No abstract

The effects of serotonin or 5-hydroxytryptamine (5-HT) on Na absorption and intracellular free Ca (Cai) in isolated chicken enterocytes was examined. The rate of initial 22Na uptake was inhibited by 50% after 90 s of stimulation with 5-HT (10(-5) M), an effect that was not additive with amiloride (10(-3) M) and was transient (less than 10 min). 5-HT similarly decreased intracellular pH (pHi) in cells loaded with the pH indicator carboxyfluorescein, an effect that was also transient, not additive with amiloride, and Na dependent. The ED50 of this effect was approximately 10(-8) M. 5-HT also stimulated a transient increase in Cai as determined by quin2 fluorescence. A maximal increase of 60 nM occurred significantly before the peak change in pHi, but the total duration of response was similar in each case. In the absence of extracellular Ca, the 5-HT effects on pHi and Cai still persisted. In cells loaded with the Ca-buffering agent MAPTAM, the 5-HT (10(-5) M) inhibitory effect of 22Na influx was partially inhibited. We conclude that 5-HT directly inhibits Na absorption by isolated enterocytes by releasing endogenous Ca, which subsequently causes an inhibition of amiloride-sensitive Na+-H+ exchange.

In rabbit proximal colon, in vitro addition of phorbol 12,13-dibutyrate (PDB, 10(-7) M) to the serosal bathing medium inhibits mucosal (m)-to-serosal (s) unidirectional Na flux (JsmNa) without altering JsmNa or unidirectional Cl fluxes. Similar results were obtained when amiloride (2 X 10(-4) M) was added to the mucosal bathing medium. No additivity of effect was seen when tissues were exposed to both agents. Measurements with carboxyfluorescein reveal that the two agents cause equal decreases of intracellular pH (pHi), an effect that is dependent on the presence of extracellular Na (Na replacement also decreases pHi). No additivity of pHi effects is seen when both agents are added together. To determine the membrane site of this PDB-inhibitable Na-H exchange, Na influx across the luminal border of proximal colon was measured and was found to be inhibited equally by PDB and amiloride. We conclude that PDB, by activation of protein kinase C, inhibits electro-neutral amiloride-sensitive Na-H exchange in the luminal membrane of proximal colon.

Aberrant production of IL-21 by T cells is critical for the development of type 1 diabetes (T1D) in NOD mice. The pathogenic effects of IL-21 are partly due to its ability to promote the generation of TH-17 cells. Interferon Regulatory Factor (IRF4) is a crucial regulator of IL-17 and IL-21 production. We recently found that the serine-threonine kinase ROCK2 phosphorylates IRF4 and regulates its ability to control IL-17 and IL-21 production. Here we show that NOD T cells aberrantly activate ROCK2. We furthermore demonstrate that ROCK inhibition corrects the abnormal IRF4 function in NOD T cells and diminishes their production of IL-17 and IL-21. Importantly, administration of a ROCK inhibitor to NOD mice protects against diabetes development. These studies thus support the idea that ROCK2 is inappropriately activated in NOD T cells and that ROCK kinases could represent important therapeutic targets for the treatment of T1D.

No abstract