Suppression of presynaptic calcium currents by hypoxia in hippocampal tissue slices.

We tested the hypothesis that suppression of inward calcium current in presynaptic terminals is the cause of failure of synaptic transmission early during cerebral hypoxia. Postsynaptic responses in CA1 zone of hippocampal tissue slices were blocked either by the combined administration of 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 3-((+-)-2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (CPP) or by lowering extracellular calcium concentration ([Ca2+]o). Repetitive orthodromic activation of central neurons caused transient decrease of [Ca2+]o (measured by ion selective microelectrodes) in neuropil, attributable to influx of Ca2+ in presynaptic terminals. Presynaptic [Ca2+]o responses were rapidly and reversibly suppressed when oxygen was withdrawn from hippocampal tissue slices. The 'resting' baseline level of [Ca2+]o declined at first gradually, then precipitously as in spreading depression (SD). Presynaptic volleys during high frequency train stimulation were also depressed somewhat before SD began. We conclude that (1) presynaptic Ca2+ currents fail during hypoxia, perhaps because 'resting' intracellular free Ca2+ activity is increased and, in part, also because of partial failure of presynaptic impulse conduction; (2) the influx of Ca2+ into brain cells in hypoxic spreading depression is not mediated by glutamate/aspartate dependent channels.     

Na(+) and K(+) concentrations, extra- and intracellular voltages, and the effect of TTX in hypoxic rat hippocampal slices

Severe hypoxia causes rapid depolarization of CA1 neurons and glial cells that resembles spreading depression (SD). In brain slices in vitro, the SD-like depolarization and the associated irreversible loss of function can be postponed, but not prevented, by blockade of Na(+) currents by tetrodotoxin (TTX). To investigate the role of Na(+) flux, we made recordings from the CA1 region in hippocampal slices in the presence and absence of TTX. We measured membrane changes in single CA1 pyramidal neurons simultaneously with extracellular DC potential (V(o)) and either extracellular [K(+)] or [Na(+)]; alternatively, we simultaneously recorded [Na(+)](o), [K(+)](o), and V(o). Confirming previous reports, early during hypoxia, before SD onset, [K(+)](o) began to rise, whereas [Na(+)](o) still remained normal and V(o) showed a slight, gradual, negative shift; neurons first hyperpolarized and then began to gradually depolarize. The SD-like abrupt negative DeltaV(o) corresponded to a near complete depolarization of pyramidal neurons and an 89% decrease in input resistance. [K(+)](o) increased by 47 mM and [Na(+)](o) dropped by 91 mM. Changes in intracellular Na(+) and K(+) concentrations, estimated on the basis of the measured extracellular ion levels and the relative volume fractions of the neuronal, glial, and extracellular compartment, were much more moderate. Because [Na(+)](o) dropped more than [K(+)](o) increased, simple exchange of Na(+) for K(+) cannot account for these ionic changes. The apparent imbalance of charge could be made up by Cl(-) influx into neurons paralleling Na(+) flux and release of Mg(2+) from cells. The hypoxia-induced changes in interneurons resembled those observed in pyramidal neurons. Astrocytes responded with an initial slow depolarization as [K(+)](o) rose. It was followed by a rapid but incomplete depolarization as soon as SD occurred, which could be accounted for by the reduced ratio, [K(+)](i)/[K(+)](o). TTX (1 microM) markedly postponed SD, but the SD-related changes in [K(+)](o) and [Na(+)](o) were only reduced by 23 and 12%, respectively. In TTX-treated pyramidal neurons, the delayed SD-like depolarization took off from a more positive level, but the final depolarized intracellular potential and input resistance were not different from control. We conclude that TTX-sensitive channels mediate only a fraction of the Na(+) influx, and that some of the K(+) is released in exchange for Na(+). Even though TTX-sensitive Na(+) currents are not essential for the self-regenerative membrane changes during hypoxic SD, in control solutions their activation may trigger the transition from gradual to rapid depolarization of neurons, thereby synchronizing the SD-like event.     

Conditions for the triggering of spreading depression studied with computer simulations

In spite of five decades of study, the biophysics of spreading depression (SD) is incompletely understood. Earlier we have modeled seizures and SD, and we have shown that currents through ion channels normally present in neuron membranes can generate SD-like depolarization. In the present study, we define the conditions for triggering SD and the parameters that influence its course in a model of a hippocampal pyramidal cell with more complete representation of ions and channels than the previous version. "Leak" conductances for Na(+), K(+), and Cl(-) and an ion pump were present in the membrane of the entire cell; fast inactivating voltage dependent conductances for sodium and potassium in the soma; "persistent" conductances in soma and apical dendrite, and K(+)- and voltage-dependent N-methyl-D-aspartate (NMDA)-controlled conductance in the apical dendrite. The neuron was surrounded by restricted interstitial space and by a "glia-endothelium" system of extracellular ion regulation bounded by a membrane having leak conductances and an ion pump. Ion fluxes and concentration changes were continuously computed as well as osmotic cell volume changes. As long as reuptake into the neuron and "buffering" by glia kept pace with K(+) released from the neuron, stimulating current applied to the soma evoked repetitive firing that stopped when stimulation ceased. When glial uptake was reduced, K(+) released from neurons could accumulate in the interstitium and keep the neuron depolarized so that strong depolarizing pulses injected into the soma were followed either by afterdischarge or SD. SD-like depolarization was ignited when depolarization spreading into the apical dendrite, activated persistent Na(+) current and NMDA-controlled current. With membrane parameters constant, varying the injected stimulating current influenced SD onset but neither the depolarization nor the increase in extracellular K(+). Glial "leak" conductance influenced SD duration and SD ignition point. Varying maximal conductances (representing channel density) also influenced SD onset time but not the amplitude of the depolarization. Hypoxia was simulated by turning off the Na-K exchange pump, and this resulted in SD-like depolarization. The results confirm that, once ignited, SD runs an all-or-none trajectory, the level of depolarization is governed by feedback involving ion shifts and glutamate acting on ion channels and not by the number of channels open, and SD is ignited if the net persistent membrane current in the apical dendrites turns inward.     

Fatty acid bile acid conjugates (FABACs)--new molecules for the prevention of cholesterol crystallisation in bile

BACKGROUND: Cholesterol gall stones are a frequent disease for which at present surgery is the usual therapy. Despite the importance of bile acids it has become evident that phospholipids are the main cholesterol solubilisers in bile. Even phospholipid components, such as fatty acids, have anticrystallising activity. AIM: To synthesise fatty acid bile acid conjugates (FABACs) and study their effects on cholesterol crystallisation in bile in vitro and in vivo. METHODS: FABACs were prepared by conjugation of cholic acid at position 3 with saturated fatty acids of variable chain length using an amide bond. Cholesterol crystallisation and its kinetics (crystal observation time, crystal mass) were studied in model bile, pooled enriched human bile, and fresh human bile using FABACs with saturated fatty acids of varying chain length (C-6 to C-22). Absorption of FABACs into blood and bile was tested in hamsters. Prevention of biliary cholesterol crystallisation in vivo was tested in hamsters and inbred mice. RESULTS: FABACs strongly inhibited cholesterol crystallisation in model as well as native bile. The FABACs with longer acyl chains (C-16 to C-22) were more effective. At a concentration of 5 mM, FABACs almost completely inhibited cholesterol crystallisation in fresh human bile for 21 days. FABACs were absorbed and found in both portal and heart blood of hamsters. Levels in bile were 2-3 times higher than in blood, indicating active secretion. Appreciable levels were found in the systemic circulation 24-48 hours after a single administration. Ingested FABACs completely prevented the formation of cholesterol crystals in the gall bladders of hamsters and mice fed a lithogenic diet. CONCLUSIONS: FABACs are potent inhibitors of cholesterol crystallisation in bile. They are absorbed and secreted into bile and prevent the earliest step of cholesterol gall stone formation in animals. These compounds may be of potential use in cholesterol gall stone disease in humans.     

TRH is a tonic secretagogue in growth hormone secreting but not in nonfunctioning pituitary tumors

In most patients with growth hormone (GH) secreting pituitary adenomas and clinically nonfunctioning pituitary tumors (NFPT) the intravenous injection of thyrotropin releasing hormone (TRH) augments the secretion of GH and subunits of gonadotropin hormones respectively. Similar hormone responses to TRH have been detected in rat pituitary cell lines and in primary human pituitary tumor cultures in vitro. Nevertheless the TRH effect on tumor hormonal secretion has not been well characterized. In the present study we examined TRH-induced hormone secretion in GH secreting tumors and in NFPT in vitro. Cultured cells secreted betaLH and betaFSH (NFPT) or GH (GH secreting adenomas) up to 14 days in culture. In NFPT TRH (10(-8) mol/l) elicited peak betaLH and betaFSH secretion at 60 to 90 min, with no further increase at 24 h. TRH-stimulated GH secretion peaked at 90-120 min, and decreased after 3 h, but a secondary rise occurred after 24 h of incubation. Chronic daily exposure to TRH followed by an acute TRH challenge resulted in a further increase of GH secretion after one hour. In contrast, acute TRH administration following chronic exposure did not elicit increased P-subunits secretion in NFPT. Coadministration of cycloheximide did not change TRH induced beta-subunits secretion in NFPT. However, when it was administered 24 h prior to TRH, it blocked both basal and TRH induced beta-subunits levels in NFPT. Cycloheximide had no effect on basal or stimulated GH secretion when administered concomitantly or 24 h before TRH. Incubation of cultured GH secreting tumors with cycloheximide during 5 days blocked both basal and TRH stimulated GH secretion, thus indicating dependency on protein synthesis during the chronic, secondary phase. Since the acute secretion was not affected by coadministration of cycloheximide, these early increases in hormone levels apparently reflect the release of stored hormone. In summary, GH secreting adenomas and NFPT differ significantly in their hormonal response to continuous exposure to TRH. The mechanisms underlying the sustained effect of TRH on GH secretion in vitro remain to be investigated. If endogenous TRH exerts a similar continuous effect it may contribute to the disregulated GH secretion in acromegaly.     

Channel shutdown: a response of hippocampal neurons to adverse environments

Stretch-activated ion channels have been discovered in the membrane of many types of cells, but their presence in neurons is uncertain. We used freshly dissociated rat hippocampal neurons to study the effect of hypotonic swelling but, surprisingly, the isolated neurons did not swell. Voltage-dependent whole-cell membrane currents mediated by K⁺, Na⁺ and Ca²⁺ were rapidly and reversibly suppressed during sudden exposure to strongly hypo-osmotic, hyper-osmotic or glucose deficient solutions. The amplitudes of the sustained components of K⁺ and Ca²⁺ currents were more depressed than transient currents, but the rate of decay of transient K⁺ current greatly accelerated. The voltage dependence of activation and of steady state inactivation of residual K⁺ and Ca²⁺ currents were not shifted. The current holding membrane potential at −70 mV and therefore the conductance at that voltage were unchanged or somewhat decreased. Capacitive (charging) membrane current was not affected. Changes in tail current suggested moderate loss of cytosolic K⁺ in some but not in all cells. We conclude that channel shutdown is a uniform response of neuron somata and proximal dendrites to various adverse environments. Hypothetically we propose that swelling was prevented in anisosmotic conditions because membrane water permeability decreased.