We have examined the role of inositol 3,4,5,6-tetrakisphosphate [Ins(3,4,5,6)P4] in the control of Cl- current in CFPAC-1 cells. Intracellular Ins(3,4,5,6)P4 had no effect on basal current, but it produced a five- to sevenfold reduction in the Cl- current stimulated by either 2 microM extracellular ATP or by 1 microM extracellular thapsigargin. The half-maximally effective dose of Ins(3,4,5,6)P4 was 2.9 microM, and 4 microM blocked >80% of the ATP-activated current. In contrast, 10 microM Ins(1,4,5,6)P4, Ins(1,3,4,5)P4, or Ins(1,3,4,6)P4 enhanced rather than inhibited the ATP-activated Cl- current, although Ins(1,4,5,6)P4 only acted transiently. These stimulatory effects were Ca2+ dependent and largely inhibited by coapplication of equimolar Ins(3,4,5,6)P4. Inositol 1,3,4,5,6-pentakisphosphate, the precursor of Ins(3,4,5,6)P4, did not affect Cl- current. These data consolidate and extend the hypothesis that Ins(3,4,5,6)P4 is an important intracellular regulator of Cl- current in epithelial cells.

No abstract

Neuropeptide Y (NPY) potently inhibits excitatory synaptic transmission in the hippocampus, acting predominantly via a presynaptic Y(2) receptor. Recent reports that the Y(5) receptor may mediate the anticonvulsant actions of NPY in vivo prompted us to test the hypothesis that Y(5) receptors inhibit synaptic excitation in the hippocampal slice and, furthermore, that they are effective in an in vitro model of anticonvulsant action. Two putative Y(5) receptor-preferring agonists inhibited excitatory postsynaptic currents (EPSCs) evoked by stimulation of stratum radiatum in pyramidal cells. We recorded initially from area CA1 pyramidal cells, but subsequently switched to cells from the subiculum, where a much greater frequency of response was observed to Y(5) agonist application. Both D-Trp(32)NPY (1 microM) and [ahx(8-20)]Pro(34)NPY (3 microM), a centrally truncated, Y(1)/Y(5) agonist we synthesized, inhibited stimulus-evoked EPSCs in subicular pyramidal cells by 44.0 +/- 5.7% and 51.3 +/- 3.5% (mean +/- SE), in 37 and 58% of cells, respectively. By contrast, the less selective centrally truncated agonist, [ahx(8-20)] NPY (1 microM), was more potent (66.4 +/- 4.1% inhibition) and more widely effective, suppressing the EPSC in 86% of subicular neurons. The site of action of all NPY agonists tested was most probably presynaptic, because agonist application caused no changes in postsynaptic membrane properties. The selective Y(1) antagonist, BIBP3226 (1 microM), did not reduce the effect of either more selective agonist, indicating that they activated presynaptic Y(5) receptors. Y(5) receptor-mediated synaptic inhibition was more frequently observed in slices from younger animals, whereas the nonselective agonist appeared equally effective at all ages tested. Because of the similarity with the previously reported actions of Y(2) receptors, we tested the ability of Y(5) receptor agonists to suppress stimulus train-induced bursting (STIB), an in vitro model of ictaform activity, in both area CA3 and the subiculum. Neither [ahx(8-20)]Pro(34)NPY nor D-Trp(32)NPY were significantly effective in suppressing or shortening STIB-induced afterdischarge, with <20% of slices responding to these agonists in recordings from CA3 and none in subiculum. By contrast, 1 microM each of [ahx(8-20)]NPY, the Y(2) agonist, [ahx(5-24)]NPY, and particularly NPY itself suppressed the afterdischarge in area CA3 and the subiculum, as reported earlier. We conclude that Y(5) receptors appear to regulate excitability to some degree in the subiculum of young rats, but their contribution is relatively small compared with those of Y(2) receptors, declines with age, and is insufficient to block or significantly attenuate STIB-induced afterdischarges.

No abstract

We have studied the regulation of Ca 2؉ -dependent chloride (Cl Ca ) channels in a human pancreatoma epithelial cell line (CFPAC-1), which does not express functional cAMP-dependent cystic fibrosis transmembrane conductance regulator chloride channels. In cell-free patches from these cells, physiological Ca 2؉ concentrations activated a single class of 1-picosiemens Cl ؊ -selective channels. The same channels were also stimulated by a purified type II calmodulin-dependent protein kinase (CaMKII), and in cell-attached patches by purinergic agonists. In whole-cell recordings, both Ca 2؉ -and CaMKII-dependent mechanisms contributed to chloride channel stimulation by Ca 2؉ , but the CaMKII-dependent pathway was selectively inhibited by inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P 4 ). This inhibitory effect of Ins(3,4,5,6)P 4 on Cl Ca channel stimulation by CaMKII was reduced by raising [Ca 2؉ ] and prevented by inhibition of protein phosphatase activity with 100 nM okadaic acid. These data provide a new context for understanding the physiological relevance of Ins(3,4,5,6)P 4 in the longer term regulation of Ca 2؉ -dependent Cl ؊ fluxes in epithelial cells.