in SIP cells. By analyzing the transcriptomes, we have identified a unique set of PDGFRα+ cell signature genes, growth factors, transcription factors, epigenetic enzymes/regulators, receptors, protein kinases/phosphatases, and ion channels/transporters. We demonstrated that the low voltage-dependent T-type Ca2+ channel Cacna1g gene was particularly expressed in PDGFRα+ cells in the intestinal serosal layer in mice. Expression of this gene was significantly induced in the hyperplasic PDGFRα+ cells of obstructed small intestine in mice. This gene was also over-expressed in colorectal cancer, Crohn’s disease, and diverticulitis in human patients. Taken together, our data suggest that Cacna1g exclusively expressed in serosal PDGFRα+ cells is a new pathological marker for gastrointestinal diseases.