Cytoplasmic segment interactions in the alpha1-subunit of the rat Na+, K+-atpase.

The currently accepted topographical model for the organization of the alpha-subunit of the Na+, K+-ATPase in the membrane considers that the protein has ten transmembrane segments and six cytoplasmic loops. Evidence of interaction between the cytoplasmic regions may contribute to a better understanding of the structure/function relationship of this protein. In this study, the first four cytoplasmic segments (C1, C2, C3 and C4) of the rat alpha1 subunit were expressed in Escherichia Coli. The large cytoplasmic loop between transmembrane segments four and five (C3) retained its native structure as demonstrated by the ability of ATP to protect against chemical modification by Fluorescein 5-isothiocyanate (FITC). Interaction studies were conducted by an overlay assay (Far Western blots) and surface plasmon resonance technology. We observed that C3 interacts with the N-terminal segment of the Na+, K+-ATPase, C1; and that both C1 and C3 interact with the cytoplasmic segments C2 and C4.

A role for L-type calcium channels in developmental regulation of transmitter phenotype in primary sensory neurons.

To examine the influence of activity-dependent cues on differentiation of primary afferent neurons, we investigated the short- and long-term effects of depolarization and calcium influx on expression of transmitter traits in sensory ganglion cell cultures. We focused on expression of tyrosine hydroxylase (TH), a marker for dopaminergic neurons, in developing petrosal ganglion (PG), nodose ganglion, and dorsal root ganglion neurons grown in the presence or absence of depolarizing concentrations of KCl. Exposure to 40 mM KCl increased the proportion of TH-immunoreactive neurons in all three ganglia in a developmentally regulated manner that corresponded to the temporal pattern of dopaminergic expression in vivo. PG neurons, for example, were most responsive to elevated KCl on embryonic day 16.5 (E16.5), the age at which the dopaminergic phenotype is first detectable in vivo. However, KCl was relatively ineffective at increasing TH expression in neonatal PG, indicating a critical period for induction of this phenotype by depolarization. Detailed analysis of TH induction in PG neurons demonstrated that, although N-type calcium channels carried the majority of the high voltage-activated calcium current, only L-type calcium channel blockade inhibited the effect of elevated KCl. Further studies revealed that after removal of high KCl, neurons remained sensitized to subsequent stimulation for >1 week. Specifically, cultures exposed to KCl beginning on E16.5 (the conditioning stimulus), then returned to control medium, and subsequently re-exposed to elevated KCl after 9 d (the test stimulus) contained fourfold more TH-positive neurons than did cultures exposed to the test stimulus alone. Moreover, blockade of L-type calcium channels during the conditioning stimulus completely abolished long-term potentiation of the TH response to elevated KCl. These findings demonstrate a novel role for L-type calcium channels in activity-dependent plasticity of transmitter expression in sensory neurons and indicate that exposure to depolarizing stimuli during early development may alter neuronal response properties at later ages.

Serotonin inhibits epileptiform discharge by activation of 5-HT1A receptors in CA1 pyramidal neurons.

The anti-epileptiform effect of serotonin was characterized in cellular models of epilepsy using electrophysiological recording techniques. In the bicuculline model, both serotonin (20 microM) and its 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT, 10 microM) completely blocked the epileptiform discharge and caused membrane hyperpolarization and reduction in input resistance. These effects were completely antagonized by the 5-HT1A receptor antagonist N-t-butyl-3(4-[2-methoxyphenyl]piperazin-1-yl)-2-phenyl-propanamid e(WAY 100135) (10 microM). Epileptiform discharge induced by positive current injection was also blocked by serotonin. The presence of WAY 100135 renders serotonin ineffective in the same model. In the bicuculline model, epileptiform discharge blocked by serotonin reappeared and was also intensified when BaCl2 was added to the medium. To rule out the possibility of serotonin-induced hyperpolarization strengthening the inhibitory effect of endogenous Mg2+ on glutamate N-methyl-D-aspartic acid (NMDA) receptor we studied the antiepileptic effect of serotonin in the 0 Mg2+ model. Spontaneous activity and evoked bursts seen with the 0 Mg2+ model were completely blocked by serotonin. WAY 100135 completely antagonized serotonin effects in this model as well. This study provides evidence suggesting that in rat CA1 pyramidal neurons, serotonin can inhibit epileptiform activity in a variety of accepted epilepsy cellular models and that inhibition of epileptiform bursts by serotonin may be mediated by activation of the 5-HT1A receptor subtype.

Effects of serotonin on induced epileptiform activity in CA1 pyramidal neurons of genetically epilepsy prone rats.

The seizure susceptibility in genetically epilepsy prone rats (GEPRs) is reported to be caused by abnormalities in several neurotransmitter systems including the serotonergic system. Among the reported abnormalities is a decrease in brain serotonin content. Therefore, we examined the effects of exogenous serotonin on brain slices from the severe seizure strain of GEPRs (GEPR-9s). We employed conventional electrophysiological techniques to record from CA1 pyramidal neurons of hippocampi of GEPR-9s. The membrane resting potential and input resistance of the GEPR-9 CA1 pyramidal neurons were not different from those of the Sprague-Dawley rats. Serotonin (20 microM) inhibited the directly and synaptically evoked action potentials in GEPR-9 CA1 neurons, as it did in the Sprague Dawley neurons, but only in some and not all of the neurons tested (blocked the directly evoked potentials in 57% and synaptically evoked potentials in 33.3% of the total neurons). This inhibition was also accompanied by hyperpolarization and reduction of membrane input resistance. In the bicuculline-treated brain slices of the GEPR-9, serotonin inhibited the epileptiform bursts causing concurrent hyperpolarization and reduction in membrane input resistance. The effects of the selective serotonin 5-HT1A receptor agonist, 8-OH-DPAT (20 microM) on GEPR-9 pyramidal CA1 neurons were similar to those of serotonin, except the magnitude of hyperpolarization and reduction of membrane input resistance were less than those produced by serotonin. We conclude that the apparent decrease in sensitivity of the GEPR-9 CA1 pyramidal neurons to serotonin may represent a deficiency of serotonin 5-HT1A receptor.

Induction and maintenance of ganglionic long-term potentiation require activation of 5-hydroxytryptamine (5-HT3) receptors.

1. An extracellular recording technique was used to study the effects of 5-hydroxytryptamine (5-HT, serotonin) on the tetanus-induced long-term potentiation (LTP) of the nicotinic pathway of transmission in the superior cervical ganglion (SCG) of the rat. The postganglionic compound action potential (CAP), made submaximal by treatment with hexamethonium (O.4 mM), was used as an index of transmission in the ganglion. 2. Serotonin (10 microM) markedly enhanced the magnitude of LTP without affecting the post-tetanic potentiation (PTP). The serotonin (2-30 microM) concentration-response curve for LTP was bell shaped as no enhancement was seen with 30 microM serotonin. This may largely be due to activation of a 5-HT1 receptor subtype and not to desensitization. 3. When superfused before tetanus, the 5-HT1A receptor agonist 8-hydroxydipropylamino-tetralin (8-OH-DPAT, 5 microM) prevented the expression of LTP without affecting PTP. 4. Pretreatment of ganglia with the 5-HT2 receptor agonist R-(+)-dimethoxy-4-iodoamphetamine (R-(+)-DOI, 1 microM) enhanced the tetanus-induced LTP. Similar treatment with the 5-HT2 receptor antagonist ketanserin (3 microM) had no significant effect on LTP. 5. Pretreatment of ganglia with the 5-HT3 receptor agonist 1-m-(chlorophenyl) biguanide (m-CPGB, 1 microM), markedly increased (300%) the tetanus-induced LTP. Similar pretreatment with the 5-HT3 receptor antagonist 3-tropanyl-3,5-dichlorobenzoate (MDL 72222, 0.5 microM) completely prevented the expression of LTP. Fully expressed LTP was reversibly blocked by MDL 72222 when applied during the maintenance phase of LTP. 6. Tetanic stimulation of monoamine-depleted ganglia (from reserpine-pretreated rats, 3 mg kg-1 for 24 h) failed to induced LTP. 7. In monoamine-depleted ganglia, tetanus preceded by superfusion with m-CPBG readily induced LTP. MDL 72222 completely blocked this LTP. However in these ganglia tetanus failed to induced LTP when m-CPBG was given 2 min (during PTP) or 1 h after tetanus. 8. Tetanic stimulation of monoamine-depleted ganglia in the presence of R-(+)-DOI failed to induced LTP. 9. We conclude that tetanus-induced LTP of the SCG of the rat requires activation of 5-HT3 receptors both for induction and maintenance.