Expression of Shal potassium channel subunits in the adult and developing cochlear nucleus of the mouse.

The pattern of expression of potassium (K(+)) channel subunits is thought to contribute to the establishment of the unique discharge characteristics exhibited by cochlear nucleus (CN) neurons. This study describes the developmental distribution of mRNA for the three Shal channel subunits Kv4.1, Kv4.2 and Kv4.3 within the mouse CN, as assessed with in situ hybridization and RT-PCR techniques. Kv4.1 was not present in CN at any age. Kv4.2 mRNA was detectable as early as postnatal day 2 (P2) in all CN subdivisions, and continued to be constitutively expressed throughout development. Kv4.2 was abundantly expressed in a variety of CN cell types, including all of the major projection neuron classes (i.e., octopus, bushy, stellate, fusiform, and giant cells). In contrast, Kv4.3 was expressed at lower levels and by fewer cell types. Kv4.3-labeled cells were more prevalent in ventral subdivisions than in the dorsal CN. Kv4.3 expression was significantly delayed developmentally in comparison to Kv4.2, as it was detectable only after P14. Although the techniques employed in this study detect mRNA and not protein, it can be inferred from the differential distribution of Kv4 transcripts that CN neurons selectively regulate the expression of Shal K(+) channels among individual neurons throughout development.

C-type natriuretic peptide attenuates evoked dopamine efflux by influencing Goalpha.

-Natriuretic peptides suppress adrenergic neurotransmission by a mechanism sensitive to pertussis toxin, suggesting that GTP-binding proteins are involved in the response. The major GTP-binding proteins present in the pheochromocytoma (PC12) cells used in this report are Goalpha and Gialpha2. We tested the hypothesis that the more abundant GTP-binding protein, Goalpha, mediates natriuretic peptide effects in PC12 cells by selectively ablating Goalpha from the cells with antisense oligodeoxynucleotides. The results indicate that a selective ablation of Goalpha with this technique eliminated C-type natriuretic peptide (CNP) effects and suppressed dopamine efflux evoked by a depolarizing stimulus. However, the activation of guanylyl cyclase (GC) by CNP was sustained after the Goalpha ablation. Further, Nomega-nitro-L-arginine methyl ester suppressed evoked dopamine efflux equally in the presence and absence of Goalpha. These results suggest that CNP attenuates evoked catecholamine efflux from PC12 cells by a mechanism requiring Goalpha but independent of GC activation.

Atrial natriuretic peptide inhibits evoked catecholamine release by altering sensitivity to calcium.

Natriuretic peptides are cyclized peptides produced by cardiovascular and neural tissues. These peptides inhibit various secretory responses such as the release of renin, aldosterone and autonomic neurotransmitters. This report tests the hypothesis that atrial natriuretic peptide reduces dopamine efflux from an adrenergic cell line, rat pheochromocytoma cells, by suppressing intracellular calcium concentrations. The L-type calcium channel inhibitor, nifedipine, markedly suppressed dopamine release from depolarized PC12 cells, suggesting that calcium entering through this channel was the predominant stimulus for dopamine efflux. Atrial natriuretic peptide maximally reduced depolarization-evoked dopamine release 20 +/- 3% at a concentration of 100 nM and this effect was abolished by nifedipine, but not by pretreatment with the N-type calcium channel inhibitor, omega-conotoxin, or an inhibitor of calcium-induced calcium release, ryanodine. In cells loaded with Fura-2, atrial natriuretic peptide both augmented depolarization-induced increases of intracellular free calcium concentrations and accelerated the depolarization-induced quenching of the Fura-2 signal by manganese, findings consistent with enhanced conductivity of calcium channels. Dopamine efflux induced by either the calcium ionophore, A23187, or staphylococcal alpha toxin was attenuated by atrial natriuretic peptide. Additionally, a natriuretic peptide interacting solely with the natriuretic peptide C receptor in these cells, C-type natriuretic peptide, also suppressed calcium-induced dopamine efflux in permeabilized cells. These data are consistent with natriuretic peptides attenuating catecholamine exocytosis in response to calcium but inconsistent with the neuromodulatory effect resulting from a reduction in intracellular calcium concentrations within pheochromocytoma cells.

C-type natriuretic peptide neuromodulates via "clearance" receptors.

A recently discovered endogenous autacoid, C-type natriuretic peptide, was tested in a pheochromocytoma (PC12) cell line for effects on 1) catecholamine release induced by a depolarizing stimulus, 2) guanylyl and adenylyl cyclase activities, and 3) specific 125I-labeled atrial natriuretic peptide (ANP) binding. C-type natriuretic peptide suppressed evoked neurotransmitter release in the absence of guanylyl cyclase activation or adenylyl cyclase inhibition; however, both a "clearance" (ANP-C) receptor binding agent, des-[Gln18Ser19Gly20Leu21Gly22]-ANF-(4-23)-NH2 (cANF), and pertussis toxin prevented this neuromodulatory effect. The C-type natriuretic peptide preferentially bound to receptors that also bound cANF. The results suggest that C-type natriuretic peptide suppressed evoked neurotransmitter efflux by binding to ANP-C receptors coupled to a pertussis toxin-sensitive process; furthermore, the neuromodulatory effect of C-type natriuretic peptide occurred independently of guanylyl cyclase activation or adenylyl cyclase inhibition. The novel aspects of these findings are 1) neuromodulatory effects of C-type natriuretic peptide, 2) guanylyl cyclase-independent actions of C-type natriuretic peptide, and 3) ANP-C receptors mediating C-type natriuretic peptide actions.

Soluble factors from rabbit spleen cells kill and lyse Treponema pallidum in vitro.

Antibody and complement immobilize (kill) Treponema pallidum in vitro. Recent evidence also documents immobilization by soluble factors released by activated macrophages and lymphocytes. Immune-mediated lysis of treponemes, however, has not been reported. The findings in this paper focus on apparent treponemal lysis by rabbit splenic cell preparations. Using cells from animals infected testicularly for 9 to 12 days, unfractionated splenic preparations, as well as adherent and nonadherent preparations, killed and lysed T. pallidum. Phagocytosis alone could not explain the detrimental effects of adherent cells. When cytochalasin B was used to block phagocytosis, decreases in treponemal numbers were still detected. In related studies, immune rabbit sera did not enhance treponemicidal activity of the adherent cells. To assess the specificity of these reactions, T. pallidum was incubated with two monocyte-like cell lines (human U937 and mouse P388D1). Neither cell line was detrimental, and treponemal numbers were not lowered. The soluble nature of the treponemicidal factors from adherent and nonadherent preparations was shown by physically separating these cells from the organisms and demonstrating treponemal killing and lysis. In summary, clearance of T. pallidum from infected tissues is probably at least partially attributed to macrophage phagocytosis. Our findings suggest another mechanism involving lytic factors secreted by activated adherent and nonadherent cells.

Macrophage-mediated suppression of con A-induced IL-2 production in spleen cells from syphilitic rabbits.

Blast transformation studies have indicated a diminished T cell response in spleen cell preparations from rabbits infected with Treponema pallidum. IL-2 synthesis by T lymphocytes is required for proliferation of these cells. Thus, Con A-induced IL-2 generation was measured in syphilitic animals infected for 9 to 14 days. IL-2 production in the infected rabbits was only one-half that observed for uninfected rabbits. This marked decrease in IL-2 was not caused by decreased IL-1 secretion by adherent cells from infected animals because similar levels were found in both infected and uninfected splenic cultures. This decrease was also not caused by an increase in infected spleen cell adsorption of IL-2; similar numbers of receptors for this IL were present in Con A-stimulated infected and uninfected splenic preparations. The inhibited IL-2 production in infected spleen cells was reversed upon removal of the adherent cells and also elevated upon addition of indomethacin to the cultures. PGE levels were also elevated in splenic cultures from infected animals. Finally, IL-2 synthesis, when evaluated at various days postinfection, showed that at 4 days, splenic cells generated twice as much IL-2 as uninfected cells. At 9 to 14 days, IL-2 levels were dramatically decreased (50% lower than that observed in uninfected cultures), and suppression of IL-2 by adherent cells was observed as late as 35 days post-infection. We propose that premature down regulation (suppression) of IL-2 secretion is mediated by adherent cells via a cyclo-oxygenase product, most likely PGE. These results may explain why most, but not all, treponemes are cleared during infection, and why the secondary manifestations of the disease occur.