Origin of Rhythmic Calcium Transients in the ICC-MP of the Mouse Small Intestine

Abstract

Interstitial cells of Cajal associated with the myenteric plexus (ICC-MP) are pacemaker cells of the small intestine, producing the characteristic omnipresent electrical slow waves, which orchestrate peristaltic motor activity and are associated with rhythmic intracellular calcium oscillations. Our objective was to elucidate the origins of the calcium transients. We hypothesized that calcium oscillations in the ICC-MP are primarily regulated by the sarcoplasmic reticulum (SR) calcium release system. With the use of calcium imaging, study of the effect of T-type calcium channel blocker mibefradil revealed that T-type channels did not play a major role in generating the calcium transients. 2-Aminoethoxydiphenyl borate, an inositol 1,4,5 trisphosphate receptor (IP 3R) inhibitor, and U73122, a phospholipase C inhibitor, both drastically decreased the frequency of calcium oscillations, suggesting a major role of IP 3 and IP3-induced calcium release from the SR. Immunohistochemistry proved the expression of IP 3R type I (IP3R-I), but not type II (IP 3R-II) and type III (IP3R-III) in ICC-MP, indicating the involvement of the IP 3R-I subtype in calcium release from the SR. Cyclopiazonic acid, a SR/endoplasmic reticulum calcium ATPase pump inhibitor, strongly reduced or abolished calcium oscillations. The Na-Ca exchanger (NCX) in reverse mode is likely involved in refilling the SR because the NCX inhibitor KB-R7943 markedly reduced the frequency of calcium oscillations. Immunohistochemistry revealed 100% colocalization of NCX and c-Kit in ICC-MP. Testing a mitochondrial NCX inhibitor, we were unable to show an essential role for mitochondria in regulating calcium oscillations in the ICC-MP. In summary, ongoing IP 3 synthesis and IP3-induced calcium release from the SR, via the IP 3R-I, are the major drivers of the calcium transients associated with ICC pacemaker activity. This suggests that a biochemical clock intrinsic to ICC determines the pacemaker frequency, which is likely directly linked to kinetics of the IP 3-activated SR calcium channel and IP3 metabolism. interstitial cells of Cajal; myenteric plexus; intestinal pacemaker activity; sarcoplasmic reticulum; inositol 1,4,5 trisphosphate receptor; phospholipase c INTERSTITIAL CELLS OF CAJAL (ICC) are pacemaker cells (33, 56) responsible for initiating and propagating electrical slow waves (13) essential for several motor patterns of the gastrointestinal tract (23, 51). The ICC associated with the myenteric plexus of the small intestine (ICC-MP) spontaneously oscillate between high and low intracellular calcium (57, 59) at the same frequency as their electrical pacemaker activity (45). Before calcium oscillations were demonstrated, intracellular calcium

Mitochondrial calcium handling within the interstitial cells of Cajal

“…7 suggests that [IP 3 ] below 100 nM would set depletion times around the lower experimentally observed cpm, consistent with inhibited phospholipase C lowering IP 3 production and Ca 2ϩ oscillation frequencies (45). ICC [IP 3 ] are not well known, but Xenopus laevis levels change from tens of nanomolar to up over a micromolar (46); the ICC may conceivably display similar ranges.…”

“…Alternatively, if only the LetM1 antiporter is turned off in the PMU, no significant increases occur at all, suggesting that both the LetM1 and the MCU are required for effective MT Ca 2ϩ removal. Notably, location of the MCU either within the ER-MT gap or the PMU proper exposes them to different Ca 2ϩ levels (up to 45 concentration. The plasma membrane NCX is also noted with a rather high density in the ICC (45), and our estimate based on cardiac cell NCX activity may also be too low (see Appendix, section Other fluxes J SERCA , J PMNCX , J PMCA , J PMLeak , J MT ).…”

“…These include ER Ca 2ϩ depletion responses like SOCE, ion influxes, and membrane depolarizations triggering channels such as the ANO1 (30). We also excluded PM NCX reversal modes suggested to be involved in SW (45) or voltage-gated channels like the Ca 2ϩ T-type implicated in entrainment of spatially distinct pacing sites (42,72). These results are thus limited to mechanisms generating observed intracellular Ca 2ϩ oscillations before membrane channel flux (55,69).…”

“…This is most salient here with sealing of the ER-MT gap that is physiologically unlikely. Also, a fixed [IP 3 ] excludes known dynamic interactions with Ca 2ϩ as in negative feedback effects (57) with implications for whether [IP 3 ] themselves are oscillating as suggested (45,72). Nevertheless, [IP 3 ] and several other key aspects such as SERCA or NCX density in the ICC await experimental determination.…”

“…It is known that Ca 2ϩ transport via the intracellular inositol-trisphosphate (IP 3 ) receptors (IP 3 R) on the ER into the cytosol (73,78) and mitochondria (MT) uptake of cytosolic Ca 2ϩ (38,78) is involved in generation of SW activity. Disruption of MT sodium-calcium exchangers (NCX) reduces the frequency of the Ca 2ϩ oscillations (45), albeit modestly and eventually on a time scale of minutes; notably, the MT NCX are also essential to the function of store-operated Ca 2ϩ entry (SOCE) (52). We thus hypothesize that depletions of the ER Ca 2ϩ reservoir and subsequent activation of SOCE are fundamental to pacemaking of the ICC.…”