CFTR is functionally active in GnRH-expressing GT1-7 hypothalamic neurons.

We have demonstrated the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, mRNA, and protein within the rat and human brains, in areas regulating sexual differentiation and function. We have found that GT1-7, a gonadotropin-releasing hormone (GnRH)-secreting hypothalamic neuronal cell line, expresses the CFTR gene, mRNA, and protein and cAMP-dependent (36)Cl efflux. A linear 7-pS Cl- conductance, which is stimulated by ATP and cAMP analogs and inhibited by glibenclamide, consistent with CFTR activity, has been identified in GT1-7 cells. Antisense oligo(dN) generated against exon 10 of the CFTR gene transcript (mRNA) inhibit GnRH secretion into media [312 +/- 73, 850 +/- 150, 963 +/- 304, and 912 +/- 74 pg GnRH/4 x 10(6) cells for antisense, sense, missense, and no oligo(dN), respectively; P < 0. 029 for antisense oligo(dN)-treated vs. normal cells]. No changes in intracellular synthesis of GnRH were noted [1,400 +/- 371 and 1,395 +/- 384 pg GnRH/4 x 10(6) cells for antisense and sense oligo(dN), respectively]. Antisense oligo(dN), but not sense or missense oligo(dN), inhibited cAMP-dependent 36Cl efflux. The expression of CFTR protein, detected by Western blotting, was also inhibited 68% by preincubation of cells with antisense oligo(dN). GT1-7 hypothalamic neurons express the CFTR gene, mRNA, and protein, which modulate neurosecretion. Abnormal neuropeptide vesicle trafficking by mutant CFTR may help to explain some of the diverse manifestations of cystic fibrosis.

Prostaglandin F2alpha stimulates CFTR activity by PKA- and PKC-dependent phosphorylation.

The cystic fibrosis transmembrane conductance regulator (CFTR) can be activated by protein kinase A (PKA)- or protein kinase C (PKC)-dependent phosphorylation. To understand how activation of both kinases affects CFTR activity, transfected NIH/3T3 cells were stimulated with forskolin (FSK), phorbol myristate acetate (PMA), or prostaglandin F2alpha (PGF). PGF stimulates inositol trisphosphate and cAMP production in NIH/3T3 cells. As measured by I- efflux, maximal CFTR activity with PGF and FSK was equivalent and fivefold greater than that with PMA. Both PGF and PMA had additive effects on FSK-dependent CFTR activity. PMA did not increase cellular cAMP, and maximal PGF-dependent CFTR activity occurred with approximately 20% of the cellular cAMP observed with FSK-dependent activation. Staurosporine, but not H-89, inhibited CFTR activation and in vivo phosphorylation at low PGF concentrations. In contrast, at high PGF concentrations, CFTR activation and in vivo phosphorylation were inhibited by H-89. As judged by protease digestion, the sites of in vivo CFTR phosphorylation with FSK and PMA differed. For PGF, the data were most consistent with in vivo CFTR phosphorylation by PKA and PKC. Our data suggest that activation of PKC can enhance PKA-dependent CFTR activation.

Dopamine-stimulated changes in activated calpain I in rat hippocampal slices.

Activated calpain I immunoreactivity (76 kDa band) was detected in membranes prepared from rat brain hippocampal slices using a polyclonal antiserum raised against an N-terminus peptide of the cleaved subunit of calpain I. While basal levels of activated calpain I were stable over incubation times, 1 nM dopamine (DA) produced an initial 32% increase (5 min) in the 76 kDa protein followed by a 53% decrease in this band at 20 min of incubation. The DA-induced changes in activated calpain I immunoreactivity were blocked by the calpain inhibitor peptide, N-acetyl-Leu-Leu-norleucinal(100 microM) or by EGTA. Basal levels of the 76 kDa band were not affected by the calpain inhibitor. These changes in activated calpain I, elicited by DA, are in accord with the DA-induced decreases in the levels of the calpain substrate, gamma PKC (Yurko-Mauro and Friedman; J Cell Biochem [Abstr] 180:80, 1994; J Neurochem 65: 1622-1630, 1995) and suggest that DA activates this Ca(++)-dependent protease in its regulation of neuronal signal transduction.

Dopamine receptor stimulation decreases cytosolic gamma protein kinase C immunoreactivity in rat hippocampal slices: evidence for increased Ca(2+)-dependent proteolysis.

The effect of dopamine (DA) receptor stimulation on the distribution of gamma protein kinase C (gamma PKC) in hippocampal slices was assessed. Nanomolar concentrations of DA decreased cytosolic gamma PKC (56%) without altering membrane gamma PKC levels, resulting in decreased total gamma PKC immunoreactivity. The maximal decrease in cytosolic gamma PKC occurred at 20 min of incubation and was significantly blocked by the D1 DA antagonist SCH 23390 (10(-6) M) but not by the D2 antagonist sulpiride (10(-5) M). The D1 agonists SKF 38393 and A 77636 mimicked the effect of DA with similar responses produced at 10 microM and 1 nM, respectively. The D2 agonist quinpirole had no effect on gamma PKC immunoreactivity, thus indicating that this dopaminergic response is mediated through a D1-like receptor. DA had no effect on alpha, delta, or zeta PKC isozyme immunoreactivity in the same hippocampal preparations. The DA-induced decrease in cytosolic gamma PKC immunoreactivity was blocked by the Ca(2+)-dependent protease inhibitor N-acetyl-Leu-Leu-norleucinal (100 microM) and by the inorganic Ca2+ channel blocker Co2+. The data suggest that DA stimulates a D1-like DA receptor, which increases the influx of Ca2+ and activates the Ca(2+)-dependent proteolysis of gamma PKC.