[Show abstract][Hide abstract] ABSTRACT: Genome-scale expression data on the absolute numbers of gene isoforms offers essential clues in cellular functions and biological processes. Smooth muscle cells (SMCs) perform a unique contractile function through expression of specific genes controlled by serum response factor (SRF), a transcription factor that binds to DNA sites known as the CArG boxes. To identify SRF-regulated genes specifically expressed in SMCs, we isolated SMC populations from mouse small intestine and colon, obtained their transcriptomes, and constructed an interactive SMC genome and CArGome browser. To our knowledge, this is the first online resource that provides a comprehensive library of all genetic transcripts expressed in primary SMCs. The browser also serves as the first genome-wide map of SRF binding sites. The browser analysis revealed novel SMC-specific transcriptional variants and SRF target genes, which provided new and unique insights into the cellular and biological functions of the cells in gastrointestinal (GI) physiology. The SRF target genes in SMCs, which were discovered in silico, were confirmed by proteomic analysis of SMC-specific Srf knockout mice. Our genome browser offers a new perspective into the alternative expression of genes in the context of SRF binding sites in SMCs and provides a valuable reference for future functional studies.
PLoS ONE 08/2015; DOI:10.1371/journal.pone.0133751 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In cerebral artery myocytes, close proximity of the sarcoplasmic reticulum (SR) and plasma membrane (PM) creates microdomains where Ca2+ released from the SR attains a concentration sufficient to activate large-conductance Ca2+-activated K+ (BK) and melastatin transient receptor potential 4 (TRPM4) channels; essential regulators of membrane excitability. Microtubules organize the SR in cardiac and skeletal muscle cells, but it is not known if they serve this function in smooth muscle cells. Here, we test the hypothesis that microtubules maintain the SR architecture forming Ca2+ microdomains essential for BK and TRPM4 channel activity. Using membrane- and tubulin-specific fluorescent dyes, we observed distinct microtubule arches beneath the peripheral SR proximal to the PM in contractile cerebral artery smooth muscle cells. Nocodazole, an inhibitor of microtubule polymerization, disrupted these subcellular structures and increased the distance between the SR and PM. Using high-speed, high-resolution confocal Ca2+ imaging, we found that microtubule depolymerization altered the spatiotemporal properties of localized Ca2+ signaling events. Nocodazole treatment also resulted in the loss of Ca2+-dependent TRPM4 and BK channel activity in perforated whole-cell patch clamp recordings and diminished contractility in pressure myography experiments. We conclude that the microtubule network is essential for maintaining the SR architecture and Ca2+ microdomains necessary for the activation of BK and TRPM4 channels in cerebral artery myocytes.
[Show abstract][Hide abstract] ABSTRACT: Gastric peristalsis begins in the orad corpus and propagates to the pylorus. Directionality of peristalsis depends upon orderly generation and propagation of electrical slow waves and a frequency gradient between proximal and distal pacemakers. We sought to understand how chronotropic agonists affect coupling between corpus and antrum.
Electrophysiological and imaging techniques were used to investigate regulation of gastric slow wave frequency by muscarinic agonists in mice. We also investigated the expression and role of cholinesterases in regulating slow wave frequency and motor patterns in the stomach.
Both acetycholinesterase (Ache) and butyrylcholine esterase (Bche) are expressed in gastric muscles and AChE is localized to var-icose processes of motor neurons. Inhibition of AChE in the absence of stimulation increased slow wave frequency in corpus and throughout muscle strips containing corpus and antrum. CCh caused depolarization and increased slow wave frequency. Stimulation of cholinergic neurons increased slow wave frequency but did not cause depolarization. Neostigmine (1 μM) in-creased slow wave frequency, but uncoupling between corpus and antrum was not detected. Motility mapping of contractile activity in gastric muscles showed similar effects of enteric nerve stimulation on the frequency and propagation of slow waves, but neostigmine (> 1 μM) caused aberrant contractile frequency and propagation and ectopic pacemaking.
Our data show that slow wave uncoupling is difficult to assess with electrical recording from a single or double sites and sug-gest that efficient metabolism of ACh released from motor neurons is an extremely important regulator of slow wave frequency and propagation and gastric motility patterns.
Journal of neurogastroenterology and motility 03/2015; 21(2):200-16. DOI:10.5056/jnm14120 · 2.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Growing evidence suggests important roles for specialized platelet-derived growth factor-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 was 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 eGFP driven off 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 lacked 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 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 c-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.
Copyright 2015 by The Society for the Study of Reproduction.
Biology of Reproduction 03/2015; 92(4). DOI:10.1095/biolreprod.114.124388 · 3.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Interstitial cells, known as PDGFRα+ cells, are closely associated with varicosities of enteric motor neurons and suggested to mediate purinergic hyperpolarization responses in smooth muscles of the gastrointestinal (GI) tract, but this concept has not been demonstrated directly in intact muscles. We used confocal microscopy to monitor Ca2+ transients in neurons and post-junctional cells of the murine colon evoked by exogenous purines or electrical field stimulation (EFS) of enteric neurons. EFS (1-20 Hz) caused Ca2+ transients in enteric motor nerve processes and then in PDGFRα+ cells shortly after the onset of stimulation: (latency from EFS was 280 ms at 10 Hz). Responses in smooth muscle cells (SMCs) were typically a small decrease in Ca2+ fluorescence just after the initiation of Ca2+ transients in PDGFRα+ cells. Upon cessation of EFS, several fast Ca2+ transients were noted in SMCs (rebound excitation). Strong correlation was noted in the temporal characteristics of Ca2+ transients evoked in PDGFRα+ cells by EFS and inhibitory junction potentials (IJPs) recorded with intracellular microelectrodes. Ca2+ transients and IJPs elicited by EFS were blocked by MRS-2500, a P2Y1 antagonist, and absent in P2ry1(-/-) mice. PDGFRα+ cells expressed gap junction genes, and gap junction uncouplers, 18β-glycyrrhetinic acid (18β-GA) and octanol, blocked Ca2+ transients in SMCs, but not in neurons or PDGFRα+ cells. IJPs recorded from SMCs were also blocked. These findings demonstrate direct innervation of PDGFRα+ cells by motor neurons. PDGFRα+ cells are primary targets for purinergic neurotransmitter(s) in enteric inhibitory neurotransmission. Hyperpolarization responses are conducted to SMCs via gap junctions.This article is protected by copyright. All rights reserved
The Journal of Physiology 01/2015; 593(8). DOI:10.1113/jphysiol.2014.287599 · 4.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Protease-activated receptors (PARs) are G protein-coupled receptors activated by proteolytic cleavage at their amino termini by serine proteases. PAR activation contributes to the inflammatory response in the gastrointestinal (GI) tract and alters GI motility, but little is known about the specific cells within the tunica muscularis that express PARs and the mechanisms leading to contractile responses. Using real time PCR, we found PARs to be expressed in smooth muscle cells (SMCs), interstitial cells of Cajal (ICC) and platelet-derived growth factor receptor α positive (PDGFRα+) cells. The latter cell-type showed dominant expression of F2r (encodes PAR1) and F2rl1 (encodes PAR2). Contractile and intracellular electrical activities were measured to characterize the integrated responses to PAR activation in whole muscles. Cells were isolated and ICC and PDGFRα+ cells were identified by constitutive expression of fluorescent reporters. Thrombin (PAR1 agonist) and trypsin (PAR2 agonist) caused biphasic responses in colonic muscles: transient hyperpolarization and relaxation followed by repolarization and excitation. The inhibitory phase was blocked by apamin, revealing a distinct excitatory component. Patch clamp studies showed that the inhibitory response was mediated by activation of small conductance calcium-activated K+ channels in PDGFRα+ cells, and the excitatory response was mediated by activation of a Cl− conductance in ICC. SMC contributed little to PAR responses in colonic muscles. In summary, PARs regulate the excitability of colonic muscles; different conductances are activated in each cell-type of the SMC/ICC/PDGFRα+ cells (SIP) syncytium. Motor responses to PAR agonists are integrated responses of the SIP syncytium.This article is protected by copyright. All rights reserved
The Journal of Physiology 12/2014; 593(5). DOI:10.1113/jphysiol.2014.285148 · 4.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Proceedings of the National Academy of Sciences 10/2014; DOI:10.1073/pnas.1409078111 · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effector cells and second messengers participating in nitrergic neuromuscular transmission (NMT) were investigated in the mouse internal anal sphincter (IAS). Protein expression of guanylate cyclase (GCα, GCβ) and cyclic GMP dependent protein kinase I (cGKI) were examined in cryostat sections with dual labeling immunohistochemical techniques in PDGFRα(+) cells, interstitial cells of Cajal (ICC) and smooth muscle cells (SMC). Gene expression levels were determined with qPCR of dispersed cells from Pdgfrα(egfp/+), Kit(copGFP/+) and smMHC(Cre-egfp) mice sorted with FACS. The relative gene and protein expression levels of GCα and GCβ were: PDGFRα(+) cells>ICC>SMC. In contrast, cGKI gene expression sequence was SMC=ICC>PDGFRα(+) cells while cGKI protein expression sequence was neurons>SMC>ICC=PDGFRα(+) cells. The functional role of cGKI was investigated in cGKI(-/-) mice. Relaxation with 8-Br-cGMP was greatly reduced in cGKI(-/-) mice while responses to sodium nitroprusside (SNP) were partially reduced and forskolin responses were unchanged. A nitrergic relaxation occurred with nerve stimulation (NS, 5Hz, 60s) in cGKI(+/+) and cGKI(-/-) mice although there was a small reduction in the cGKI(-/-) mouse. L-NNA abolished responses during the first 20-30s of NS in both animals. The GC inhibitor ODQ greatly reduced or abolished SNP and nitrergic NS responses in both animals. These data confirm an essential role for GC in NO-induced relaxation in the IAS. However, the expression of GC and cGKI by all three cell types suggests that each may participate in coordinating muscular responses to NO. The persistence of nitrergic NMT in the cGKI(-/-) mouse suggests the presence of a significant GC-dependent, cGKI-independent pathway.
[Show abstract][Hide abstract] ABSTRACT: Four types of electrical activity were recorded and related to cell structure by intracellular recording and dye injection into impaled cells in muscles of rabbit small intestine. The specific cell-types from which recordings were made were longitudinal smooth muscle cells (LSMCs), circular smooth muscle cells (CSMCs), interstitial cells of Cajal distributed in the myenteric region (ICC-MY) and fibroblast-like cells (FLCs). Slow waves (slow wavesSMC) were recorded from LSMCs and CSMCs. Slow waves (slow wavesICC) were of greatest amplitude (>50 mV) and highest maximum rate-of-rise (> 10 V s−1) in ICC. The dominant activity in FLCs was spontaneous transient hyperpolarizations (STHs), with maximum amplitudes more than 30 mV. STHs were often superimposed upon small amplitude slow waves (slow wavesFLC). STHs displayed a cyclical patterned discharge irrespective of background slow wave activity. STHs were inhibited by MRS2500 (3 μM), a P2Y1 antagonist, and abolished by apamin (0.3 μM), a blocker of small conductance Ca2+-activated K+ (SK) channels. Small amplitude STHs (< 15 mV) were detected in smooth muscle layers, whereas STHs were not resolved in cells identified as ICC-MY. Electrical field stimulation (EFS) evoked purinergic inhibitory junction potentials (IJPs) in CSMCs. Purinergic IJPs were not recorded from ICC-MY. These results suggest that FLCs may regulate smooth muscle excitability in the rabbit small intestine via generation of rhythmic apamin-sensitive STHs. Stimulation of P2Y1 receptors modulates the amplitudes of STHs. Our results also suggest that purinergic inhibitory motor neurons regulate the motility of the rabbit small intestine by causing IJPs in FLCs that conduct to CSMCs.This article is protected by copyright. All rights reserved
The Journal of Physiology 09/2014; 592(21). DOI:10.1113/jphysiol.2014.276337 · 4.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Enteric inhibitory neurotransmission is an important feature of the neural regulation of gastrointestinal (GI) 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 upon both of these assumptions and suggested another cell type, PDGFRα(+) cells, as 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 MRS2500, a P2Y1 antagonist. ADP, MRS2365, β-NAD and ADPR (P2Y1 agonists) hyperpolarized PDGFRα(+) cells, and these responses were blocked by MRS2500. ADPR 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 an SK channel agonist, CyPPA, consistent with the low expression and current density of SK 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.
[Show abstract][Hide abstract] ABSTRACT: Smooth muscles are complex tissues containing a variety of cells in addition to muscle cells. Interstitial cells of mesenchymal origin interact with and form electrical connectivity with smooth muscle cells in many organs, and these cells provide important regulatory functions. For example, in the gastrointestinal tract, interstitial cells of Cajal (ICC) and PDGFRα(+) cells have been described, in detail, and represent distinct classes of cells with unique ultrastructure, molecular phenotypes, and functions. Smooth muscle cells are electrically coupled to ICC and PDGFRα(+) cells, forming an integrated unit called the SIP syncytium. SIP cells express a variety of receptors and ion channels, and conductance changes in any type of SIP cell affect the excitability and responses of the syncytium. SIP cells are known to provide pacemaker activity, propagation pathways for slow waves, transduction of inputs from motor neurons, and mechanosensitivity. Loss of interstitial cells has been associated with motor disorders of the gut. Interstitial cells are also found in a variety of other smooth muscles; however, in most cases, the physiological and pathophysiological roles for these cells have not been clearly defined. This review describes structural, functional, and molecular features of interstitial cells and discusses their contributions in determining the behaviors of smooth muscle tissues.
[Show abstract][Hide abstract] ABSTRACT: Smooth muscle layers of the gastrointestinal tract consist of a heterogeneous population of cells that include enteric neurons, several classes of interstitial cells of mesenchymal origin, a variety of immune cells and smooth muscle cells (SMCs). Over the last number of years the complexity of the interactions between these cell types has begun to emerge. For example, interstitial cells, consisting of both interstitial cells of Cajal (ICC) and platelet-derived growth factor receptor alpha (PDGFRα+) cells generate pacemaker activity throughout the gastrointestinal (GI) tract and also transduce enteric motor nerve signals and mechanosensitivity to adjacent SMCs. ICC and PDGFRα+ cells are electrically coupled to SMCs possibly via gap junctions forming a multicellular functional syncytium termed the SIP syncytium. Cells that make up the SIP syncytium are highly specialized containing unique receptors, ion channels and intracellular signaling pathways that regulate the excitability of GI muscles. The unique role of these cells in coordinating GI motility is evident by the altered motility patterns in animal models where interstitial cell networks are disrupted. Although considerable advances have been made in recent years on our understanding of the roles of these cells within the SIP syncytium, the full physiological functions of these cells and the consequences of their disruption in GI muscles have not been clearly defined. This review gives a synopsis of the history of interstitial cell discovery and highlights recent advances in structural, molecular expression and functional roles of these cells in the GI tract.
Journal of neurogastroenterology and motility 06/2014; 20(3). DOI:10.5056/jnm14060 · 2.70 Impact Factor