Targeting acute ischemic stroke with a calcium-sensitive opener of maxi-K potassium channels.

During ischemic stroke, neurons at risk are exposed to pathologically high levels of intracellular calcium (Ca++), initiating a fatal biochemical cascade. To protect these neurons, we have developed openers of large-conductance, Ca++-activated (maxi-K or BK) potassium channels, thereby augmenting an endogenous mechanism for regulating Ca++ entry and membrane potential. The novel fluoro-oxindoles BMS-204352 and racemic compound 1 are potent, effective and uniquely Ca++-sensitive openers of maxi-K channels. In rat models of permanent large-vessel stroke, BMS-204352 provided significant levels of cortical neuroprotection when administered two hours after the onset of occlusion, but had no effects on blood pressure or cerebral blood flow. This novel approach may restrict Ca++ entry in neurons at risk while having minimal side effects.

Differential effects of coadministration of fluoxetine and WAY-100635 on serotonergic neurotransmission in vivo: sensitivity to sequence of injections.

Serotonin 5-HT(1A) receptor antagonists potentiate the effects of serotonin reuptake inhibitors on extracellular serotonin levels in a variety of brain regions. These effects are quite variable, however, with reports indicating potentiations of anywhere from 100-1900%. One factor that might impact the magnitude of such potentiations is the timing of administration of the two agents; reports in which the reuptake inhibitor is given prior to the serotonin receptor antagonist consistently report larger potentiations than reports in which the antagonist is given first. To test this relationship directly, microdialysis and electrophysiology studies were performed to assess the magnitude of increase in extracellular serotonin and changes in cellular activity produced by the serotonin reuptake inhibitor fluoxetine and the 5-HT(1A) receptor antagonist WAY-100635 under various dosing regimens. In microdialysis studies, when WAY-100635 (0.5 mg/kg s.c.) was administered 80 min after fluoxetine (10 mg/kg i.p.) the increase in serotonin was more than twice that observed when the compounds were coadministered. In electrophysiology studies in vivo, WAY-100635 reversed the depression of cell firing produced by fluoxetine when administered 30 min after fluoxetine, but when the two compounds were coadministered, a depression in firing rate was observed comparable to that produced by fluoxetine alone. In contrast, slice recording studies showed that WAY-100635 blocked the effects of fluoxetine regardless of the order of administration. These results indicate that fluoxetine and WAY-100635 can interact in a fashion not predicted by the currently accepted model. It is likely that neuronal circuitry outside of the raphe nuclei underlies this relationship.

A reexamination of the role of GABA in the mammalian suprachiasmatic nucleus.

Three independent electrophysiological approaches in hypothalamic slices were used to test the hypothesis that gamma-amino butyric acid (GABA)A receptor activation excites suprachiasmatic nucleus (SCN) neurons during the subjective day, consistent with a recent report. First, multiple-unit recordings during either the subjective day or night showed that GABA or muscimol inhibited firing activity of the SCN population in a dose-dependent manner. Second, cell-attached recordings during the subjective day demonstrated an inhibitory effect of bath- or microapplied GABA on action currents of single SCN neurons. Third, gramicidin perforated-patch recordings showed that bicuculline increased the spontaneous firing rate during the subjective day. Therefore, electrophysiological data obtained by three different experimental methods provide evidence that GABA is inhibitory rather than excitatory during the subjective day.

Phase shifting of circadian rhythms and depression of neuronal activity in the rat suprachiasmatic nucleus by neuropeptide Y: mediation by different receptor subtypes.

Neuropeptide Y (NPY) has been implicated in the phase shifting of circadian rhythms in the hypothalamic suprachiasmatic nucleus (SCN). Using long-term, multiple-neuron recordings, we examined the direct effects and phase-shifting properties of NPY application in rat SCN slices in vitro (n = 453). Application of NPY and peptide YY to SCN slices at circadian time (CT) 7.5-8.5 produced concentration-dependent, reversible inhibition of cell firing and a subsequent significant phase advance. Several lines of evidence indicated that these two effects of NPY were mediated by different receptors. NPY-induced inhibition and phase shifting had different concentration-response relationships and very different phase-response relationships. NPY-induced phase advances, but not inhibition, were blocked by the GABAA antagonist bicuculline, suggesting that NPY-mediated modulation of GABA may be an underlying mechanism whereby NPY phase shifts the circadian clock. Application of the Y2 receptor agonists NPY 13-36 and (Cys2,8-aminooctanoic acid5,24,D-Cys27)-NPY advanced the peak of the circadian rhythm but did not inhibit cell firing. The Y1 and Y5 agonist [Leu31,Pro34]-NPY evoked a substantial inhibition of discharge but did not generate a phase shift. NPY-induced inhibition was not blocked by the specific Y1 antagonist BIBP-3226; the antagonist also had no effect on the timing of the peak of the circadian rhythm. Application of the Y5 agonist [D-Trp32]-NPY produced only direct neuronal inhibition. These are the first data to indicate that at least two functional populations of NPY receptors exist in the SCN, distinguishable on the basis of pharmacology, each mediating a different physiological response to NPY application.

Molecular dissection of two distinct actions of melatonin on the suprachiasmatic circadian clock.

The pineal hormone melatonin elicits two effects on the suprachiasmatic nuclei (SCN): acute neuronal inhibition and phase-shifting. Melatonin evokes its biological effects through G protein-coupled receptors. Since the Mel1a melatonin receptor may transduce the major neurobiological actions of melatonin in mammals, we examined whether it mediates both melatonin effects on SCN function by using mice with targeted disruption of the Mel1a receptor. The Mel1a receptor accounts for all detectable, high affinity melatonin binding in mouse brain. Functionally, this receptor is necessary for the acute inhibitory action of melatonin on the SCN. Melatonin-induced phase shifts, however, are only modestly altered in the receptor-deficient mice; pertussis toxin still blocks melatonin-induced phase shifts in Mel1a receptor-deficient mice. The other melatonin receptor subtype, the Mel1b receptor, is expressed in mouse SCN, implicating it in the phase-shifting response. The results provide a molecular basis for two distinct, mechanistically separable effects of melatonin on SCN physiology.

Extreme derangements of acid-base balance in exercise: advantages and limitations of the Stewart analysis.

The acid-base analysis method described by Stewart (1981) was applied to the greyhound, an animal that undergoes large changes in intra- and extracellular hydrogen ion concentrations during a race. Increases in plasma [H+] especially during the first 15 min of recovery, induced by increases in lactate concentration in the plasma, were reduced by lowering of PCO2 (hyperventilation) and removal of Cl- from the plasma. [H+] calculated by the Stewart method is similar to that measured directly with a pH electrode when the strong ion difference is within 10 meq/L of resting values (approximately 40 meq/L); thus the measured independent variables were sufficient to account for the [H+] using the Stewart analysis. When the strong ion difference became lower than 30 meq/L, increased variability between measured and calculated [H+] occurred. An error analysis demonstrated the importance of minimizing measurement error of all independent variables, including as many strong and weak electrolytes as possible in the analyses, using the most accurate dissociation constants possible, and understanding the dissociation behavior of the weak electrolytes, especially the plasma proteins, when using the Stewart analysis. The Stewart method of analyzing acid-base balance can contribute to improved training methods for obtaining maximum exercise performance.

Fluid, electrolyte, and packed cell volume shifts in racing greyhounds.

Arterial blood samples were obtained at rest, just before, and 5 minutes after a 704-m race, to quantify changes in hematologic variables, plasma electrolyte and protein concentrations, osmolality, and acid/base variables. Changes in plasma volume were estimated from the change in plasma protein concentration. Immediately prior to the race, plasma volume decreased by 10% from rest and total circulating RBC volume increased by 60%, attributable to increased RBC number rather than size. Increases in blood volume (VB) by 24% and PCV by 29% also were detected before the race. Five minutes after the race, plasma volume was 21% below the resting value and total circulating RBC volume had increased 73% above the resting value, resulting in a 40% increase in PCV. Contraction of the spleen appeared responsible for increased PCV and VB before the race and maintenance of VB after the race. Plasma chloride concentration was the same before and after the race; the chloride content of the plasma decreased by the same fraction (22%) as did the plasma volume, indicating Cl- loss from the plasma. Plasma Na+ content decreased by a smaller fraction (13%), causing Na+ concentration to increase from 151 mEq/L at rest to 167 mEq/L after the race. Assuming that Na+ concentration was the same throughout the extracellular fluid, H2O likely moved into the intracellular compartment. As a consequence of these changes, the inorganic strong ion difference in plasma increased by about 16 mEq/L, tending to minimize the acid/base disturbance induced by the 33 mEq/L increase in lactate concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid-base changes in the running greyhound: contributing variables.

To determine the factors responsible for changes in [H+] during and after sprint exercise in the racing greyhound, Stewart's quantitative acid-base analysis was applied to arterial blood plasma samples taken at rest, at 8-s intervals during exercise, and at various intervals up to 30 min after a 402-m spring (approximately 30 s) on the track. [Na+], [K+], [Cl-], [total Ca], [lactate], [albumin], [Pi], PCO2, and pH were measured, and the [H+] was calculated from Stewart's equations. This short sprint caused all measured variables to change significantly. Maximal changes were strong ion difference decreased from 36.7 meq/l at rest to 16.1 meq/l; [albumin] increased from 3.1 g/dl at rest to 3.7 g/dl; PCO2, after decreasing from 39.6 Torr at rest to 27.9 Torr immediately prerace, increased during exercise to 42.8 Torr and then again decreased to near 20 Torr during most of recovery; and [H+] rose from 36.6 neq/l at rest to a peak of 76.6 neq/l. The [H+] calculated using Stewart's analysis was not significantly different from that directly measured. In addition to the increase in lactate and the change in PCO2, changes in [albumin], [Na+], and [Cl-] also influenced [H+] during and after sprint exercise in the running greyhound.

Mechanism of exercise-induced hypoxemia in horses.

Arterial hypoxemia has been reported in horses during heavy exercise, but its mechanism has not been determined. With the use of the multiple inert gas elimination technique, we studied five horses, each on two separate occasions, to determine the physiological basis of the hypoxemia that developed during horizontal treadmill exercise at speeds of 4, 10, 12, and 13-14 m/s. Mean, blood temperature-corrected, arterial PO2 fell from 89.4 Torr at rest to 80.7 and 72.1 Torr at 12 and 13-14 m/s, respectively, whereas corresponding PaCO2 values were 40.3, 40.3, and 39.2 Torr. Alveolar-arterial PO2 differences (AaDO2) thus increased from 11.4 Torr at rest to 24.9 and 30.7 Torr at 12 and 13-14 m/s. In 8 of the 10 studies there was no change in ventilation-perfusion (VA/Q) relationships with exercise (despite bronchoscopic evidence of airway bleeding in 3) and total shunt was always less than 1% of the cardiac output. Below 10 m/s, the AaDO2 was due only to VA/Q mismatch, but at higher speeds, diffusion limitation of O2 uptake was increasingly evident, accounting for 76% of the AaDO2 at 13-14 m/s. Most of the exercise-induced hypoxemia is thus the result of diffusion limitation with a smaller contribution from VA/Q inequality and essentially none from shunting.

O2 transport in the horse during rest and exercise.

We studied mechanisms of O2 transport in 6 adult (2-5 year old) horses at rest and during steady-state exercise on a treadmill (0% slope) at 12 m/s (a submaximal gallop). Oxygen consumption was measured using an open-flow system. Arterial and mixed venous blood samples were simultaneously obtained for measurement of O2 content and hemoglobin concentration. VO2 increased from 1.5 +/- 0.2 L/min at rest to 46.2 +/- 4.8 L/min during exercise. HR increased from a resting value of 36.9 +/- 2.5 bpm to 196.5 +/- 10.9 bpm and the arterio-venous O2 content difference (a-v O2) increased from 4.2 +/- 0.8 ml O2/100 ml blood to 20.3 +/- 1.6 ml O2/100 ml blood. The 30.4-fold increase in oxygen consumption in the horse at submaximal VO2 versus only a 10-fold increase in man at VO2 max demonstrates the marked ability of the horse to transfer O2 at each step in the O2 transport pathway.