Orexin A but not orexin B rapidly enters brain from blood by simple diffusion. (1/209)

We determined the ability of orexin A and orexin B, recently discovered endogenous appetite enhancers, to cross the blood-brain barrier (BBB) of mice. Multiple time-regression analysis showed that an i.v. bolus of 125I-orexin A rapidly entered the brain from the blood, with an influx rate (Ki = 2.5 +/- 0.3 x 10(-4) ml/g.min) many times faster than that of the 99mTc-albumin control. This relatively rapid rate of entry was not reduced by administration of excess orexin A (or leptin) or by fasting for 22 h, even when penetration into only the hypothalamus was measured. Lack of saturability also was shown by perfusion in blood-free buffer. HPLC revealed that most of the injected 125I-orexin A reached the brain as intact peptide. Capillary depletion studies showed that the administered peptide did not remain bound to the endothelial cells comprising the BBB but reached the brain parenchyma. Efflux of 125I-orexin A from the brain occurred at the same rate as 99mTc-albumin. The octanol/buffer partition coefficient of 0.232 showed that orexin A was highly lipophilic, whereas the value for orexin B was only 0.030. Orexin B, moreover, was rapidly degraded in blood, so no 125I-orexin B could be detected in intact form in brain when injected peripherally. Thus, although orexin B is rapidly metabolized in blood and has low lipophilicity, orexin A rapidly crosses the BBB from blood to reach brain tissue by the process of simple diffusion.  (+info)

Dynamics of correlated genetic systems. IV. Multilocus effects of ethanol stress environments. (2/209)

Four replicate populations of Drosophila melanogaster, two reared on medium supplemented with ethanol and two reared on standard medium, were electrophoretically monitored for 28 generations. During the first 12 generations, allelic, genotypic and gametic frequencies were determined for eight polymorphic enzymes: GOT, alpha-GPDH, MDH, ADH, TO, E6, Ec and ODH. Samples from generation 18 and 28 were electrophoretically typed for ADH and alpha-GPDH. In addition, samples from generation 27 were analyzed for the presence of inversion heterozygotes. The experimental results showed rapid gene-frequency divergence between control and treatment populations at the Adh locus in a direction consistent with the activity hierarchy of Adh genotypes. Gene-frequency divergence between control and treatment populations also occurred at the alpha-Gpdh locus, although the agreement among replicates appeared to have broken down by generation 28. No differential gene-frequency change occurred at any of the six remaining marker loci. Furthermore, values of linkage disequilibria among all linked pairs of genes were initially small and remained small throughout the course of the experiment. Taking these facts into account, it is argued that the gene-frequency response observed at ADH is most probably caused by selection at the Adh locus. The gene frequency response at alpha-Gpdh can also be be accounted for in terms of the effect of ethanol on energy metabolism, although other explanations cannot be excluded.  (+info)

Entry of CART into brain is rapid but not inhibited by excess CART or leptin. (3/209)

Cocaine- and amphetamine-regulated transcript (CART) is a new anorectic peptide found in the brain and periphery. It is closely associated with leptin, an anorectic agent saturably transported across the blood-brain barrier (BBB). Using multiple time-regression analysis, we found that CART has a rapid rate of entry into brain from blood. However, there was no self-inhibition with CART, even when perfused in blood-free buffer or in fasted mice, showing a lack of saturation. HPLC showed that at least 58% of the injected CART reached brain tissue in intact form, and capillary depletion with and without washout showed that the CART was not bound to endothelial cells or adherent to vascular components. There was no evidence for an efflux system out of the brain for CART. Thus CART can cross the BBB from blood to brain, but its rapid rate of entry is not inhibited by excess CART or leptin.  (+info)

Lipid-dependent activation of protein kinase C-alpha by normal alcohols. (4/209)

Significant stimulation of protein kinase C-alpha (PKCalpha) by n-alcohols was observed in characterized lipid systems composed of phosphatidylcholine/phosphatidylserine/dioleoylglycerol (PC/PS/DO). The logarithm of the alcohol concentrations to achieve half-maximal PKC stimulation (ED(50)) and of the maximal PKC stimulation by alcohols were both linear functions of alcohol chain length, consistent with the Meyer-Overton effect. Binding of phorbol esters to PKC was not significantly affected by octanol. Octanol increased, up to 4-fold, the affinity of PKC binding to the lipid bilayers in both the absence and presence of DO. However, octanol increased PKC activity much more significantly than it enhanced binding of the enzyme to the lipid bilayers, suggesting that the stimulation of PKC is not merely a reflection of the increase in PKC bilayer binding affinity. (31)P NMR experiments did not reveal formation of non-lamellar phases with octanol. Differential scanning calorimetry suggested that alcohols, like diacylglycerol, induce formation of compositionally distinct domains and the maximal enzyme activity with alcohol resided roughly in the putative domain-coexistence region. These results suggest that alcohols are mimicking diacylglycerol in activating PKC, not by binding to the high affinity phorbol ester binding site, but by altering lipid structure and by enhancing PKC-bilayer binding.  (+info)

Precision of the pacemaker nucleus in a weakly electric fish: network versus cellular influences. (5/209)

We investigated the relative influence of cellular and network properties on the extreme spike timing precision observed in the medullary pacemaker nucleus (Pn) of the weakly electric fish Apteronotus leptorhynchus. Of all known biological rhythms, the electric organ discharge of this and related species is the most temporally precise, with a coefficient of variation (CV = standard deviation/mean period) of 2 x 10(-4) and standard deviation (SD) of 0.12-1.0 micros. The timing of the electric organ discharge is commanded by neurons of the Pn, individual cells of which we show in an in vitro preparation to have only a slightly lesser degree of precision. Among the 100-150 Pn neurons, dye injection into a pacemaker cell resulted in dye coupling in one to five other pacemaker cells and one to three relay cells, consistent with previous results. Relay cell fills, however, showed profuse dendrites and contacts never seen before: relay cell dendrites dye-coupled to one to seven pacemaker and one to seven relay cells. Moderate (0.1-10 nA) intracellular current injection had no effect on a neuron's spiking period, and only slightly modulated its spike amplitude, but could reset the spike phase. In contrast, massive hyperpolarizing current injections (15-25 nA) could force the cell to skip spikes. The relative timing of subthreshold and full spikes suggested that at least some pacemaker cells are likely to be intrinsic oscillators. The relative amplitudes of the subthreshold and full spikes gave a lower bound to the gap junctional coupling coefficient of 0.01-0.08. Three drugs, called gap junction blockers for their mode of action in other preparations, caused immediate and substantial reduction in frequency, altered the phase lag between pairs of neurons, and later caused the spike amplitude to drop, without altering the spike timing precision. Thus we conclude that the high precision of the normal Pn rhythm does not require maximal gap junction conductances between neurons that have ordinary cellular precision. Rather, the spiking precision can be explained as an intrinsic cellular property while the gap junctions act to frequency- and phase-lock the network oscillations.  (+info)

Multiple agents potentiate alpha1-adrenoceptor-induced conduction depression in canine cardiac purkinje fibers. (6/209)

BACKGROUND: Halothane more so than isoflurane potentiates an alpha1-adrenoceptor (alpha1-AR)-mediated action of epinephrine that abnormally slows conduction in Purkinje fibers and may facilitate reentrant arrhythmias. This adverse drug interaction was further evaluated by examining conduction responses to epinephrine in combination with thiopental and propofol, which "sensitize" or reduce the dose of epinephrine required to induce arrhythmias in the heart, and with etomidate, which does not, and responses to epinephrine with verapamil, lidocaine, and l-palmitoyl carnitine, a potential ischemic metabolite. METHODS: Action potentials and conduction times were measured in vitro using two microelectrodes in groups of canine Purkinje fibers stimulated at 150 pulses/min. Conduction was evaluated each minute after exposure to 5 microm epinephrine (or phenylephrine) alone or with the test drugs. Changes in the rate of phase 0 depolarization (Vmax) and the electrotonic spread of intracellular current were measured during exposure to epinephrine with octanol to evaluate the role of inhibition of active and passive (intercellular coupling) membrane properties in the transient depression of conduction velocity. RESULTS: Lidocaine (20 microm) and octanol (0.2 mm) potentiated alpha1-AR-induced conduction depression like halothane (0.4 mm), with maximum depression at 3-5 min of agonist exposure, no decrease of Vmax, and little accentuation at a rapid (250 vs. 150 pulses/min) stimulation rate. Thiopental (95 microm), propofol (50 microm), and verapamil (2 microm) similarly potentiated epinephrine responses, whereas etomidate (10 microm) did not. Between groups, the decrease of velocity induced by epinephrine in the presence of (10 microm) l-palmitoyl carnitine (-18%) was significantly greater than that resulting from epinephrine alone (-6%; 0.05 +info)

Identification of sites of incorporation in the nicotinic acetylcholine receptor of a photoactivatible general anesthetic. (7/209)

Most general anesthetics including long chain aliphatic alcohols act as noncompetitive antagonists of the nicotinic acetylcholine receptor (nAChR). To locate the sites of interaction of a long chain alcohol with the Torpedo nAChR, we have used the photoactivatible alcohol 3-[(3)H]azioctanol, which inhibits the nAChR and photoincorporates into nAChR subunits. At 1 and 275 microm, 3-[(3)H]azioctanol photoincorporated into nAChR subunits with increased incorporation in the alpha-subunit in the desensitized state. The incorporation into the alpha-subunit was mapped to two large proteolytic fragments. One fragment of approximately 20 kDa (alpha V8-20), containing the M1, M2, and M3 transmembrane segments, showed enhanced incorporation in the presence of agonist whereas the other of approximately 10 kDa (alpha V8-10), containing the M4 transmembrane segment, did not show agonist-induced incorporation of label. Within alpha V8-20, the primary site of incorporation was alpha Glu-262 at the C-terminal end of alpha M2, labeled preferentially in the desensitized state. The incorporation at alpha Glu-262 approached saturation between 1 microm, with approximately 6% labeled, and 275 microm, with approximately 30% labeled. Low level incorporation was seen in residues at the agonist binding site and the protein-lipid interface at approximately 1% of the levels in alpha Glu-262. Therefore, the primary binding site of 3-azioctanol is within the ion channel with additional lower affinity interactions within the agonist binding site and at the protein-lipid interface.  (+info)

Mahogany (1377-1428) enters brain by a saturable transport system. (8/209)

The mouse mahogany gene encodes a protein that is involved in the suppression of diet-induced obesity. We studied the ability of its widely conserved C-terminal fragment to cross the blood-brain barrier (BBB) in mice. Multiple-time regression analysis showed that the entry rate (K(i)) of (125)I-mahogany (1377-1428) from blood-to-brain was 5.5 x 10(-4) ml/g. min. After coinjection of unlabeled mahogany (1377-1428), the K(i) was significantly decreased, showing the self-inhibition characteristic of a saturable transport mechanism. The excess mahogany (1377-1428) did not change the influx rate of (99m)Tcalbumin, the vascular control, indicating a lack of disruption of the BBB. Statistically significant cross-inhibition was not seen with agouti-related protein (83-132), melanin-concentrating hormone, epidermal growth factor, leptin, a melanocortin-4 receptor antagonist, or alpha-melanocyte-stimulating hormone. HPLC showed that most of the injected (125)I-mahogany (1377-1428) reached the brain intact, and capillary depletion with washout showed that most of it reached the parenchyma. There was no brain-to-blood efflux system for mahogany (1377-1428) but rather retention after i.c.v. administration, and the octanol/buffer partition coefficient showed low lipophilicity. Thus, the results show that the C-terminal peptide product encoded by the mahogany gene crosses the BBB by a transport mechanism that is saturable. The ability of this system to be regulated indicates the therapeutic potential of mahogany (1377-1428) in the treatment of obesity.  (+info)