Brain mitochondrial ATP-insensitive large conductance Ca⁺²-activated K⁺ channel properties are altered in a rat model of amyloid-ß neurotoxicity.

Mitochondrial dysfunction is a hallmark of amyloid-beta (Aß)-induced neuronal toxicity in Alzheimer's disease (AD). However, the underlying mechanism of how Aß affects mitochondrial function remains uncertain. Because mitochondrial potassium channels have been involved in several mitochondrial functions including cytoprotection, apoptosis and calcium homeostasis, a study was undertaken to investigate whether the gating behavior of the mitochondrial ATP- and ChTx-insensitive-IbTx-sensitive Ca(2+)-activated potassium channel (mitoBKCa) is altered in a rat model of Aß neurotoxicity. Aß1-42 (4 µg/µl) was intracerebroventricularly injected in male Wistar rats (220-250 g). Brain Aß accumulation was confirmed two weeks later on the basis of an immunohistochemistry staining assay, and physiological impacts measured in passive avoidance task cognitive performance experiments. Brain mitochondrial inner membranes were then extracted and membrane vesicles prepared for channel incorporation into bilayer lipid. Purity of the cell fraction was confirmed by Western blot using specific markers of mitochondria, plasma membrane, endoplasmic reticulum, and Golgi. Our results first provide evidence for differences in mitoBKCa ion permeation properties with channels coming from Aß vesicle preparations characterized by an inward rectifying I-V curve, in contrast to control mitoBKCa channels which showed a linear I-V relationship under the same ionic conditions (200 mM cis/50mM trans). More importantly the open probability of channels from Aß vesicles appeared 1.5 to 2.5 smaller compared to controls, the most significant decrease being observed at depolarizing potentials (30 mV to 50 mV). Because BKCa-ß4 subunit has been documented to shift the BKCa channel voltage dependence curve, a Western blot analysis was undertaken where expression of mitoBKCa α and ß4 subunits was estimated using anti-α and ß4 subunit antibodies. Our results indicated a significant increase in mitoBKCa-ß4 subunit expression coupled to a decrease in the expression of α subunit. Our results thus demonstrate a modification in the mitoBKCa channel gating properties in membrane preparations coming from a rat model of Aß neurotoxicity, an effect potentially linked to a change in mitoBKCa-ß4 and -α subunits expression or increased ROS production due to an enhanced Aß mitochondrial accumulation. Our results may provide new insights into the cellular mechanisms underlying mitochondrial dysfunctions in Aß neurotoxicity.

Impairment of brain mitochondrial charybdotoxin- and ATP-insensitive BK channel activities in diabetes.

Existing evidence indicates an impairment of mitochondrial functions and alterations in potassium channel activities in diabetes. Because mitochondrial potassium channels have been involved in several mitochondrial functions including cytoprotection, apoptosis and calcium homeostasis, a study was carried out to consider whether the gating behavior of the mitochondrial ATP- and ChTx-insensitive Ca(2+)-activated potassium channel (mitoBKCa) is altered in a streptozotocin (STZ) model of diabetes. Using ion channel incorporation of brain mitochondrial inner membrane into the bilayer lipid membrane, we provide in this work evidence for modifications of the mitoBKCa ion permeation properties with channels from vesicles preparations coming from diabetic rats characterized by a significant decrease in conductance. More importantly, the open probability of channels from diabetic rats was reduced 1.5-2.5 fold compared to control, the most significant decrease being observed at depolarizing potentials. Because BKCa ß4 subunit has been documented to left shift the BKCa channel voltage dependence curve in high Ca(2+) conditions, a Western blot analysis was undertaken where the expression of mitoBKCa α and ß4 subunits was estimated using of anti-α and ß4 subunit antibodies. Our results indicated a significant decrease in mitoBKCa ß4 subunit expression coupled to a decrease in the expression of α subunit, an observation compatible with the observed decrease in Ca(2+) sensitivity. Our results thus demonstrate a modification in the mitoBKCa channel gating properties in membrane preparations coming from STZ model of diabetic rats, an effect potentially linked to a change in mitoBKCa ß4 and α subunits expression and/or to an increase in reactive oxygen species production in high glucose conditions.

Gastric acid secretion induced by paraventricular nucleus microinjection of orexin A is mediated through activation of neuropeptide Yergic system.

UNLABELLED: Very recently, we have reported that the modulatory effect of PVN on gastric acid secretion may be mediated through the orexin fibers and/or orexin-responsive neurons. In this study, we address the hypothesis which demonstrates the existence of a putative orexin A - neuropeptide Y Y1/Y5 receptors interaction to increase gastric acid secretion in pyloric-ligated conscious rats. Male Wistar rats were implanted with guide canula directed to the PVN and lateral ventricle. Intracerebroventricular (ICV) microinjections of GR-231118 (Y1 receptor antagonist) and CGP-71683 (Y5 receptor antagonist) on gastric acid secretion were considered. The effect of pretreatment with Y1 receptor antagonist, GR-231118, and Y5 receptor antagonist, CGP-71683, on PVN orexin A-induced acid secretion was assessed. Gastric acid secretion was measured using the pylorus-ligation method, and the amount of gastric acid was determined by titration with 0.01N NaOH to a pH of 7.0. KEY RESULTS: ICV microinjections of GR-231118 and CGP-71683 decreased acid secretion by 25±0.05% and 67±0.02%, respectively. ICV microinjections of GR-231118 and CGP-71683 inhibited effects of PVN-injected orexin-A on acid secretion. We suggest that Y1 and Y5 receptors stimulate gastric acid secretion and the stimulatory effect of PVN orexin receptors on gastric acid secretion may be mediated via interactions, at least in part, through activation of Y1 and Y5 receptors. These neural pathways may play key roles in the orexinergic action of orexins in the cephalic phase of gastric acid secretion.

Effects of orexin and glucose microinjected into the hypothalamic paraventricular nucleus on gastric acid secretion in conscious rats.

BACKGROUND: Orexin-A is a novel peptide that appears to play a role in regulation of gastric acid secretion. However, little is known about sites of its action. In addition, evidences suggest that some of orexin-A neurons respond to glucose. In this study, we address the hypothesis which demonstrates that orexin-A and glucose act in the hypothalamic paraventricular nucleus (PVN) to increase gastric acid secretion and juice volume in pyloric-ligated conscious rats. METHODS: Male Wistar rats were implanted with guide canula directed to the PVN. Orexin-A (3-10 µg), glucose (350-750 ng) SB334867 (6-20 µg) were microinjected. The effect of pretreatment with an orexin-1 receptor antagonist, SB334867, on orexin-A and D-glucose induced acid secretion was assessed. Gastric acid secretion was measured using the pylorus-ligation method, and the amount of gastric acid was determined by titration with 0.01 N NaOH to a pH of 7.0. KEY RESULTS: Intraparaventricular injection of orexin-A or D-glucose stimulated gastric acid secretion in a dose-dependent manner. The PVN injections of orexin-A receptor antagonist, SB334867, were associated with gastric acid secretion decrease and inhibited effects of PVN-injected orexin-A. Orexin-stimulated gastric acid secretion was decreased (~40%) after PVN lesions. Glucose-stimulated gastric acid secretion was also suppressed by intraperitoneal (IP) injection of SB334867. In addition, it was observed that co-injection of orexin-A and glucose at ineffective doses increased gastric secretion significantly. CONCLUSIONS & INFERENCES: We suggest that orexin-A and glucose effects on the PVN stimulate gastric acid secretion. This stimulatory effect is probably mediated by orexin-1 receptors.

How many types of large conductance Ca⁺²-activated potassium channels exist in brain mitochondrial inner membrane: evidence for a new mitochondrial large conductance Ca²âº-activated potassium channel in brain mitochondria.

Recently, we have reported electropharmacological properties of a charybdotoxin (ChTx)- and ATP-insensitive-iberiotoxin (IbTx)-sensitive large conductance Ca⁺²-activated potassium (BKCa) channel in almost purified brain mitochondrial inner membrane vesicles. In this work, we report the single-channel characterization of a new BK channel from rat brain mitochondrial inner membrane (mitochondrial large conductance Ca²âº-activated potassium channel, mitoBKCa channel) incorporated into a planar lipid bilayer. The channel conductance was 565 pS in 200 mM KCl cis/50 mM KCl trans. The channel open probability appeared voltage-independent at -40 to +40 mV. Adding 10 mM 4-aminopyridine (4-AP) at positive and negative potentials and 2.5 mM ATP at positive voltages inhibited the channel activities. Notably, addition of 70 µM glibenclamide to the cis side had no effect on the channel behavior. Hence, it can be concluded that there are, at least, two different types of mitoBKCa channels in brain mitochondrial membrane, and the IbTx-, ChTx-, and ATP-sensitive mitoBKCa channels may be activated during the decline of cell metabolism.

Electro-pharmacological profile of a mitochondrial inner membrane big-potassium channel from rat brain.

Recent studies have indicated a calcium-activated large conductance potassium channel in rat brain mitochondrial inner membrane (mitoBK channel). Accordingly, we have characterized the functional and pharmacological profile of a BK channel from rat brain mitochondria in the present study. Brain mitochondrial inner membrane preparations were subjected to SDS-PAGE analysis and channel protein reconstitution into planar lipid bilayers. Western blotting and antibodies directed against various cellular proteins revealed that mitochondrial inner membrane fractions did not contain specific proteins of the other subcellular compartments except a very small fraction of endoplasmic reticulum. Channel incorporation into planar lipid bilayers revealed a voltage dependent 211 pS potassium channel with a voltage for half activation (V(1/2)) of 11.4±1.1mV and an effective gating charge z(d) of 4.7±0.9. Gating and conducting behaviors of this channel were unaffected by the addition of 2.5mM ATP, and 500 nM charybdotoxin (ChTx), but the channel appeared sensitive to 100 nM iberiotoxin (IbTx). Adding 10mM TEA at positive potentials and 10mM 4-AP at negative or positive voltages inhibited the channel activities. These results demonstrate that the mitoBK channel, present in brain mitochondrial inner membrane, displays different pharmacological properties than those classically described for plasma membrane, especially in regard to its sensitivity to iberiotoxin and charybdotoxin sensitivity.

Role of the ventromedial hypothalamic orexin-1 receptors in regulation of gastric Acid secretion in conscious rats.

Orexins play an important role on the central nervous system to modulate gastric acid secretion. The orexin receptors are distributed within the hypothalamus, and expression of orexin-1 receptors (OX1R) is greatest in the anterior hypothalamus and ventromedial nucleus. Therefore, we hypothesised that ventromedial hypothalamic OX1R may be involved in the control of gastric acid secretion. To address this question, we examined the effects of orexin-A and a selective OX1R antagonist, SB-3345867, on gastric acid secretion in pyloric-ligated conscious rats. Intraventromedial injection of orexin-A (0.5-2 microg/microl) stimulated gastric acid secretion in a dose-dependent manner. This stimulatory effect of orexin-A persisted over 3 h. In some experiments, SB-3345867 (10 mg/kg i.p.) was administered 30 min before orexin-A or saline injections. We found that i.p. injection of SB-334867 suppressed stimulated gastric acid secretion induced by orexin-A (2 microg/microl). Atropine (5 mg/kg) also inhibited the stimulatory effect of central injection of orexin-A on acid secretion. In conclusion, the present study suggests that endogenous orexin-A acts on the ventromedial hypothalamus to stimulates acid secretion. This stimulatory effect is probably mediated through OX1R.

Characterization of a chloride-selective channel from rough endoplasmic reticulum membranes of rat hepatocytes: evidence for a block by phosphate.

We have characterized the conduction and blocking properties of a chloride channel from rough endoplasmic reticulum membranes of rat hepatocytes after incorporation into a planar lipid bilayer. Our experiments revealed the existence of a channel with a mean conductance of 164 +/- 5 pS in symmetrical 200 mm KCl solutions. We determined that the channel was ten times more permeable for Cl- than for K+, calculated from the reversal potential using the Goldman-Hodgkin-Katz equation. The channel was voltage dependent, with an open probability value ranging from 0.9 at -20 mV to 0.4 at +60 mV. In addition to its fully open state, the channel could also enter a flickering state, which appeared to involve rapid transitions to zero current level. Our results showed a decrease of the channel mean open time combined with an increase of the channel mean closed time at positive potentials. An analysis of the dwell time distributions for the open and closed intervals led to the conclusion that the observed fluctuation pattern was compatible with a kinetic scheme containing a single open state and a minimum of three closed states. The permeability sequence for test halides determined from reversal potentials was Br- > Cl- > I- approximately F-. The voltage dependence of the open probability was modified by the presence of halides in trans with a sequence reflecting the permeability sequence, suggesting that permeant anions such as Br- and Cl- have access to an internal site capable of controlling channel gating. Adding NPPB to the cis chamber inhibited the channel activity by increasing fast flickering and generating long silent periods, whereas channel activity was not affected by 50 microM DNDS in trans. The channel was reversibly inhibited by adding phosphate to the trans chamber. The inhibitory effect of phosphate was voltage-dependent and could be reversed by addition of Cl-. Our results suggest that channel block involves the interaction of HPO2-4 with a site located at 70% of the membrane span.

Differential effects of dopamine agonists on locomotion in intact and reserpine-treated mice.

1. Apomorphine and bromocriptine induced a dose-dependent locomotion in mice. The responses of both drugs were decreased by SCH 23390 or sulpiride pretreatment. 2. Locomotor activity induced by apomorphine was increased and that of bromocriptine was decreased by reserpine. 3. SKF 38393 or quinpirole also induced locomotion. The response of SKF 38393 was decreased by reserpine. 4. Combination of SKF 38393 with bromocriptine induced a significant locomotor activity different from that of SKF 38393 or bromocriptine in reserpinized animals. 5. Combination of quinpirole with bromocriptine even decreased the response of bromocriptine in intact animals. 6. In conclusion, bromocriptine needs intact dopaminergic neurons and activation of D-1 receptor for expression of locomotion. High doses of quinpirole may induce locomotor activity possibly through D-1/D-2 receptor activation and quinpirole may potentiate the effect of bromocriptine on autoreceptor for inducing sedation.