Lauren E. Peri

Differential expression of genes related to purinergic signaling in smooth muscle cells, PDGFRα-positive cells, and interstitial cells of Cajal in the murine colon.

Purinergic signaling provides regulation of colonic motility. Smooth muscle cells (SMC), interstitial cells of Cajal (ICC), and platelet‐derived growth factor receptor α‐positive (PDGFRα+) cells are electrically coupled and form a functional (SIP) syncytium that constitutes the receptive field for motor neurotransmitters in the tunica muscularis. Each cell type in the SIP syncytium has specialized functions in mediating motor neurotransmission. We compared gene transcripts for purinergic receptors and membrane‐bound enzymes for purine degradation expressed by each cell type of the SIP syncytium.

Functional expression of SK channels in murine detrusor PDGFRα+ cells

•  SK currents have been recorded from detrusor smooth muscle cells, but current density at physiological holding potentials is negligible. •  We discovered a new class of interstitial cell in the bladder that were identified using antibodies against platelet‐derived growth factor receptor‐α(PDGFRα+ cells). •  SK3 channel transcripts and protein are highly expressed in PDGFRα+ cells in comparison to smooth muscle cells. •  Current density attributable to SK‐like currents is much higher in PDGFRα+ cells than in smooth muscle cells. Single channel currents, consistent with the conductance and Ca2+ sensitivity of SK3 channels were measured in PDGFRα+ cells. •  The abundance of SK3 channels in PDGFRα+ cells in detrusor muscles suggests that PDGFRα+ cells, not SMCs, provide apamin‐sensitive regulation of detrusor excitability.

Basally activated nonselective cation currents regulate the resting membrane potential in human and monkey colonic smooth muscle

Resting membrane potential (RMP) plays an important role in determining the basal excitability of gastrointestinal smooth muscle. The RMP in colonic muscles is significantly less negative than the equilibrium potential of K(+), suggesting that it is regulated not only by K(+) conductances but by inward conductances such as Na(+) and/or Ca(2+). We investigated the contribution of nonselective cation channels (NSCC) to the RMP in human and monkey colonic smooth muscle cells (SMC) using voltage- and current-clamp techniques. Qualitative reverse transcriptase-polymerase chain reaction was performed to examine potential molecular candidates for these channels among the transient receptor potential (TRP) channel superfamily. Spontaneous transient inward currents and holding currents were recorded in human and monkey SMC. Replacement of extracellular Na(+) with equimolar tetraethylammonium or Ca(2+) with Mn(2+) inhibited basally activated nonselective cation currents. Trivalent cations inhibited these channels. Under current clamp, replacement of extracellular Na(+) with N-methyl-D-glucamine or addition of trivalent cations caused hyperpolarization. Three unitary conductances of NSCC were observed in human and monkey colonic SMC. Molecular candidates for basally active NSCC were TRPC1, C3, C4, C7, M2, M4, M6, M7, V1, and V2 in human and monkey SMC. Comparison of the biophysical properties of these TRP channels with basally active NSCC (bI(NSCC)) suggests that TRPM4 and specific TRPC heteromultimer combinations may underlie the three single-channel conductances of bI(NSCC). In conclusion, these findings suggest that basally activated NSCC contribute to the RMP in human and monkey colonic SMC and therefore may play an important role in determining basal excitability of colonic smooth muscle.

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

Platelet‐derived growth factor receptor‐α‐positive (PDGFRα+) interstitial cells in detrusor muscles may participate in post‐junctional responses to neurotransmitters. PDGFRα+ interstitial cells express purinergic receptors (P2Y) and small conductance Ca2+‐activated K+ channels (mainly SK3). ATP elicited large amplitude outward currents and hyperpolarization in PDGFRα+ cells. SK channel blockers and a P2Y1 receptor antagonist blocked responses to ATP. ATP elicited only minor responses in PDGFRα+ cells of P2ry1−/− mice. ATP elicited transient inward currents in smooth muscle cells and purinergic receptor (P2X) agonists had no effect on PDGFRα+ cells. A specific P2Y1 receptor blocker decreased electrical field stimulation‐induced relaxation. Our findings provide an explanation for the purinergic relaxation of detrusor muscles and describe a novel mechanism for inhibitory regulation of bladder muscles that may control detrusor excitability during the filling phase.

Nitrergic neuromuscular transmission in the mouse internal anal sphincter is accomplished by multiple pathways and postjunctional effector cells

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 quantitative PCR 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 whereas cGKI protein expression sequence was neurons > SMC ≫ ICC = PDGFRα(+) cells. The functional role of cGKI was investigated in cGKI(-/-) mice. Relaxation with 8-bromo (8-Br)-cGMP was greatly reduced in cGKI(-/-) mice whereas responses to sodium nitroprusside (SNP) were partially reduced and forskolin responses were unchanged. A nitrergic relaxation occurred with nerve stimulation (NS, 5 Hz, 60 s) in cGKI(+/+) and cGKI(-/-) mice although there was a small reduction in the cGKI(-/-) mouse. N(ω)-nitro-l-arginine (l-NNA) abolished responses during the first 20-30 s 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.

Identification of histamine receptors and effects of histamine on murine and simian colonic excitability

Background  Inflammatory responses can include recruitment of cells of hematopoietic origin to the tunica muscularis. These cells can secrete a variety of factors which can reset the gain of smooth muscle cells (SMC) and influence motor patterns. Histamine (H), a major mediator in inflammation, is released by mast cells and exerts diverse effects in SMC by binding to H receptors. The profiles of H receptor expression in animal models used to study inflammatory diseases are unknown.

Regulation of Gastric Electrical and Mechanical Activity by Cholinesterases in Mice

Background/Aims 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. Methods 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. Results Both acetycholinesterase (Ache) and butyrylcholine esterase (Bche) are expressed in gastric muscles and AChE is localized to varicose 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) increased 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. Conclusions Our data show that slow wave uncoupling is difficult to assess with electrical recording from a single or double sites and suggest that efficient metabolism of ACh released from motor neurons is an extremely important regulator of slow wave frequency and propagation and gastric motility patterns.

A Novel Class of Interstitial Cells in the Mouse and Monkey Female Reproductive Tracts

ABSTRACT 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.

Excitatory Neuronal Responses of Ca2+ Transients in Interstitial Cells of Cajal in the Small Intestine

Abstract Interstitial cells of Cajal (ICC) regulate smooth muscle excitability and motility in the gastrointestinal (GI) tract. ICC in the deep muscular plexus (ICC-DMP) of the small intestine are aligned closely with varicosities of enteric motor neurons and thought to transduce neural responses. ICC-DMP generate Ca2+ transients that activate Ca2+ activated Cl- channels and generate electrophysiological responses. We tested the hypothesis that excitatory neurotransmitters regulate Ca2+ transients in ICC-DMP as a means of regulating intestinal muscles. High-resolution confocal microscopy was used to image Ca2+ transients in ICC-DMP within murine small intestinal muscles with cell-specific expression of GCaMP3. Intrinsic nerves were stimulated by electrical field stimulation (EFS). ICC-DMP exhibited ongoing Ca2+ transients before stimuli were applied. EFS caused initial suppression of Ca2+ transients, followed by escape during sustained stimulation, and large increases in Ca2+ transients after cessation of stimulation. Basal Ca2+ activity and the excitatory phases of Ca2+ responses to EFS were inhibited by atropine and neurokinin 1 receptor (NK1) antagonists, but not by NK2 receptor antagonists. Exogenous ACh and substance P (SP) increased Ca2+ transients, atropine and NK1 antagonists decreased Ca2+ transients. Neurokinins appear to be released spontaneously (tonic excitation) in small intestinal muscles and are the dominant excitatory neurotransmitters. Subcellular regulation of Ca2+ release events in ICC-DMP may be a means by which excitatory neurotransmission organizes intestinal motility patterns.

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.