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Tooth impaction is defined as any tooth that fails to erupt into a normal functional position and remains unerupted beyond the time at which it should normally erupt. Reports of impaction and eruption failure in primary teeth are relatively rare compared to permanent teeth. We report 2 rare cases where the second premolar was located on the occlusal side of the impacted mandibular second primary molar. In the first case, the succedaneous permanent tooth erupted after extraction of the primary tooth, fenestration, and traction. In the second case, the succedaneous permanent tooth erupted without fenestration or traction. Although the etiology of the tooth displacement was unknown in both cases, inhibition of the eruptive movement of the primary molar may have been associated with displacement of the succedaneous permanent premolar.

Regional odontodysplasia is a rare, severe, and nonhereditary developmental disorder in tooth formation and involves epithelial and mesenchymal-derived dental tissue. On radiographs, affected teeth have an abnormal morphology, a hypoplastic crown, and only a faint outline of hard tissue, a condition termed “ghost teeth.” We report clinical and radiographic findings from two children with regional odontodysplasia. Using computed tomography (CT), we calculated attenuation coefficients (i.e., Hounsfield units) for affected teeth and assessed the condition of dental follicles. To measure density, regions of interest were delimited and CT values were calculated. In our two patients, the CT values for enamel were lower in affected teeth than in sound teeth, while CT values for dentin were similar for affected and sound teeth. The average CT value for dental follicles in affected teeth was about 65 to 120, which suggests that dense fibrous connective tissues or hard tissue-like structures might be present in dental follicles. Analysis of CT values may be quite useful in the diagnosis and treatment of regional odontodysplasia.

In summary, the voltage-sensitive sodium channel from eel electroplax provides an optimal preparation for biochemical and biophysical studies of molecular structure and gating. We have demonstrated that the purified and reconstituted protein is capable of functioning normally, exhibiting, among other properties, voltage-dependent activation and inactivation gating mechanisms. We have been able to recreate the classical electrophysiological studies in which inactivation gating can be removed by proteolytic modification of the cytoplasmic surface of the molecule, and have mapped the probable site of modification to the peptide segment lying between subunit domains III and IV. We have demonstrated that the reconstituted protein undergoes interactions with the lidocaine derivative QX-314 which, at low concentrations, results in paradoxical activation of the channel and a facilitation of modification by oxidizing reagents that remove inactivation gating.

Ba ions caused an intense and prolonged discharge of melanocyte-stimulating hormone (MSH) from perifused neurointermediate lobes of mouse pituitaries and dispersed pars intermedia cells. The effect persisted in chronically cultured lobes or cells. It did not require Ca, but, like the Ca-dependent response to excess K, was blocked by cyanide combined with glucose lack. The secretagogue effect of Ba was blocked or prevented by Co or by excess Ca, both of which can reduce inward Ba currents through Ca channels. Prior exposure to excess K partially reduced the secretagogue effect of Ba, suggesting that depolarization caused some inactivation of Ba current. In contrast to Ba, excess K elicited secretion that was transient, and prior exposure of preparations to excess K (in the absence of Ca) profoundly suppressed the secretagogue effect of Ca. The evidence is consistent with the view that inward Ca current rapidly inactivates in these cells. It is concluded that Ba ions have a potent and persistent direct secretagogue effect on the melanotrophs that may reflect, in part, their ability to penetrate Ca channels more easily than Ca ions. The strong secretagogue effects of Ba on melanotrophs may be of considerable utility in studies on MSH secretion since a physiological secretagogue has yet to be discovered. Moreover, since the responses of melanotrophs (and other endocrine cells) to Ba can be distinguished from those of various other secretory cells and neurones, it is suggested that Ba may provide a tool for characterizing the distinctive membrane properties of the Ba-responsive endocrine cells.

Current pulses applied to isolated neurointermediate lobes of mice increased output of melanocyte-stimulating hormone. This response was dependent on extracellular calcium over a wide range of stimulus intensities and thus appears to be a true secretory response from the melanotrophs. Since substantial responses persisted in the presence of tetrodotoxin, much of the effect seems to be independent of Na spiking.

At equilibrium, voltage-sensitive sodium channels normally are closed at all potentials. They open transiently in response to changes in membrane voltage or chronically under the influence of certain neurotoxins. Covalent modifications that result in chronic opening may help identify molecular domains involved in conductance regulation. Here, the purified sodium channel from electric eel electroplax, reconstituted in artificial liposomes, has been used to screen for such modifications. When the liposomes were treated with the alkaloid neurotoxin batrachotoxin, sodium-selective ion fluxes were produced, with permeability ratios PNa greater than PTl greater than PK greater than PRb greater than PCs. When the liposomes were treated with either of two oxidizing reagents (N-bromoacetamide or N-bromosuccinimide), or with Pronase or trypsin, ion-selective fluxes also were stimulated. These were blocked by tetrodotoxin and the anesthetic QX-314 in a manner suggesting that only modification of the cytoplasmic protein surface resulted in stimulation. Limited exposure to trypsin resulted in strong flux activation, with the concomitant appearance of peptide fragments with masses of approximately equal to 130, 70, and 38 kDa and fragments with masses of 45 and 24 kDa appearing later. We propose that characterization of these fragments may allow identification of channel domains important for inactivation gating.

We describe the isolation and characterization of a cDNA encoding the alpha subunit of a new voltage-sensitive sodium channel, microI, from rat skeletal muscle. The 1840 amino acid microI peptide is homologous to alpha subunits from rat brain, but, like the protein from eel electroplax, lacks an extended (approximately 200) amino acid segment between homologous domains I and II. Northern blot analysis indicates that the 8.5 kb microI transcript is preferentially expressed in skeletal muscle. Sodium channels expressed in Xenopus oocytes from synthetic RNA encoding microI are blocked by tetrodotoxin and mu-conotoxin at concentrations near 5 nM. The expressed sodium channels have gating kinetics similar to the native channels in rat muscle fibers, except that inactivation occurs more slowly.

A fluorescence assay for measuring Na channel activation in liposomes containing voltage-sensitive Na channels isolated from Electrophorus electricus is described. The assay is based on transport of a heavy-metal cation, T1+, through the activated channel to quench fluorescence of an internalized, water-soluble chromophore. The channel is "locked" in a chronically opened configuration with alkaloid neurotoxins such as veratridine or batrachotoxin. Diffusion potentials are used to amplify the signal, and enlarged liposomes (greater than 8000 A) result in time courses extended to the range of seconds. Analysis of the kinetics of quenching yields parameters that behave as linear functions of channel activation and reflect vesicle size and channel abundance. The k1/2's for activation by veratridine and batrachotoxin were 5 microM and 169 nM, respectively, and that for tetrodotoxin blockade was 4 nM. Externally applied QX-222 and tetrodotoxin each acted to partially block the stimulated signal, as expected for compounds that act on oppositely oriented channels in the membrane. Single-channel conductances estimated with either veratridine or batrachotoxin ranged between 0.6 and 40.7 pS, corresponding to transport numbers of (1.2 X 10(5)) to (8.1 X 10(6)) ions s-1 channel-1 under the conditions of assay. The assay is approximately 100-fold more sensitive than radiotracer influx assays, requiring 1 fmol of protein per time course.

The functional reconstitution of the voltage-regulated Na channel purified from the electroplax of Electrophorus electricus is described. Reconstitution was achieved by removing detergent with Bio-Beads SM-2 followed by freeze-thaw-sonication in the presence of added liposomes. This preparation displayed heat-stable binding of 3H-labeled tetrodotoxin (TTX) (Kd = 33 nM). 22Na+ influx was stimulated 2- to 5-fold by alkaloid neurotoxins and blocked by TTX. Veratridine activated Na+ influx with a K1/2 of 18 microM, and this activation was blocked by TTX precisely in parallel with specific [3H]TTX binding. Batrachotoxin stimulated 22Na+ flux more effectively than did veratridine. No effect of the peptide anemone toxin II was found. Insertion of the Na channel into membranes resulted in 60-70% of the TTX-binding sites facing the vesicle exterior. Thus, external TTX partially inhibited flux, whereas blockade was complete when TTX was also equilibrated with the vesicle interior. The lipid-soluble local anesthetics tetracaine and dibucaine inhibited flux completely. QX-222, a charged derivative of lidocaine, blocked only a fraction of the channels, apparently those oriented inside-out. Purified samples were predominantly composed of the Mr 260,000-300,000 glycopeptide but contained variable quantities of smaller peptides. Veratridine-dependent flux and peptide compositions were determined on fractions across a gel filtration column profile. Stimulated flux codistributed only with the large peptide.

Since melanotrophs are electrically active and exhibit spontaneous Na spikes, a study was made of the effects, on melanotroph secretion, of drugs known to influence electrical properties. The output of melanocyte-stimulating hormone was measured from perifused neurointermediate lobes of mice or melanotrophs dispersed from such lobes of mice or rats. Veratridine (200 microM), which is known to increase Na permeability in a variety of cells, caused a large, although transient, increase in secretion from the melanotrophs that required extracellular Ca2+ and was blocked by the Na-channel blocker tetrodotoxin (1 microM). Tetraethylammonium (10 mM), which blocks K channels and thus prolongs the duration of the action potential in many cells, also stimulated secretion in the melanotrophs in a Ca-dependent manner. This response was not, however, blocked by tetrodotoxin, and is thus not attributable to prolongation of Na spikes in these cells. Moreover, tetrodotoxin did not inhibit basal secretion. The stimulant effect of veratridine on secretion in melanotrophs and its suppression by tetrodotoxin suggests that voltage-dependent Na channels can participate in the regulation of hormone output in these cells of the pituitary pars intermedia. However, the apparent lack of effect of tetrodotoxin on basal secretion suggests that the spontaneous Na spikes previously observed in these cells are not required for promoting the Ca influx which other evidence shows is important for basal secretion.

Recent immunohistochemical evidence from the rat, indicating that gamma-aminobutyric acid (GABA)-containing fibres of central nervous origin project to the pars intermedia of the pituitary1,2, prompts inquiry into the function of this innervation. There is electrophysiological evidence that GABA acts directly on melanotrophs isolated from rat, through bicuculline-blockable receptors, to increase Cl- conductance and thereby drive the membrane potential towards the Cl- equilibrium potential in these cells, resulting in depolarization at rest or reduction of the depolarization caused by excess K+ (ref. 3). As voltage-dependent Ca channels can participate in the regulation of secretion in these cells4, we have now examined the effect of GABA on hormone output and find that it first stimulates and then inhibits spontaneous secretion of melanocyte-stimulating hormone (MSH) and inhibits K+-evoked secretion. Moreover, our pharmacological evidence suggests that similar receptors are involved in the secretory and the electrophysiological responses. A function of the GABAergic innervation may therefore be to regulate hormone output by acting directly on the melanotrophs, and this regulation may be affected by the changes in electrical properties induced by GABA.

The tetrodotoxin-binding protein purified from electroplax of Electrophorus electricus has been reincorporated into multilamellar vesicles that were used for patch recording. When excised patches of these reconstituted membranes were voltage clamped in the absence of neurotoxins, voltage-dependent single-channel currents were recorded. These displayed properties qualitatively and quantitatively similar to those reported for Na channels from nerve and muscle cells, including uniform single-channel conductances of the appropriate magnitude (approximately equal to 11 pS in 95 mM Na+), mean open times of approximately equal to 1.9 msec, and 7-fold selectively for Na+ over K+. Currents averaged from many depolarizations showed initial voltage-dependent activation and subsequent inactivation. In the presence of batrachotoxin, channels were observed with markedly different properties, including conductances of 20-25 pS (95 mM Na+), mean open times of approximately equal to 28 msec, and no indication of inactivation. Collectively, these findings indicate that the tetrodotoxin-binding protein of electroplax is a voltage-regulated sodium channel.