Sean M. Ward

Acetylcholine is a predominant excitatory neurotransmitter in the gastrointestinal (GI) tract and targets M ₂ and M ₃ receptors. We used cell-specific, conditional M ₃ knockout mice and found that responses to cholinergic neurotransmission in gastric fundus were unaffected in smooth muscle cell knockouts and greatly depressed in knockouts in interstitial cells of Cajal (ICCs). The results suggest that responses to cholinergic neurotransmission are mediated through M ₃ receptors expressed by ICCs and not directly through SMCs.

The physiological roles of newly described PDGFRα ⁺ interstitial cells in neurotransmission within the gastrointestinal (GI) tract have predominantly come from studies on isolated cells. Here we used an inhibitor of PDGFRα, crenolanib, to examine the effects of PDGFRα ⁺ cells in enteric inhibitory neurotransmission. Crenolanib caused loss of PDGFRα ⁺ cells and neurally evoked fast inhibitory junction potentials associated with purine neurotransmission, providing evidence for the function of PDGFRα ⁺ cells within intact tissues of the GI tract.

Prostaglandins are lipid mediators that have complex actions on gastrointestinal motility that are highly dependent on the expression of the receptor subtypes where they exert their actions. PGE₂ has inhibitory or excitatory effects on circular or longitudinal muscle layers of the small intestine. Despite many studies of the effects of prostaglandins on tissue contractility, little is known about the specific receptors eliciting these effects. The present study examines functional receptor expression in the small intestine.

The physiological roles of interstitial cells of Cajal (ICC) throughout the gastrointestinal (GI) tract have been derived predominantly from studies of mice. We sought to determine whether reduction in ICC in the rat, a commonly used animal for studies of GI motor functions, leads to functional deficits. Ws/Ws rats display reduced ICC leading to a disruption in pacemaker activity and neuroeffector responses. Our results provide additional evidence for the functions of ICC in the GI tract.

Smooth muscle cells (SMCs), Interstitial cells of Cajal (ICC) and Platelet‐derived growth factor receptor α positive (PDGFRα ⁺ ) cells form an integrated, electrical syncytium within the gastrointestinal (GI) muscular tissues known as the SIP syncytium. Immunohistochemical analysis of gastric corpus muscles showed that c‐KIT ⁺ /ANO1 ⁺ ICC‐IM and PDGFRα ⁺ cells were closely apposed to one another in the same anatomical niches. We used intracellular microelectrode recording from corpus muscle bundles to characterize the roles of intramuscular ICC and PDGFRα ⁺ cells in conditioning membrane potentials of gastric muscles. In muscle bundles, that have a relatively higher input impedance than larger muscle strips or sheets, we recorded an ongoing discharge of stochastic fluctuations in membrane potential, previously called unitary potentials or spontaneous transient depolarizations (STDs) and spontaneous transient hyperpolarizations (STHs). We reasoned that STDs should be blocked by antagonists of ANO1, the signature conductance of ICC. Activation of ANO1 has been shown to generate spontaneous transient inward currents (STICs), which are the basis for STDs. Ani9 reduced membrane noise and caused hyperpolarization, but this agent did not block the fluctuations in membrane potential quantitatively. Apamin, an antagonist of small conductance Ca ²⁺ ‐activated K ⁺ channels (SK3), the signature conductance in PDGFRα ⁺ cells, further reduced membrane noise and caused depolarization. Reversing the order of channel antagonists reversed the sequence of depolarization and hyperpolarization. These experiments show that the ongoing discharge of STDs and STHs by ICC and PDGFRα ⁺ cells, respectively, exerts conditioning effects on membrane potentials in the SIP syncytium that would effectively regulate the excitability of SMCs.

Pulmonary arterial (PA) smooth muscle cells (PASMC) generate vascular tone in response to agonists coupled to G_q-protein receptor signaling. Such agonists stimulate oscillating calcium waves, the frequency of which drives the strength of contraction. These Ca²⁺ events are modulated by a variety of ion channels including voltage-gated calcium channels (Ca_V1.2), the Tmem16a or Anoctamin-1 (ANO1)-encoded calcium-activated chloride (CaCC) channel, and Ca²⁺ release from the sarcoplasmic reticulum through inositol-trisphosphate receptors (IP₃R). Although these calcium events have been characterized, it is unclear how these calcium oscillations underly a sustained contraction in these muscle cells. We used smooth muscle–specific ablation of ANO1 and pharmacological tools to establish the role of ANO1, Ca_V1.2, and IP₃R in the contractile and intracellular Ca²⁺ signaling properties of mouse PA smooth muscle expressing the Ca²⁺ biosensor GCaMP3 or GCaMP6. Pharmacological block or genetic ablation of ANO1 or inhibition of Ca_V1.2 or IP₃R, or Ca²⁺ store depletion equally inhibited 5-HT-induced tone and intracellular Ca²⁺ waves. Coimmunoprecipitation experiments showed that an anti-ANO1 antibody was able to pull down both Ca_V1.2 and IP₃R. Confocal and superresolution nanomicroscopy showed that ANO1 coassembles with both Ca_V1.2 and IP₃R at or near the plasma membrane of PASMC from wild-type mice. We conclude that the stable 5-HT-induced PA contraction results from the integration of stochastic and localized Ca²⁺ events supported by a microenvironment comprising ANO1, Ca_V1.2, and IP₃R. In this model, ANO1 and Ca_V1.2 would indirectly support cyclical Ca²⁺ release events from IP₃R and propagation of intracellular Ca²⁺ waves.

The cell types and conductance that contribute to normal cardiac functions remain under investigation. We used mice that express an enhanced green fluorescent protein (eGFP)‐histone 2B fusion protein driven off the cell‐specific endogenous promoter for Pdgfra to investigate the distribution and functional role of PDGFRα ⁺ cells in the heart. Cardiac PDGFRα ⁺ cells were widely distributed within the endomysium of atria, ventricle, and sino‐atrial node (SAN) tissues. PDGFRα ⁺ cells formed a discrete network of cells, lying in close apposition to neighboring cardiac myocytes in mouse and Cynomolgus monkey ( Macaca fascicularis ) hearts. Expression of eGFP in nuclei allowed unequivocal identification of these cells following enzymatic dispersion of muscle tissues. FACS purification of PDGFRα ⁺ cells from the SAN and analysis of gene transcripts by qPCR revealed that they were a distinct population of cells that expressed gap junction transcripts, Gja1 and Gjc1 . Cardiac PDGFRα ⁺ cells generated spontaneous transient inward currents (STICs) and spontaneous transient depolarizations (STDs) that reversed at 0 mV. Reversal potential was maintained when E _Cl = −40 mV. [Na ⁺ ] _o replacement and FTY720 abolished STICs, suggesting they were due to a non‐selective cation conductance (NSCC) carried by TRPM7. PDGFRα ⁺ cells also express β ₂ ‐adrenoceptor gene transcripts, Adrb2 . Zinterol, a selective β ₂ ‐receptor agonist, increased the amplitude and frequency of STICs, suggesting these cells could contribute to adrenergic regulation of cardiac excitability. PDGFRα ⁺ cells in cardiac muscles generate inward currents via an NSCC. STICs generated by these cells may contribute to the integrated membrane potentials of cardiac muscles, possibly affecting the frequency of pacemaker activity.

Enteric neurotransmission is critical for coordinating motility throughout the gastrointestinal (GI) tract. However, there is considerable controversy regarding the cells that are responsible for the transduction of these neural inputs. In the present study, utilization of a cell‐specific calcium biosensor GCaMP6f, the spontaneous activity and neuroeffector responses of intramuscular ICC (ICC‐IM) to motor neural inputs was examined. Simultaneous intracellular microelectrode recordings and high‐speed video‐imaging during nerve stimulation was used to reveal the temporal relationship between changes in intracellular Ca²⁺ and post‐junctional electrical responses to neural stimulation. ICC‐IM were highly active, generating intracellular Ca²⁺‐transients that occurred stochastically, from multiple independent sites in single ICC‐IM. Ca²⁺‐transients were not entrained in single ICC‐IM or between neighbouring ICC‐IM. Activation of enteric motor neurons produced a dominant inhibitory response that abolished Ca²⁺‐transients in ICC‐IM. This inhibitory response was often preceded by a summation of Ca²⁺‐transients that led to a global rise in Ca²⁺. Individual ICC‐IM responded to nerve stimulation by a global rise in Ca²⁺ followed by inhibition of Ca²⁺‐transients. The inhibition of Ca²⁺‐transients was blocked by the nitric oxide synthase antagonist l‐NNA. The global rise in intracellular Ca²⁺ was inhibited by the muscarinic antagonist, atropine. Simultaneous intracellular microelectrode recordings with video‐imaging revealed that the rise in Ca²⁺ was temporally associated with rapid excitatory junction potentials and the inhibition of Ca²⁺‐transients with inhibitory junction potentials. These data support the premise of serial innervation of ICC‐IM in excitatory and inhibitory neuroeffector transmission in the proximal stomach. image Key points: The cells responsible for mediating enteric neuroeffector transmission remain controversial. In the stomach intramuscular interstitial cells of Cajal (ICC‐IM) were the first ICC reported to receive cholinergic and nitrergic neural inputs. Utilization of a cell specific calcium biosensor, GCaMP6f, the activity, and neuroeffector responses of ICC‐IM were examined. ICC‐IM were highly active, generating stochastic intracellular Ca²⁺‐transients. Stimulation of enteric motor nerves abolished Ca²⁺‐transients in ICC‐IM. This inhibitory response was preceded by a global rise in intracellular Ca²⁺. Individual ICC‐IM responded to nerve stimulation with a rise in Ca²⁺ followed by inhibition of Ca²⁺‐transients. Inhibition of Ca²⁺‐transients was blocked by the nitric oxide synthase antagonist l‐NNA. The global rise in Ca²⁺ was inhibited by the muscarinic antagonist atropine. Simultaneous intracellular recordings with video imaging revealed that the global rise in intracellular Ca²⁺ and inhibition of Ca²⁺‐transients was temporally associated with rapid excitatory junction potentials followed by more sustained inhibitory junction potentials. The data presented support the premise of serial innervation of ICC‐IM in excitatory and inhibitory neuroeffector transmission in the proximal stomach.

Obesity is a critical risk factor of several life-threatening diseases and the prevalence in adults has dramatically increased over the past ten years. In the USA the age-adjusted prevalence of obesity in adults was 42.4%, i.e., with a body mass index (BMI, weight (kg)/height (m)²) that exceeds 30 kg/m². Obese individuals are at the higher risk of obesity-related diseases, co-morbid conditions, lower quality of life, and increased mortality more than those in the normal BMI range i.e., 18.5–24.9 kg/m². Surgical treatment continues to be the most efficient and scientifically successful treatment for obese patients. Sleeve gastrectomy or vertical sleeve gastrectomy (VSG) is a relatively new gastric procedure to reduce body weight but is now the most popular bariatric operation. To date there have been few studies examining the changes in the cellular components and pacemaker activity that occur in the gastric wall following VSG and whether normal gastric activity recovers following VSG. In the present study we used a murine model to investigate the chronological changes of gastric excitability including electrophysiological, molecular and morphological changes in the gastric musculature following VSG. There is a significant disruption in specialized interstitial cells of Cajal in the gastric antrum following sleeve gastrectomy. This is associated with a loss of gastric pacemaker activity and post-junctional neuroeffector responses. Over a 4-month recovery period there was a gradual return in interstitial cells of Cajal networks, pacemaker activity and neural responses. These data describe for the first time the changes in gastric interstitial cells of Cajal networks, pacemaker activity and neuroeffector responses and the time-dependent recovery of ICC networks and normalization of motor activity and neural responses following VSG.

Gangliosides are sialic acid-containing glycosphingolipids, which are the most abundant family of glycolipids in eukaryotes. Gangliosides have been suggested to be important lipid molecules required for the control of cellular procedures, such as cell differentiation, proliferation, and signaling. GD1a is expressed in interstitial cells during ovarian maturation in mice and exogenous GD1a is important to oocyte maturation, monospermic fertilization, and embryonic development. In this context, GM1 is known to influence signaling pathways in cells and is important in sperm–oocyte interactions and sperm maturation processes, such as capacitation. GM3 is expressed in the vertebrate oocyte cytoplasm, and exogenously added GM3 induces apoptosis and DNA injury during in vitro oocyte maturation and embryogenesis. As a consequence of this, ganglioside GT1b and GM1 decrease DNA fragmentation and act as H₂O₂ inhibitors on germ cells and preimplantation embryos. This review describes the functional roles of gangliosides in spermatozoa, oocytes, and early embryonic development.