Modulation of BK Channels by Ethanol.

In alcohol-naïve systems, ethanol (<100mM) exposure of calcium-gated BK channels perturbs physiology and behavior. Brief (several minutes) ethanol exposure usually leads to increased BK current, which results from ethanol interaction with a pocket mapped to the BK channel-forming slo1 protein cytosolic tail domain. The importance of this region in ethanol-induced intoxication has been independently supported by an unbiased screen of Caenorhabditis elegans slo1 mutants. However, ethanol-induced BK activation is not universal as refractoriness and inhibition have been reported. The final effect depends on many factors, including intracellular calcium levels, slo1 isoform, BK beta subunit composition, posttranslational modification of BK proteins, channel lipid microenvironment, and type of ethanol administration. Studies in Drosophila melanogaster, C. elegans, and rodents show that protracted/repeated ethanol administration leads to tolerance to ethanol-induced modification of BK-driven physiology and behavior. Unveiling the mechanisms underlying tolerance is of major importance, as tolerance to ethanol has been proposed as predictor of risk for alcoholism.

Motor, cytoarchitectural and biochemical assessment of pharmacological neuroprotection against CNS damage induced by neonatal exposure to ionizing radiation.

Exposure of neonatal rats to a 5 Gy single dose of X-irradiation induces permanent abnormalities in cerebellar cortex cytoarchitecture and neurochemistry and motor function. This rodent model constitutes an useful tool to evaluate morphological, neurochemical and motor changes induced by ionizing radiation and the possible restorative effects of potential or clearly established neuroprotective drugs. After selection and administration of a neuroprotective agent to neonatally irradiated rats, quantitative evaluations of motor behavior (gait), cerebellar cortex cytoarchitecture and cerebellar monoamine levels are performed. Data are compared to those of both saline-injected, X-irradiated, and saline-injected, sham-irradiated controls. Evaluation of data from the different experimental groups is performed at postnatal days 30 and 90. After this postnatal interval, radiation-induced damage of cerebellar function in nonprotected rodents is considered to be permanent. The longitudinal evaluation of various parameters in the different experimental groups through a multidisciplinary approach, allows determination of the variables that are more sensitive to X-irradiation-induced damage and/or neuroprotective agent-induced restoration. Given the well-known correspondence in cerebellar developmental stages between rodents and humans, this model and related studies bring health-related implications, considering the accidental or therapeutic exposure of developing human beings to ionizing radiation.

A plasticizer released from IV drip chambers elevates calcium levels in neurosecretory terminals.

We report that intracellular calcium levels rise in mammalian neurosecretory terminals and in cultured pheochromocytoma cells during acute exposure to physiological medium incubated in IV drip chambers. The agent responsible for this effect is shown to be di(2-ethylhexyl)phthalate (DEHP). DEHP (800 nM) added to saline solution caused a rise in [Ca(2+)](i) similar to that elicited by the contaminant-containing solution. The extraction of this contaminant from the IV drip chamber, as measured by spectrophotometry, was time-dependent and was markedly accelerated by the presence of 50 mM ethanol in the solution. Larger [Ca(2+)](i) increases were observed in terminals exposed to solutions incubated in IV drip chambers for greater durations. The rise in calcium requires transmembrane calcium flux through membrane channels, as the response is blocked by either 100 microM cadmium or by lowering the extracellular free Ca(2+) concentration to 10 microM. Our results suggest that acute alterations in intracellular calcium should be considered in addition to long-term effects when determining the safety of phthalate-containing plastics and that laboratory researchers using plastic perfusion materials consider this potential source of artifactual results.

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.

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 mV per 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 microM 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.

Ethanol potentiation of calcium-activated potassium channels reconstituted into planar lipid bilayers.

We examined the actions of ethanol on the single channel properties of large conductance Ca2+-activated K+ (BK) channels isolated from skeletal muscle T-tubule membranes and incorporated into planar lipid bilayer membranes. We have taken advantage of this preparation, because it lacks most elements of cellular complexity, including cytoplasmic constituents and complex membrane lipid composition and architecture, to examine the minimum requirements for the effects of alcohol. Clinically relevant concentrations (25-200 mM) of ethanol increased the activity of BK channels incorporated into bilayers composed of phosphatidylethanolamine (PE) alone or PE and phosphatidylserine. The potentiation of channel activity by ethanol was attributable predominantly to a decrease in the average amount of time spent in closed states. Ethanol did not significantly affect the current amplitude-voltage relationship for BK channels, indicating that channel conductance for K+ was unaffected by the drug. Although base-line characteristics of BK channels incorporated into bilayers composed only of PE differed from those of channels in PE/ phosphatidylserine in a manner expected from the change in bilayer charges, the actions of ethanol on channel activity were qualitatively similar in the different lipid environments. The effects of ethanol on single channel properties of BK channels in the planar bilayer are very similar to those reported for the action of ethanol on neurohypophysial BK channels studied in native membrane, and for cloned BK channels expressed in Xenopus laevis oocytes, which suggests that ethanol's site and mechanism of action are preserved in this greatly simplified preparation.

Ethanol increases the activity of Ca(++)-dependent K+ (mslo) channels: functional interaction with cytosolic Ca++.

Ethanol (EtOH) reversibly activates large conductance, Ca(++)-activated K+ (BK) channels in rat neurohypophysial terminals, an effect that probably contributes to the inhibition of vasopressin release by this drug. Heterogeneity in the terminal channel population makes it difficult to determine the mechanisms underlying this activation. Here, we report the effects of EtOH on the steady-state activity of BK channels cloned from mouse brain (mslo, alpha subunit) and expressed in Xenopus oocytes. EtOH reversibly increased mslo channel activity in excised patches, showing a potency (EC50 = 24 mM) similar to that reported using native channels. EtOH activation was observed under conditions that make it highly improbable that it is mediated by freely diffusible second messengers, or secondary to G-protein modulation. Rather, it probably results from a functional interaction between the drug and the channel alpha subunit. Activation occurred without increase in the number of functional channels present in the patch and resulted from actions that were a function of EtOH concentration: at < or = 10 mM, activation was due to a decrease in the channel mean closed time, whereas between 25 and 100 mM, activation was due to both a decrease in the mean closed time and an increase in the mean open time. The characteristic high unitary conductance and ionic selectivity of BK channels were unaltered by the drug. Whereas the voltage dependence of channel gating remained unchanged, channel activation mediated by the response of the Ca(++)-sensing site(s) to increases in the concentration of intracellular calcium, [Ca++]ic, was reduced by EtOH. This finding is consistent with EtOH and [Ca++]ic behaving functionally as partial and full agonists of mslo channels, respectively. Because the potentiation of mslo activity by the drug decreased as Ca++ levels were increased, EtOH-activation of BK channels would be most evident when [Ca++]ic is near resting levels, rather than during periods of high activity and Ca++ influx.

A novel large conductance, nonselective cation channel in pheochromocytoma (PC12) cells.

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+ approximately K+ > Li+, and is practically impermeable to Cl-. The channel activity-voltage relationship is bell-shaped, showing maximal activation at approximately -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 ( approximately 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.

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.

Ethanol increases the activity of large conductance, Ca(2+)-activated K+ channels in isolated neurohypophysial terminals.

Large conductance, Ca(2+)-activated K+ channels are believed to underlie interburst intervals and, thus, contribute to the control of hormone release from neurohypophysial terminals. Because ethanol inhibits the release of vasopressin and oxytocin, we studied its effects on large conductance, Ca(2+)-activated K+ channels from these terminals using patch-clamp techniques. Ethanol (10-100 mM) applied to the cytosolic surface of excised, inside-out patches reversibly increases channel activity in a concentration-dependent manner, reaching a plateau at 50-100 mM. This activation is not mediated by freely diffusible cytosolic second messengers or the release of Ca2+ from intracellular stores. Rather, it likely reflects a direct interaction of ethanol with the channel protein or a closely associated component. Neither the unitary conductance nor the characteristics of the voltage-current relationship are modified by the drug. The increase of channel activity by ethanol results from a modification of channel gating properties: the contribution of long openings to the total time spent in the open state is increased, the average duration of the fast openings is slightly increased, and long closures disappear in the presence of the drug. The activation of large conductance, Ca(2+)-activated K+ channels by ethanol, in conjunction with the previously reported inhibition of voltage-dependent Ca2+ channels, can explain the reduced release of vasopressin and oxytocin after ethanol ingestion.

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 A's, 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.

A home telecare management system.

The increasing tendency to discharge chronic patients from hospital, as well as the growing expectation of improved quality of life for elderly and disabled people at home, was the original motivation for the development of a home telecare management system. The system allows a service centre to perform remote monitoring of biological signals and other data via the public telephone network, as well as to manage different emergency situations arising at home. The system is part of the FU-funded EPIC project (European Prototype for Integrated Care). It was tested in Belfast (Northern Ireland) and is currently being installed in Torre del Mar (Spain). This paper describes the system design and preliminary evaluation. The results indicate that the system operators find it highly acceptable in terms of efficiency, effectiveness, helpfulness, control and learnability. Integration of home telecare data with community-care information systems is essential if data captured at home are to be incorporated into the care process effectively.

Membrane stretch directly activates large conductance Ca(2+)-activated K+ channels in mesenteric artery smooth muscle cells.

Large-conductance, Ca(2+)-activated K+ channels were identified in single smooth muscle cells freshly isolated from rabbit superior mesenteric artery. They typically showed a reversal potential close to 0 mV in excised, inside-out patches in symmetric 130 mmol/L [K+] with a unitary conductance of 260 pS, and increased activity at more positive potentials and/or when [Ca2+] was raised at the cytosolic surface of the membrane. Both in cell-attached and in excised, inside-out configurations, stretching the membrane patch by applying suction to the back of the patch pipette increased the activity of these channels without changing either the unitary conductance or the voltage sensitivity of the channel. Stretch activation was repeatedly seen in inside-out patches when both surfaces were bathed with a 0 Ca2+ solution containing 2 or 5 mmol/L EGTA to chelate trace amounts of Ca2+, making it highly improbable that stretch activation could be secondary to a stretch-induced flux of Ca2+. Consequently, stretch activation of large-conductance, Ca(2+)-activated K+ channels in mesenteric artery smooth muscle cells seems to be due to a direct effect of stretch on the channel itself or on some closely associated, membrane-bound entity.

Motor abnormalities and changes in the noradrenaline content and the cytoarchitecture of developing cerebellum following X-irradiation at birth.

We have studied the developmental time-course of changes in the noradrenaline (NA) content of cerebellum (CE), cytoarchitecture of the cerebellar cortex, and motor abnormalities induced by the exposure of the cephalic end of rats to a single dose (5 Gy) of X-irradiation immediately after birth. At all ages examined, i.e., from postnatal (PN) d 5 to 90, CE from exposed animals show a marked atrophy, with an agranular cortex that has lost its layered structure. Purkinje cells are scattered at all depths in the cortex, and their primary dendrite is randomly oriented. The motor syndrome includes dystonia-like movements, a fine tremor, and an ataxic gait. Being progressive, the abnormal movements are evident from PN d 10, and fully developed by d 30. On the other hand, no differences in cerebellar NA content between X-irradiated rats and age-matched nonirradiated controls were detected from PN d 5 to 60. However, at PN d 90 a significant increase in NA content of CE from exposed animals is found when compared to either age-matched controls (+36%, p < 0.01), or data from irradiated rats obtained at PN d 5 to 60 (p < 0.01). These results indicate a temporal dissociation between the motor and cytoarchitectural abnormalities and the increase in cerebellar NA content produced by a single dose of X-rays at birth. The late increase in cerebellar NA content might represent a compensatory response of noradrenergic terminals to an altered information flow out of the cerebellar cortex induced by the ionizing noxa.

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

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.(ABSTRACT TRUNCATED AT 250 WORDS)

[Endocarditis and splenic abscess from Salmonella no typhi]. / Endocarditis y absceso esplénico por Salmonella no typhi.

Several focal forms of infection by Salmonella no typhi, as the endocarditis, are very rare and the coexistence of splenic abscess is even more rare. We present a case in which the simultaneous presence of both infections was observed and we review the literature with special emphasis in the therapeutical aspects.

Ambulatory blood pressure monitoring and diastolic left ventricular abnormalities in established and borderline hypertension

In order to evaluate left ventricular diastolic function by means of Doppler echocardiography in borderline and established hypertension, identified by office and ABPM, compared with normotensives, 54 subjects: 15 normotensives, 11 borderlines and 28 nontreated mild to moderate essential hypertensives were studied. Age and weight were similar among groups. Established hypertensives showed higher left ventricular mass index (p < 0.05), peak velocity of late left ventricular filling (peak A; p < 0.01), ratio peak A/peak velocity of early ventricular filling, peak E (p < 0.01), velocity time integral of systolic atrial volume (p < 0.001), deceleration half time of peak early diastolic inflow velocity (p < 0.05), left ventricular isovolumic relaxation period (IRP; p < 0.01) than normotensives and lower Doppler indexes of early diastolic left ventricular filling (p < 0.01), peak filling rat normalized to mitral stroke volume (PFRn; p < 0.01) than normotensives. Although borderline hypertensives showed intermediate LVM and Doppler indexes between hypertensives and normotensives only IRP (p < 0.05) and PFRn (p < 0.05) were significant different to normotensives. In conclusion, established hypertension leads to abnormalities in left ventricular diastolic function which can be detected by Doppler echocardiography. In borderline hypertension, the left ventricular diastolic abnormalities are predominantly related to the active process of early diastole. Therefore, these indexes may be early markers of left ventricular dysfunction in hypertension.

Ambulatory blood pressure monitoring and diastolic left ventricular abnormalities in established and borderline hypertension

In order to evaluate left ventricular diastolic function by means of Doppler echocardiography in borderline and established hypertension, identified by office and ABPM, compared with normotensives, 54 subjects: 15 normotensives, 11 borderlines and 28 nontreated mild to moderate essential hypertensives were studied. Age and weight were similar among groups. Established hypertensives showed higher left ventricular mass index (p < 0.05), peak velocity of late left ventricular filling (peak A; p < 0.01), ratio peak A/peak velocity of early ventricular filling, peak E (p < 0.01), velocity time integral of systolic atrial volume (p < 0.001), deceleration half time of peak early diastolic inflow velocity (p < 0.05), left ventricular isovolumic relaxation period (IRP; p < 0.01) than normotensives and lower Doppler indexes of early diastolic left ventricular filling (p < 0.01), peak filling rat normalized to mitral stroke volume (PFRn; p < 0.01) than normotensives. Although borderline hypertensives showed intermediate LVM and Doppler indexes between hypertensives and normotensives only IRP (p < 0.05) and PFRn (p < 0.05) were significant different to normotensives. In conclusion, established hypertension leads to abnormalities in left ventricular diastolic function which can be detected by Doppler echocardiography. In borderline hypertension, the left ventricular diastolic abnormalities are predominantly related to the active process of early diastole. Therefore, these indexes may be early markers of left ventricular dysfunction in hypertension.(Au)

Ambulatory blood pressure monitoring and diastolic left ventricular abnormalities in established and borderline hypertension.

In order to evaluate left ventricular diastolic function by means of Doppler echocardiography in borderline and established hypertension, identified by office and ABPM, compared with normotensives, 54 subjects: 15 normotensives, 11 borderlines and 28 nontreated mild to moderate essential hypertensives were studied. Age and weight were similar among groups. Established hypertensives showed higher left ventricular mass index (p < 0.05), peak velocity of late left ventricular filling (peak A; p < 0.01), ratio peak A/peak velocity of early ventricular filling, peak E (p < 0.01), velocity time integral of systolic atrial volume (p < 0.001), deceleration half time of peak early diastolic inflow velocity (p < 0.05), left ventricular isovolumic relaxation period (IRP; p < 0.01) than normotensives and lower Doppler indexes of early diastolic left ventricular filling (p < 0.01), peak filling rat normalized to mitral stroke volume (PFRn; p < 0.01) than normotensives. Although borderline hypertensives showed intermediate LVM and Doppler indexes between hypertensives and normotensives only IRP (p < 0.05) and PFRn (p < 0.05) were significant different to normotensives. In conclusion, established hypertension leads to abnormalities in left ventricular diastolic function which can be detected by Doppler echocardiography. In borderline hypertension, the left ventricular diastolic abnormalities are predominantly related to the active process of early diastole. Therefore, these indexes may be early markers of left ventricular dysfunction in hypertension.