PDGFRα+ Interstitial Cells are Effector Cells of PACAP Signaling in Mouse and Human Colon.

BACKGROUND & AIMS: Platelet-derived growth factor receptor α (PDGFRα)-positive interstitial cells (PIC) are interposed between enteric nerve fibers and smooth muscle cells (SMCs) in the tunica muscularis of the gastrointestinal tract. PIC have robust expression of small conductance Ca2+ activated K+ channels 3 (SK3 channels) and transduce inhibitory inputs from purinergic and sympathetic nerves in mouse and human colon. We investigated whether PIC also express pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, PAC1 (PAC1R), and are involved in mediating inhibitory regulation of colonic contractions by PACAP in mouse and human colons. METHODS: Gene expression analysis, Ca2+ imaging, and contractile experiments were performed on mouse colonic muscles. Ca2+ imaging, intracellular electrical recordings, and contractile experiments were performed on human colonic muscles. RESULTS: Adcyap1r1 (encoding PAC1R) is highly expressed in mouse PIC. Interstitial cells of Cajal (ICC) and SMCs expressed far lower levels of Adcyap1r. Vipr1 and Vipr2 were expressed at low levels in PIC, ICC, and SMCs. PACAP elicited Ca2+ transients in mouse PIC and inhibited spontaneous phasic contractions via SK channels. In human colonic muscles, PAC1R agonists elicited Ca2+ transients in PIC, hyperpolarized SMCs through SK channels and inhibited spontaneous phasic contractions. CONCLUSIONS: PIC of mouse and human colon utilize PAC1R-SK channel signal pathway to inhibit colonic contractions in response to PACAP. Effects of PACAP are in addition to the previously described purinergic and sympathetic inputs to PIC. Thus, PIC integrate inhibitory inputs from at least 3 neurotransmitters and utilize several types of receptors to activate SK channels and regulate colonic contractile behaviors.

A novel postsynaptic signal pathway of sympathetic neural regulation of murine colonic motility.

Transcriptome data revealed α1 adrenoceptors (ARs) expression in platelet-derived growth factor receptor α+ cells (PDGFRα+ cells) in murine colonic musculature. The role of PDGFRα+ cells in sympathetic neural regulation of murine colonic motility was investigated. Norepinephrine (NE), via α1A ARs, activated a small conductance Ca2+ -activated K+ (SK) conductance, evoked outward currents and hyperpolarized PDGFRα+ cells (the α1A AR-SK channel signal pathway). α1 AR agonists increased intracellular Ca2+ transients in PDGFRα+ cells and inhibited spontaneous phasic contractions (SPCs) of colonic muscle through activation of a SK conductance. Sympathetic nerve stimulation inhibited both contractions of distal colon and propulsive contractions represented by the colonic migrating motor complexes (CMMCs) via the α1A AR-SK channel signal pathway. Postsynaptic signaling through α1A ARs in PDGFRα+ cells is a novel mechanism that conveys part of stress responses in the colon. PDGFRα+ cells appear to be a primary effector of sympathetic neural regulation of murine colonic motility.

The functional role of intramuscular interstitial cells of Cajal in the stomach.

Intramuscular interstitial cells of Cajal (ICC-IM) are found within the smooth muscle layers of the stomach. ICC-IM are mainly spindle shaped cells with bipolar processes orientated along the long axis of surrounding smooth muscle cells. ICC-IM make close contacts with nerve varicosities and form gap junctions with neighbouring smooth muscle cells, indicating that ICC-IM mediate enteric motor neurotransmission. These morphological properties of ICC-IM are similar throughout the stomach. However, the electrical properties of these cells differ from region to region. In the fundus, ICC-IM generate spontaneous transient depolarizations (STDs), resulting in an ongoing discharge of unitary potentials in the smooth muscle cells. ICC-IM in the corpus generate slow waves and as they fire at the highest frequency they serve as the dominant pacemaker cells in the stomach. On the other hand, ICC-IM in the antrum generate the secondary component of slow waves triggered by the initial component that propagates passively from myenteric ICC (ICC-MY). Thus, the different electrical properties of ICC-IM play a critical role in creating the distinct functions of the proximal and distal regions of the stomach such that the fundus acts as a reservoir of food, the corpus as a dominant pacemaker region, while the antrum acts as a region for mixing and propulsion of food.

Interstitial cells of Cajal generate spontaneous transient depolarizations in the rat gastric fundus.

Intracellular recordings were made from isolated circular muscle bundles of rat gastric fundus. The majority of cells generated an ongoing discharge of electrical activity that were 15 min) resulted in the spread of dye between CSMC, between ICC-IM, and between CSMC and ICC-IM. Two types of STDs were observed, regularly occurring continuous STDs and irregular noisy bursting STDs. The amplitude of STDs varied between the two types of STDs. Single units summed to develop STDs with a maximum amplitude of 30 mV. Sodium nitroprusside (3 microM) induced membrane hyperpolarization and abolished unitary potentials generated by CSMC. In contrast, the amplitude of STDs generated by ICC-IM was increased with membrane hyperpolarization. Hyperpolarization induced by pinacidil (10 microM) also increased the amplitude of STDs and enhanced dV/dt(max). These observations indicate that STDs generated in ICC-IM spread passively to the adjacent CSMC to evoke the discharge of unitary potentials in the gastric fundus.

Role of K(+) channels in the regulation of electrical spontaneous activity of the mouse small intestine.

The roles of K(+) channels in the regulation of slow waves and pacemaker potentials recorded from mouse small intestine were investigated using intracellular recording techniques in the presence of nifedipine. Iberiotoxin (0.1 microM) and charybdotoxin (0.1 microM) had no effect on the generation of slow waves recorded from circular smooth muscle cells. Apamin (0.3 microM) depolarized the membrane and decreased the amplitude of early, rapid repolarization of slow waves, without altering the amplitude, frequency, duration, or maximum rate of rise of the initial upstroke phase (dV/dt(max)). The early, rapid repolarization was enhanced by phenylephrine (15 microM). 4-Aminopyridine (4-AP, 5 mM) depolarized the membrane and increased the amplitude and dV/dt(max) of slow waves. Both apamin and 4-AP depolarized the membrane and decreased the amplitude and dV/dt(max) of pacemaker potentials recorded from interstitial cells of Cajal distributed in the myenteric region (ICC-MY). Membrane depolarization with a high-K(+) solution decreased the amplitude and dV/dt(max) of slow waves. These results suggest that apamin-sensitive K(+) conductance and 4-AP-sensitive K(+) conductance may contribute to the resting membrane potential of circular smooth muscle cells. The early, rapid repolarization of slow waves appears to result from the opening of apamin-sensitive K(+) conductance. 4-AP-sensitive K(+) conductance is likely to be activated in the initial upstroke component (primary component) of slow waves. In ICC-MY, membrane depolarization induced by apamin or 4-AP may result from electrotonic spread from smooth muscle cells.

Effects of temperature on pacemaker potentials in the mouse small intestine.

The effects of temperature on the generation of pacemaker potentials recorded from myenteric interstitial cells of Cajal (ICC-MY) distributed in the mouse small intestine were investigated using intracellular recording techniques. In response to increasing temperatures in the range of 26-40 degrees C, the frequency and maximum rate of rise (dV/dt (max)) of pacemaker potentials were increased while their duration was decreased. The resting membrane potential and amplitude of the pacemaker potentials were not affected by change in temperature. Elevation of temperature decreased the amplitude, duration, and rise time of unitary potentials generated spontaneously during intervals between the pacemaker potentials. Metabolic inhibition (KCN and iodoacetic acid) decreased the frequency of pacemaker potentials with no alteration to the amplitude and dV/dt (max). Cyclopiazonic acid (3 muM), an inhibitor of the internal Ca(2+) pump, abolished pacemaker potentials in low-temperature conditions (<29 degrees C) but not at high-temperature conditions (>38 degrees C). These results suggest that the primary and plateau components of pacemaker potentials have different temperature sensitivities: the primary component is highly temperature-sensitive and is activated at higher temperatures, while the plateau component is formed by activation of temperature-insensitive mechanisms. The results also suggest that the mitochondria-induced intracellular Ca(2+) handling system seems to be involved in the initiation of the generation of pacemaker potentials but not in their configuration.

Inhibitory actions of cilostazol on electrical responses of smooth muscle isolated from the guinea-pig stomach antrum.

We have investigated the effects of cilostazol, a type III phosphodiesterase inhibitor, on the electrical responses of smooth muscle tissue isolated from the guinea-pig stomach antrum. Cilostazol (10(-5) M) inhibited slow waves recorded from circular muscle cells, but did not significantly alter the pacemaker potentials and follower potentials recorded from myenteric interstitial cells and longitudinal muscle cells respectively. Slow potentials generated in isolated circular muscle bundles without attached myenteric interstitial cells were inhibited by cilostazol (>10(-7) M), while all membrane activities were abolished by 10(-5) M cilostazol. In circular muscle bundles, the input resistance of smooth muscle cells and the refractory period for the generation of slow potentials were not altered during the inhibition of spontaneous activity with cilostazol. While cilostazol at 10(-7) and 10(-6) M did not elevate the tissue content of cyclic AMP, at 10(-5) M cyclic AMP was elevated by about 30%. A similar elevation was also produced by 10(-7) M forskolin. The content of cyclic AMP was not significantly increased in preparations stimulated with 10(-3) M caffeine. The potency for inhibiting slow waves was in the order caffeine (10(-3) M) > forskolin (10(-7) M) > cilostazol (10(-5) M). The frequency of slow waves was decreased by caffeine or forskolin but not by cilostazol, while the duration was reduced by caffeine but not by cilostazol or forskolin. Follower potentials were modulated by caffeine and forskolin, but not by cilostazol: the duration was reduced by caffeine, the frequency was reduced by caffeine or forskolin, and the amplitude was not significantly altered by any of them. The results indicate that cilostazol has high selectivity in inhibiting the activity of circular muscle much more than that of longitudinal muscle or pacemaker cells, with no causal relation to the tissue content of cyclic AMP as appears to be the case for the inhibitory actions of caffeine and forskolin.

Components of pacemaker potentials recorded from the guinea pig stomach antrum.

Pacemaker potentials recorded intracellularly from the guinea pig stomach consisted of initial primary and following plateau components. Inhibition of the internal Ca2+ store pump with cyclopiazonic acid depolarized the membrane and inhibited the plateau component of pacemaker potentials. 2-aminoethoxydiphenyl borate (an inhibitor of IP3-induced Ca2+ release) and carbonyl cyanide m-chlorophenyl-hydrazone (a mitochondrial protonophore) depolarized the membrane and abolished pacemaker potentials. Low [Ca2+]o solution reduced the frequency and rate of rise of pacemaker potentials, and the effects were mimicked by BAPTA-AM (an intracellular Ca2+ chelator). 4,4-diisothiocyanatostilbene-2,2-disulphonic acid and low [Cl-]o solution inhibited the plateau component of pacemaker potentials. Depolarization of the membrane with high [K+]o solutions increased the frequency and reduced the dV/dt(max) of pacemaker potentials. During high-[K+]o-induced depolarization, cyclopiazonic acid abolished pacemaker potentials. Caffeine, forskolin, papaverine, 8-bromo-cGMP and (+/-)S-nitroso-N-acetylpenicillamine (SNAP) inhibited the plateau component, with no alteration of the primary component. It is concluded that the primary and plateau components of pacemaker potentials are related to voltage-gated Ca2+ influx and Ca2+-activated Cl- channels, respectively, and cyclic nucleotides inhibit mainly the latter. Pacemaker potentials may be generated by the release of Ca2+ from internal stores through excitation of inositol 1,4,5-trisphosphate receptors, coupled with Ca2+ uptake into mitochondria.

Excitation of smooth muscles isolated from the guinea-pig gastric antrum in response to depolarization.

In small segments of circular smooth muscle bundle isolated from the guinea-pig gastric antrum, depolarization of the tissue with intracellular current stimuli evoked regenerative slow potentials after a refractory period of 5-10 s. The refractory period changed inversely with the amplitude and duration of the stimulating depolarization. Thapsigargin (an inhibitor of calcium-ATPase at internal stores), 2-aminoethoxydiphenyl borate (2-APB, an inhibitor of inositol 1,4,5-trisphosphate (IP3)-receptor-mediated Ca2+ release), and carbonyl cyanide m-chlorophenyl-hydrazone (a mitochondrial protonophore) reduced the amplitude of slow potentials, with no significant alteration of the refractory period. Bisindolylmaleimide I or chelerythrine (inhibitors of protein kinase C, PKC) increased the refractory period and inhibited the amplitude of slow potentials. These results indicate that the refractory period and amplitude of slow potentials are related to the activation of PKC and the amount of Ca2+ released from the internal stores through activation of IP3 receptors, respectively. Acetylcholine (ACh) reduced the refractory period and increased the amplitude of slow potentials: the former was antagonized by chelerythrine and the latter by 2-APB. The results suggest that ACh has dual actions; stimulation of the metabolism of inositol phosphate and activation of PKC. Phorbol-12-myristate-13-acetate, a selective stimulant of PKC, at low concentrations (< 10 nM) mimicked the actions of ACh and at high concentrations reduced the frequency of slow potentials and increased the refractory period. The possible involvement of the concentration-dependent differences in the actions of phorbol ester on the translocation of PKC was considered.