Alejandro M. Dopico

Rat supraoptic magnocellular neurones show distinct large conductance, Ca2+-activated K+ channel subtypes in cell bodies versus nerve endings

1 Large conductance, Ca2+‐activated K+ (BK) channels were identified in freshly dissociated rat supraoptic neurones using patch clamp techniques. 2 The single channel conductance of cell body BK channels, recorded from inside‐out patches in symmetric 145 mM K+, was 246.1 pS, compared with 213 pS in nerve ending BK channels (P < 0.01). 3 At low open probability (Po), the reciprocal of the slope in the ln(NPo)‐voltage relationship (N, number of available channels in the patch) for cell body and nerve ending channels were similar: 11 vs. 14 mVper e‐fold change in NPo, respectively. 4 At 40 mV, the [Ca2+]i producing half‐maximal activation was 273 nM, as opposed to > 1.53 μM for the neurohypophysial channel, indicating the higher Ca2+ sensitivity of the cell body isochannel. 5 Cell body BK channels showed fast kinetics (open time constant, 8.5 ms; fast closed time constant, 1.6 and slow closed time constant, 12.7 ms), identifying them as ‘type I’ isochannels, as opposed to the slow gating (type II) of neurohypophysial BK channels. 6 Cell body BK activity was reduced by 10 nM charybdotoxin (NPo, 37 % of control), or 10 nM iberiotoxin (NPo, 5 % of control), whereas neurohypophysial BK channels are insensitive to charybdotoxin at concentrations as high as 360 nM. 7 Whilst blockade of nerve ending BK channels markedly slowed the repolarization of evoked single spikes, blockade of cell body channels was without effect on repolarization of evoked single spikes. 8 Ethanol reversibly increased neurohypophysial BK channel activity (EC50, 22 mM; maximal effect, 100 mM). In contrast, ethanol (up to 100 mM) failed to increase cell body BK channel activity. 9 In conclusion, we have characterized BK channels in supraoptic neuronal cell bodies, and demonstrated that they display different electrophysiological and pharmacological properties from their counterparts in the nerve endings.

Direct effects of fatty acids and other charged lipids on ion channel activity in smooth muscle cells

A variety of fatty acids increase the activity of certain types of K+ channels. This effect is not dependent on the three enzymatic pathways that convert arachidonic acid to various bioactive oxygenated metabolites. One type of K+ channel in toad stomach smooth muscle cell membranes in activated by fatty acids and other single chain lipids which possess both a negatively charged head group and a sufficiently hydrophobic acyl chain. Neutral lipids have no effect on K+ channel activity, while positively charged lipids with a sufficiently hydrophobic acyl chain suppress channel activity. Acyl Coenzyme As, which do not flip across the bilayer, act only from the cytosolic surface of the membrane, suggesting that the binding site for channel activation is also located there. This fatty acid-activated channel is also activated by membrane stretch. Moreover, this mechanical response is either mediated or modulated by fatty acids. Thus, fatty acids and other charged single chain lipids may comprise another class of first or second messenger molecules that target ion channels.

GM1 ganglioside treatment protects against long-term neurotoxic effects of neonatal X-irradiation on cerebellar cortex cytoarchitecture and motor function

Exposure of neonatal rats to a 5 Gy dose of X-irradiation induces permanent abnormalities in cerebellar cortex cytoarchitecture (disarrangement of Purkinje cells, reduction of thickness of granular cortex) and neurochemistry (late increase in noradrenaline levels), and motor function (ataxic gait). The neuroprotective effects of gangliosides have been demonstrated using a variety of CNS injuries, including mechanical, electrolytic, neurotoxic, ischemic, and surgical lesions. Here, we evaluated whether systemically administered GM1 ganglioside protects against the long-term CNS abnormalities induced by a single exposure to ionizing radiation in the early post-natal period. Thus, neonatal rats were exposed to 5 Gy X-irradiation, and subcutaneously injected with one dose (30 mg/kg weight) of GM1 on h after exposure followed by three daily doses. Both at post-natal days 30 and 90, gait and cerebellar cytoarchitecture in X-irradiated rats were significantly impaired when compared to age-matched controls. By contrast, both at post-natal days 30 and 90, gait in X-irradiated rats that were treated with GM1 was not significantly different from that in non-irradiated animals. Furthermore, at post-natal day 90, cerebellar cytoarchitecture was still well preserved in GM1-treated, X-irradiated animals. GM1 failed to modify the radiation-induced increase in cerebellar noradrenaline levels. Present data indicate that exogenous GM1, repeatedly administered after neonatal X-irradiation, produces a long-term radioprotection, demonstrated at both cytoarchitectural and motor levels.

Alcohol and the Release of Vasopressin and Oxytocin

Both clinical observations and basic research studies performed during the course of many years have provided evidence that ethanol (ETOH) affects the release of vasopressin (AVP) and oxytocin (OT) from the neurohypophysis. In addition to the well-known diuresis that occurs after ETOH intake1-3several reports demonstrated that ETOH ingestion results in a reduction of plasma AVP levels in mammals.4-6In addition, the amount of OT released into plasma during human labor is reduced after intravenous ETOH administration.7

Neurochemical characterization of the alterations in the noradrenergic afferents to the cerebellum of adult rats exposed to X-irradiation at birth.

Abstract: A single dose of x‐irradiation was applied on the cephalic end of newborn rats, and the alterations in the noradrenergic afferents to the cerebellum were studied 180 days later. A net increase in the noradrenaline content of cerebellum was found (122% of nonirradiated controls). The response of noradrenaline content to reserpine injection (0.9 mg/kg, i.p.) was similar in exposed and control rats. Likewise, the 3H release induced by Ro 4‐1284 from cerebellar cortex slices labeled with [3H]noradrenaline was unmodified by x‐rays, although a mild increase in the spontaneous efflux of 3H was found. The retention of 3H by the slices was reduced in exposed animals (58% of controls). Both the in vitro activity of tyrosine hydroxylase and the accumulation of L‐3,4‐dihydroxyphenylalanine (L‐DOPA) were not significantly different between x‐treated rats and controls. In contrast, monoamine oxidase activity was markedly reduced in x‐irradiated cerebellum (38% of controls). The x‐ray‐induced decrease in cerebellar weight (—60%) resulted in marked increases in noradrenaline concentration (223%), tyrosine hydroxylase activity per milligram of protein (206%), and 3H retention (50%). The accumulation of L‐DOPA per gram of tissue was also increased at every time considered. These data indicate that x‐irradiation at birth produces a cerebellar loss not completely shared by the noradrenergic afferents, and a permanent imbalance between the noradrenergic afferent input and its target cells might eventually result. In spite of the enhanced noradrenaline content, the lack of increase in maximal tyrosine hydroxylase activity and 3H retention seems to indicate that a long‐term sprouting of the noradrenergic terminals in the cerebellum induced by the ionizing treatment is unlikely.

Activities of monoamine oxidase-A and -B in adult rat cerebellum following neonatal X-irradiation

The activities of monoamine oxidases, MAO-A and MAO-B, were separately determined in the cerebellum (CE) from adult rats neonatally exposed to 5 Gy X-irradiation. They were found to be markedly reduced: 58% and 66% of values from nonirradiated, littermate controls. Since the specific activities of both isoenzymes (per mg tissue weight) were not significantly different from controls, the reduction of activity per CE is basically explained by the irradiation-induced cerebellar atrophy. The unmodified MAO-A specific activity makes it highly improbable that the increase in the cerebellar noradrenaline content, characteristic of neonatally X-irradiated rats, could be due to a decreased neuronal metabolism of noradrenaline by this enzyme.

Molecular Targets Underlying Ethanol-Mediated Reduction of Hormone Release from Neurohypophysial Nerve Terminals

In developing a model system in which to study the molecular basis for the acute and chronic actions of ethanol in the nervous system, our basic philosophy has been: 1) to work with a relevant molecular target (i.e., a mediator of a behavioral or physiological consequence of ethanol ingestion), which is 2) amenable to analysis at the molecular level, and in which 3) we can identify the biophysical parameters responsible for acute modulation by the drug. It will also be possible to follow alterations in the function and ethanol response of this target during chronic exposure of the animal to ethanol, and the development of various forms of tolerance in those systems or behaviors subserved by the target molecule.

A Novel Large Conductance, Nonselective Cation Channel in Pheochromocytoma (PC12) Cells

Abstract. A new type of nonselective cation channel was identified and characterized in pheochromocytoma (PC12) cells using inside-out and cell-attached patch-clamp recordings. The channel shows a large unitary conductance (274 pS in symmetric 145 mm K+) and selectivity for Na+≈ K+ > Li+, and is practically impermeable to Cl−. The channel activity-voltage relationship is bell-shaped, showing maximal activation at ≈−10 mV. The overall activity of this channel is unmodified by [Na+]ic, or [Ca++]ic. However, increases in [Ca++]ic lead to a decrease in the unitary current amplitude. In addition, overall activity is mildly increased when suction is applied to the back of the patch pipette. Together, these characteristics distinguish the present channel from all other large conductance nonselective cation channels reported so far in a variety of preparations. The frequency of appearance of this channel type is similar in undifferentiated and NGF-treated PC12 cells (≈8–27% of patches). The combination of large conductance, permeability to Na+, and existence of conducting states at negative potentials, may provide a significant pathway for inward current and depolarization in PC12 cells.